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Bulbine Natalensis and Bulbine Frutescens promote cutaneous wound healing
Abstract
Ethnopharmacological relevance:
The gel from the leaves of Bulbine natalensis (BN) and Bulbine frutescens (BF) is commonly used as a traditional medicine in South Africa for the treatment of skin wounds and burns. Treatment with both leaf gel extracts has previously been demonstrated to increase tensile strength and protein and DNA content in pig dermal wounds. This study examined the effect of the leaf gel extracts in vivo on histology of wound healing in pigs to elucidate the mechanism of increased tensile strength.
Materials and methods:
Mirror imaged wounds on the dorsum of 12 post weaning female pigs were treated with either BN or BF, biopsied at days 2, 4, 7, 10 and 16 post-wounding and fixed. Sections of wound tissue were then stained with haematoxylin and eosin and Mallory's stain to analyse the general morphology and collagen arrangement; and smooth muscle actin, vascular endothelial growth factor (VEGF) and transforming growth factor β (TGFβ) receptors were immunolocalised.
Results:
Histological analysis of the wound tissue in the study indicated earlier wound contraction and collagen deposition in both treatment groups with re-organisation of the collagen (indicating collagen maturation) evident as early as at day 10.
Conclusion:
The results of this study suggests that the leaf extracts increase tensile strength by increasing fibroplasia, differentiation of fibroblasts into myofibroblasts, and increased collagen deposition and maturation. This study further validates the use of the Bulbine leaf gels for the treatment of skin wounds.
... Previous investigations on the effects of gel extracted from B. frutescens and B. natalensis on cutaneous wounds in pigs showed an increase in wound contraction and tensile strength (Pather et al., 2011). In a follow-up study it was shown that the gel extracts increased wound tensile strength by increasing fibroplasia, differentiation of fibroplasts into myofibroplasts and increased collagen deposition as well as maturation (Pather and Kramer, -2012). ...
... The gel material from fresh B. frutescens leaves was extracted according to the method described by Pather and Kramer (2012) by cutting off and removing the outer rind and scooping the exposed gel from the fresh leaves. In brief, leaves of freshly collected B. frutescens plants were washed and wiped with 70% ethanol. ...
... Similarly, significantly higher migration rate ratio values were obtained for the two concentrations of B. frutescens gel extract when compared to the untreated control. The in vitro wound healing results obtained in this study for B. frutescens extracts are generally in accordance with the in vivo wound healing results obtained in previous studies (Pather et al., 2011;Pather and Kramer, 2012). ...
... Studies have also demonstrated the ability of antioxidants to reduce the progression of HIV to AIDS (Kashou and Agarwal, 2011;Sharma, 2014), a property which can be attributed to the ability of the virus to tolerate and replicate in oxidative environments (Allard et al., 1998). Bulbine frutescens, the plant of interest in the present study, is traditionally used to treat conditions that are closely associated with HIV/AIDS (Kambizi and Afolayan, 2001), and promotes wound healing (Pather and Kramer, 2012). Inflammation is one of the mechanisms that wards off infections during the wound healing process (Akbik et al., 2014). ...
... Flavonoid compounds with reported anti-HIV activity include myricetin (Pasetto et al., 2014), xanthohumol (Wang et al., 2004), scutellarin (Zhang et al., 2005) and methoxyflavone (Casano et al., 2010). The reported wound healing and antimicrobial properties of B. frutescens (Pather and Kramer, 2012) indicates the presence of bioactive compounds in the plant with antiinfective properties and the same compounds could be responsible for the in vitro anti-HIV potential of the plant. B. frutescens extract also exhibited good free radical scavenging potential in the present study. ...
... B. frutescens extract also exhibited good free radical scavenging potential in the present study. The plant is popularly used as a wound healing agent (Pather and Kramer, 2012). Wounds reportedly activate the production of free radicals which contributes to the inflammation of wounds (Akbik et al., 2014). ...
Bulbine frutescens, a plant native to southern Africa, is used traditionally in the management of conditions related to HIV/AIDS and to promote wound healing. This study investigated the in vitro anti-HIV and antioxidant potential of B. frutescens in order to determine its possible contribution to alleviating HIV/AIDS and its traditional success as a wound healing agent. The extract of the aerial parts of B. frutescens was tested for in vitro inhibitory activity on HIV-1 protease (PR), reverse transcriptase (RT) and integrase enzymes all of which have an essential role in the HIV replication cycle. Since antioxidants contribute to wound healing, the plant extract was studied for 2,2-diphenyl-1-picryl hydrazyl (DPPH), ferric reducing power and hydrogen peroxide (H2O2) scavenging activity. In addition, phytochemical analysis of the extract was conducted using standard procedures. The extract demonstrated good inhibitory activity against HIV-1 PR and RT with IC50 values of 0.18±0.01 and 0.52±0.03mg/mL, respectively. It also demonstrated potent antioxidant activity with IC50 values of 17.94±0.72 and 26.21±0.39μg/mL for DPPH and H2O2, respectively. The reducing power of B. frutescens extract was found to be concentration dependent. Qualitative phytochemical screening revealed the presence of phenols, alkaloids and flavonoids. The content of one of the major component detected, flavonoids, was 71.60±0.05mg QE/g extract. This study demonstrates that B. frutescens extract has anti-HIV potential and the traditional use of the plant as a wound healing agent could be through its high antioxidant activity.
... 40 Antimicrobial, anti-inflammatory, antioxidant, woundhealing, and other activities affecting skin parameters received renewed attention in the 2010s and have since remained the focus of investigations. 27,43,44,[48][49][50][51][52][53][54][55][56][57][58] Coopoosamy (2011) showed moderate activity against grampositive bacteria with acetone and ethyl acetate extracts of Bulbine species but low to no activity with water extracts, 27 implying polarity of the active compounds, which was confirmed in experiments conducted by Mzindle (2017). 53 Bulbine anthraquinones were tested for antimicrobial activity, and bulbnatalonoside A showed a moderate inhibitory effect against methicillin-resistant Staphylococcus aureus (MRSA). ...
... 43,44 In an experimental setting, Pather et al. (2011 and 2012) demonstrated a wound-healing effect of B. natalensis and B. frutescens leaf gels (specifically, an increase in tensile strength by increasing fibroplasia, collagen deposition, and maturation). 48,49 Other effects investigated include antidiabetic, 59-63 androgenic, 64,65 antiproliferative, 66,67 and anti-HIV. 68 An aqueous extract of B. frutescens whole plant was shown to increase glucose utilization significantly more than insulin in C2C12 cells, and to promote glucose uptake in Chang liver cells (HeLa). ...
... Anti-cancer frutescens have all been used to treat wounds, burns and various skinrelated diseases (Thring and Weitz, 2006;Appidi et al., 2008;Pather et al., 2011;Pather and Kramer, 2012;Adebayo and Amoo, 2019;Moteetee et al., 2019). Bulbine natalensis and B. frutescens were tested for their effect on cutaneous wounds of pigs. ...
... Wound contraction increased significantly when compared to the corresponding untreated wounds and the tensile strength of the wounds treated with the leaf gels was significantly stronger than that of the untreated wounds, with significant increases in collagen, protein and DNA content (Pather et al., 2011). Histological analysis of wounds on pigs tested with the same plants showed earlier wound contraction and collagen deposition with re-organization of collagen, suggesting increased tensile strength by increasing fibroplasia, differentiation of fibroblasts into myofibroblasts, and increased collagen deposition and maturation (Pather and Kramer, 2012). These studies therefore support the traditional use of Bulbine species to treat wounds. ...
Ethnopharmacological relevance
The genus Bulbine (Asphodelaceae) is spread across Southern Africa and Australia and has been traditionally used for various medicinal applications such as treating skin diseases, burns, diarrhoea, and sexually transmitted diseases.
Aim of this review
The aim is to present a critical review of the ethnomedicinally important species of the genus Bulbine with a comprehensive overview of their chemical constituents and biological activities.
Materials and methods
This paper is an overview of literature published on the genus Bulbine in the last six decades with regards to phytochemical composition and their respective pharmacological potentials with the aid of data obtained from the search engine Google Scholar with string searches performed using keywords to obtain relevant publications from scientific databases including ACS Journals, PubMed, Science Direct, SciELO, Sci Finder, Springer, Tailor & Francis, The Plant List Database, Web of Science and Wiley.
Results
The literature survey reveals that only 12 species in the genus Bulbine have been reported to be used traditionally with scientific records of ethnomedicinal usage Anthraquinones appeared as the most abundant phytochemicals in the genus. Other isolated/detected metabolites include isofuranonaphthoquinones, flavonoids, and triterpenoids. Promising pharmacological activities have been reported by members of the genus with antiplasmodial, antitrypanosomal, antiviral, antioxidant, anticancer, anti-inflammatory and anti-microbial activity, potent wound healing properties as well as improved reproduction.
Conclusions
This review showed the traditional uses of this genus and its preventative and curative properties in the management of the listed diseases providing support from bioassays of the tested compounds and extracts. State-of-the-art analytical techniques are required for the characterisation and quantification of the compounds within the genus. The efficacy of the therapeutic potential of the Bulbine species need to be further confirmed with pre-clinical and clinical studies.
... Bulbine frutescens (Asphodelaceae) is commonly used as a traditional medicinal plant in South Africa for the treatment of skin wounds and burns [10]. Very few literatures are available on B. frutescens which generally include phytochemical isolation and preliminary screening of biological activity of the plant. ...
... Briefly, the cells (1 × 10 4 cells/well) were seeded in a 96 well microtiter plate (100 μl/ well) in triplicate. BME and BHE treatment were done for 24, 48 and 72 h at different concentrations (10,30,50,70, and 100 μg/ml). After incubation, 10 μl of MTT solution (5 mg/ml) was added to each well and incubated for 4 h at 37°C. ...
