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Cissus quadrangularis extract enhances biomineralization through up-regulation of MAPK-dependent alkaline phosphatase activity in osteoblasts
Abstract
Cissus quadrangularis Linn. has been implicated as therapeutic agent for enhancing bone healing. Though its osteogenic activity has been suggested, the underlying mechanism still remains unclear. In the present study, the effects of ethanol extract of C. quadrangularis (CQ-E) on osteoblast differentiation and function were analyzed using murine osteoblastic cells. The results indicated that mRNA expressions of osteoblast-related genes were not affected by the CQ-E treatment. However, alkaline phosphatase (ALP) activity and the extent of mineralized nodules were significantly increased in treated cells compared with controls. The addition of an extracellular regulated kinase 1/2 inhibitor, a Jun N-terminal kinase 1/2/3 inhibitor and a p38 mitogen-activated protein kinase (MAPK) inhibitor resulted in significantly decreased ALP activity, preferentially by p38 MAPK inhibitor. These results suggested that CQ-E may regulate osteoblastic activity by enhancing ALP activity and mineralization process, and the increased ALP activity effect of CQ-E is likely mediated by MAPK-dependent pathway.
... Increased ALP activity and mineralization are markers of RUNX2 transcription, which further induces osteoblastogenesis. [14] These findings (enhanced ALP activity and mineralization) were also observed when CQ hexane fraction (CQ-H) was used in a study with osteoblast cell culture [15] . A comparative study between CQ-H and CQ dichloromethane fraction (CQ-D) on mouse pre-osteoblast cell line reported CQ-H as more effective extract showing early and enhanced mineralization. ...
... CQ upregulates the m-RNA expression of IGFs, which act as mitogenic and differentiative factors for bone cells through an autocrine/paracrine mechanism [24] . In bone marrow MSCs cell culture and murine osteoblastic cell line ALP levels, proliferation and differentiation of MSCs, RUNX2 expression [3,12,14,15] In ovariectomized, osteotomized and implant operated rats serum ALP levels, mechanical strength, microarchitecture, thickness of bones, cartilage tissue formation, osteoblast proliferation, bone formation [16][17][18][19][20] Bone mineralization Deposition of calcium salts in the extracellular matrix by differentiated osteoblasts. ...
... In bone marrow MSCs cell culture and murine osteoblastic cell line extracellular matrix calcification, mineralization nodules, Collagen I, levels of osterix, osteocalcin and osteopontin [3,12,14,15] . ...
... Maternal treatment with C. q is effective against diabetes-induced delayed fetal skeletal ossification 3,4 . The effect of C. q in inducing alkaline phosphatase (ALP) activity was found to be mediated through MAPK activity in murine osteoblastic cells 5 . ...
... C. quandrangularis builds up the chemical composition of the fractured bone namely mucopolysachrides, collagen, calcium (Ca) and phosphorus (P) 6 . Petroleum ether extracts of C. q stimulated osteoblastogenesis and mineralization in bone marrow mesenchymal cells (BMSC) and murine osteoblastic cell lines 4,5 . In vitro studies have shown that ethanolic extracts of C. q increased mRNA and proteins related to the bone formation pathway and (Insulin like growth factor) IGF-I, IGF-II, and IGF binding protein 7,8 . ...
... (3) by enhancing osteoblastogenesis: the components in green tea support osteoblastogenesis by increasing OB survival, proliferation, differentiation and bone formation, (4) by suppressing osteoclastogenesis: The bioactive components in green tea decreases the action of OCs in vivo and reduce osteoclastogenesis in vitro. The effects of green tea include suppressing bone resorption, increasing apoptosis of OCs, and inhibiting the formation of OCs, and (5) probably through osteoimmunological action: GTPs may modulate osteoimmunological activity first, by inhibiting differentiation of OCs through RANKL signaling, and second, by modulating the production of cytokines by immune cells 34 . Matrix metallo protein (MMP)-2 and MMP-9 activities were lower in theaflavin-3,3'-digallate (TFDG). ...
... 11 In addition, proliferation activity observed by osteoblasts treated with C. quadrangularis was via the upregulation of the MAPK pathway. 30 Downstream cellular responses such as proliferation, inflammation, and apoptosis as a result of MAPK activation Kanwar et al are strongly associated to various stimuli. Proinflammatory cytokines such as IL-1β, and TNF-α, NO, and cyclooxygenases play essential roles in the activation of p38 MAPK and c-Jun N-terminal kinases (JNK) pathways. ...
... 40 Previous work with C. quadrangularis has shown increased ALP activity with different osteoblastic cells line. 11,30,41 It was shown that C. quadrangularis and its components were able to increase production of ALP by inducing proliferation, differentiation, and growth of osteoblasts cells. Results from our study correlated with these finding where it was shown that, as compared to the other treatments, C. quadrangularis increased levels of ALP in serum. ...
... 44 Possible mechanisms for the heightened therapeutic activity observed by these herbals is suggested to be due to their interactions with the transcriptional factor Runx2, 20 increased production of IGF, 45 and through the regulation of the MAPK signaling pathways. 30 Further, our study demonstrates for the first time the interaction of these herbals with collagenases, nitric oxides, prostaglandins, and apoptotic proteins. ...
Introduction: Inflammatory mediators are key players in the pathogenesis of osteoarthritis (OA) and bone destruction. Conventional drugs suppress symptomatic activity and have no therapeutic influence on disease. Cissus quadrangularis and Withania somnifera are widely used for the treatment of bone fractures and wounds; however, the cellular and molecular mechanisms regulated by these herbals are still unclear.
Methods: We established an in vitro OA culture model by exposing human chondrocytes to proinflammatory cytokine and interleukin (IL)-1β for 36 hours prior to treatment with the herbals: C. quadrangularis, W. somnifera, and the combination of the two herbals. Cell viability, toxicity, and gene expression of OA modifying agents were examined. In addition, expression of survivin, which is crucial for cell growth, was analyzed. In vivo work on osteotomized rats studied the bone and cartilage regenerative effects of C. quadrangularis, W. somnifera, and the combination therapy.
Results: Exposure of chondrocytes to IL-1β induced significant toxicity and cell death. However, herbal treatment alleviated IL-1β induced cell toxicity and upregulated cell growth and proliferation. C. quadrangularis inhibited gene expression of cytokines and matrix metalloproteinases, known to aggravate cartilage and bone destruction, and augmented expression of survivin by inhibiting p38 MAPK. Interestingly, osteotomized rats treated with C. quadrangularis drastically enhanced alkaline phosphatase and cartilage tissue formation as compared to untreated, W. somnifera only, or the combination of both herbals.
Conclusion: Our findings demonstrate for the first time the signaling mechanisms regulated by C. quadrangularis and W. somnifera in OA and osteogenesis. We suggest that the chondroprotective effects and regenerative ability of these herbals are via the upregulation of survivin that exerts inhibitory effects on the p38 MAPK signaling pathway. These findings thus validate C. quadrangularis as a potential therapeutic for rheumatic disorders.
... Cq is a perennial herb with pharmacological properties of that include excellent antioxidant properties, free radical scavenging potential, antibacterial activity, antiosteoporotic activity, antitumor activity, analgesic activity, antipyretic activity, and bone fracture healing activity [10]. Studies suggest that Cq regulates osteoblastic activity by enhancing alkaline phosphatase activity and mineralization process [11]. Thus, the aim of this study is to assess the osseointegration of Ti surface coated with novel Cissus quandrangularis Chitosan Hydrogel (CqChH) compared to Commercially pure (Cp) Ti implants. ...
... Parisuthiman et al., illustrated in their study that the ethanolic extract of Cq may exert a regulatory effect on osteoblastic activity by enhancing alkaline phosphatase activity, a crucial marker associated with osteoblast differentiation and the mineralization process. These findings are congruent with the observations made in our study [11]. Similarly, Singh et al., conducted a human clinical evaluation in which they compared the ability of a Cq extract, a Moringa olifera extract and a product containing a 4:1:2 ratio of Cissus, Asparagus and Moringa extracts (Osteoseal TM ) to hasten mandibular fracture bone healing and confirmed that the Cq extract alone shortened the duration of bone healing, demonstrating clinical efficacy in decreasing fixation time [26]. ...
Background
Titanium (Ti) implants has been criticized for the tiring wait for osseointegration, often making the patient reconsider implant treatment. Surface treated Ti implants are emerging as a promising solution with superior osseointegration, early loading protocols and shortened period of edentulousness. The aim of this study is to assess the osseointegration of Ti surface coated with novel Cissus quandrangularis Chitosan Hydrogel (CqChH) compared to Commercially pure (Cp) implants.
Methods
24 Cp Ti implants were divided into 2 subgroups (n = 12). The test group consisted of Ti implants surface treated with the novel hydrogel and control group consisted of Cp Ti implants. 3 % CqChH was prepared and was coated on the Ti implants prior to placement in the femur and tibial heads of rabbits. Implant Stability Quotient (ISQ) was recorded at the 6th and 12th week. Animals were sacrificed and subjected to Removal Torque Quotient (RTQ). The samples were retrieved en bloc and stained for histopathologic analysis. The collected data was subjected to statistical analysis using Unpaired student t-Test.
Results
At the end of 6th week CqChH coated implants did not show any statistically significant difference in both ISQ and RTQ values compared to Cp ones. However, at the end of the 12th week CqChH coated implants demonstrated significantly higher ISQ (73.91 ± 4.39) and RTQ (75.96 ± 14.10) compared to Cp ones.
Conclusions
This study demonstrated that the novel hydrogel coating applied to the implant's surface exhibited not only enhanced bone regeneration but also elicited a new bone formation.
... Recent studies have reported anti-osteoporotic activity of CQ in rats (Aswar et al., 2012;Potu et al., 2009a;Shirwaikar et al., 2003), mice (Banu et al., 2012) and rabbits (Stohs & Ray, 2013). CQ extracts have reported to increase calcification during differentiation and mineralization of mesenchymal stem cells into osteoblasts by enhancing alkaline phosphatase activity via mitogen activated protein kinase (MAPK) pathway (Parisuthiman et al., 2009;Potu et al., 2009b). In another study, CQ extract is reported to enhance differentiation and mineralization of human SaOS-2 cell line via insulin like growth factors (IGF) pathway (Muthusami et al., 2011). ...
... Comparable to our results, Muthusami and colleagues have reported 1 and 10μg/ml as mitogenic doses of ethanolic extract of CQ for human osteoblast SaOS-2 cell line (Muthusami et al., 2011). In another study, ethanolic extract has been reported to affect in dose-dependent manner, with 10, 50 and 100 μg/ml as mitogenic doses for MC mouse osteoblast cell line (Parisuthiman et al., 2009). Another study has reported 100, 200 and 300 μg/ml as mitogenic doses of petroleum ether extract of CQ for rat mesenchymal stem cells (Potu et al., 2009b). ...
In continuation of the investigation of osteogenic potential of solvent fractions of ethanolic extract of Cissus quadrangularis (CQ), an ancient medicinal plant, most notably known for its bone‐healing properties, to isolate and identify antiosteoporotic compounds. In the current study, we report the effect of hexane fraction (CQ‐H) and dichloromethane fraction (CQ‐D) of CQ on the differentiation and mineralization of mouse preosteoblast cell line MC3T3‐E1 (subclone 4). Growth, viability, and proliferation assays revealed that low concentrations (0.1, 1, and 100 ng/ml) of both solvent fractions were nontoxic, whereas higher concentrations were toxic to the cells. Differentiation and mineralization of MC3T3‐E1 with nontoxic concentrations of CQ‐D and CQ‐H revealed that CQ‐D delayed the mineralization of MC3T3‐E1 cells. However, early and enhanced mineralization was observed in cultures treated with nontoxic concentrations of CQ‐H, as indicated by Von Kossa staining and expression profile of osteoblast marker genes such as osterix, Runx2, alkaline phosphatase (ALP), collagen (Col1a1), integrin‐related bone sialoprotein (IBSP), osteopontin (OPN), and osteocalcin (OCN). These findings suggest CQ‐H as the most efficacious solvent fraction for further investigation to isolate and identify the active compounds in CQ‐H.
... Runx2 is a key transcription factor regulating premature osteogenesis and delayed mineralization of osteoblasts (13). Previously, in vitro studies have revealed that CQ extract has shown proliferative effects in osteoblastic MC3T3-E1 and SaOS-2 cell lines, other than anti-proliferative and apoptotic activities in human skin carcinoma A431 cells (14)(15)(16). On the basis of these reports, we speculated that CQ might have biphasic effects (proliferative and anti-proliferative) depending upon dose and time factors. Reactive oxygen species (ROS) also play an essential role in cell proliferation and cell cycle progression, depending upon dose and time of exposure (17,18). ...
... On the other hand, decreased cell viability and ALP activity here, were observed at higher CQ concentrations (Fig. 2). In agreement with previous reports, our study also indicated that higher concentrations of CQ extract decreased osteoblast proliferation over longer exposure, suggesting that higher concentration probably reduced bone formation by reducing osteoblast differentiation, accompanied by cell proliferation (14,15). Mineralized nodule formation is a characteristic feature of late-stage osteoblastic differentiation (28). ...