Breast cancer (BCa) is the most commonly diagnosed lethal cancer in women worldwide. Notch signaling pathway is directly linked to BCa recurrence and aggressiveness. Natural remedies are becoming a prime choice to overcome against cancer due to lesser side effect and cost-effectiveness. Bulbine frutescens (Asphodelaceae), a traditional medicinal plant in South Africa possess bioactive flavonoids and terpenoids. Polar (methanol) and non-polar (hexane) B. frutescens plant extracts were prepared. GC–MS analysis revealed the differential presence of secondary metabolites in both methanolic and hexane extracts. We hereby first time evaluated the anticancer potential of B. frutescens methanolic and hexane extract in triple-negative and luminal BCa cells. B. frutescens extracts significantly decreased cell viability (IC50 4.8–28.4 μg/ml) and induced cell cycle arrest at G1 phase in MDA-MB-231 and T47D cells as confirmed by spectrophotometry and flow cytometry technique. RT-PCR analysis of cell cycle (cyclin D1, CDK4, and p21) and apoptosis modulating genes (caspase 3, Bcl2 and survivin) revealed upexpression of p21, and caspase 3, and down expression of cyclin D1, CDK4, Bcl2 and survivin genes in extract-treated BCa cells. Fluorescence spectrophotometry and confocal microscopy showed B. frutescens induced nuclear morphology and mitochondrial integrity disruption, and increased reactive oxygen species production in MDA-MB-231 and T47D cells. Flow cytometric apoptosis analysis of B. frutescens extracts treated MDA-MB-231 cells showed ≈13% increase in early apoptotic population in comparison to non-treated cells. Dual-Luciferase Reporter assay confirmed notch promoter inhibitory activity of B. frutescens extracts. Moreover, RTPCR analysis showed down regulation of notch responsive genes (Hes1 and Hey1) at transcription levels in extract-treated BCa cells. Western Blot analysis showed increased procaspase 3 protein expression in extract-treated BCa cells. In all the assays methanolic extract showed better anti-cancer properties. Literature-based identification of methanol soluble phytochemicals in B. frutescens and in silico docking study revealed Bulbineloneside D as a potent ϒ-secretase enzyme inhibitor. In comparison to standard notch inhibitor, lead phytochemical showed two additional hydrophobic interactions with Ala80 and Leu81 amino acids. In conclusion, B. frutescens phytochemicals have cell cycle arrest, ROS production, apoptosis induction, and mitochondria membrane potential disruption efficacy in breast cancer cells. B. frutescens phytochemicals have the ability to downregulate the notch signaling pathway in triple-negative and luminal breast cancer cells.
... 13 Other traditional uses of B. frutescens include treating diarrhea, ringworms, herpes and insect bites using dried leaf bases. 14,15 Bulbine frutescens has not been evaluated for its effect on eczema-associated symptoms; therefore, this study aimed to determine whether extracts prepared from Bulbine frutescens alleviated skin damage through wound healing and reduced the production of histamine. ...
Background
Atopic dermatitis (eczema) is an inflammatory skin condition with synthetic treatments that induce adverse effects and are ineffective. One of the proposed causes for the development of the condition is the outside-in hypothesis, which states that eczema is caused by a disruption in the skin barrier. These disruptions include developing dry cracked skin, which promotes the production of histamine. Bulbine frutescens (BF) is traditionally used to treat wounds and eczema; however, limited research has been conducted to scientifically validate this. Furthermore, gold nanoparticles (AuNPs) have been used to repair damaged skin; however, no research has been conducted on AuNPs synthesized using BF.
Purpose
The study aimed to determine whether BF alleviated skin damage through wound healing, reducing the production of histamine and investigate whether AuNPs synthesized using BF would enhance biological activity.
Methods
Four extracts and four synthesized AuNPs were prepared using BF and their antiproliferative and wound healing properties against human keratinocyte cells (HaCaT) were evaluated. Thereafter, the selected samples antiproliferative activity and antihistamine activity against phorbol 12-myristate 13-acetate (PMA) stimulated granulocytes were evaluated.
Results
Of the eight samples, the freeze-dried leaf juice (BFE; p < 0.01) extract and its AuNPs (BFEAuNPs; p < 0.05) displayed significant wound closure at 100 µg/mL and were further evaluated. The selected samples displayed a fifty percent inhibitory concentration (IC50) of >200 µg/mL against PMA stimulated granulocytes. Compared to the untreated (media with PMA) control (0.30 ± 0.02 ng/mL), BFEAuNPs significantly inhibited histamine production at a concentration of 100 (p < 0.01) and 50 µg/mL (p < 0.001).
Conclusion
BFE and BFEAuNPs stimulated wound closure, while BFEAuNPs significantly inhibited histamine production. Further investigation into BFEAuNPs in vivo wound healing activity and whether it can target histamine-associated receptors on mast cells as a potential mechanism of action should be considered.
... [19] Accumulation of collagen increases the hexosamine levels as hexosamine helps in electrostatic bonding by providing collagen sites. [20] Thus, it may be presumed that application of IMQ to rats resulted in the development of psoriatic-like skin lesions and deformation of normal skin structure, which may be due to the decreased levels of collagen and hexosamine. ...
The plant Begonia roxburghii (Miq.) A.DC (Begoniaceae) is a shrub widely distributed in the Himalayan regions of North East India. In Meghalaya and Assam states of India, the plant is regularly used as vegetables and the roots of the plant are traditionally used in the treatment of diarrhoea, bronchitis, dysentery, candidiasis, colds, in digestive disorders and liver problems. The present investigation was undertaken for the first time to report the complete cytomorphological and quality control profile of the roots from the plant. The study included morphological, microscopical, physicochemical and nutritional analysis. Further, the root extract was also subjected to phytochemical screening and quantitative estimation of various identified phytoconstituents and HPTLC standardization. Morphologically, the roots appeared brown, bulb shaped and tapering towards base, while microscopical examination of the root showed the presence of cortical cells with brown content, cork cambium, lignified xylem fibers, simple or compound starch grains and clustered oxalate crystals. All the physicochemical parameters, heavy metals, microbial counts were found to be within the limits of WHO guidelines, while aflatoxins were absent and nutritional analysis showed the presence of essential elements viz. Ca, Cu, Fe, K, Mg and Zn. The phytochemical standardization revealed the roots extract to be highly rich in flavonoids and tannins, while rutin quantified using HPTLC was found to be 6.83% w/w. Thus, the quality control profile developed through this study could be used as referential source for further research and also to minimize adulteration.
... Mocktar (2000) [10] confirmed antimicrobial activities of methanolic extracts of B. frutescens, B. narcissifolia, and B. abyssinica in a battery of tests against Klebsiella pneumoniae, Staphylococcus aureus, Salmonella typhi, and Shigella flexneri and demonstrated that a B. frutescens root extract inhibited Candida albicans. In an experimental setting, Pather et al. (2011 and [11][12] demonstrated a wound-healing effect of B. natalensis and B. frutescens leaf gels (specifically, an increase in tensile strength by increasing fibroplasia, collagen deposition, and maturation). Other effects investigated include antidiabetic, androgenic, antiproliferative and anti-HIV. ...
The aim of this work was to evaluate if the use of Effective microorganisms on succulent plants such as Bulbine frutescens can improve plant growth, germination of seeds and increse mineral content of vegetative tissues. The experiments, started in January 2021, were conducted in the greenhouses of CREA-OF in Pescia (Pt) on Bulbine frutescens. The plants were placed in ø 12 cm pots; 30 plants per thesis, divided into 3 replicas of 10 plants each. The experimental groups were: i) group control, irrigated with water and substrate previously fertilized; ii) group with Effective microorganisms (EM) irrigated with
water and substrate previously fertilized. The experiment at the greenhouse of CREA-OF in Pescia showed a significant improvement of agronomic parameters and physical, chemical and microbiological characteristics analyzed on Bulbine frutescens plants treated with microbial biostimulants, in particular Effective microorganisms (EM). In the trial, there was a significant improvement in the leaves number per plant, new shoots, inflorescences number, fresh vegetative weight, root weight and inflorescence
weight. In addition, the use of EM resulted in a lowering of the growing medium pH, a significant increase in the microbial colonization of the treated medium, an increase in the number of germinated seeds, and a reduction in the average germination time. Chemically, there was a significant increase in nitrogen, phosphorus and potassium content in the plant tissues of Bulbine frutescens. Knowledge of the raw materials that make up biofertilizers is essential to understanding the actual functionality based on
the plants you want to grow. The study and understanding of the application of Effective
microorganisms can play an important role when it comes to productivity, sustainability, quality, growth and defense of plants from biotic and abiotic stresses
... 6 The jelly-like juice from the leaf is used as a remedy for burns, skin rashes, eczema, insect bites, itching, skin redness and as a promoter of wound healing. 7 In contrast, the plant's root is used traditionally for treating diabetes, where the infusion of freshly boiled roots is taken orally three times. As for BN, there is a dearth of scientific data on its therapeutic target, but it has been reported that aqueous BF extracts increase glucose utilization in skeletal muscle and liver cell lines. ...
Aims
Bulbine natalensis (BN) and Bulbine frutescens (BF) are recommended in South African traditional medicine to treat diabetes, but their modes of action are unknown. This study assessed the phenolic acid profiles, mineral composition and in vitro functional effects of BN and BF to better understand their glucose lowering capabilities.
Methods
Phenolic acid and mineral composition of BN and BF methanolic extracts were determined by HPLC and inductively coupled plasma optical emission spectroscopy respectively. Antioxidant capacity was assessed by potassium ferricyanide reducing power and 2,2-diphenyl-2-picrylhydrazyl radical scavenging assays, and inhibition of alpha-amylase, alpha-glucosidase, pancreatic lipase and DPP4 was evaluated by standard enzyme assays. The effects of BN and BF extracts on insulin secretion were investigated using static incubations of isolated mouse islets and molecular docking analysis was used to identify interactions of BN and BF with partners that could mediate stimulatory effects on insulin secretion.
Results
Methanolic extracts of BN and BF contained high concentrations of protocatechuic and gallic acids, and high levels of Zn, Mn and Cr. The extracts inhibited alpha-glucosidase, alpha-amylase, pancreatic lipase and DPP4 activities, and they also inhibited free radical generation. Both extracts significantly potentiated glucose-stimulated insulin secretion without significantly affecting basal insulin secretion or islet cell viability. Protocatechuic acid, the most abundant phenolic acid in the extracts, showed high affinity for PKA, PKC, DPP4 and CaMK II in the docking analysis.
Conclusions
BN and BF have multiple beneficial effects on glucoregulatory pathways and they, or their derivatives, could be developed to treat type 2 diabetes.
... [19] Accumulation of collagen increases the hexosamine levels as hexosamine helps in electrostatic bonding by providing collagen sites. [20] Thus, it may be presumed that application of IMQ to rats resulted in the development of psoriatic-like skin lesions and deformation of normal skin structure, which may be due to the decreased levels of collagen and hexosamine. ...
Objective:
The present investigation was undertaken to develop a psoriatic-like skin inflammation rat model using imiquimod (IMQ) as an inducing agent.
Materials and methods:
The hairs of the back dorsal portion of the Wistar rats were removed and 80, 100, and 120 mg of IMQ cream (5% w/w) for 10 consecutive days was applied to different groups of rats. Further, psoriasis area severity index was used for calculating the psoriatic score, which included scoring of erythema, scaling, and thickening. Various biochemical parameters, pro-inflammatory cytokines, vascular endothelial growth factor (VEGF), and histopathological examination were also performed.
Results:
The results demonstrated signs of erythema, scaling, and thickening on group applied with 120 mg and 100 mg of IMQ along with ear thickening. Biochemical evaluation revealed a significant increase in the granulation tissue weight followed by significant decrease in the levels of collagen and hexosamine. The antioxidant parameters superoxide dismutase and catalase were found to decline, while nitric oxide and lipid peroxidation were significantly elevated in skin lesions, also supported by increased pro-inflammatory cytokines expression, i.e., interleukin (IL)-1 β, IL-6, IL-17, tumor necrosis factor-α, and VEGF. Histopathological studies revealed a disturbed natural structure along with increased epidermal proliferation, abnormal differentiation with increased number of keratinocytes in the psoriatic skin tissue.
Conclusion:
From the overall study, we have successfully developed a psoriatic-like skin inflammation rat model for the first time on Wistar strain using IMQ as an inducing agent.
... Fase remodeling (maturasi) merupakan fase terakhir dari proses penyembuhan, pada hari ke 21sampai 1,5 tahun kemudian setelah terluka. Kolagen berperan penting untuk reepitelisasi matriks seluler dan interaksi inter seluler sehingga memperkuat dan mengintegrasikan matriks pada luka (14) serta membantu homeostatis pada akhir penyembuhan luka (15) . Kolagen merupakan komponen utama jaringan granulasi dan sintesisnya berasal dari TGF-β1 dependent pada fibroblas yang penting untuk penyembuhan luka (16) . ...
... B. latifolia) MilL. Bulbine frutescens, Hypoxis hemerocallidea and Hypoxis colchicifolia Bak. were reported in vitro, except for Hypoxis colchicifolia which has been studied both in vitro and in vivo [23,[47][48][49][50][51]. It was suggested that the anti-diabetic molecules in B. abyssinica could be carvone, quercetin or psoralen [49]. ...
The use of medicinal plants for the management of diabetes mellitus is on the rise in the developing countries, including South Africa. There is increasing scientific evidence that supports the claims by the traditional healers. In this review, we compare the families of previously reported anti-diabetic plants in the Eastern Cape by rating the anti-diabetic activity, mode of action and also highlight their therapeutic potentials based on the available evidence on their pharmacology and toxicity. Forty-five plants mentioned in ethnobotanical surveys were subjected to a comprehensive literature search in the available electronic databases such as PubMed, ScienceDirect, Google Scholar and Elsevier, by using “plant name” and “family” as the keywords for the primary searches to determine the plants that have been scientifically investigated for anti-diabetic activity. The search returned 25 families with Asteraceae highly reported, followed by Asphodelaceae and Alliaceae. Most of the plants have been studied for their anti-diabetic potentials in vivo and/or in vitro, with most of the plants having a higher percentage of insulin release and inhibition against carbohydrate digesting enzymes as compared with insulin mimetic and peripheral glucose uptake. Almost all the investigated plants also inhibit oxidative stress as part of their hypoglycemic activity with less toxicity. However, the isolation of their bioactive molecules is still lacking. This review provides a resource to enable thorough assessments of the therapeutic profiles of available medicinal plants used for the management of diabetes in the Eastern Cape, South Africa. Further studies such as the identification of the active ingredients of potent plants still need to be carried out; this may lead to new molecules in drug discovery and development.
... The enhanced levels of collagen (hydroxyproline) are stabilized by hexosamine, which provides collagen sites for electrostatic bonding. Collagen critically controls the healing process by making up a major portion of connective tissue and also manages its construction, deposition and ensuing evolution therefore; higher concentration of hydroxyproline and hexosamine is directly proportional to faster healing (Pather and Kramer, 2012). The above stated functions of collagen are complimented by hyaluronic acid, which does not participate in its synthesis or maintenance but instead it is involved in water retention, nutrient exchange, cell differentiation and cell mobilization (Chen et al., 2014). ...
Ethnopharmacological relevance:
The plant Solanum xanthocarpum Schrad. & Wendl. (Solanaceae) is one of the members of the dashamula (ten roots) in Ayurvedic system of medicine. The stem and fruits are used as an antipyretic, antiasthmatic and is prescribed in skin infections and for relief in burning sensation in the feet accompanied by vesicular eruptions.
Objective:
To scientifically validate the anti-psoriatic potential of Solanum xanthocarpum stem in Imiquimod-induced psoriatic mice model MATERIALS AND METHODS: Ethanolic stems extract of Solanum xanthocarpum (ESX) was first subjected to phytochemical screening and quantification of identified phytoconstituents, which was further standardized with the help of HPTLC using chlorogenic acid as a marker. The extract was then subjected to acute oral toxicity and skin irritability study for determining the safety profile of the extract. Imiquimod-induced psoriatic mouse model was then performed to check the efficacy of extract against psoriasis, where treatment was carried out for 15 days both topically (Gel at 2.5, 5 and 10%) as well as orally (at 100, 200 and 400mg/kg p.o.) and their Psoriasis Area Severity Index (PASI) was calculated. The study also included determination of levels of of TNF-α, IL-1β, IL-6 and IL-17 in the animal tissues, which further included biochemical evaluations such as total collagen, hexosamine, hyaluronic acid DNA, protein antioxidant profiles such as lipid peroxidation, nitric oxide, superoxide dismutase and catalase along with histopathological studies of the tissues.
Result:
ESX showed the presence of mainly phenols, tannins, flavonoids, alkaloids and carbohydrates, while chlorogenic acid was reported to be 3.49% w/w. The imiquimod-induced psoriatic mouse model, depicted a potent anti-psoriatic activity of the extract both topical (10%) and oral (200 and 400mg/kg p.o., as evident through PASI grading The effect was found to be more prominent in case of topically treated as compared to orally treated mice. The results also showed a significant inhibition in the expression of TNF-α, IL-1β, IL-6 and IL-17 in treated animal tissues and also showed significant restoration of the altered biochemical parameters along with reduced hyperkeratinisation as observed through histopathology.
Conclusion:
The study scientifically justified the anti-psoriatic activity of the ESX, which may be attributed to inhibition in the expression of cytokines such as TNF-α, IL-1β, IL-6 and IL-17. Further, the observed antioxidant, antimicrobial and cellular proliferative activities may act as a contributing factor in treatment of psoriasis, which may be attributed to the presence of chlorogenic acid along with other phytochemicals in combination.
... Since, collagen critically controls the healing process by making up a major portion of connective tissue and also manages its construction, deposition and ensuing evolution; a higher concentration of hydroxyproline and hexosamine, therefore, promote faster wound healing. Collagen is also vital for re-epithelialisation of cellularmatrix and inter-cellular interactions thereby strengthening and integrating the wound matrix (Pather and Kramer, 2012). Collagen not only provides the tissue matrix with strength and integrity, but it also caters to the homeostatic demands in the latter portion of wound healing timeline (Roy et al., 2012). ...
_Leea macrophylla_ Roxb. ex Hornem. (Leeaceae) commonly known as Hastikarnapalasa is mainly distributed throughout the tropical parts of India. Traditionally, the plant is found to be effective against guinea worm, ringworm and is applied to sores and wounds.
The present study aims to validate traditional wound healing claim of _Leea macrophylla_ scientifically.
Box–Behnken design (BBD) was used to optimize the extraction process. The optimized root tuber extract of _Leea macrophylla_ was standardized with chlorogenic acid by HPLC for the first time. Both oral and topical routes were selected as administrative means for the wound healing study using excision and incision wound model. For topical treatment bioadhesive gel was formulated and characterized for mechanical and physical characteristics by texture profile analysis (TPA) and scanning electron microscopy (SEM). The effect on wound healing was also assessed by evaluating antioxidant enzymes viz. glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), free radicals lipid peroxidation (LPO) and nitric oxide (NO), inflammatory marker myeloperoxidase (MPO), collagen markers hydroxyproline, hexosamine and hexuronic acid along with the histopathological examination. Furthermore, the effect on the level of the proinflammatory cytokines interleukin-1β (IL-1β), interleukin −6 (IL-6), tumor necrosis factor - α (TNF-α) and growth factor, vascular endothelial growth factor (VEGF) were determined. The expression of cell proliferation nuclear marker Ki-67 was also analyzed by Western blot analysis.
With mesh openings Sieve no. 20, semi polar nature of solvent (92.5:7.5 ethanol-water blend) and extraction time of 18 h, substantially greater extraction efficiency (29%) and phenolic yield (181.54 mg/g) were obtained. The content of chlorogenic acid in ethanol extracts of _Leea macrophylla_ was obtained as 9.01% w/w. In incision model, oral treatment with 500 mg/kg ethanolic extract increased wound breaking strength by 23.41% while bioadhesive gel (5% w/v) showed a higher increase of 44.68%. Topical application produced complete wound contraction in 20 days against 22 days taken by oral treatment. Topical treatment also produced a significant (p<0.05) increase in antioxidants glutathione, superoxide dismutase and catalase whereas the level of enzymes lipid peroxidation and nitric oxide and inflammatory markers myeloperoxidase were reduced. Further advantageous effects were reflected by significantly (p<0.05) increased levels of hydroxyproline, hexosamine and hexuronic acid. Favorable effects on the level of proinflammatory cytokines interleukin-1β, interleukin −6, tumor necrosis factor - α and growth factor, vascular endothelial growth factor were also observed. The wound healing potential of _Leea macrophylla_ was further supported by its ability to promote cell proliferation during wound healing as demonstrated by Western blot analysis of proliferation marker Ki-67.
The study justified traditional use of _Leea macrophylla_ in wound healing and demonstrated that the bioadhesive gel of ethanolic extract produced faster and more significant healing as compared to oral treatment.
... Although the plant is widely recognized for its ornamental value owing to colorful (yellow) flowers and succulent leaves, is also known for its use in traditional medicinal practice. The leaf exudate of B. frutescens is used for the treatment of various ailments, particularly for wound healing and esthetic purposes (Rabe and van Staden, 1997;Abegaz et al., 2002;Pather et al., 2011;Pather and Kramer, 2012). The gel extract has recently been patented as a promoter of wound healing (Lusunzi and Karuso, 2001). ...
Phytochemical investigation of the dichloromethane/methanol (1:1) extract of the roots of Bulbine frutescens led to the isolation of a new xanthone, 8-hydroxy-6-methylxanthone-1-carboxylic acid (1) and a new phenylanthraquinone, 6',8-O-dimethylknipholone (2) along with six known compounds. The structures were elucidated on the basis of NMR and MS spectral data analyses. The structure of compound 1 was confirmed through X-ray crystallography which was then used as a reference to propose the revision of the structures of six seco-anthraquinones into xanthones. The isolated compounds were evaluated for cytotoxicity against human cervix carcinoma KB-3-1 cells with the phenylanthraquinone knipholone being the most active (IC50 = 0.43 mu M). Two semi-synthetic knipholone derivatives, knipholone Mannich base and knipholone-1,3-oxazine, were prepared and tested for cytotoxic activity; both showed moderate activities (IC50 value of 1.89 and 2.50 mu M, respectively).