This report highlights phytoconstituents present in Cissus quadrangularis (CQ) extract and examines biphasic (proliferative and anti-proliferative) effects of its extract on bone cell proliferation, differentiation, mineralization, ROS generation, cell cycle progression and Runx2 gene expression in primary rat osteoblasts.
Phytoconstituents were identified using gas chromatography-mass spectroscopy (GC-MS). Osteoblasts were exposed to different concentrations (10-100 μg/ml) of CQ extract and cell proliferation and cell differentiation were investigated at different periods of time. Subsequently, intracellular ROS intensity, apoptosis and matrix mineralization of osteoblasts were evaluated. We performed flow cytometry for DNA content and real-time PCR for Runx2 gene expression analysis.
CQ extract's approximately 40 bioactive compounds of fatty acids, hydrocarbons, vitamins and steroidal derivatives were identified. Osteoblasts exposed to varying concentrations of extract exhibited biphasic variation in cell proliferation and differentiation as a function of dose and time. Moreover, lower concentrations (10-50 μg/ml) of extract slightly reduced ROS intensity, although they enhanced matrix mineralization, DNA content in S phase of the cell cycle, and levels of Runx2 expression. However, higher concentrations (75-100 μg/ml) considerably induced the ROS intensity and nuclear condensation in osteoblasts, while it reduced mineralization level, proportion of cells in S phase and Runx2 level of the osteogenic gene.
These findings suggest that CQ extract revealed concentration-dependent biphasic effects, which would contribute notably to future assessment of pre-clinical efficacy and safety studies.
© 2015 John Wiley & Sons Ltd.
... The supernatants were stored at À 20 1C until the determination of ALP activity. Owen and pan (2008) and Parisuthiman et al. (2009)) The ALP activity was analyzed by determining the enzyme activity required to convert p-nitrophenyl phosphate (p-NPP) to its yellow product, p-nitrophenol (p-NP). A 50-μL aliquot of supernatant was mixed with 150 μL of a p-NPP substrate solution (10 mM p-NPP in 1 mM MgCl 2 and 100 mM Tris buffer, adjusted to pH 10.3 and pre-warmed to 37 1C before use) in a 96-well plate. ...
... c interchangeable. phytoestrogen-rich fraction of CQ ethanolic extract, defined as standardized 2.5% friedelin, possessed mild-to-moderate estrogenic activity in ovariectomized rats (Aswar et al., 2010), and the speculated mechanisms of action of the CQ extract were estrogenic-related signaling pathways (Deng et al., 2008;Hawse et al., 2008;Muthusami et al., 2011a;Muthusami et al., 2011b;Parisuthiman et al., 2009). Together, these studies demonstrate that the CQ plant possesses multi-target synergistic activity on bone formation via both estrogenic-dependent and -independent pathways. ...
... The supernatants were stored at À 20 1C until the determination of ALP activity. Owen and pan (2008) and Parisuthiman et al. (2009)) The ALP activity was analyzed by determining the enzyme activity required to convert p-nitrophenyl phosphate (p-NPP) to its yellow product, p-nitrophenol (p-NP). A 50-μL aliquot of supernatant was mixed with 150 μL of a p-NPP substrate solution (10 mM p-NPP in 1 mM MgCl 2 and 100 mM Tris buffer, adjusted to pH 10.3 and pre-warmed to 37 1C before use) in a 96-well plate. ...
... c interchangeable. phytoestrogen-rich fraction of CQ ethanolic extract, defined as standardized 2.5% friedelin, possessed mild-to-moderate estrogenic activity in ovariectomized rats (Aswar et al., 2010), and the speculated mechanisms of action of the CQ extract were estrogenic-related signaling pathways (Deng et al., 2008;Hawse et al., 2008;Muthusami et al., 2011a;Muthusami et al., 2011b;Parisuthiman et al., 2009). Together, these studies demonstrate that the CQ plant possesses multi-target synergistic activity on bone formation via both estrogenic-dependent and -independent pathways. ...
... In the present study, LG was shown to inhibit the p38MAPK signaling pathway, thus demonstrating that the inhibitory effect induced by LG is via future science group the activation of LRP mechanisms. Furthermore, other studies that correlate with the present study regarding the signaling pathways activated by the components of LG include work performed by Singh et al. [48], Ichikawa et al. [6] and Parisuthiman et al. [49]. Work by Singh and Ichikawa reported the ability of W. somnifera to modulate various pathways, and found that it had direct inhibitory effects on the NF-kB, JNK and AP-1 pathways. ...
... Work by Singh and Ichikawa reported the ability of W. somnifera to modulate various pathways, and found that it had direct inhibitory effects on the NF-kB, JNK and AP-1 pathways. Similarly, C. quadrangularis, another component of LG, was found to significantly increase bone mineralization and alkaline phosphatase activity by impeding the activation of the JNK, ERK1/2 and p38MAPK pathways [49]. In addition, chitosan has been shown to modulate inflammatory diseases by inhibiting the expression of TNF-a and IL-6, and through the inhibition of NF-kB signaling [50]. ...
Aim:
This study aimed to evaluate the antiarthritic and chondroprotective potentials of Lakshadi Guggul (LG) and Cissus quadrangularis encapsulated in novel alginate-enclosed chitosan-calcium phosphate nanocarriers (NCs) both in vitro in primary human chondrocytes and in vivo in mice with collagen-induced arthritis.
Materials & methods:
Chondrocytes exposed to IL-1β and osteoarthritis chondrocytes grown in an ex vivo inflammation-based coculture were incubated with different concentrations of herbals, and cell modulatory activities were determined. For in vivo studies, herbals and their encapsulated nanoformulations were administered orally to DBA/1 mice with collagen-induced arthritis.
Results:
C. quadrangularis and LG showed enhanced chondroprotective and proliferative activity in IL-1β-exposed primary chondrocytes, with LG showing the highest therapeutic potency. LG increased viability, proliferative and mitogenic activity, and inhibited cell apoptosis and mitochondrial depolarization. In vivo studies with LG and alginate-enclosed chitosan-calcium phosphate LG NCs revealed cartilage regenerative activity in those administered with the nanoformulation. The NCs were nontoxic to mice, reduced joint swelling and paw volume, and inhibited gene expression of MMPs and cytokines.
Conclusion:
The promising results from this study reveal, for the first time, the novel polymeric NC encapsulating LG as a potential therapeutic for rheumatic diseases.
... CQ is used to treat various bone disorders and can also be used as a preventive measure for disorders that lead to decreased bone mineral density 21 In ovariectomized rats, it was found that CQ increases apoptosis of osteoclasts thereby preventing boneloss. The same study has also reported the ability of CQ in converting procollagen to collagen 22 Another study involving ethanolic extract of CQ has shown increased osteoblastic activity enabled through MAPkinase pathway 24 . ...
Cissus quadrangularis (CQ) is a widely known herb in India and southern part of India in particular. The bone healing ability of CQ is outlined in Ayurvedic medicine and other ancient medical texts. Bone loss in elderly especially osteoporosis is a public health problem across the globe. Bioactive molecules present in medicinal plants like CQ helps not only in bone healing but also in improving overall health. CQ is suggested as a plant herb in treatment of bone fractures and bone loss as it is safe or usage and side effects are not identified so far.However, research on the effect of CQ in humans are few and far. This review attempts to give a clear picture on the available literature that describes the osteogenic role of CQ in improving bone health and healing fractures in general and maxillofacial fractures in particular. Also, the role of CQ in green synthesis of bone scaffolds and its osteogenic potential in regeneration of bone using scaffolds are also discussed.
... Many modern-day studies validate the use of C. quadrangularis as a potent anti-osteoporotic drug. 10,11 Bhat and Chowdhary showed the osteogenic potential of the C. quadrangularis extract in vitro. 12 C. quadrangularis has lately been applied in dentistry as well mainly for mandibular fracture healing. ...
Aim:
The aim of the study was to evaluate the osteogenic potential of Cissus quadrangularis (CQ) hydrogel in enhancing the osseointegration of titanium to the bone in an experimental rabbit model.
Materials and methods:
Six adult male New Zealand white rabbits were used in this study. A total of 24 implants (12 coated test implants and 12 uncoated control implants) were placed in these 6 rabbits. A polyethylene glycol (PEG) hydrogel was prepared with the C. quadrangularis hydrogel in which the test implants were coated. Each rabbit was operated on both hind legs and one implant, each, was placed in the femur and tibia. Hence, one rabbit received four implants [two test implants (HG coated) and two control implants (uncoated)]. The animals were sacrificed after 4 weeks, and the specimens were histomorphometrically analyzed. The bone-to-implant contact (BIC) and the bone area fraction occupancy (BAFO) were calculated using Image J analysis.
Results:
The statistically analyzed values which were obtained by paired t-test, revealed that the average mean values were higher in the test implants (coated) than the control implants (uncoated). The BIC values of the test implants were not significantly different from the control implants in the case of both femur and tibia (p >0.05). The test implants showed significantly increased BAFO values in femur (p <0.05). However, the BAFO values of test implants in tibia did not vary significantly from the control implants.
Conclusion:
Based on the findings of the study, the authors conclude that the coating of C. quadrangularis hydrogel enhances the osseointegration of titanium implants to bone. The further studies need to be designed to check the osseointegrative potential of C. quadrangularis.
Clinical significance:
The findings of this study suggest that the C. quadrangularis hydrogel is a potent osteogenic material that can reduce the osseointegration period and thus enhance the patient compliance toward implant treatment.
... Plants have served as the central provenance of traditional medicine for healing and persist as a relevant resource of inspiration for several pharmaceutical formulations (Anand et al. 2019). Recent years witnessed a significant research interest in investigating herbal medicine as a potential alternative therapeutic option (Parisuthiman et al. 2009). ...
Contemporary demand calls for a high restorative index as an indispensable requirement for bone tissue engineering scaffolds, where therapeutic agents of natural origin function as a modulator for new bone formation become of utmost importance. The study presents a systematic investigation of the edible stem part of Cissus quadrangularis (CQ) as a natural resource of bioactive metabolites capable of invoking early biomineralization and osteogenesis in vitro. Phytochemical screening of CQ stem extracts (sequential solvent extraction: polarity hexane\chloroform\ethyl acetate\methanol\water) was performed by qualitative and quantitative assays, which are substantiated by UV-Visible and FTIR spectroscopic analyses. Cytocompatibility and proliferation index of the extracts and their effect on cellular architecture were investigated by MTT assay (different time points) and F-actin/DAPI staining, respectively, in Human Osteosarcoma (HOS) cells. MTT results exemplified profound cell proliferation index for hexane (HE) and aqueous (WE) extracts (24 and 48 h), while Alizarin Red S and von-Kossa staining validated early biomineralization (day 7). The activity of the early bone marker, Alkaline phosphatase (ALP), on day 7 authenticates the effective osteogenic potential of HE and WE compared to other extracts, configuring abundance of bone regenerative phytochemicals in HE and WE and presenting ample opportunities for customized bone tissue engineering.
... Typical constituents of these extracts are stilbenoids like quadrangularin A and pallidol (Figure 9). Extracts of C. quadrangularis have been experimentally demonstrated to accelerate the healing of fractured jaw bones in an animal model (Brahmkshatriya et al., 2015), to alleviate bone deterioration in osteotomized rats via p38 MAPK signalling (Kanwar et al., 2015), to up-regulate the matrix mineralization of human osteoblast like SaOS-2 cells (Muthusami et al., 2011), and to enhance biomineralization through up-regulation of MAPKdependent alkaline phosphatase activity in osteoblasts (Parisuthiman et al., 2009). The validity of the traditional use of the same plant for increasing sperm production has also been experimentally validated (Neuwinger, 1996). ...
Traditional medicines embody knowledge on medicinal plants that has been accumulated through cultural evolution over millennia. In the latter half of the 20th century, two approaches to medicinal plant research have been established: the “Bench to Bedside” and the “Bedside to Bench” approaches which serve primarily for the development of more efficient therapeutics. Here, we propose a third, novel approach: from “Tradition to Pathogenesis” which aims to understand the pathogenesis of diseases based on the cultural evolution of their respective empirical treatments. We analyse multiple examples of diseases where the acting mechanism of traditional treatments across multiple cultures points to the pathogenesis of the respective disease. E.g., many cultures traditionally treat rheumatism with anti-bacterial botanical drugs, which is at odds with our current understanding that rheumatism is an aseptic inflammation. Furthermore, gastric ailments have traditionally been treated with anti-infectious botanical drugs indicating local infections, as demonstrated by the discovery of Helicobacter pylori as a common cause of gastric ulcer. Understanding traditional treatments can thus help to elucidate the pathogenesis of the disease.
... Plants have served as the central provenance of traditional medicine for healing and persist as a relevant resource of inspiration for several pharmaceutical formulations (Anand et al. 2019). Recent years witnessed a significant research interest in investigating herbal medicine as a potential alternative therapeutic option (Parisuthiman et al. 2009). ...