Bulbine frutescens is an evergreen perennial that is native to Southern Africa, but can be found in Eastern, Western and Northern Cape. B. frutescens is known for its ethnobotanical use and has been used by traditional healers for years. The jelly-like juice found in the leaves has been discovered to alleviate burns, blisters, insect bites and cracked skin. Furthermore, when the leaves are boiled and strained, the tea can be drunk to treat colds and arthritis. The gel found within the leaves has anti-clotting potential and can be used to reduce acne, mouth ulcers and ringworms. In vitro studies that have been conducted indicated that B. frutescens displayed antibacterial, anti-cancer and anti-HIV activity. However, further research into the potential wound healing properties should be investigated as minimal information regarding this was found.
In the present review, the ethnomedicinal uses, the phytochemistry, and the pharmacological effects of established cytotoxic plants of the flora of Africa have been reported. The best botanical source of potential anticancer agents, as well as the best cytotoxic phytochemicals, were also identified and highlighted. The relationship between various health conditions in association with cancer was also given. It was shown that few evidenced cytotoxic are used to treat cancer as well as diseases with cancer-related symptoms, such as inflammatory diseases, parasitic infections, microbial and viral infections, cardiovascular diseases, central nervous system disorders, diabetes, and digestive system disorders. A total of 111 cytotoxic African medicinal plants belonging to 46 families are reported. Up to 50 potent African medicinal plants were identified as potentially suitable to fight at least one type of cancer. A total of 20 plants were identified to be suitable to fight the drug resistance of at least one type of cancer. One hundred and eight phytochemicals from African medicinal plants were identified as potentially suitable to fight at least one type of cancer. Phytochemicals identified as potent cytotoxic agents included 48 phenolics, 28 terpenoids, 26 alkaloids, 4 carbohydrates, and 2 fatty acids. A total of 42 phytoconstituents were identified to be suitable to fight the drug resistance of at least one type of cancer. This review constitutes a good collection for scientists, especially since it identifies new botanicals and phytochemicals that could undergo further in-depth studies to discover novel and powerful anti-cancer drugs. It also identifies the best classes of plant cytotoxic molecules, which could better guide scientists in the search for new anticancer agents.
Background:
Medicinal plants are regarded as a large source of phytochemicals that may have anticancer properties. This could lead to the development of innovative drugs or alternative therapy against cancer.
Objective:
This study was designed to determine the antioxidant and cytotoxicity effect of 5 selected indigenous South African medicinal plants namely; Bulbine frutescens, Bulbine natalensis, Chlorophytum comosum, Kniphofia uvaria, and Tulbaghia violacea.
Method:
Phytochemical extracts namely; methanol, 50%, 100% ethanol, and water extracts were prepared from the root and shoot of the plants. The antioxidant effect of methanol extracts of the plant materials was performed using a DPPH assay. A preliminary cytotoxicity screening of the phytochemical extracts in the human colon (Caco-2), cervical (HeLa), and hepatocellular (HepG2) cell lines were determined followed by the half-maximal inhibitory concentration (IC50) using MTT assay.
Result:
The methanol root extract of B. natalensis and B. frutescens (33.20% and 26.33% respectively) and shoot extract of K. uvaria (17.10%) showed the highest antioxidant. Out of the 5 plants, only 100% ethanol extract of C. comosum, K. uvaria, and T. violacea caused more than 80% cytotoxicity in HepG2 and Caco-2 cell lines. The shoot of B. frutescens (10.43 µg/ml), K. uvaria (23.0 µg/ml), and root of C. comosum (23.77 µg/ml) were the most active with the highest cytotoxicity.
Conclusion:
C. comosum, K. uvaria, and T. violacea possess significant cytotoxicity that is promising in developing alternative drugs against colon and liver cancers. Our results provided new pieces of evidence for antioxidant and cytotoxic activities of these plants which could be useful for developing new anticancer therapies.
Scientific studies of Aloe vera have tentatively explained therapeutic claims from a mechanistic perspective. Furthermore, in vitro outcomes demonstrate that the breakage of acemannan chains into smaller fragments enhances biological effects. These fragments can intravenously boost vaccine efficacy or entrain the immune system to attack cancer cells by mannose receptor agonism of macrophage or dendritic cells. With oral consumption, epithelialisation also occurs at injured sites in the small intestine or colon. The main advantage of dietary acemannan is the attenuation of the digestive process, increasing satiety, and slowing the release of sugars from starches. In the colon, acemannan is digested by microbes into short-chain fatty acids that are absorbed and augment the sensation of satiety and confer a host of other health benefits. In topical applications, an acemannan/chitosan combination accelerates the closure of wounds by promoting granular tissue formation, which creates a barrier between macrophages or neutrophils and the wound dressing. This causes M2 polarisation, reversal of inflammation, and acceleration of the re-epithelialisation process. This review summarises and explains the current pharmacodynamic paradigm in the context of acemannan in topical, oral, and intravenous applications. However, due to contradictory results in the literature, further research is required to provide scientific evidence to confirm or nullify these claims.
The rhizomes of Bulbine natalensis furnished six previously unreported anthraquinone derivatives (1-6), together with eight known specialized metabolites. Their structures were determined by interpretation of 1D and 2D NMR and HRESIMS data. The absolute configurations of compounds 1-6 were determined by specific rotation and circular dichroism experiments. The isolated compounds were evaluated for antimicrobial activities, and compound 1 was found to be a moderate inhibitor (IC50 0.02 μM) against methicillin-resistant Staphylococcus aureus (MRSA).
Deregulated signaling pathways/processes result in uncontrolled cell proliferation and cell survival. Adaptation to this condition often resulted in chemotherapeutic resistance in cancer cells. According to the concept of various signaling involvement in the development of cancer, fractional inhibition of numerous targets can be more productive than single one. Bulbine frutescens is a traditional medicinal plant of South Africa, has been preliminarily reported for anti-proliferative and anti-drug resistance property in cancer cells. Anticancer and mode of action of various phytochemicals present in the plant not yet has been studied. The present in silico study was designed to assess the potential B. frutescens phytochemicals as novel natural inhibitors of signaling proteins involved in cancer. Furthermore, drug-likeness property, ADME/T (absorption, distribution, metabolism, excretion/toxicity) properties, interaction ability with xenobiotic enzymes and biological activities were predicted by using various computational tools. For the first time Palmidin C, AC1NSTKE, Bulbine-knipholone and 4′-Demethylknipholone 2′-β-D-glucopyranoside compounds have been reported for their anticancer potential. These phytochemicals have better potential to inhibit NFkB, TGF-β, PI3K, and JAK2 proteins than their respective standard inhibitors. Lead phytochemicals showed better ADME/T, drug-likeness, xenobiotic enzymes interactions and biological activities. In conclusion, Palmidin C, AC1NSTKE and Bulbine-knipholone are the novel potent anticancer phytochemicals and need to be studied further in vitro and in vivo.
Accumulating evidence indicates that there is extensive crosstalk between integrins and TGF-beta signalling. TGF-beta affects integrin-mediated cell adhesion and migration by regulating the expression of integrins, their ligands and integrin-associated proteins. Conversely, several integrins directly control TGF-beta activation. In addition, a number of integrins can interfere with both Smad-dependent and Smad-independent TGF-beta signalling in different ways, including the regulation of the expression of TGF-beta signalling pathway components, the physical association of integrins with TGF-beta receptors and the modulation of downstream effectors. Reciprocal TGF-beta-integrin signalling is implicated in normal physiology, as well as in a variety of pathological processes including systemic sclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cancer; thus, integrins could provide attractive therapeutic targets to interfere with TGF-beta signalling in these processes.
Persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury is a characteristic but poorly understood feature of normal wound healing. It results in extravasation of fibrinogen that clots to form fibrin, which serves as a provisional matrix and promotes angiogenesis and scar formation. We present evidence indicating that vascular permeability factor (VPF; also known as vascular endothelial growth factor) may be responsible for the hyperpermeable state, as well as the angiogenesis, that are characteristic of healing wounds. Hyperpermeable blood vessels were identified in healing split-thickness guinea pig and rat punch biopsy skin wounds by their capacity to extravasate circulating macromolecular tracers (colloidal carbon, fluoresceinated dextran). Vascular permeability was maximal at 2-3 d, but persisted as late as 7 d after wounding. Leaky vessels were found initially at the wound edges and later in the subepidermal granulation tissue as keratinocytes migrated to cover the denuded wound surface. Angiogenesis was also prominent within this 7-d interval. In situ hybridization revealed that greatly increased amounts of VPF mRNA were expressed by keratinocytes, initially those at the wound edge, and, at later intervals, keratinocytes that migrated to cover the wound surface; occasional mononuclear cells also expressed VPF mRNA. Secreted VPF was detected by immunofluoroassay of medium from cultured human keratinocytes. These data identify keratinocytes as an important source of VPF gene transcript and protein, correlate VPF expression with persistent vascular hyperpermeability and angiogenesis, and suggest that VPF is an important cytokine in wound healing.
A series of studies has shown that application of transforming growth factor β (TGF-β) to a wound has a beneficial effect,
especially in animals with wound healing disorders. In this study we have investigated the regulation of TGF-β1, β2, and β3
and their receptors during the repair process. We found a large induction of all three TGF-β isoforms and also of TGF-β types
I and II receptors, although the time course of induction and the absolute expression levels were different for these genes.
Furthermore, each TGF-β isoform had distinct sites of expression in the wound. Systemic treatment with glucocorticoids significantly
altered the expression levels of TGF-βs and TGF-β receptors. Whereas expression of TGF-β1, TGF-β2, and TGF-β type II receptor
was suppressed by glucocorticoids in normal and wounded skin, expression of TGF-β3 and TGF-β receptor type I mRNA was stimulated.
These findings provide an explanation for the beneficial effect of exogenous TGF-β in the treatment of impaired wound healing
in glucocorticoid-treated animals. Furthermore, they suggest that a disturbed balance between the levels of the three TGF-β
isoforms and their receptors might underlie the wound healing defect seen in glucocorticoid-treated animals.