Contemporary demand calls for a high restorative index as an indispensable requirement for bone tissue engineering scaffolds, where therapeutic agents of natural origin function as a modulator for new bone formation become of utmost importance. The study presents a systematic investigation of the edible stem part of Cissus quadrangularis (CQ) as a natural resource of bioactive metabolites capable of invoking early biomineralization and osteogenesis in vitro. Phytochemical screening of CQ stem extracts (sequential solvent extraction: polarity hexane<chloroform<ethyl acetate<methanol<water) was performed by qualitative and quantitative assays, which are substantiated by UV-Visible and FTIR spectroscopic analyses. Cytocompatibility and proliferation index of the extracts and their effect on cellular architecture were investigated by MTT assay (different time points) and F-actin/DAPI staining, respectively, in Human Osteosarcoma (HOS) cells. MTT results exemplified profound cell proliferation index for hexane (HE) and aqueous (WE) extracts (24 and 48 h), while Alizarin Red S and von-Kossa staining validated early biomineralization (day 7). The activity of the early bone marker, Alkaline phosphatase (ALP), on day 7 authenticates the effective osteogenic potential of HE and WE compared to other extracts, configuring abundance of bone regenerative phytochemicals in HE and WE and present ample opportunities for customized bone tissue engineering.
... Both these actions benefit healing of fractures. C.Q acts by up regulating MAPK dependant alkaline phosphatase activity in osteoblasts which leads to increase biomineralization [5]. There is increased DNA synthesis, increased matrix mineralization of human osteoblast like SaOS-2 cells. ...
... The dosedependent effect is in correlation with previous studies on crude CQ extracts and CQ-solvent fractions that have reported similar findings with lower non-cytotoxic doses. [21][22][23][24][25][26][27] To validate the observation of cell viability analysis, effect of non-cytotoxic concentrations of CQ-B was observed on metabolic activity, proliferation and growth parameters of 3T3-L1 cells and selected concentrations (0.0001, 0.01, 0.1 and 10 μg/ml) were found to be non-detrimental for the cells (Fig. 1). Doubling time of the cells was found to be 24.1h with specific growth rate of 0.013. ...
Obesity is marked by the buildup of fat in adipose tissue that increases body weight and the risk of many associated health problems, including diabetes and cardiovascular disease. Treatment options for obesity are limited, and available medications have many side effects. Thus there is a great need to find alternative medicines for treating obesity. This study explores the anti-adipogenic potential of the n-butanol fraction of Cissus quadrangularis (CQ-B) on 3T3-L1 mouse preadipocyte cell line. The expression of various lipogenic marker genes such as adiponectin, peroxisome proliferator-activated receptor gamma, leptin, fatty acid-binding proteins, sterol regulatory element-binding proteins, fetal alcohol syndrome, steroyl-CoA desaturase-1, lipoproteins, acetyl-CoA carboxylase alpha, and acetyl-CoA carboxylase beta were variously significantly downregulated. After establishing the anti-adipogenic potential of CQ-B, it was fractionated to isolate anti-adipogenic compounds. We observed significant reduction in neutral lipid content of differentiated cells treated with various fractions of CQ-B. Gas chromatography-mass spectrometry analysis revealed the presence of thirteen compounds with reported anti-adipogenic activities. Further studies to purify these compounds can offer efficacious and viable treatment options for obesity and related complications.
... Ethanolic extracts of CQ supported bone formation in fetal rats when administered to the pregnant rats [14]. In addition, the ethanol extract of the CQ enhanced alkaline phosphatase (ALP) activity, a classical marker for bone formation, in murine osteoblasts occurs via the MAPK (mitogen-activated protein kinase) signaling pathway [15]. By using an osteoblastic cell line (SaOS-2) as a model, the CQ extract showed its ability to promote cellular proliferation and matrix calcification by promoting the expression of IGF-I, II and IGFBP-3 [16,17]. ...
Background:
Cissus quadrangularis Linn. (CQ) has been used in Indian and Thai traditional medicine for healing bone fractures because of numerous active ingredients in CQ. It is still unclear which compounds are the active ingredients for bone formation.
Methods:
The molecular docking technique, the ethanolic extraction along with hexane fractionation, and an in vitro experiment with a human osteoblast cell line (MG-63) were used to narrow down the active compounds, to prepare the CQ extract, and to test biological activities, respectively.
Results:
The molecular docking technique revealed that quercetin and β-sitosterol had highest and lowest potential to bind to estrogen receptors, respectively. Compared to the crude ethanol extract (P1), the ethanolic fraction (P2) was enriched with rutin and quercetin at 65.36 ± 0.75 and 1.06 ± 0.12 mg/g, respectively. Alkaline phosphatase (ALP) activity was significantly enhanced in osteoblasts exposed to the P2 in both tested concentrations. The amount of hydroxyproline was slightly increased in the P1 treatment, while osteocalcin was inhibited. Moreover, the P2 significantly activated osteoprotegerin (OPG) and inhibited receptor activator of nuclear factor κ ligand (RANKL) expression.
Conclusions:
Taken together, the enriched rutin and quercetin fraction of CQ triggered the molecules involved in bone formation and the molecules inhibiting bone resorption.
... Therefore, it is necessary to quest alternative therapies (natural) that have minimal side effects. Currently, medicines with natural ingredients are efficacious as an alternative choice because herbal medicines are cheaper and safer than synthetic chemical drugs [7]. Research on the utilization of natural ingredients for the prevention and treatment of osteoporosis has also been widely conducted [8]. ...
Aim: This study aimed to determine the potential of honey as anti-osteoporosis by evaluating its effectiveness in increasing bone impact strength and cortical thickness, through scanning electron microscopy (SEM) examination.
Materials and Methods: Forty-five female rats at 3 months of age, weighing 150-200 g were used in the study. They were placed in individual cages and adapted to food and environment for 10 days. On the 11th day, after the animals were adapted for 10 days, the animals were randomly divided into five treatment groups (n=9): Sham operation group (SH); ovariohysterectomized (OVX) group with no treatment; OVX with treatment Apis dorsata 1 g/kg BW (AD-1); OVX with treatment A. dorsata 2 g/kg BW (AD-2); and OVX with treatment A. dorsata 4 g/kg BW (AD-3). Furthermore, those nine rats in each treatment group were divided into three groups. Three of them were observed at months 1st, 2nd, and 3rd so that in each observation taken three rats in each treatment group. At the end of the study, the rats were euthanized and necropsy for taking their second femoral bone, i.e. dexter region for examining their bone impact strength, while the sinister region was used for measure the cortical thickness of the femoral diaphysis and examining their bone microarchitecture using SEM analysis.
Results: Based on results of the ANOVA test, the cortical thickness measurements of femoral diaphyseal can be seen that from month 1 to month 3 the lowest result was found in the group of rats that were OVX-I. Meanwhile, the highest result was found in the group of rats that were not OVX (SH-III). It was significantly different from the other treatment groups (p0.05) from that in the group of rats was not OVX in month 1 (SH-I). The results of the bone impact strength measurement from month 1 to month 3 indicated that the groups of rats were OVX without the administration of honey supplements had the lowest value. The highest bone impact strength was found in the group of rats that was not OVX, but not significantly different (p>0.05) with the groups of rats that were OVX administered honey supplement with a dose of 2 g/kg BW (AD-2) and 4 g/kg BW (AD-3).
Conclusion: The supplement of honey A. dorsata at doses of 2 g/kg BW in the group of rats was that OVX can inhibit the decreasing of the cortical bone thickness and repair damage in microarchitecture to generate bone impact strength. As a result, bones are not easily broken.
... Various in vivo studies have been conducted on CQ and its various extracts that report its fracture healing properties in animal models (Chopra, Patel, & Awadhiya, 1976;Deka, Lahon, Saikia, & Mukit, 1994;Parsad & Udupa, 1963;Pathomwichaiwat et al., 2014;Stohs & Ray, 2012) and describe it as antiosteoporotic agent (Aswar, Mohan, & Bodhankar, 2012;Banu et al., 2012;Potu et al., 2009Potu et al., , 2010Potu, Nampurath, Rao, & Bhat, 2011;Shirwaikar, Khan, & Malini, 2003). Effect of various extracts of CQ has been studied on mesenchymal stem cells (Kumar et al., 2010;Parisuthiman, Singhatanadgit, Dechatiwongse, & Koontongkaew, 2009;Potu et al., 2009), SaOS-2 cells (Muthusami et al., 2011), MC3T3-E1 (Pathomwichaiwat et al., 2014;Tasadduq et al., 2017), which suggest that CQ has potential to be used as antiosteoporotic drug. ...
In a sequel to investigate osteogenic potential of ethanolic extract of Cissus quadrangularis (CQ), the present study reports the osteoblast differentiation and mineralization potential of ethyl acetate (CQ‐EA) and butanol (CQ‐B) extracts of CQ on mouse pre‐osteoblast cell line MC3T3‐E1 (sub‐clone 4) with an objective to isolate an antiosteoporotic compound. Growth curve, proliferation, and viability assays showed that both the extracts were nontoxic to the cells even at high concentration (100 µg/ml). The cell proliferation was enhanced at low concentrations (0.1 µg/ml and 1 µg/ml) of both the extracts. They also upregulated the osteoblast differentiation and mineralization processes in MC3T3‐E1 cells as reflected by expression profile of osteoblast marker genes such as RUNX2, Osterix, Collagen (COL1A1), Alkaline Phosphatase (ALP), Integrin‐related Bone Sialoprotein (IBSP), Osteopontin (OPN), and Osteocalcin (OCN). CQ‐EA treatment resulted in early differentiation and mineralization as compared with the CQ‐B treatment. These findings suggest that low concentrations of CQ‐EA and CQ‐B have proliferative and osteogenic properties. CQ‐EA, however, is more potent osteogenic than CQ‐B. The present study reports the osteoblast differentiation and mineralization potential of ethyl acetate (CQ‐EA) and butanol (CQ‐B) extracts of Cissus quadrangularis (CQ) on mouse pre‐osteoblast cell line MC3T3‐E1 (sub‐clone 4) with an objective to isolate an antiosteoporotic compound. We have concluded in this part of the study that the CQ‐EA treatment resulted in early differentiation and mineralization as compared with the CQ‐B treatment. These findings suggest that low concentrations of CQ‐EA and CQ‐B have proliferative and osteogenic properties. CQ‐EA, however, is more potent osteogenic than CQ‐B.
... Although abovementioned studies were on ovariectomized animals suggestive more of estrogenic potential instead of effect on bone healing, a previous report in 2009 showed that EE increases biomineralization through upregulation of mitogenactivated protein kinasedependent alkaline phosphatase activity in osteoblasts. [26] Cissus is reported to contain certain stilbenes such as resveratrol, some of which are known to have a beneficial effect in bone health. [7,27] However, the quantities reported earlier suggest that the presence of these compounds is minimal. ...
Background: Cissus quadrangularis (CQ) L. reported to contain 3ketosteroids and have bone health benefits.
Aim: This study aimed at establishing the relationship between the ketosteroid content and anabolic as well as bone health promoting activities of various Cissus extracts in well established
orchidectomized (ORX) rat model.
Materials and Methods: Supercritical carbon dioxide, ethyl acetate, and aqueous extracts (AE) of CQ L. were prepared and
standardized for ketosteroid content by two methods used in commerce. Moreover, ketosteroid standardized extracts of this plant were evaluated for anabolic activity in rats in well established ORX rat model.
Results: The increase in the absolute weight was appreciable in the CQAE treated group. Similarly, with respect to bone parameters, a similar trend was seen. The mean bone density, strength, and calcium content were found to be highest in the group treated with CQAE compared to groups treated with other extracts. This study reveals for the first time that 3ketosteroids
are not linked to the beneficial activities on bone and highlights
the need for extensive characterization of biological active principles from CQ L.
... An in vitro studies [30], [31], [32] showed that Curcumin could stop osteoclastogenesis through suppression of RANKL activity completely, another in vivo studies [33], [34], [35], [36], [37] showed that using Curcumin could limit bone loss, improve remodeling and improve bone strength in osteoporosis animal models. For Cissus Quadrangularis an in vitro studies [38], [39], [40] showed its ability in promoting osteoblast proliferation and differentiation and as a potent antiosteoporotic substance in osteoporotic animal model [41], [42], [43]. Add to promote healing of bone fracture [44]. ...
... Estrogen juga merangsang ekpresi dari osteoprotegerin (OPG) dan TGF-β oleh osteoblas dan sel stroma, yang selanjutnya berfungsi menghambat resorpsi tulang dan mempercepat/merangsang apoptosis osteoklas (Bell, 2003 (Dimitriou et al., 2005;Schmidt-Bleek et al., 2009). (Parisuthiman et al., 2009;Potu et al., 2009;Sabri dkk., 2009;Shirwaikar et al., 2003;Deka et al., 1994) (Nikolaou et al, 2009;Kyllönen et al, 2015). ...
This study aims to determine the potential of the Cissus quadrangularis extract to decreased the expression of IL-1 β so that it can to inhibit bone resorption and osteoporosis fracture healing is not delayed union. Forty rats adapted for 1 week, and then divided into four groups with 10 replications. P0 (sham-operated + Osteotomy + CMC Na); P1 (ovariectomy + Osteotomy + CMC Na); P2 (ovariectomy + Osteotomy + Raloxifene 5.4 mg /kg); P3 (ovariectomy + Osteotomy + CQ 750 mg / kg). Osteoporosis induced is made by bilateral ovariectomy in rats, then to make sure that osteoporosis is already happened, 8 weeks post-ovariectomy performed radiology examinations on the femur, then the osteotomy action. IL1-β expression observations performed in at the 2nd week and 6th week after osteotomy through the immunohistochemistry examination. The results of immunohistochemistry examination in the 2nd week showed an increased expression of IL1-β in all groups of rat that did ovariectomy (P1, P2 and P3) were significantly different (p <0.05) with a rat group that didn’t ovariectomy, as well P2 dan P3 were significantly different with P1. The Results of immunohistochemistry examination in the 6th week showed the samepattern with the 2nd week and there is increased IL1-β expression in all groups of rat compared with the results of the examination in the 2nd week. Based on these results it can be concluded that Cissus quadrangularis extract andRaloxifen can decreased the expression of IL-1 β, and the results that don’t significantly different, so that these both materials can to inhibit bone resorption and osteoporosis fracture healing don’t get delayed union.
... Furthermore, the role of CQ in RANK-RANKL expression and osteoclast differentiation and activity needs to be identified. Recently, it has been shown that the CQ-mediated increase in osteoblast activity may be mediated through a MAPKdependent pathway [24] . ...
Cissus quadrangularis belongs to the taxonomic group Magnoliopsida and family Vitaceae. Cissus quadrangularis is an ancient medicinal plant native to the hotter parts of Ceylon and India. It has been used by common folk in India for promoting the fracture healing process. It was prescribed in the ancient Ayurvedic texts as a general tonic and analgesic, with specific bone fracture healing properties. This review gives a brief idea about its botanical description, phytochemistry, Osteoblastogenesis activity and its molecular healing mechanisms in osteoporosis.
... Although above-mentioned studies were on ovariectomized animals suggestive more of estrogenic potential instead of effect on bone healing, a previous report in 2009 showed that EE increases bio-mineralization through upregulation of mitogen-activated protein kinase-dependent alkaline phosphatase activity in osteoblasts. [26] Cissus is reported to contain certain stilbenes such as resveratrol, some of which are known to have a beneficial effect in bone health. [7,27] However, the quantities reported earlier suggest that the presence of these compounds is minimal. ...
Background: Cissus quadrangularis (CQ) L. reported to contain 3-ketosteroids and have bone health benefits. Aim: This study aimed at establishing the relationship between the ketosteroid content and anabolic as well as bone health-promoting activities of various Cissus extracts in well-established orchidectomized (ORX) rat model. Materials and Methods: Supercritical carbon dioxide, ethyl acetate, and aqueous extracts (AE) of CQ L. were prepared and standardized for ketosteroid content by two methods used in commerce. Moreover, ketosteroid standardized extracts of this plant were evaluated for anabolic activity in rats in well-established ORX rat model. Results: The increase in the absolute weight was appreciable in the CQ-AE treated group. Similarly, with respect to bone parameters, a similar trend was seen. The mean bone density, strength, and calcium content were found to be highest in the group treated with CQ-AE compared to groups treated with other extracts. This study reveals for the first time that 3-ketosteroids are not linked to the beneficial activities on bone and highlights the need for extensive characterization of biological active principles from CQ L. Conclusion: In light of the above estimation studies, we believe that current standardization of Cissus extraction “3-ketosteroids” is incorrect. We also did not find any report suggesting the presence of androgenic steroids in this plant and hence the characterization based on “3-ketosteroids” is scientifically incorrect. This study highlights the insufficient understanding of biological active principles from CQ L. and underlines the need for extensive bioactivity guided studies.
SUMMARY
Cissus quadrangularis (CQ) L. reported to contain 3-ketosteroids and have bone health benefits We did not find correlation between ketosteroid content obtained by conventional methods and its biological effect Studies indicate that claims of ketosteroid content need not necessarily correlate to biological effects and hence warrants extensive phytochemical characterization of biological active principles from CQ L.
Abbreviations used: CQ: Cissus quadrangularis, ORX: Orchidectomized, AE: Aqueous extract, EE: Ethyl acetate extract, SFE: Supercritical fluid extract.
Atul N. Jadhav
... The observed increase in the ALP activity in the present study might be mediated through ß carotene and/or quercetin present in the extract. The increase in the activities of ALP in the femur of OVX rats is consistent with the in vitro studies where C. quadrangularis extract has promoted the activity of ALP in mouse osteoblasts like cells [48]. We earlier reported the positive regulation of C. quadrangularis on the proliferation, differentiation, and matrix mineralization [49] and IGF system components of human osteoblast like SaOS-2 cells [50]. ...
Methods:
Twenty four female adult rats, 90days old showing regular estrous cycles were used for the present study. The animals were divided into two groups. The Group-1 rats (n=6) were sham operated and Group-II rats were bilaterally ovariectomized (n=18) and treated with C. quadrangularis for sixty days (100mg/kg body weight and 250mg/kg body weight). After sixty days, the rats were killed, femora were dissected out, minced and homogenized in Tris-HCl buffer (pH 7.4) and the supernatant was collected and used for biochemical assays.
Results:
Ovariectomy registered a decrease (p<0.05) in the activities of SOD, GPx, GST, ALP, collagen content and increased (p<0.05) the activities of TRAP and lipid peroxidation. Simultaneous administration of C. quadrangularis maintained the enzyme activities in ovariectomized rats.
Conclusion:
C. quadrangularis, a natural herb may be used to treat the estrogen deficiency/menopause onset and ovariectomy induced oxidative stress.
... Although abovementioned studies were on ovariectomized animals suggestive more of estrogenic potential instead of effect on bone healing, a previous report in 2009 showed that EE increases biomineralization through upregulation of mitogenactivated protein kinasedependent alkaline phosphatase activity in osteoblasts. [26] Cissus is reported to contain certain stilbenes such as resveratrol, some of which are known to have a beneficial effect in bone health. [7,27] However, the quantities reported earlier suggest that the presence of these compounds is minimal. ...
... In another mechanistic study, it was demonstrated that the ethanolic extract of Cissus may regulate osteoblastic activity by enhancing alkaline phosphatase activity, which is associated with osteoblast differentiation, and the mineralization process via a p38 mitogen-activated protein kinase-dependent pathway (Parisuthiman et al., 2011). Thus, some information exists regarding the anti-osteoblastic activity of Cissus extract, and multiple mechanisms of action may be involved. ...
Extracts of Salacia reticulata Wight (Hypocrataceae) roots, stems, and leaves have been used in Asia for hundreds of years for the folkloric treatment of diabetes and other health problems. Constituents that have been identified as exhibiting anti-diabetic effects include salacinol, kotalanol, ponkorinol, salaprinol, and their corresponding de-0-sulfonated compounds. Mangiferin, kotalagenin 16-acetate and various proanthocyanidin oligomers have also been isolated. Studies indicate that Salacia extracts modulate multiple targets that influence carbohydrate and lipid metabolism including α-glucosidase, aldose reductase, pancreatic lipase, peroxisomal proliferator-activated receptor-α, glucose transporter-4 mediated glucose uptake, and angiotensin II type 1 receptor. Furthermore, Salacia extracts exhibit free radical scavenging, antioxidant and hepatoprotectant activities. In human studies, Salacia extracts have been shown to decrease plasma glucose and insulin levels, decrease HbA1c, and modulate serum lipid levels with no adverse effects being reported. Similar results have been demonstrated in rat and mouse models as well as in vitro systems. Safety of S. reticulata and other Salacia species as S. oblonga and S. chinensis in rats and mice indicate that extracts are exceedingly safe. No clinical studies have examined the effects of Salacia extracts on human weight loss, although weight loss and decreases in weight gain have been demonstrated in animal models. Because of the large number of pharmacologically active compounds, it is difficult to establish standards for extracts. © 2015 The Authors. Phytotheraphy Research published by John Wiley & Sons Ltd.
© 2015 The Authors. Phytotheraphy Research published by John Wiley & Sons Ltd.
... Fresh CQ stem used as a paste to cover the fracture area of patients suffering from broken bones has been shown to reduce the total time required for fracture healing and to increase the rate of clinical improvement of symptoms, such as pain, tenderness, and swelling, compared to the standard technique alone [18]. In vitro studies have shown that the bone protective effect of CQ extracts involves the stimulation of all processes in osteoblast development, including proliferation, differentiation, and mineralization [19,20]. ...
Background: Osteoporosis becomes a major health problem in aging populations, and this disease increases the risk of bone fractures, leading to disability and mortality. Cissus quadrangularis L. (CQ) has been reported to have beneficial effects on bone metabolism; however, there has been no investigation to identify the active compounds responsible for this activity. Objective: Sequential extracts (hexane, dichloromethane, ethanol, and water) and freeze-dried CQ juice were investigated to determine their effects on bone metabolism in an ovariectomized (ovx) mouse model. Methods: Six-week-old ICR mice were divided into eight groups: sham-operated, ovx-control, estradiol (E 2)-treated and five CQ-treated ovx-mouse groups (n = 3). The CQ extracts were orally administered at a dose equivalent to 5g of crude powder/kg/day for 8 weeks. Bone mineral densities (BMD) of the femur and tibia, serum levels of osteocalcin (bone formation marker) and TRAP5b (bone resorption marker), and histomorphological change of lumbar spine were determined at the end of the experiment. Results: The BMD of the femur and tibia in the hexane-treated group were elevated to the same level as those of sham-operated group. This BMDs correlated with restoration of the trabecular bone of the lumbar spine, which was only observed in the hexane-treated group. These results were also supported by the lowest serum levels of osteocalcin and TRAP5b observed in this group, compared to the ovx-control and E 2-treated groups, representing a decrease in the bone turnover rate. Neither signs of abnormality nor pathological changes of internal organs were observed after the experiment. Conclusion: The hexane extract possessed antiosteoporotic activity in ovariectomized mice without any toxicity throughout the experiment. Therefore, the hexane extract is the most interesting for further bioassay-guided purification of pharmacologically active compounds.
... Ethanol extract of the plant reportedly demonstrated anti-osteoporotic effect in ovariectomized rats (Shirwaikar et al., 2003). Extract of the plant has also been shown to enhance biomineralization in osteoblasts (Parisuthiman et al., 2009). ...
Folk medicinal practitioners (Kavirajes) are possibly the most ancient practitioners of traditional medicine in Bangladesh and in general are the primary health-care providers to a majority of the rural population and
a substantial segment of the urban population in the country. The major characteristic that separates the folk
medicinal practitioners from other systems of existing medicinal practices is their almost exclusive use of simple preparations of medicinal plants for treatment of various ailments. Since the population of Bangladesh is primarily rural, village Kavirajes form the major unit from whom ethnomedicinal data can be obtained. The objective of the present study was to conduct a randomized ethnomedicinal survey among the Kavirajes of four villages, Kalakandi, Gorashal, Kadamtoli, and Gunjar, all villages being situated in Daudkandi sub-district of Comilla district in Bangladesh. Informed consent was obtained from the Kavirajes and surveys were carried out with the help of a semi-structured questionnaire and the guided field-walk method, where the Kavirajes took the interviewers to places from where they collected their medicinal plants, pointed out the plants and described their uses. All plant specimens were collected and identified at the Bangladesh National Herbarium. It was observed that the Kavirajes of the four villages surveyed used 44 plant species distributed into 32 families. The Lamiaceae family contributed 4 plants, followed by the Leguminosae, Rutaceae, and Solanaceae families with 3 plants each. Leaves constituted the major plant part used (45.3%), followed by roots (13.2%), and whole plants, fruits, and seeds (7.5% each). The various ailments treated included respiratory tract problems, gastrointestinal disorders, sexual problems, fever, cardiovascular disorders, mental disease, diabetes, loss of hair, vomiting, menstrual problems, skin disorders, hepatic disorders, piles, leprosy, calcium deficiency, dental diseases, cracked foot, bleeding, insect bites, mumps, rabies, chicken pox, body ache, and bone fracture.Cumulatively, the plants obtained in the present survey present considerable potential for further scientific research towards discovery of lead compounds and more efficacious drugs.
... The extracts of C. quadrangularis stem showed anti-inflammatory properties [60][61][62] and were used in enhancing osteoblast proliferation, bone fracture healing, ossification of fetal bone and increasing the thickness of trabecular bone [63][64][65]. The exact molecular mechanism involved in C. quadrangularis-promoted osteogenesis is still to be elucidated, despite some mechanisms have been proposed. ...
Dental biomaterials and natural products represent two of the main growing research fields, revealing plant-derived compounds may play a role not only as nutraceuticals in affecting oral health, but also in improving physico-chemical properties of biomaterials used in dentistry. Therefore, our aim was to collect all available data concerning the utilization of plant polysaccharides, proteins and extracts rich in bioactive phytochemicals in enhancing performance of dental biomaterials. Although compelling evidences are suggestive of a great potential of plant products in promoting material-tissue/cell interface, to date, only few authors have investigated their use in development of innovative dental biomaterials. A small number of studies have reported plant extract-based titanium implant coatings and periodontal regenerative materials. To the best of our knowledge, this review is the first to deal with this topic, highlighting a general lack of research findings in an interesting field which still needs to be investigated.
... Fresh CQ stem used as a paste to cover the fracture area of patients suffering from broken bones has been shown to reduce the total time required for fracture healing and to increase the rate of clinical improvement of symptoms, such as pain, tenderness, and swelling, compared to the standard technique alone [18]. In vitro studies have shown that the bone protective effect of CQ extracts involves the stimulation of all processes in osteoblast development, including proliferation, differentiation, and mineralization [19,20]. ...