Transforming growth factor (TGF)-beta isoforms (TGF-beta 1, -beta 2, and -beta 3) regulate cell growth and differentiation and have critical regulatory roles in the process of tissue repair and remodeling. Signal transduction for TGF-beta function is transmitted by a heteromeric complex of receptors consisting of two serine/threonine kinase transmembrane proteins (RI and RII). We have previously shown that each TGF-beta isoform is widely expressed in a distinct spatial and temporal pattern throughout the processes of excisional and incisional wound repair. As the presence of TGF-beta receptors determines cellular responsiveness, we have currently examined, by immunohistochemistry, the localization of RI (ALK-1, ALK-5) and RII throughout repair of full-thickness excisional wounds up to 21 days after wounding. The expression of RI (ALK-5) and RII co-localized in both the unwounded and wounded skin and was present in the same cell types as TGF-beta ligands. However, immunoreactivity for TGF-beta receptors, throughout repair, occurred 1 to 5 days later than TGF-beta isoform immunostaining. This implies that the presence of TGF-beta ligands may up-regulate TGF-beta receptors for function and/or may reflect a lag due to local processing of latent TGF-beta. As observed for the immunohistochemical localization of TGF-beta isoforms in unwounded skin, RI and RII were expressed throughout the four layers of the epidermis, showing a wavy pattern of slight to moderate immunostaining, and hair follicles, sweat glands, and sebaceous glands were moderately immunoreactive. The extracellular matrix, fibroblasts, and blood vessels in the dermis were not immunoreactive. After injury, as observed for TGF-beta ligands, RI and RII expression was increased in the epidermis adjacent to the wound and the epithelium migrating over the wound was completely devoid of TGF-beta receptor immunoreactivity until re-epithelialization was completed by day 7 after wounding. The dermis was only slightly immunoreactive for RI and RII until day 5 when, immediately under the wound, immunostaining for fibroblasts, connective tissue cells, and newly forming vasculature began to increase and remained intense until day 14. Consistent with the role for TGF-beta in scarring, numerous fibroblasts, ostensibly active in the production of extracellular matrix components, continued to be slightly immunoreactive for RI and RII at 21 days. The ALK-1 (TSR-1) type I receptor, which binds both activin and TGF-beta, showed slight immunostaining early in repair (days 1 to 7) that progressively became more intense later in repair after day 10 and through day 21. This suggests that there may be a switch to a different type I receptor, implying different functions for the ALK-1 and ALK-5 receptors. The concomitant expression of TGF-beta isoforms and their signal-transducing receptors denote potential spatial and temporal activity of TGF-beta. Thus, although TGF-beta ligand is present, TGF-beta would not function in wound repair until a later time when RI and RII appear. This information should aid in the development of receptor antagonists as a therapeutic approach to scarring and fibrosis. In addition, these studies underscore the importance of defining the expression of proteins in vivo to establish a basis for the analysis of mechanisms in vitro.
In the present study we have analyzed and compared, by immunohistochemistry and in situ hybridization, the expression pattern of the R4/ALK5 transforming growth factor (TGF)-beta type I receptor (RI) and the TGF-beta type II receptor (RII) in normal human skin, in wounded skin at various stages during the transition of wound granulation tissue to scar, and in long-persisting post-burn hypertrophic scars. In normal human skin, expression of RI and RII was clearly visible in the epidermis, in epidermal appendages, and in vascular cells, although only a small number of dermal fibroblasts revealed detectable levels of TGF-beta receptor expression. In contrast, granulation tissue fibroblasts showed strong expression of both TGF-beta receptor types, although in normal-healing excisional wounds their density decreased during granulation tissue remodeling. However, in post-burn hypertrophic scars, RI- and RII-overexpressing fibroblasts were found in high densities up to 20 months after injury. From these findings we suggest that the repair process of deep wounds involves the transformation of a subset of fibroblastic cells toward an increased TGF-beta responsiveness and a transient accumulation of these cells at the wound site. In addition, our study provides evidence that excessive scarring is associated with a failure to eliminate TGF-beta receptor-overexpressing fibroblasts during granulation tissue remodeling, which leads to a persistent autocrine, positive feedback loop that results in over-production of matrix proteins and subsequent fibrosis.
Angiogenesis is an essential component of normal wound repair, yet the primary mediators of wound angiogenesis have not been well described. The current study characterizes the contribution of vascular endothelial cell growth factor (VEGF) to the angiogenic environment of human surgical wounds. Surgical wound fluid samples (n = 70) were collected daily for up to 7 postoperative days (POD) from 14 patients undergoing mastectomy or neck dissection. VEGF levels in surgical wound fluid were lowest on POD 0, approximating values of serum, but increased steadily through POD 7. An opposite pattern was noted for basic fibroblast growth factor-2. Fibroblast growth factor-2, which has been previously described as a wound angiogenic factor, exhibited highest levels at POD 0, declining to near serum levels by POD 3. Surgical wound fluid form all time points stimulated marked endothelial cell chemotaxis and induced a brisk neovascular response in the rat corneal micropocket angiogenesis assay. Antibody neutralization of VEGF did not affect the in vitro chemotactic or the in vivo angiogenic activity early wound samples (POD 0). In contrast, VEGF neutralization significantly attenuated both chemotactic activity (mean decrease 76 +/- 13%, P < 0.01) and angiogenic activity (5 of 5 samples affected) of later wound samples (POD 3 and 6). The results suggest a model of wound angiogenesis in which an initial angiogenic stimulus is supplied by fibroblast growth factor-2, followed by a subsequent and more prolonged angiogenic stimulus mediated by VEGF.
To clarify interleukin (IL)-6 roles in wound healing, we prepared skin excisions in wild-type (WT) and IL-6-deficient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte infiltration was evident at wound sites. Furthermore, the re-epithelialization rate was approximately 80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL-1, chemokines, adhesion molecules, transforming growth factor-beta1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was significantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte infiltration, re-epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti-IL-6 monoclonal antibody significantly delayed wound closure in WT mice. These observations suggest that IL-6 has crucial roles in wound healing, probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation.
In this study, we examined the impact of matrix metalloproteinases (MMP) on epithelialization, granulation tissue development, wound contraction, and alpha-smooth muscle actin (ASMA) expression during cutaneous wound repair through systemic administration of the synthetic broad-spectrum MMP inhibitor GM 6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide). Four full-thickness excisional wounds (50 mm2) on the back of 22 young female Sprague-Dawley rats, 12 treated with GM 6001 100 mg/kg and 10 with vehicle, were allowed to heal by secondary intention. GM 6001-treated wounds were minimally resurfaced with neoepithelium, despite unaltered keratinocyte proliferation in wound edges, whereas control wounds were completely covered with 3-7 cell layers of parakeratinized epithelium on post-wounding day 7. Hydroxyproline concentration, a marker of collagen, and cell proliferation in granulation tissue did not differ significantly between GM 6001-treated and control groups. Impaired wound contraction (P < 0.01) was associated with a dramatic reduction of ASMA-positive myofibroblasts in granulation tissue of GM 6001 wounds. This was not due to GM6001 blocking transforming growth factor-beta1 (TGF-beta1)-induced myofibroblast differentiation since GM 6001 did not inhibit TGF-beta1-induced ASMA expression and force generation in cultured rat dermal fibroblasts. The profound impairment of skin repair by the nonselective MMP inhibitor GM 6001 suggests that keratinocyte resurfacing, wound contraction, and granulation tissue organization are highly MMP-dependent processes.
Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 [N-(2,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide], K6PC-5, [N-(1,3-dihydroxypropyl-2-hexyl-3-oxo-decanamide] and K6PC-9 (N-ethanol-2-hexyl-3-oxo-decanamide) [corrected] These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.
We determined the effects of helium-neon (He-Ne) laser irradiation on wound healing dynamics in mice treated with steroidal and non-steroidal anti-inflammatory agents. Male albino mice, 28-32 g, were randomized into 6 groups of 6 animals each: control (C), He-Ne laser (L), dexamethasone (D), D + L, celecoxib (X), and X + L. D and X were injected im at doses of 5 and 22 mg/kg, respectively, 24 h before the experiment. A 1-cm long surgical wound was made with a scalpel on the abdomens of the mice. Animals from groups L, D + L and X + L were exposed to 4 J (cm(2))-1 day-1 of He-Ne laser for 12 s and were sacrificed on days 1, 2, or 3 after the procedure, when skin samples were taken for histological examination. A significant increase of collagen synthesis was observed in group L compared with C (168 +/- 20 vs 63 +/- 8 mm(2)). The basal cellularity values on day 1 were: C = 763 +/- 47, L = 1116 +/- 85, D = 376 +/- 24, D + L = 698 +/- 31, X = 453 +/- 29, X + L = 639 +/- 32 U/mm(2). These data show that application of L increases while D and X decrease the inflammatory cellularity compared with C. They also show that L restores the diminished cellularity induced by the anti-inflammatory drugs. We suggest that He-Ne laser promotes collagen formation and restores the baseline cellularity after pharmacological inhibition, indicating new perspectives for laser therapy aiming to increase the healing process when anti-inflammatory drugs are used.
Keloids are benign fibroproliferative diseases of unknown aetiology. They occur as a result of derangement of the normal wound healing process in susceptible individuals. Although several factors have been postulated in the aetiopathogenesis of this condition, there has been growing evidence to suggest a role for Transforming Growth Factor beta (TGFbeta) family members in its pathogenesis. TGFbeta has also been found to be associated with fibrotic diseases affecting different organs of the body including liver, kidney, lung as well as skin. In this review article, we will discuss the morphology and mechanism of action of TGFbeta and its isoforms and present the most up to date literature discussing the role of TGFbeta isoforms, their receptors, and intracellular signalling pathways (the SMAD pathway) in the pathogenesis of keloid disease. Understanding the role of TGFbeta in keloid disease could lead to the development of clinically useful therapeutic modalities for treatment of this condition.
Persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury is a characteristic but poorly understood feature of normal wound healing. It results in extravasation of fibrinogen that clots to form fibrin, which serves as a provisional matrix and promotes angiogenesis and scar formation. We present evidence indicating that vascular permeability factor (VPF; also known as vascular endothelial growth factor) may be responsible for the hyperpermeable state, as well as the angiogenesis, that are characteristic of healing wounds. Hyperpermeable blood vessels were identified in healing split-thickness guinea pig and rat punch biopsy skin wounds by their capacity to extravasate circulating macromolecular tracers (colloidal carbon, fluoresceinated dextran). Vascular permeability was maximal at 2-3 d, but persisted as late as 7 d after wounding. Leaky vessels were found initially at the wound edges and later in the subepidermal granulation tissue as keratinocytes migrated to cover the denuded wound surface. Angiogenesis was also prominent within this 7-d interval. In situ hybridization revealed that greatly increased amounts of VPF mRNA were expressed by keratinocytes, initially those at the wound edge, and, at later intervals, keratinocytes that migrated to cover the wound surface; occasional mononuclear cells also expressed VPF mRNA. Secreted VPF was detected by immunofluoroassay of medium from cultured human keratinocytes. These data identify keratinocytes as an important source of VPF gene transcript and protein, correlate VPF expression with persistent vascular hyperpermeability and angiogenesis, and suggest that VPF is an important cytokine in wound healing.
To clarify interleukin (IL)-6 roles in wound healing, we prepared skin excisions in wild-type (WT) and IL-6-deficient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte infiltration was evident at wound sites. Furthermore, the re-epithelialization rate was ∼80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL-1, chemokines, adhesion molecules, transforming growth factorβ1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was significantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte infiltration, re-epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti-IL-6 monoclonal antibody significantly delayed wound closure in WT mice. These observations suggest that IL-6 has crucial roles in wound healing, probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation.