Background: Osteoporosis becomes a major health problem in aging populations, and this disease increases the risk of bone fractures, leading to disability and mortality. Cissus quadrangularis L. (CQ) has been reported to have beneficial effects on bone metabolism; however, there has been no investigation to identify the active compounds responsible for this activity. Objective: Sequential extracts (hexane, dichloromethane, ethanol, and water) and freeze-dried CQ juice were investigated to determine their effects on bone metabolism in an ovariectomized (ovx) mouse model. Methods: Six-week-old ICR mice were divided into eight groups: sham-operated, ovx-control, estradiol (E2)-treated and five CQ-treated ovx-mouse groups (n = 3). The CQ extracts were orally administered at a dose equivalent to 5g of crude powder/kg/day for 8 weeks. Bone mineral densities (BMD) of the femur and tibia, serum levels of osteocalcin (bone formation marker) and TRAP5b (bone resorption marker), and histomorphological change of lumbar spine were determined at the end of the experiment. Results: The BMD of the femur and tibia in the hexane-treated group were elevated to the same level as those of sham-operated group. This BMDs correlated with restoration of the trabecular bone of the lumbar spine, which was only observed in the hexane-treated group. These results were also supported by the lowest serum levels of osteocalcin and TRAP5b observed in this group, compared to the ovx-control and E2-treated groups, representing a decrease in the bone turnover rate. Neither signs of abnormality nor pathological changes of internal organs were observed after the experiment. Conclusion: The hexane extract possessed antiosteoporotic activity in ovariectomized mice without any toxicity throughout the experiment. Therefore, the hexane extract is the most interesting for further bioassay-guided purification of pharmacologically active compounds.
... The steroids can stimulate the proliferation and differentiation of osteoblasts and thereby increases the bone growth (Chagin, Chrysis, Takigawa, Ritzen, & Savendahl, 2006). The action of CQ on the enhanced mineralization of osteoblasts is likely mediated (Parisuthiman & Singhatanadgit, 2009;Prasad & Udupa, 1972). Also the anabolic steroidal principles show a marked influence in the rate of fracture healing by influencing early regeneration of all connective tissues involved in the healing and quicker mineralization of the callus (Shirwaiker et al., 2003). ...
A novel “herbal scaffold” (Alg/O-CMC/CQ-E scaffold) was fabricated by incorporating medicinal plant Cissus quadrangularis (CQ) extract with natural biopolymers alginate (Alg) and O-carboxymethyl chitosan (O-CMC) by lyophilization technique The prepared composite scaffolds were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction analysis (XRD). Preliminary cytocompatibility studies with human mesenchymal stem cells (hMSCs) supports the biocompatible nature of the composite scaffolds. There was a significant difference in initial cell attachment and proliferation on the herbal scaffolds compared to scaffolds fabricated without extract. Moreover, the hybrid scaffold favoured a substantially enhanced differentiation of hMSCs to osteoblasts, even without osteogenic media supplements, followed by increased calcified mineral deposition within two weeks of incubation. Hence, our primary investigation of physico-chemical and biological properties of the herbal scaffolds suggests that this osteoinductive scaffold could serve as a potential candidate for bone tissue engineering therapeutics.
... Its effect was initially compared to vitamin C, where it was revealed that Cissus is better than vitamin C. 8 Cissus has shown increased levels of mucopolysaccharides and faster differentiation of callus in fractures. 9 Parisuthiman et al. 10 have shown the osteogenic effect of ethanolic extract of Cissus. Muthusami et al. 11 have systematically analysed the osteogenic action of Cissus at the molecular level. ...
Porous polymeric scaffolds are extensively studied for delivery of bone growth factors. Since phytochemicals are known to produce changes in cell signalling and other metabolic pathways, osteogenic phytochemicals, that is, extracts of Cissus quadrangularis and Butea monosperma, are incorporated into sulphonated poly(aryl ether ketone) sponges. The results have shown that the scaffolds with phytochemicals enhanced the proliferation and alkaline phosphatase activity of the cells compared to cells treated on scaffolds without phytochemicals. Hence, these phytochemicals can be evaluated to augment, if not substitute the use of bone morphogenetic proteins in scaffolds.
... In another mechanistic study, it was demonstrated that the ethanolic extract of Cissus may regulate osteoblastic activity by enhancing alkaline phosphatase activity, which is associated with osteoblast differentiation, and the mineralization process via a p38 mitogen-activated protein kinase-dependent pathway (Parisuthiman et al., 2011). Thus, some information exists regarding the anti-osteoblastic activity of Cissus extract, and multiple mechanisms of action may be involved. ...
Extracts and powders of Cissus quadrangularis have been used for many years to promote bone and tissues healing, as an analgesic, to treat infections, as an anabolic, and to promote weight loss and weight management. This review summarizes the studies in animals, humans and in vitro systems that have been conducted to determine the efficacy and safety of various Cissus preparations. Animal and in vitro studies provide support for the use of Cissus in promoting bone fracture healing and as an anti-osteoporotic. Several human studies support the use of Cissus extracts in weight management. No studies have been conducted demonstrating that Cissus exhibits anabolic and body building activities. Based on studies to date, Cissus extracts appear to be exceedingly safe and free of adverse effects at the doses commonly used. A wide variety of chemical constituents have been isolated and identified from Cissus extracts, including steroids, flavonoids, stilbenes, iridoids, triterpenes and gallic acid derivatives. However, in few cases have specific physiological effects been related to identifiable constituents. Better standardization of extracts and more well-controlled human studies are required. Copyright © 2012 John Wiley & Sons, Ltd.
Treatment therapies used to manage osteoporosis are associated with severe side effects. So worldwide herbs are widely studied to develop alternative safe & effective treatments. Cissus quadrangularis (CQ) has a significant role in bone health and fracture healing. It is documented that its extracts increase osteoblastic differentiation & mineralization. Currently, Cissus quadrangularis is available in the form of tablets in the market for oral delivery. But these conventional forms are associated with poor bioavailability. There is a need for a novel drug delivery system with improving oral bioavailability. Therefore, a Cissus quadrangularis-loaded self-emulsifying drug delivery system (CQ-SEDDS) was developed which disperses rapidly in the gastrointestinal fluids, yielding nano-emulsions containing a solubilized drug. This solubilized form of the drug can be easily absorbed through lymphatic pathways and bypass the hepatic first-pass effect. The emulsification efficiency, zeta potential, globule size, in-vitro dissolution, ex-vivo, in-vivo and bone marker studies were performed to assess the absorption and permeation potential of CQ incorporated in SEDDS. CQ-SEDDS with excipients Tween 80, Cremophor RH40, Transcutol HP & α-Tocopherol acetate had shown about 76% enhancement in the bioavailability of active constituents of CQ. This study provided the pre-clinical data of CQ-SEDDS using osteoporotic rat model studies.
Novel tissue regeneration strategies are constantly being developed worldwide. Research on bone regeneration is noteworthy, as many promising new approaches have been documented with novel strategies currently under investigation. Innovative biomaterials that allow the coordinated and well-controlled repair of bone fractures and bone loss are being designed to reduce the need for autologous or allogeneic bone grafts eventually. The current engineering technologies permit the construction of synthetic, complex, biomimetic biomaterials with properties nearly as good as those of natural bone with good biocompatibility. To ensure that all these requirements meet, bioactive molecules are coupled to structural scaffolding constituents to form a final product with the desired physical, chemical, and biological properties. Bioactive molecules that have been used to promote bone regeneration include protein growth factors, peptides, amino acids, hormones, lipids, and flavonoids. Various strategies have been adapted to investigate the coupling of bioactive molecules with scaffolding materials to sustain activity and allow controlled release. The current manuscript is a thorough survey of the strategies that have been exploited for the delivery of biomolecules for bone regeneration purposes, from choosing the bioactive molecule to selecting the optimal strategy to synthesize the scaffold and assessing the advantages and disadvantages of various delivery strategies.
Developing biofunctionalized ceramic bone substitutes with phytobioactives for their sustained delivery is highly desired to enhance the osteo-active potential of ceramic bone substitutes, reduce the systemic toxicity of synthetic drugs, and increase the bioavailability of phytobioactives. The present work highlights the local delivery of phytobioactives of Cissus quadrangularis (CQ) through nano-hydroxyapatite (nHAP) based ceramic nano-cement. The phytoconstituent profiling represented the optimized CQ fraction to be rich in osteogenic polyphenols and flavonoids like quercetin, resveratrol, and their glucosides. Further, CQ phytobioactives-based formulation was biocompatible, increased bone formation, calcium deposition, proliferation, and migration of cells with simultaneous alleviation of cellular oxidative stress. In the in vivo critical-sized bone defect model, enhanced formation of highly mineralized tissue (BV mm3) in CQ phytobioactives functionalized nano-cement (10.5 ± 2 mm3) were observed compared to the control group (6.5 ± 1.2 mm3). Moreover, the addition of CQ phytobioactives to the bone nano-cement increased the fractional bone volume (BV/TV%) to 21 ± 4.2% compared to 13.1 ± 2.5% in non-functionalized nano-cement. The results demonstrated nHAP-based nano-cement as a carrier for phytobioactives which could be a promising approach for neo-bone formation in different bone defect conditions.
Cissus quadrangularis L. is a Vitaceae succulent plant that grows in tropical and subtropical xeric forests. The most frequent traumatic injury in humans is a fractured bone. Bone fracture repair is a regenerative process that mimics many of the biological events that occur during embryonic skeletal development. It usually results in effective healing and repair of the injured bone. Alternative medicine and folklore techniques have long employed Phyto-bioactive to treat bone disorders. Bioactive plant extracts help bone repair by acting on various signal transduction pathways. The stem of CQ (Cissus quadrangularis) possesses interesting pharmacological and nutritional qualities that help to maintain skeletal health. The powder and extracts of CQ have been utilized for several years as an analgesic, and anabolic, to treat infections, enhance bone and tissue healing, and help with weight reduction and management. Animal and in vitro assays have validated the utilize of Cissus in enhancing bone fracture recovery and as an anti-osteoporotic. Public health concern osteoporosis is associated with a high incidence of mortality and morbidity. Locally synthesized in the bone, growth factors regulate biological functions including stimulating bone growth. Locally formed IGF-I or IGF-II signaling via the Insulin-like growth factor receptor (IGF-IR) in osteoblast is thought to be critical for bone remodeling and normal growth. Western blotting revealed a rise in IGF-IR protein levels in cells that had been treated with C. quadrangularis. These findings suggest that C. quadrangularis has a favorable effect on the IGF system components of human osteoblasts such as SaOS-2 cells.
This paper identifies and provides the first detailed assessment of hormetic dose responses by bone marrow stem cells (BMSCs) from a broad range of animal models and humans with particular emphasis on cell renewal (proliferation), cell differentiation and enhancing resilience to inflammatory stress. Such hormetic dose responses are commonly reported, being induced by a broad range of chemicals, including pharmaceuticals (e.g., caffeine, dexamethasone, nicotine), dietary supplements (e.g., curcumin, Ginkgo biloba, green tea extracts. resveratrol, sulforaphane), endogenous agents (e.g., hydrogen sulfide, interleukin 10), environmental contaminants (e.g., arsenic, PFOS) and physical stressor agents (e.g., EMF, shockwaves). Hormetic dose responses reported here for BMSCs are similar to those induced with other stem cell types [e.g., adipose-derived stem cells (ADSCs), dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), neuro stem cells (NSCs), embryonic stem cells (ESC)], indicating a substantial degree of generality for hormetic responses in stem cells. The paper assesses both the underlying mechanistic foundations of BMSC hormetic responses and their potential therapeutic implications.
Ethnopharmacological relevance
Cissus quadrangularis L. is a perennial herb of the Vitaceae family and is utilized comprehensively as a medicinal herb in most tropical regions by various names. This herb is documented to possess a wide-ranging ethnomedicinal uses in malaria, fever, epilepsy, gout, piles, skin diseases, colic, etc.
Aim of the review
A organized summary of the botany, traditional uses, phytochemistry, pharmacology, toxicology, available marketed formulations and filed patents were presented to explore the future therapeutic potential and scientific potential of this herb.
Materials and methods
For a review of the literature, various databases were searched, including PubMed, EMBASE, and Scopus etc. From, total 408 records of this herb, we have screened 155 articles consist of desired information and available as full text. Present manuscript is structured from comprehensive information on this herb from screened 155 records. Plant taxonomy was confirmed to the database “The Plant List”.
Results
Phytochemical assessment as a whole indicated the presence of flavonoids, triterpenoids, alkaloids, saponins, iridoids, stilbenes, vitamins, steroids, and glycosides. A toxicity study revealed that its LD50 value is above 3000 mg/kg in animals indicating its safety. A variety of pharmacological studies of aerial parts of this herb by different extracts have demonstrated analgesic, anti-inflammatory, anticonvulsant, antimicrobial, anticancer, anti-osteoporotic activity and other bone-related disorders to justify its name as Hadjod. Still, the herb has been utilized in clinical practice and several patents were filed in India and US for its antiosteoporotic property.
Conclusion
The studies on Cissus quadrangularis Linn. are extensive, but gaps still remain. The molecular mechanism, structure-activity relationship, potential synergistic and antagonistic effects of these components needs to be further elucidated. These findings suggest the need for further research on this herb for the management of several other chronic ailments.
Background: Diabetes has emerged to be an undesired public health concern, one of four priority Non-Communicable Diseases (NCDs) that call for immediate universal attention. While adherence to drug is well
practiced among diabetics, dietary regimen, especially macronutrient distribution, is not strictly followed.
Objectives: The study was embarked to identify the food frequency pattern and macronutrient consumption of Type-2 diabetics with Metabolic Syndrome. Methods: A sample of 166 Type-2 diabetics with metabolic syndrome (MS) were selected from a diabetic clinic in a tertiary hospital through purposive sampling based on inclusion/exclusion criteria and after signing an informed consent. Baseline characteristics of the selected Type-2 diabetics were elicited using a developed and validated questionnaire. Food frequency pattern and macronutrient intake were assessed. Results: The macronutrient consumption of the Type-2 Diabetics (T2D)
deviated from the ICMR guidelines for T2D by exceeding in total calorie consumption which was supplied by the excess carbohydrate consumption. The mean Diet Diversity Score (DDS) was slightly raised in the female (6.3±1.0) T2D with MS compared to male (5.7 ±0.6) subjects. An overall majority (80.8%) of the subjects had an average DDS. The scores reflect a moderate diversity in the daily diet of the Type-2 Diabetics with MS.
Conclusion: Food frequency pattern and Macronutrient consumption of the T2D showed higher consumption of carbohydrates compared to recommended values. Critical attention needs to be drawn towards the quality of macronutrients, specifically, carbohydrates and fats, and their quantity, in managing the total calories in routine diet.
Key words: Type-2 diabetes, Metabolic Syndrome, macronutrient consumption
Growing demand in bone tissue replacement has shifted treatment strategy from pursuing traditional bone-bone grafting to tissue replacement with bioactive biomaterials. Constructs that exhibit the ability to support the bone structure while encouraging tissue regeneration, integration, and replacement represent the future of bone tissue engineering. The present study aimed to understand the osteogenic and mechanical effects of binder-jet 3D printed, porous β-tricalcium phosphate scaffolds modified with a natural polymer/drug coating of polydopamine and Cissus Quadrangularis extract. Compression testing was used to determine the effect the polydopamine coating process had on the mechanical strength of the scaffolds. 3D printed scaffolds without and with polydopamine coatings fractured at 3.84 ± 1.01 MPa and 3.88 ± 0.51 MPa, respectively, suggesting no detrimental effect on strength due to the coating process. The osteogenic potential of the extract-loaded coating was tested in vitro, under static and dynamic flow conditions, and in vivo in a rat distal femur model. Osteoblast cell cultures showed polydopamine resulted in increased proliferation and alkaline phosphatase expression under dynamic flow, which was further enhanced by the addition of Cissus Quadrangularis extract. Histological analysis of implanted scaffolds showed significantly more new bone growth throughout the implant pores at 4 weeks post-op in polydopamine and extract-loaded implants compared to pure β-tricalcium phosphate. These results indicate that implants modified with polydopamine and Cissus Quadrangularis extract considerably outperform pure β-tricalcium phosphate scaffolds in facilitating early bone formation and ingrowth.
Medicinal plants are attracting considerable interest as a potential therapeutic agent for bone tissue regeneration. Cissus quadrangularis L. is also a medicinal plant known with its osteogenic activity. In this study, a phytochemical scaffold was produced by incorporating Cissus quadrangularis with chitosan/Na-carboxymethyl cellulose blend by lyophilization technique. The effect of Cissus quadrangularis loading on the mechanical, morphological, chemical, and degradation properties as well as in vitro cytotoxicity, cell proliferation, and differentiation of the composites was investigated. Scanning electron microscopy images showed that porous Cissus quadrangularis–loaded scaffolds were obtained with an average pore size of 148–209 µm which is appropriate for bone regeneration. Cissus quadrangularis incorporation enhanced the compression modulus of scaffolds from 76 to 654 kPa. In vitro cell culture results indicated that Cissus quadrangularis/chitosan/Na-carboxymethyl cellulose scaffolds provided a favorable substrate for the osteoblast adhesion, proliferation, and mineralization. Results supported the osteoinductive property of the Cissus quadrangularis extract–incorporated scaffolds even without osteogenic media supplement. Cissus quadrangularis extract increased the alkaline phosphatase activity of the SaOS-2 cells on scaffolds on 7th and 14th days of incubation. The investigation of characterization and cell culture studies suggest that Cissus quadrangularis–loaded osteoinductive Cissus quadrangularis/chitosan/Na-carboxymethyl cellulose scaffold can serve as a potential biomaterial for bone tissue engineering applications.
p>The aim of this study was to evaluate the effect and mechanism of action of ethanolic extract of Cissus quadrangularis Linn. stem in an in vitro system using MG-63 (osteoblast-like) cells. Cells were pre-exposed to high glucose (45 mmol/l). Insulin was used as positive control. To determine whether C. quadrangularis (CQ) extract could prevent hyperglycaemia induced oxidative stress, and apoptosis we performed reactive oxygen species generation using 2’,7’-dichlorofluorescin diacetate and propidium iodide staining for apoptosis. For cell proliferation, we carried out cell viability (MTT) assay. Further, alkaline phosphate assay and alizarin staining were done to study the matrix mineralization. High glucose (45 mmol/l) significantly ( P < 0.001) inhibited the cell viability and matrix mineralization, and induced apoptosis as compared to physiological control. C. quadrangularis extract as well as insulin treatment significantly (p < 0.001 versus high glucose) restored the cell viability, cell differentiation and bone nodule formation. Thus, ethanolic extract of C. quadrangularis stem prevents the antiproliferative effect of high glucose.
Dhaka Univ. J. Pharm. Sci. 16(2): 159-164, 2017 (December)</p
Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti-osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre-osteoblast cell line, MC3T3-E1. Ethanolic extract of CQ (CQ-E) was found to affect growth kinetics of MC3T3-E1 cells in a dosage-dependent manner. High concentrations of CQ-E (more than 10μg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1μg/ml were non-toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ-E. CQ-E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose-dependent effect of CQ-E with lower concentrations exhibiting anabolic and osteogenic properties.
Ormocarpum cochinchinense has been traditionally used for curing bone fracture in the villages of Tamil Nadu, India. Significant healing of bone fractures was observed in experiments with the albino Wistar rats. Animals treated orally as well as topically showed healing effects within 7 days by radiological examination and those treated topically alone had lower healing effects. Biochemical analysis of minerals and enzymes associated with bone healing showed a positive trend with serum Ca, inorganic phosphorus, and alkaline phosphatase concentrations in animals treated orally and topically. The concentration of Ca was reduced on the seventh day and increased on the fourteenth and twenty-first days.
Since ancient age nature has been a source of medicinal agents and many of the traditional drugs have been isolated from natural source. Research focus on the natural source has been increased recently due to its minimum side effects. Articles reveal that almost more than 15000 plants have been used by different ethnic communities in India. Many active compounds have been isolated from the plants through various extraction method using different solvents and these are pharmacologically active. The isolated chemical constituents from Cissus quadrangularis extract, which plays major role including gallic acid derivatives, steroids, iridoids, flavonoids, stilbenes and triterpenes. This review is concentrated on the different extracts of Cissus quadrangularis and its activity against numerous pathophysiological effects. Versatile activity of this plant has revealed it as a valuable medicinal plant.
Osteoporosis is a systemic disorder that affects entire skeleton, which is a metabolic bone disease characterized by low bone mass and microarchitectural deterioration of the skeleton, leading to enhanced bone fragility and a consequent increase in fracture risk. In Ayurveda, it can be correlated with Asthi-Majjakshaya. Basti (therapeutic enema) is the prime therapy for Asthi related diseases and Asthi Shrinkhala (Cissus quadrangularis) is the drug which is being used for strengthening of bone by traditional Vaidya since long. It has been selected for oral administration. In clinical trial, 12 patients treated with Majja Basti along with Asthi Shrinkhala pulp capsules and results are very encouraging.
A study was undertaken to evaluate the effect of methanolic extract of Cissus quadrangularis Linn (CQ) on the healing process of experimentally fractured radius-ulna of dog. CQ treated animals revealed faster initiation of healing process than the control animals on radiological and histopathological examinations. The treated group also revealed a decrease in serum calcium level to a greater extent than the control group. Healing was almost complete on 21st day of fracture in the treated animals and remained incomplete in the control animals. No significant alteration of serum calcium level was observed on 21st day of fracture in both the groups.
Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important
role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role
of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped
retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells)
or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation
were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization
by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin,
and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices
were also inhibited. In Erk1DN cells, expression of αβ1, αvβ3, and αvβ5 integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited
in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but
also are important for osteoblast adhesion, spreading, migration, and integrin expression.
The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. These include RUNX2, Osterix (OSX), ATF4 and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations and precedes the branching of chondrogenic and osteogenic lineages. Given its central role in bone development, it is not surprising that RUNX2 is subject to a variety of controls. These include posttranslational modification, especially phosphorylation, and interactions with accessory nuclear factors. Specific examples of RUNX2 regulation to be reviewed include phosphorylation by the ERK/MAP kinase pathway and interactions with DLX5. RUNX2 is regulated via phosphorylation of critical serine residues in the proline/serine/threonine domain. In vivo, the transgenic expression of constitutively active MAP kinase in osteoblasts accelerated skeletal development, while a dominant-negative MAPK retarded development in a RUNX2-dependent manner. DLX5-RUNX2 complexes can be detected in osteoblasts and this interaction plays a critical role in maintaining osteoblast-specific expression of the bone sialoprotein gene. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation as mesenchymal progenitors progress down the osteoblast lineage.
The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. We previously identified an osteoblast-specific cis-acting element, termed OSE2, in the Osteocalcin promoter. We have now cloned the cDNA encoding Osf2/Cbfa1, the protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Finally, forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation.
The signaling mechanisms responsible for the regulation of alkaline phosphatase (ALP) activity by exogenous factors in osteoblast-like cells remain poorly understood. Among various agents, epinephrine was recently found to increase ALP activity in differentiating MC3T3-E1 cells by stimulating alpha1 adrenergic receptors coupled to Gi proteins. In the present study, we investigated the role of both ERK2 and p38 mitogen-activated protein (MAP) kinases in mediating this response in MC3T3-E1 cells. Our results indicate that both MAP kinases are transiently stimulated by epinephrine in differentiating cells via a pertussis toxin sensitive mechanism. The role of each MAP kinase pathway in mediating the stimulation of ALP activity by epinephrine was investigated using specific inhibitors. The MEK inhibitor PD98059, blocked ERK2 activity induced by epinephrine but had no effect on the stimulation of ALP activity. In contrast, low concentrations of SB203580, a specific inhibitor of the p38 MAP kinase, completely blunted this cellular response. However, this inhibitor had no influence on the stimulation of ALP activity induced by ascorbic acid. In conclusion, the results of this study suggest distinct roles for ERK and p38 MAP kinase pathways in regulating activity of MC3T3-E1 osteoblastic cells. The ERK pathway is likely involved in the control of cell proliferation whereas the p38 MAP kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors.
Ethanol extract of Cissus quadrangularis was evaluated for its anti-osteoporotic activity in ovariectomized rat model of osteoporosis at two different dose levels of 500 and 750 mg/kg per day. Healthy female albino rats were divided into five groups of six animals each. First group was sham operated and served as control. All the remaining groups were ovariectomized. Group 2 was fed with equivolume of saline and served as ovariectomized control. Groups 3-5 were orally treated with Raloxifen (5.4 mg/kg) and ethanol extract of Cissus quadrangularis (500 and 750 mg/kg), respectively. The findings assessed on the basis of biomechanical, biochemical and histopathological parameters showed that the ethanol extract of the plant had a definite antiosteoporotic effect.
Cissus quadrangularis (family: Vitaceae) is well known for the treatment of gastric disorders in traditional medicine, owing to its rich source of carotenoids, triterpenoids and ascorbic acid, and has received considerable attention regarding its role in human nutrition. In the search of new potential antiulcer agents, the present study evaluated the ethanol extract of Cissus quadrangularis (CQE) against the gastric toxicity induced by aspirin in rats. The optimum protective dose of 500 mg/kg of extract was selected by the pretreatment of gastric ulcers with different doses of CQE (250, 500 and 750 mg/kg) for 7 days which showed ulcer protection by 40, 71.2 and 72.6%, respectively, as compared to ranitidine (RTD) (30 mg/kg) by 71.9% in the aspirin model. In addition, results have shown that administration of aspirin increases lipid peroxidation status, xanthine oxidase (XO), myeloperoxidase and decrease in selenium-glutathione peroxidase activities in the gastric mucosa, resulting in mucosal damage at both cellular and subcellular level. Pretreatment with CQE ameliorated the observed effect significantly in the gastric mucosa of ulcerated rats. These findings suggest that the gastroprotective activity of CQE could be mediated possibly through its antioxidant effect as well as by the attenuation of the oxidative mechanism and neutrophil infiltration.
Once considered a problem of developed countries, obesity and obesity-related complications (such as metabolic syndrome) are rapidly spreading around the globe. The purpose of the present study was to investigate the use of a Cissus quadrangularis formulation in the management of metabolic syndrome, particularly weight loss and central obesity.