Among many molecules known to influence wound healing, transforming growth factor β1 (TGFβ1) has the broadest spectrum of actions, affecting all cell types that are involved in all stages of wound healing. Both positive and negative effects of TGFβ1 on wound healing have been reported. However, the underlying mechanisms are largely unknown. We observed that endogenous TGFβ1 was elevated in a narrow window of time after injury, and transgenic mice constitutively overexpressing wild-type TGFβ1 in keratinocytes (K5.TGFβ1wt) exhibited a significant delay in full-thickness wound healing as compared to non-transgenic mice. Delayed wound healing was associated with profound inflammation throughout all stages of wound healing in K5.TGFβ1wt mice. Our data suggest that excessive and prolonged TGFβ1 at the wound site does not benefit wound healing, which is partially owing to its pro-inflammatory effect. Future studies need to be conducted to assess whether tightly regulated TGFβ1 expression will benefit wound healing. To this end, we have developed a gene-switch TGFβ1 transgenic system that allows TGFβ1 induction in keratinocytes temporally with desired levels. These mice will provide a tool to study stage-specific effects of TGFβ1 on cutaneous wound healing.Journal of Investigative Dermatology Symposium Proceedings (2006) 0, 000–000. doi:10.1038/sj.jidsymp.5650004
Growth factors and cytokines are the vital mediators of both normal physiological and pathophysiological processes. This review will highlight the contrasting effects of certain growth factors and cytokines involved in a normal physiological process such as wound healing and in a pathological condition such as atherosclerosis. In addition, this review will also discuss the future of these signalling molecules in wound healing and atherosclerosis research.
Degradation of angiogenic mediators might be an underlying cause of chronic wounds. To test this hypothesis, we evaluated the expression and integrity of vascular endothelial growth factor, a potent angiogenic mediator, and its receptors, Fit-1 and KDR, in chronic venous leg ulcerations. Immunohistochemical, in situ hybridization, and semiquantitative reverse transcriptase polymerase chain reaction analyses all indicate that expression of vascular endothelial growth factor is elevated in ulcerative tissue, with vascular endothelial growth factor mRNA being especially pronounced in the hyperplastic epithelium of the wound margin. Flt-1 and KDR protein and mRNA were detected in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds. Although increased expression of vascular endothelial growth factor protein was detected in the epidermis, the intensity of this staining was weak compared with the epidermal staining in psoriatic lesions and compared with the strong vascular endothelial growth factor mRNA signal in chronic wounds and psoriasis. To analyze whether this apparent decrease in immunoreactivity could be the result of degradation of vascular endothelial growth factor by proteolytic activities from the wound environment, we examined the stability of recombinant vascular endothelial growth factor in wound fluid from chronic leg ulcers. As demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, incubation of rVEGF165 with chronic, but not acute, wound fluid resulted in rapid proteolytic degradation of rVEGF165. Protease inhibitor studies indicate that serine proteases, such as plasmin, are involved in this degradation. Together, our data show that, although vascular endothelial growth factor expression is elevated in chronic wounds, increased proteolytic activity in this environment results in its degradation, which may contribute to an impaired wound healing response.
The medical literature describes numerous in vitro and in vivo wound-healing models. The selection of an animal model depends on a number of factors including availability, cost, ease of handling, investigator familiarity, and anatomical/functional similarity to humans. Small mammals are frequently used for wound healing studies, however, these mammals differ from humans in a number of anatomical and physiological ways. Anatomically and physiologically, pig skin is more similar to human skin. The many similarities between man and pig would lead one to believe that the pig should make an excellent animal model for human wound healing. The purpose of this paper is to review the existing literature for evidence of this supposition and determine how well the various models correlate to human wound healing. Studies of wound dressings, topical antimicrobials, and growth factors are examined. Over 180 articles were utilized for this comparative review. Our conclusion is that the porcine model is an excellent tool for the evaluation of therapeutic agents destined for use in human wounds.
The positive influence of Aloe vera, a tropical cactus, on the healing of full-thickness wounds in diabetic rats is reported. Full-thickness excision/incision wounds were created on the back of rats, and treated either by topical application on the wound surface or by oral administration of the Aloe vera gel to the rat. Wound granulation tissues were removed on various days and the collagen, hexosamine, total protein and DNA contents were determined, in addition to the rates of wound contraction and period of epithelialization. Measurements of tensile strength were made on treated/untreated incision wounds. The results indicated that Aloe vera treatment of wounds in diabetic rats may enhance the process of wound healing by influencing phases such as inflammation, fibroplasia, collagen synthesis and maturation, and wound contraction. These effects may be due to the reported hypoglycemic effects of the aloe gel.
The influence of Aloe vera (L.) Burman f. on the glycosaminoglycan (GAG) components of the matrix in a healing wound was studied. Wound healing is a dynamic and complex sequence of events of which the major one is the synthesis of extracellular matrix components. The early stage of wound healing is characterized by the laying down of a provisional matrix, which is then followed by the formation of granulation tissue and synthesis of collagen and elastin. The provisional matrix or the ground substance consists of GAGs and proteoglycans (PGs), which are protein GAG conjugates. In the present work, we have studied the influence of Aloe vera on the content of GAG and its types in the granulation tissue of healing wounds. We have also reported the levels of a few enzymes involved in matrix metabolism. The amount of ground substance synthesized was found to be higher in the treated wounds, and in particular, hyaluronic acid and dermatan sulphate levels were increased. The levels of the reported glycohydrolases were elevated on treatment with Aloe vera, indicating increased turnover of the matrix. Both topical and oral treatments with Aloe vera were found to have a positive influence on the synthesis of GAGs and thereby beneficially modulate wound healing.
Aim of the study:
In South Africa the local population relies extensively on indigenous plants in the formulation of traditional medicines to treat skin ailments. The scientific merits of many of these plants used to treat wounds and burns are yet to be validated. Bulbine natalensis and Bulbine frutescens of the Asphodelaceae family are indigenous to only southern Africa and are widely used as a skin remedy. This study aimed to explore the scientific value of these plants through investigating the in vivo biochemical effects of Bulbine natalensis and Bulbine frutescens on cutaneous wounds.
Material and methods:
Excisional and incisional wounds treated with either B. natalensis or B. frutescens and mirrored control wounds were created on the back of 12 domestic pigs. Wound contraction was recorded daily. The excisional wounds, biopsied at days 2, 4, 7, 10 and 16, were used to analyse the biochemical composition of the wounds by estimating the total amount of protein, DNA, collagen and hexosamine present. The incisional wounds, biopsied at day 16, were used to test the tensile strength of the healed wounds using a tensiometer.
Results:
Wound contraction following treatment with Bulbine natalensis on days 2, 4 and 10 (p=.004, 0.007 and 0.03, respectively), and Bulbine frutescens on day 4 (p=0.004) increased significantly when compared to the corresponding untreated wounds. The tensile strength of the wounds treated with the leaf gels was significantly stronger than that of the untreated wounds. There was also a significant increase in the collagen, protein and DNA content of the Bulbine natalensis- and Bulbine frutescens-treated wounds compared with that of the untreated wounds (collagen content: p=0.014 and 0.018; protein content: p=0.03 and 0.04; DNA content p=0.04 and 0.04; respectively) over the 16-day experimental period. Treatment with both leaf gels followed the same pattern in hexosamine content with a maximum hexosamine content on day 4 followed by a steady decrease to day 16. No significant difference between the hexosamine content of the wounds of animals treated with either Bulbine frutescens or Bulbine natalensis was found.
Conclusions:
These findings validate the traditional use of the leaf gel extracts of B. frutescens and B. natalensis in the treatment of wounds and may warrant further investigation towards producing a low-cost effective topical treatment for wounds.
Indigenous medical plants contribute significantly to a large South African population as part of a long-standing healthcare system intimately linked to folklore and for livelihood security. For the economically marginalized, access to such plants is largely through herbal markets which are part of an informal economy. Otherwise the formal natural products economy services those with a better socio-economic standing. Recently, the latter has experienced tremendous growth which largely mirrors the global cultural trend for organic naturopathies. Commercialisation of traditional plants and their contribution to the cosmeceutical, nutraceutical and pharmaceutical industries locally and abroad is reviewed. Traditional plant knowledge of southern African people is a source of inspiration for new product development. Concomitantly, an upsurge in research activities emanating from South Africa which confirms the pharmacological efficacy of these plants is fuelling a greater trust in indigenous flora. The escalating consumption of ethnomedicinals as highly valued commodities not only presents South Africa with socio-economic opportunities but also with challenges. Sustainable utilization benefiting the commodification of ethnoherbals, plus meeting aims of poverty alleviation and people empowerment, is a new paradigm in South Africa. The future sustainability of local ecosystems depends upon scientific conservation management practices that recognize the importance of involving local communities. Conservationists should remain aware and sensitive of socio-cultural dynamics within communities in order to manage natural resources.
Natural products have been the single most productive source of leads for the development of drugs. Over a 100 new products are in clinical development, particularly as anti-cancer agents and anti-infectives. Application of molecular biological techniques is increasing the availability of novel compounds that can be conveniently produced in bacteria or yeasts, and combinatorial chemistry approaches are being based on natural product scaffolds to create screening libraries that closely resemble drug-like compounds. Various screening approaches are being developed to improve the ease with which natural products can be used in drug discovery campaigns, and data mining and virtual screening techniques are also being applied to databases of natural products. It is hoped that the more efficient and effective application of natural products will improve the drug discovery process.
A new reproducibility index is developed and studied. This index is the correlation between the two readings that fall on the 45 degree line through the origin. It is simple to use and possesses desirable properties. The statistical properties of this estimate can be satisfactorily evaluated using an inverse hyperbolic tangent transformation. A Monte Carlo experiment with 5,000 runs was performed to confirm the estimate's validity. An application using actual data is given.
Granulation tissue fibroblasts (myofibroblasts) develop several ultrastructural and biochemical features of smooth muscle (SM) cells, including the presence of microfilaments bundles and the expression of alpha-SM actin, the actin isoform typical of contractile vascular SM cells. Myofibroblasts have been suggested to play a role in wound contraction and in retractile phenomena observed during fibrotic diseases. When granulation tissue evolves into a scar, myofibroblasts containing alpha-SM actin disappear, probably as a result of apoptosis. In contrast myofibroblasts expressing alpha-Sm actin persist in excessive scarring and in fibrotic conditions. The mechanisms leading to the development of myofibroblastic features remain to be investigated. Studies on the factors regulating the phenotype of myofibroblasts will be necessary for understanding their behavior in vivo, and possibly modifying this behavior during the different clinical settings.