The study was a randomized, double-blind, placebo-controlled design involving 123 overweight and obese persons (47.2% male; 52.8% female; ages 19-50). The 92 obese (BMI >30) participants were randomized into three groups; placebo, formulation/no diet, and formulation/diet (2100-2200 calories/day). The 31 overweight participants (BMI = 25-29) formed a fourth (no diet) treatment group. All participants received two daily doses of the formulation or placebo and remained on a normal or calorie-controlled diet for 8 weeks.
At the end of the trial period, statistically significant net reductions in weight and central obesity, as well as in fasting blood glucose, total cholesterol, LDL-cholesterol, triglycerides, and C-reactive protein were observed in participants who received the formulation, regardless of diet.
Cissus quadrangularis formulation appears to be useful in the management of weight loss and metabolic syndrome.
Embryonic stem (ES) cells can give rise, in vivo, to the ectodermal, endodermal, and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages, offering broad perspectives in regenerative medicine. Understanding the molecular mechanisms governing ES cell commitment is an essential challenge in this field. The mitogen-activated protein kinase (MAPK) pathways extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38MAPK are able to regulate ES commitment from early steps of the process to mature differentiated cells. Whereas the ERK pathway inhibits the self-renewal of ES cells, upon commitment this pathway is involved in the development of extraembryonic tissues, in early mesoderm differentiation, and in the formation of mature adipocytes; p38MAPK displays a large spectrum of action from neurons to adipocytes, and JNK is involved in both ectoderm and primitive endoderm differentiations. Furthermore, for a given pathway, several of these effects are isoform-dependent, revealing the complexity of the cellular response to activation of MAPK pathways. Regarding tissue regeneration, the potential outcome of systematic analysis of the function of different MAPKs in different ES cell differentiation programs is discussed.
Disclosure of potential conflicts of interest is found at the end of this article.
The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that play an essential role in signal transduction by modulating gene transcription in the nucleus in response to changes in the cellular environment. They include the extracellular signal-regulated protein kinases (ERK1 and ERK2); c-Jun N-terminal kinases (JNK1, JNK2, JNK3); p38s (p38alpha, p38beta, p38gamma, p38delta) and ERK5. The molecular events in which MAPKs function can be separated in discrete and yet interrelated steps: activation of the MAPK by their upstream kinases, changes in the subcellular localization of MAPKs, and recognition, binding and phosphorylation of MAPK downstream targets. The resulting pattern of gene expression will ultimately depend on the integration of the combinatorial signals provided by the temporal activation of each group of MAPKs. This review will focus on how the specificity of signal transmission by MAPKs is achieved by scaffolding molecules and by the presence of structural motifs in MAPKs that are dynamically regulated by phosphorylation and protein-protein interactions. We discuss also how MAPKs recognize and phosphorylate their target nuclear proteins, including transcription factors, co-activators and repressors and chromatin-remodeling molecules, thereby affecting an intricate balance of nuclear regulatory molecules that ultimately control gene expression in response to environmental cues.
Extracts of Cissus quadrangularis L. were tested for antioxidant activity by beta-carotene linoleic acid model and also by 1,1-diphenyl-2-picrylhydrazyl model. The ethyl acetate fraction of both fresh and dry stem extracts at a concentration of 100 ppm showed 64.8% antioxidant activity in the beta-carotene linoleic acid system and 61.6% in the 1,1-diphenyl-2-picrylhydrazyl system. This fraction showed the presence of sterols, vitamin C, and tannins as phytoconstituents. The antioxidant activity of methanol extract and aqueous extract were comparatively less significant than that of ethyl acetate extract, and n-hexane extract showed the least activity. The ethyl acetate extract and methanol extract of both fresh and dry stems further exhibited antimicrobial activity against Gram-positive bacteria, including Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Streptococcus species. The results of the study have implications in the use of C. quadrangularis as an antibacterial agent and more so as an antioxidant in several applications requiring these properties.
The signaling mechanisms responsible for the regulation of alkaline phosphatase (ALP) activity by exogenous factors in osteoblast-like cells remain poorly understood. Among various agents, epinephrine was recently found to increase ALP activity in differentiating MC3T3-E1 cells by stimulating α1 adrenergic receptors coupled to Gi proteins. In the present study, we investigated the role of both ERK2 and p38 mitogen-activated protein (MAP) kinases in mediating this response in MC3T3-E1 cells. Our results indicate that both MAP kinases are transiently stimulated by epinephrine in differentiating cells via a pertussis toxin sensitive mechanism. The role of each MAP kinase pathway in mediating the stimulation of ALP activity by epinephrine was investigated using specific inhibitors. The MEK inhibitor PD98059, blocked ERK2 activity induced by epinephrine but had no effect on the stimulation of ALP activity. In contrast, low concentrations of SB203580, a specific inhibitor of the p38 MAP kinase, completely blunted this cellular response. However, this inhibitor had no influence on the stimulation of ALP activity induced by ascorbic acid. In conclusion, the results of this study suggest distinct roles for ERK and p38 MAP kinase pathways in regulating activity of MC3T3-E1 osteoblastic cells. The ERK pathway is likely involved in the control of cell proliferation whereas the p38 MAP kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors.
A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3-E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA-dependent induction of a transiently transfected OCN promoter-luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone-like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. Although some strongly differentiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization.
Tissue nonspecific alkaline phosphatase (TNAP) knockout (ko) mice manifest defects in bone mineralization that mimic the phenotypic abnormalities of infantile hypophosphatasia. In this article, we have searched for phenotypic differences between calvarial osteoblasts and osteoclasts in wild-type (wt), heterozygous and homozygous TNAP null mice. In vitro release of 45Ca from calvarial bones, with and without stimulation with parathyroid hormone (PTH), revealed no functional difference between osteoclasts from the three TNAP genotypes. Studies of primary cultures of TNAP+/+, TNAP+/-, and TNAP-/- calvarial osteoblasts revealed no differences in the rate of protein synthesis or in the expression levels of messenger RNAs (mRNAs) for osteopontin (OP), osteocalcin (OC), collagen type I, core binding factor alpha1 (Cbfa 1), N-cadherin, Smad 5, and Smad 7. Release of interleukin-6 (IL-6) from calvarial osteoblasts under basal conditions and after stimulation with PTH, tumor necrosis factor alpha (TNF-alpha) or IL-1beta was similar in all genotypes. The amount of cyclic adenosine monophosphate (cAMP) accumulation also was comparable. However, although cultures of primary TNAP-/- osteoblasts were able to form cellular nodules as well as TNAP positive osteoblasts do, they lacked the ability to mineralize these nodules in vitro. Mineralization also was delayed in TNAP+/- osteoblast cultures compared with cultures of wt osteoblasts. Incubation with media supplemented with recombinant TNAP, but not with enzymatically inactive TNAP, restored mineralization in ko osteoblast cultures. Our data provide evidence that osteoblasts in TNAP null mice differentiate normally but are unable to initiate mineralization in vitro. The fact that even heterozygous osteoblasts show delayed mineralization provides a rationale for the presence of bone disease in carriers of hypophosphatasia.
Four marker constituents, namely, onocer-7-ene-3 alpha ,21 beta -diol, delta -amyrin, delta -amyrone and 3,3',4,4'-tetrahydroxybiphenyl of an Ayurvedic crude drug Cissus quadrangularis Linn. are defined for standardisation purposes. 3,3',4,4'-tetradydroxybiphenyl has been isolated for the first time from this drug. The contents of the marker constituents were quantitatively determined by HPTLC and HPLC methods in samples collected from five different geographic zones of India. Copyright (C) 2001 John Wiley & Sons, Ltd.
Osteoblasts secrete a complex extracellular matrix (ECM) containing collagenous and noncollagenous proteins, bone morphogenetic proteins (BMPs), and growth factors. Osteoblast-specific gene expression requires ascorbic acid (AA)-dependent assembly of a collagenous ECM. Matrix responsiveness requires an alpha2beta1 integrin-collagen interaction and mitogen-activated protein kinase (MAPK) activity, which phosphorylates and activates the osteoblast-specific transcription factor Cbfa1. This study examines interactions between this integrin/MAPK-mediated pathway and signals initiated by BMPs contained in the osteoblast matrix. MC3T3-E1 cells were shown to constitutively express BMP-2, BMP-4, and BMP-7. Noggin, a specific BMP inhibitor, reversibly blocked AA-induced gene expression, indicating that BMP production by MC3T3-E1 cells was necessary for differentiation. The ability of exogenously added BMP-2, BMP-4, or BMP-7 to stimulate osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs or OCN promoter activity was synergistically increased in cells that were actively synthesizing an ECM (i.e., were grown in the presence of AA). A minimum of 4 days of ECM accumulation was required for this synergistic response to be observed. Neither BMP-7, AA, nor a combination of these two treatments had major effects on Cbfa1 messenger RNA (mRNA) or protein levels, as would be expected if regulation was mainly at the posttranscriptional level. U0126, a specific inhibitor of MAPK/extracellular signal-regulated kinase (MEK), blocked AA- or BMP-7/AA-dependent gene expression in a time- and dose-dependent manner that was closely correlated with inhibition of extracellular signal-regulated kinase (ERK) phosphorylation. This work establishes that autocrine BMP production as well as integrin-mediated cell-collagen interactions are both required for osteoblast differentiation, and both these pathways require MAP kinase activity.
We have identified a novel zinc finger-containing transcription factor, called Osterix (Osx), that is specifically expressed in all developing bones. In Osx null mice, no bone formation occurs. In endochondral skeletal elements of Osx null mice, mesenchymal cells, together with osteoclasts and blood vessels, invade the mineralized cartilage matrix. However, the mesenchymal cells do not deposit bone matrix. Similarly, cells in the periosteum and in the condensed mesenchyme of membranous skeletal elements cannot differentiate into osteoblasts. These cells do, however, express Runx2/Cbfa1, another transcription factor required for bone formation. In contrast, Osx is not expressed in Runx2/Cbfa1 null mice. Thus, Osx acts downstream of Runx2/Cbfa1. Because Osx null preosteoblasts express typical chondrocyte marker genes, we propose that Runx2/Cbfa1-expressing preosteoblasts are still bipotential cells.
In the present study, we investigate the implication of the mitogen-activated protein kinases (MAPKs) Erk, p38, and JNK in mediating the effect of fetal calf serum (FCS) on the differentiation of MC3T3-E1 osteoblast-like cells. Erk is stimulated by FCS in proliferating, early-differentiating, as well as in mature cells. Activation of p38 by FCS is not detected in proliferating cells but is observed as the cells differentiate. JNK is activated in response to FCS throughout the entire differentiation process, but a maximal stimulation is observed in early differentiating cells. The roles of Erk and p38 pathways in mediating MC3T3-E1 cell differentiation was determined using specific inhibitors such as U0126 and SB203580, respectively. These experiments confirmed that the Erk pathway is essential for mediating cell proliferation in response to FCS, but indicated that this MAP kinase has little effect in regulating the differentiation of MC3T3-E1 cells. In contrast, p38 only marginally influenced proliferation, but appeared to be critical for the control of alkaline phosphatase (ALP) expression in differentiating cells. Finally, results obtained with high doses of SB203580, which also affected JNK activity, suggest that p38 and/or JNK are probably also involved in the control of type 1 collagen and osteocalcin expression in differentiating cells. The data indicate that MAPKs regulate different stages of MC3T3-E1 cell development in response to FCS. Distinct MAPK pathways seem to independently modulate osteoblastic cell proliferation and differentiation, with Erk playing an essential role in cell replication, whereas p38 is involved in the regulation of ALP expression during osteoblastic cell differentiation. JNK is also probably involved in the regulation of osteoblastic cell differentiation, but its precise role requires further investigation.
The Cbfa1/Runx2 is an important transcription factor necessary for osteoblast differentiation and bone formation. However, the signaling pathways regulating Runx2 activity are just beginning to be understood. Inconsistencies between Runx2 mRNA or protein levels and its transcriptional activity suggests that posttranslational modification and/or protein-protein interactions may regulate this factor. Runx2 can be phosphorylated and activated by the mitogen-activated protein kinase (MAPK) pathway. This pathway can be stimulated by a variety of signals including those initiated by extracellular matrix (ECM), osteogenic growth factors like bone morphogenic proteins (BMPs) and fibroblast growth factor-2 (FGF-2), mechanical loading and hormones such as parathyroid hormone (PTH). Protein kinase A (PKA) may also phosphorylate/activate Runx2 under certain conditions. In addition, Runx2 activity is enhanced by protein-protein interactions as are seen with PTH-induced Runx2/AP-1 and BMP-mediated Runx2/Smads interactions. Mechanisms for interaction with Runx2 are complex including binding of distinct components such as AP-1 factors and Smads proteins to separate DNA regions in target gene promoters and direct physical interactions between Runx2 and AP-1/Smad factors. Post-translational modifications such as phosphorylation may influence interactions between Runx2 and other nuclear factors. These findings suggest that Runx2 plays a central role in coordinating multiple signals involved in osteoblast differentiation.
The skeleton is an efficient 'servo' (feedback-controlled/steady-state) system that continuously integrates signals and responses which sustain its functions of delivering calcium while maintaining strength. In many individuals, bone mass homeostasis starts failing in midlife, leading to bone loss, osteoporosis and debilitating fractures. Recent advances, spearheaded by genetic information, offer the opportunity to stop or reverse this downhill course.