Human fetal skin heals via scarless regeneration, whereas adult skin heals with scar. Scarless repair may reflect a distinct cytokine milieu. We studied the role of the cytokine transforming growth factor beta (TGF beta) using an established model of scarless human fetal skin repair in which human fetal skin is transplanted into a subcutaneous pocket on the flank of an adult nude mouse. In this model, wounded 16-week-gestation human fetal skin heals without scar, whereas wounded adult skin heals with scar. Seven days after transplantation, incisional wounds were made in the skin grafts. In the first phase of the study, wounds were harvested from 1 hour to 4 weeks postwounding, and immunohistochemistry was performed for TGF beta (isoform nonspecific), TGF beta 1, and TGF beta 2. Scarfree wounds in the fetal skin grafts did not show TGF beta staining. In contrast, wounds in adult grafts that heal with scar demonstrated isoform nonspecific TGF beta staining from 6 hours through 21 days, TGF beta 1 from 6 hours through 21 days, and TGF beta 2 from 12 hours through 7 days. In the second phase of the study, a slow-release disk with 0.01, 0.1, 1.0, or 10 micrograms of TGF beta 1 was placed beneath the fetal skin graft at the time of wounding. Fourteen days postwounding, there was marked scarring in the fetal grafts treated with TGF beta 1, and the size of the scar was proportional to the amount of TGF beta 1 applied.(ABSTRACT TRUNCATED AT 250 WORDS)
Massive cell migration, proliferation, phenotypic differentiation, and enhanced biosynthetic activities characterize the sites of wound healing and fibrosis. Regulation of cellular functions by extracellular matrix, which consists of a dynamic assemblage of a variety of interacting molecules capable of reorganization in response to endogenous and exogenous stimuli, represents a fundamental epigenetic mechanism regulating cellular behavior and phenotype. Interactions of the individual components of extracellular matrix with specific cell surface molecules, integrin receptors, and proteoglycans initiate a cascade of signal transduction leading to varied short-term or persistent cellular responses. Extracellular matrix also serves as an important reservoir of cytokines and growth factors, thus modulating the action of a host of potent biological response modifiers by their selective, local accumulation and release. Currently known mechanisms by which extracellular matrix modulates different facets of the process of tissue remodeling after injury, which culminate either in normal wound repair or fibrosis, are discussed.
The Transforming Growth Factor beta superfamily (TGF beta) is one of the most complex groups of cytokines with widespread effects on many aspects of growth and development. The TGF beta isoforms and other family members, e.g. Activins and BMPs, have diverse effects in similar physiological situations. TGF beta is involved in the wound healing process. The three mammalian isoforms (TGF beta 1, 2 and 3) and recently other family members, e.g. Activin, have been localised in healing wounds. Manipulation of the ratios of TGF beta superfamily members, particularly the ratio of TGF beta 1 relative to TGF beta 3, reduces scarring and fibrosis. Such manipulations include reducing the levels of TGF beta 1/TGF beta 2 using neutralising antibodies or preventing the activation of TGF beta s. In chronic or impaired wounds the exogenous addition of TGF beta superfamily members accelerates aspects of the healing process. This review summarises evidence for the role of TGF beta superfamily members in wound healing and how modulation of TGF beta levels can prevent scarring and fibrosis.
Vascular endothelial growth factor (VEGF), an endothelium-specific growth factor and microvessel hypermeability factor, is expressed and secreted by several kinds of cells and is implicated in angiogenesis of tumors. The present study was performed to determine the relationship between the expression of VEGF in normal skin, benign and malignant epithelial lesions and cultured keratinocytes and the proliferative activity and degree of differentiation of keratinocytes. Skin lesions were studied immunohistochemically by staining with two anti-VEGF antibodies and secretion and production of VEGF by keratinocyte cultures were evaluated using an enzyme-linked immunosorbent assay. Low to moderate VEGF expression was observed in normal epidermis. In epithelial tumors, different reactivity patterns were observed and different areas of the same tumor expressed different amounts of VEGF. A more prominent labelling occurred in proliferative layers and/or more differentiated cells of virus-induced lesions, squamous cell carcinomas and Bowen's disease, whereas basal cell carcinomas always stained weakly for VEGF. In cultured keratinocytes, the amount of cell-associated and secreted VEGF increased with time, and the constitutively produced VEGF was mostly released extracellularly. High calcium concentrations upregulated the intracellular content of VEGF but downregulated its release. Taken together, these results showed a modulated expression and release of VEGF in relation to the stage of cell differentiation and in rapidly growing or activated keratinocytes.
The structure of porcine skin as examined by light microscopy is reviewed and its similarities to and differences from human skin are highlighted. Special imaging techniques and staining procedures are described and their use in gathering morphological information in porcine skin is discussed. Confocal laser scanning microscopy (CLSM) was employed to examine the structure of porcine skin and the findings are presented as an adjunct to the information already available in the literature. It is concluded that CLSM provides valuable additional morphological information to material examined by conventional microscopy and is useful for wound healing studies in the porcine model.
Myofibroblasts play an important role in normal wound healing. They are present transiently during tissue repair. Their differentiation from fibroblasts and their role in granulation tissues are most likely to be modulated by cytokines. As these cells are derived from normal fibroblasts, their responses to cytokines are assumed to be similar. Until now, however, the difficulties in obtaining and maintaining normal human wound healing myofibroblasts in vitro have hampered comparison. The present study was designed to determine the effect of TGF-beta 1 and IFN-gamma, two cytokines known to modulate fibroblast morphology, on wound healing myofibroblasts and to compare it to fibroblasts. Morphological and phenotypic changes were followed by light and electron microscopy (stress fibers) and immunofluorescence cytochemistry (alpha-SM actin). Functional parameters such as the capacity to synthesize collagen and collagen gel contraction were studied. Both cytokines induced a strong modification of growth rate and phenotypic and morphological parameters in fibroblasts whereas collagen synthesis was slightly changed. Furthermore, TGF-beta 1 increased contractile capacity of fibroblasts whereas IFN-gamma greatly decreased it. In myofibroblasts, TGF-beta 1 and IFN-gamma did not induce any variation of morphology or growth rate. Interestingly, a strong modulation of functional parameters was observed: collagen synthesis was highly modified and, as for fibroblasts, the contractile capacity was altered. However, inhibition of contraction by IFN-gamma was irreversible in myofibroblasts but not in fibroblasts. These results suggest that fibroblastic cells show modulated responses to cytokines according to their stage of differentiation during wound healing.
To test the influence of vascular endothelial growth factor (VEGF) on normal and ischemic wounds in a noncontractive dermal ulcer standardized model in the rabbit ear and to assay the levels of both VEGF and basic fibroblastic growth factor messenger RNA levels in normal and ischemic wounds at different intervals during the healing process.
Dermal ulcers were created in the normal and ischemic ears of 20 anesthetized young female New Zealand white rabbits. Either VEGF 121, VEGF 165 (30 microg per wound), or buffered saline solution alone was applied to each wound and covered. Wounds were harvested at day 7 or 10 and evaluated histologically. Twenty-four similar rabbits were wounded in the same manner and their untreated wounds were harvested at 1, 3, 7, and 10 days after wounding. The wounds were analyzed with reverse transcriptase polymerase chain reaction.
Histologic specimens were measured for amount of new epithelium and granulation tissue. Reverse transcriptase polymerase chain reaction was used to determine basic fibroblastic growth factor and VEGF messenger RNA expression.
Both isoforms of VEGF improved granulation tissue formation in both normal and ischemic wounds with a magnitude similar to other vulnerary agents tested in the past. Vascular endothelial growth factor application had no effect on new epithelium formation. In contrast to basic fibroblastic growth factor, VEGF messenger RNA levels were induced 4 fold by ischemia alone and 6 fold by wounding in both ischemic and normal wounds.
Vascular endothelial growth factor seems to be more important than basic fibroblastic growth factor during ischemic wound healing. Treatment of ischemic wounds with VEGF improves the deficit in wound healing produced by ischemia.
In human skin, large burned surfaces heal using two concomitant phenomena: re-epithelialization and dermal neoformation. Numerous studies report the role of interactions between keratinocytes and fibroblasts, but the relationship between wound healing myofibroblasts and keratinocytes is not clear, even though these two cell types coexist during healing. We investigated the influence of myofibroblasts on keratinocyte growth and differentiation using an in vitro skin model. A histological study was performed to determine the speed and quality of epithelialization. When the dermis was populated with fibroblasts, a continuous epidermis was formed in 7-10 days. In contrast, with wound healing myofibroblasts or without cell in dermis, the complete reepithelialization never occurred over the 10-day period studied. After 7 further days of epidermal differentiation, histology showed an epidermis more disorganized and expression of basement membrane constituents was reduced when wound healing myofibroblasts or no cells were added in the dermis instead of fibroblasts. These results suggest that wound healing myofibroblasts are not efficient to stimulate keratinocyte growth and differentiation. Treatment of fibroblasts with TGFbeta1 induced an increase of epidermal cell differentiation as seen when myofibroblasts were present. However, this cytokine did not change re-epithelialization rate and induced an increase of basement membrane matrix deposition in opposition to myofibroblasts. Thus, TGFbeta1 action is not sufficient to explain all the different keratinocyte reactions towards fibroblasts and wound healing myofibroblasts. Our conclusion is that myofibroblasts seem to have a limited role in the re-epithelialization process and might be more associated with the increased extracellular matrix secretion.
We have generated transgenic mice expressing green fluorescent protein (GFP) driven by 2.453-kb (-2,362 to +91) of the 5'-upstream region of the human vascular endothelial growth factor (VEGF) promoter to monitor changes of VEGF gene transcription in situ. Neonatal transgenic mice exhibited GFP-derived fluorescence in tissues that have been previously reported to express VEGF mRNA expression, including lung, cartilage, and brain. In normal skin during postnatal development, moderate fluorescence was observed in the upper epidermis and, more prominently, in the outer root sheath keratinocytes of hair follicles. Strong up-regulation of GFP fluorescence was observed in the hyperplastic epidermis of the wound edge at 48 hours after wounding, whereas little GFP fluorescence was detected in the dermis. In situ hybridization confirmed an identical expression pattern of VEGF mRNA in these wounds. Topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced strong VEGF-GFP expression in suprabasal epidermis. Little or no fibroblast-derived fluorescence was seen both in the wound model and after TPA application. By confocal laser microscopy, increased GFP fluorescence was detectable in the epidermis of intact mouse ear skin as early as 6 hours after topical TPA treatment. Importantly, GFP fluorescence was also measurable in the skin of living transgenic mice. These results resolve the present controversy regarding the ability of VEGF-GFP transgenic mouse models to correctly reflect established patterns of VEGF expression, and show the model to be a powerful tool for the in vivo monitoring of VEGF gene expression.
Vascular endothelial growth factor (VEGF) expression in healing wounds provokes dermal angiogenesis through complex mechanisms involving promotion of endothelial cell proliferation 1,2 and survival, 3-5 specific induction of endothelial cell gene expression, 6-10 and increased microvascular permeability. 11,12 Microvascular hyperpermeability transforms the extracellular matrix by promoting extravasation of fibrinogen, 13 fibronectin, 14 and other proteins from blood plasma. 14
Keratinocytes are increasingly recognized as key regulators of skin inflammation and remodeling, as they are capable of producing growth factors and cytokines that are important mediators in the wound healing process. We investigated the expression and distribution of TGF-beta 1 mRNA by mRNA in situ hybridization and of TGF-beta 1, TGF-beta 2, TGF-beta 3, bFGF and VEGF protein expression using immunohistochemistry in spontaneously healed, partial-thickness burns and compared this with the expression of these markers in matched unburned skin. This was done to assess their role in the remodeling phase of burn wound healing. Punch biopsies were taken from both partial-thickness burns after re-epithelialization and from matched, unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. We observed a higher expression of all mentioned growth factors in keratinocytes in scars at 1 month post-burn compared with matched unburned skin. At 4 months, keratinocytes still displayed a higher expression of TGF-beta 3 and bFGF, but the expression of TGF-beta 1, TGF-beta 2 and VEGF was normalized. The expression of TGF-beta 3 in the epidermis of hypertrophic scars was slightly higher than in normotrophic scars. At 7 months post-burn, all growth factors studied showed a normal expression on keratinocytes. Our results suggest that keratinocytes are not only involved in re-epithelialization, but also in the scar maturation. The data support the idea that keratinocytes not only respond to cytokines and growth factors in an autocrine fashion, but also exert regulatory paracrine effects on contiguous cells.
During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. A dynamic interaction occurs among endothelial cells, angiogenic cytokines, such as FGF, VEGF, TGF-beta, angiopoietin, and mast cell tryptase, and the extracellular matrix (ECM) environment. Specific endothelial cell ECM receptors are critical for these morphogenetic changes in blood vessels during wound repair. In particular, alpha(v)beta3, the integrin receptor for fibrin and fibronectin, appears to be required for wound angiogenesis: alpha(v)beta3 is expressed on the tips of angiogenic capillary sprouts invading the wound clot, and functional inhibitors of alpha(v)beta3 transiently inhibit granulation tissue formation. Recent investigations have shown that the wound ECM can regulate angiogenesis in part by modulating integrin receptor expression. mRNA levels of alpha(v)beta3 in human dermal microvascular endothelial cells either plated on fibronectin or overlaid by fibrin gel were higher than in cells plated on collagen or overlaid by collagen gel. Wound angiogenesis also appears to be regulated by endothelial cell interaction with the specific three-dimensional ECM environment in the wound space. In an in vitro model of human sprout angiogenesis, three-dimensional fibrin gel, simulating early wound clot, but not collagen gel, simulating late granulation tissue, supported capillary sprout formation. Understanding the molecular mechanisms that regulate wound angiogenesis, particularly how ECM modulates ECM receptor and angiogenic factor requirements, may provide new approaches for treating chronic wounds.
The medical literature describes numerous in vitro and in vivo wound-healing models. The selection of an animal model depends on a number of factors including availability, cost, ease of handling, investigator familiarity, and anatomical/functional similarity to humans. Small mammals are frequently used for wound healing studies, however, these mammals differ from humans in a number of anatomical and physiological ways. Anatomically and physiologically, pig skin is more similar to human skin. The many similarities between man and pig would lead one to believe that the pig should make an excellent animal model for human wound healing. The purpose of this paper is to review the existing literature for evidence of this supposition and determine how well the various models correlate to human wound healing. Studies of wound dressings, topical antimicrobials, and growth factors are examined. Over 180 articles were utilized for this comparative review. Our conclusion is that the porcine model is an excellent tool for the evaluation of therapeutic agents destined for use in human wounds.
Background:
To clarify the clinical significance of the expression of vascular endothelial growth factor (VEGF) and its receptor, kinase domain-containing receptor (KDR) in colorectal cancer, we evaluated the relationship between the expression of VEGF and KDR, and the microvessel counts and clinicopathological factors in colorectal cancer.
Methods:
A total of 259 specimens from sequential colorectal cancer patients who had undergone surgery were examined by the avidin-biotin peroxidase complex method, using anti-human VEGF, anti-human KDR, and anti-human von Willebrand factor antibodies.
Results:
The incidence of VEGF expression in the tumor cells of the patients with liver metastasis was significantly higher than that in the tumor cells of the patients without liver metastasis (67% vs 44%). The microvessel count at the tumor invasive edge in the patients whose tumor cells were positive for VEGF was significantly higher than that in the patients whose tumor cells were negative for VEGF (33.0 +/- 7.8 vs 28.0 +/- 7.9); the significant difference in microvessel counts was greater when there was a combination of VEGF and KDR expression. The overall survival rate of patients positive for VEGF was significantly (P = 0.0276) lower than that of those who were negative for VEGF. Although there was no significant difference (P = 0.0743) in the survival rates after potentially curative resection according to VEGF expression, the survival rate of the patients positive for both VEGF in tumor cells and KDR in endothelial cells was significantly (P = 0.0026) lower than that in the patients who were negative for VEGF and/or KDR. In addition, multivariate analysis revealed that the expression of both VEGF and KDR was an independent prognostic factor even after potentially curative resection.
Conclusion:
VEGF may be implicated in the definition of the malignant phenotype of colorectal cancer via tumor angiogenesis. VEGF and its receptor KDR expression in tumorous tissues could be useful prognostic factors in colorectal cancer.
Revascularization of damaged tissue is a necessary part of wound healing. With unregulated or insufficient vessel growth, healing is delayed or pathological. Angiogenesis is regulated by expression of a variety of vascular growth factors and modulators, the most widely expressed and critical of which is vascular endothelial growth factor (VEGF). This protein is secreted by tissues in response to ischemic and inflammatory stimuli and results in endothelial migration, proliferation, and increased vascular permeability. The regulation of VEGF expression during wound healing is of considerable importance since angiogenesis appears to be disturbed in abnormally healing wounds. This paper describes the current state of knowledge of VEGF expression in wounds, regulation of expression, control of isoform specificity, and the effects of VEGF expression on blood vessels as they grow in wound healing, as understood from many different pathological paradigms.
Wound healing is a complex biological process, comprised of a series of a sequential events aiming to repair injured tissue. The role of the immune system in this process is not only to recognise and combat the newly presented antigens at the site of injury, but also to participate in the debridement of the damaged area and to contribute to the process of healing. In this review, we discuss the molecules and cells of the immune system that participate in tissue repair. We describe the mechanisms of immune recognition during initial insult and the innate and adaptive immune responses to injury. Finally, we address the role of the immune system in regeneration and repair.
Among many molecules known to influence wound healing, transforming growth factor beta 1 (TGF beta 1) has the broadest spectrum of actions, affecting all cell types that are involved in all stages of wound healing. Both positive and negative effects of TGF beta 1 on wound healing have been reported. However, the underlying mechanisms are largely unknown. We observed that endogenous TGF beta 1 was elevated in a narrow window of time after injury, and transgenic mice constitutively overexpressing wild-type TGF beta 1 in keratinocytes (K5.TGF beta 1wt) exhibited a significant delay in full-thickness wound healing as compared to non-transgenic mice. Delayed wound healing was associated with profound inflammation throughout all stages of wound healing in K5.TGF beta 1wt mice. Our data suggest that excessive and prolonged TGF beta 1 at the wound site does not benefit wound healing, which is partially owing to its pro-inflammatory effect. Future studies need to be conducted to assess whether tightly regulated TGF beta 1 expression will benefit wound healing. To this end, we have developed a gene-switch TGF beta 1 transgenic system that allows TGF beta 1 induction in keratinocytes temporally with desired levels. These mice will provide a tool to study stage-specific effects of TGF beta 1 on cutaneous wound healing.
Wound healing is a complex process that can be divided into at least 3 continuous and overlapping processes: an inflammatory reaction, a proliferative process leading to tissue restoration, and, eventually, tissue remodeling. Wound healing processes are strictly regulated by multiple growth factors and cytokines released at the wound site. Although the desirable final result of coordinated healing would be the formation of tissue with a similar structure and comparable functions as with intact skin, regeneration is uncommon (with notable exceptions such as early fetal healing); healing however results in a structurally and functionally satisfactory but not identical outcome. Alterations that disrupt controlled healing processes would extend tissue damage and repair. The pathobiologic states may lead to chronic or nonhealing wounds or excessive fibrosis.
Cutaneous tissue repair aims at restoring the barrier function of the skin. To achieve this, defects need to be replaced by granulation tissue to form new connective tissue, and epithelial wound closure is required to restore the physical barrier. Different wound-healing phases are recognized, starting with an inflammation-dominated early phase giving way to granulation tissue build-up and scar remodeling after epithelial wound closure has been achieved. In the granulation tissue, mesenchymal cells are maximally activated, cells proliferate, and synthesize huge amounts of extracellular matrix. Epithelial cells also proliferate and migrate over the provisional matrix of the underlying granulation tissue, eventually closing the defect. This review focuses on the role of keratinocyte-fibroblast interactions in the wound-healing process. There is ample evidence that keratinocytes stimulate fibroblasts to synthesize growth factors, which in turn will stimulate keratinocyte proliferation in a double paracrine manner. Moreover, fibroblasts can acquire a myofibroblast phenotype under the control of keratinocytes. This depends on a finely tuned balance between a proinflammatory or a transforming growth factor (TGF)-beta-dominated environment. As the phenotype of fibroblasts from different tissues or body sites becomes better defined, we may understand their individual contribution in wound healing in more detail and possibly explain different clinical outcomes.
Theory and Practice of Histological Techniques The role of VEGF in wound healing
- J D Bancroft
- M Gamble
- D O Bates
- R O Pritchard-Jones
Bancroft, J.D., Gamble, M., 2002. Theory and Practice of Histological Techniques, 5th edition, Churchill Livingstone Publishers, Edinburgh. Bates, D.O., Pritchard-Jones, R.O., 2003. The role of VEGF in wound healing. International Journal of Lower Extremity Wounds 2, 107–120.
Zulu Medicinal Plants: an Inventory
- A Hutchings
- A H Scott
- G Lewis
- A Cunningham
Hutchings, A., Scott, A.H., Lewis, G., Cunningham, A., 1996. Zulu Medicinal Plants: an Inventory. University of Natal Press, Pietermaritzburg, South Africa.
Natural Product Pharmaceuticals: a Diverse Approach to Drug Discovery
- A L Harvey
Harvey, A.L., 2001. Natural Product Pharmaceuticals: a Diverse Approach to Drug Discovery. Scrip Reports, PJB Publications, Surry, UK.