The bone morphogenetic proteins (BMPs) are potent osteoinductive factors that accelerate osteoblast maturation, accompanied by increased cell-substrate adhesion. BMP-2 treatment of osteoblastic cells increases phosphorylation of the cytoplasmic BMP-2 signaling molecules, Smad1 and Smad5. We have previously reported that BMP-2 treatment increase cytoskeletal organization of human trabecular bone-derived osteoblast-like cells (osteoblasts), which is also accompanied by an activation of the focal adhesion kinase p125(FAK). We report here that activation of p125(FAK) occurs with the same kinetics as the phosphorylation of Smad1, suggesting that BMP-2 initiates cross-talk between Smad signaling and the adhesion-mediated signaling pathway. As an adjunct to these effects, we examined activation of mitogen-activated protein (MAP) kinase family members in response to focal adhesion contact formation. Although phosphorylated forms of all three kinases were apparent, only SAPK2alpha/p38 (p38) was activated in response to BMP-2 treatment. Inhibition of p38 kinase activity suppressed BMP-2 induced Smad1 phosphorylation, as well as its translocation to the nucleus, suggesting the integration of p38 activation with Smad1 signaling. Finally, inhibition of p38 in osteoblasts also led to the complete abrogation of BMP-2 induced osteocalcin gene expression and matrix mineralization. These findings suggest that BMP-2 must activate p38 in order to mediate osteogenic differentiation and maturation.
Signaling involved in osteoblastic cell differentiation remains largely unknown. This study further investigates mechanisms involved in BMP-2-induced osteoblastic cell differentiation. We report that BMP-2 can activate JNK and p38 in osteoblastic cells and provide evidences that these MAP kinases have distinct roles in regulating alkaline phosphatase and osteocalcin expression.
Bone morphogenetic protein (BMP)-2 exerts many of its biological effects through activation of the Smad pathway. Cooperative interactions between the Smads and the stress-activated protein kinase (SAPK) p38 and c-Jun-NH2-terminal kinase (JNK) pathways have recently been observed in TGF-beta signaling.
Activation of mitogen-activated protein (MAP) kinases by BMP-2 and the role of these signaling pathways for cell differentiation induced by BMP-2 was investigated in mouse MC3T3-E1 and primary cultured calvaria-derived osteoblastic cells using immunoprecipitation, in vitro kinase assay and Western blot analysis, as well as specific MAP kinase inhibitors.
Associated with the rapid activation of Smads, BMP-2 barely affected extracellular-signal regulated kinase (ERK) activity, whereas it induced a transient activation of p38 and JNK. The role of p38 and JNK in mediating BMP-2-induced stimulation of osteoblastic cell differentiation was evaluated using the respective specific inhibitors SB203580 and SP600125. Inhibition of p38 by SB203580 was mainly associated with decreased alkaline phosphatase (ALP) activity, whereas inhibition of JNK by SP600125 was associated with a marked reduction in osteocalcin (OC) production induced by BMP-2. Corresponding alterations in ALP and OC mRNA levels were found in cells treated with BMP-2 and inhibitors, suggesting an implication of p38 and JNK pathways in BMP-2-induced osteoblastic cell differentiation at a transcriptional level.
Data presented in this study describe p38 and JNK as new signaling pathways involved in BMP-2-induced osteoblastic cell differentiation with evidences for a distinct role of each MAP kinase in the control of alkaline phosphatase and osteocalcin expression.
Several MC3T3-E1 cell-derived clones expressing higher levels of LH2b were analyzed for their abilities to form collagen fibrils and mineralization. The clones all exhibited smaller collagen fibrils and defective matrix mineralization in vitro and in vivo, indicating a critical role of LH2b-catalyzed post-translational modifications of collagen in bone matrix formation and mineralization.
We have recently shown that lysyl hydroxylase (LH) 2b, through its action on the telopeptidyl lysine residues of collagen, regulates collagen cross-linking pathway in the osteoblastic cell line, MC3T3-E1. To further elucidate the roles of LH2b in bone physiology, the effects of overexpression of LH2b on collagen fibrillogenesis and matrix mineralization were investigated.
Several MC3T3-E1-derived osteoblastic cell clones expressing higher levels of LH2b (S clones) and two controls (i.e., MC3T3-E1 cells and those transfected with an empty vector) were cultured. MALDI-TOF mass spectrometry was used to identify the LH2b. The collagen fibrillogenesis in the cultures was characterized by transmission electron microscopy, and the ability of these clones and cells to form mineralized matrix was analyzed by both in vitro and in vivo mineralization assays.
The diameter of collagen fibrils in the S clone cultures was markedly smaller than that of the controls. The onset of matrix mineralization in the S clones was significantly delayed, and considerably fewer mineralized nodules were formed in their cultures in comparison with the controls. When transplanted into immunodeficient mice, the S clones failed to form mineralized matrices in vivo, whereas a bone-like mineralized matrix was well formed by the controls. The diameter of the collagen fibrils and the timing/extent of matrix mineralization in vitro were inversely correlated with the level of LH2b. In vitro cell differentiation was unaffected by the LH2b overexpression.
These results indicate a critical role of LH2b catalyzed post-translational modification of collagen (i.e., telopeptidyl lysine hydroxylation and subsequent cross-linking) in collagen matrix formation and mineralization in bone.
The osteogenic factors bone morphogenetic protein (BMP-7), platelet-derived growth factor (PDGF)-BB, and fibroblast growth factor (FGF-2) regulate the recruitment of osteoprogenitor cells and their proliferation and differentiation into mature osteoblasts. However, their mechanisms of action on osteoprogenitor cell growth, differentiation, and bone mineralization remain unclear. Here, we tested the hypothesis that these osteogenic agents were capable of regulating osteoblast differentiation and bone formation in vitro. Normal human bone marrow stromal (HBMS) cells were treated with BMP-7 (40 ng ml(-1)), PDGF-BB (20 ng ml(-1)), FGF-2 (20 ng ml(-1)), or FGF-2 plus BMP-7 for 28 days in a serum-containing medium with 10 mM beta-glycerophosphate and 50 microg ml(-1) ascorbic acid. BMP-7 stimulated a morphological change to cuboidal-shaped cells, increased alkaline phosphatase (ALKP) activity, bone sialoprotein (BSP) gene expression, and alizarin red S positive nodule formation. Hydroxyapatite (HA) crystal deposition in the nodules was demonstrated by Fourier transform infrared (FTIR) spectroscopy only in BMP-7- and dexamethasone (DEX)-treated cells. DEX-treated cells appeared elongated and fibroblast-like compared to BMP-7-treated cells. FGF-2 did not stimulate ALKP, and cell morphology was dystrophic. PDGF-BB had little or no effect on ALKP activity and biomineralization. Alizarin Red S staining of cells and calcium assay indicated that BMP-7, DEX, and FGF-2 enhanced calcium mineral deposition, but FTIR spectroscopic analysis demonstrated no formation of HA similar to human bone in control, PDGF-BB-, and FGF-2-treated samples. Thus, FGF-2 stimulated amorphous octacalcium phosphate mineral deposition that failed to mature into HA. Interestingly, FGF-2 abrogated BMP-7-induced ALKP activity and HA formation. Results demonstrate that BMP-7 was competent as a sole factor in the differentiation of human bone marrow stromal cells to bone-forming osteoblasts confirmed by FTIR examination of mineralized matrix. Other growth factors, PDGF, and FGF-2 were incompetent as sole factors, and FGF-2 inhibited BMP-7-stimulated osteoblast differentiation.
p38 MAPK is a conserved subfamily of MAPKs involved in inflammatory response, stress response, cell growth and survival, as well as differentiation of a variety of cell types. In this report we demonstrated that p38 MAPK played an important role in osteoblast differentiation using primary calvarial osteoblast, bone marrow osteoprecursor culture, and a murine cell line, MC3T3-E1. We found that p38 MAPK was activated as calvarial osteoblast differentiates along with extracellular signal-regulated kinases (ERKs). When p38 MAPK is inhibited with a specific inhibitor, the expression of differentiation markers, such as alkaline phosphatase and mineral deposition, were significantly reduced. MC3T3-E1 cells expressing dominant negative p38 MAPK also displayed signs of delay in ALP and mineral deposition. Differentiation of the bone marrow osteoprecursors was also impeded by the p38 MAPK inhibitor, justified by the same markers. Yet the inhibitory effects observed in calvarial osteoblasts and bone marrow osteoprogenitor cells could be partially prevailed by bone morphogenetic protein-2. Inhibition of ERKs with a specific drug did not significantly affect osteoblast differentiation even though ERK1/2 were also activated during osteoblast differentiation. These results taken together indicate that p38 MAPK, but not ERKs, is necessary for osteoblast differentiation.
Osteoporosis is a disorder in which loss of bone strength leads to fragility fractures. This review examines the fundamental pathogenetic mechanisms underlying this disorder, which include: (a) failure to achieve a skeleton of optimal strength during growth and development; (b) excessive bone resorption resulting in loss of bone mass and disruption of architecture; and (c) failure to replace lost bone due to defects in bone formation. Estrogen deficiency is known to play a critical role in the development of osteoporosis, while calcium and vitamin D deficiencies and secondary hyperparathyroidism also contribute. There are multiple mechanisms underlying the regulation of bone remodeling, and these involve not only the osteoblastic and osteoclastic cell lineages but also other marrow cells, in addition to the interaction of systemic hormones, local cytokines, growth factors, and transcription factors. Polymorphisms of a large number of genes have been associated with differences in bone mass and fragility. It is now possible to diagnose osteoporosis, assess fracture risk, and reduce that risk with antiresorptive or other available therapies. However, new and more effective approaches are likely to emerge from a better understanding of the regulators of bone cell function.
The objective of this research was to analyse the gastroprotective effect of Cissus quadrangularis extract (CQE) along with its mechanism underlying the therapeutic action against the gastric mucosal damage induced by aspirin. In this study, we investigated the effect of CQE on the course of experimentally induced gastric ulcer by analyzing the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), microvascular permeability, activity of nitric oxide synthase-2 (NOS-2), mitochondrial antioxidants, lipid peroxidation and DNA damage. A significant increase in vascular permeability, NOS-2 activity, TNF-alpha, IL-1beta levels and oxidative damage were noted in aspirin administered rats. Pretreatment with CQE (500 mg/kg bw/day) by oral gavage for 7 days significantly attenuated these biochemical changes caused by aspirin in rats. Tissue damage was showed by decreased levels of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) and an associated rise in lipid peroxidation (LPO) in mitochondria, which were reversed by CQE. In addition, CQE prevents oxidative damage of DNA by reducing DNA fragmentation indicating its block on cell death. Ulcer protection in CQE treated rats was confirmed by histoarchitecture, which was comprised of reduced size of ulcer crater and restoration of mucosal epithelium. Thus, reduced neutrophil infiltration, antiapoptotic and antioxidant action have a pivotal role in the gastroprotective effect of CQE.
Many plant-derived substances have estrogenic activities. Due to their ability to bind the estrogen receptor (ER), these compounds have the potential to counteract the deleterious effects of estrogen deficiency on bone. In this study, we investigated the in vitro effect of two widespread flavonols, quercetin and kaempferol, on alkaline phosphatase (ALP) activity in MG-63 cultured human osteoblasts. We found that both flavonols significantly increased ALP activity. This effect was markedly reduced by PD 98059, an inhibitor of the extracellular regulated kinase (ERK) pathway, and by ICI 182780, an antagonist of ERs. Western blot studies confirmed that ERK is rapidly activated in cells treated by both flavonols. Finally, ICI 182780 markedly inhibits the flavonol-induced ERK activation. The data presented in this study support the conclusion that, in MG-63 osteoblasts (i) the increase in ALP activity by flavonols involves a rapid stimulation of ERK activation but also involves the ER, and that (ii) the activation of ERK by flavonols occurs most likely downstream of the ERs activation. Taken together, these results suggest that flavonols derivatives as quercetin and kaempferol can stimulate osteoblastic activity. Such compounds may represent new pharmacological tools for the treatment of osteoporosis.
Cissus quadrangularis, a medicinal plant indigenous to Asia and Africa, is used for many ailments, especially for the treatment of hemorrhoid. The effects associated with hemorrhoid, i.e. analgesic and anti-inflammatory activities as well as the venotonic effect of the methanol extract of C. quadrangularis (CQ) were assessed in comparison with reference drugs. In the analgesic test, CQ provoked a significant reduction of the number of writhes in acetic acid-induced writhing response in mice. CQ also significantly reduced the licking time in both phases of the formalin test. The results suggest peripheral and central analgesic activity of CQ. In acute phase of inflammation CQ elicited the inhibitory effect on the edema formation of the rats' ear induced by ethyl phenylpropiolate as well as on the formation of the paw edema in rats induced by both carrageenin and arachidonic acid. It is likely that CQ is a dual inhibitor of arachidonic acid metabolism. In addition, CQ exerted venotonic effect on isolated human umbilical vein similarly to the mixture of bioflavonoids, i.e. 90% diosmin and 10% hesperidin. The results obtained confirmed the traditional use of C. quadrangularis for the treatment of pain and inflammation associated with hemorrhoid as well as reducing the size of hemorrhoids.