[Show abstract][Hide abstract] ABSTRACT: Yu Ping Feng San (YPFS), a Chinese herbal decoction comprising Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu), and Saposhnikoviae Radix (SR; Fangfeng), has been used clinically to treat inflammatory bowel diseases (IBD). Previously, we demonstrated a dual role of YPFS in regulating cytokine release in cultured macrophages. In this study, we elucidated the anti-inflammatory effect of YPFS that is mediated through modulating the expression of three key enzymes involved in IBD: inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and intestinal alkaline phosphatase (IALP). In a lipopolysaccharide (LPS)-induced chronic-inflammation model of cultured murine macrophages, YPFS treatment suppressed the activation of iNOS and COX-2 expression in a dose-dependent manner. Conversely, application of YPFS in cultured small intestinal enterocytes markedly induced the expression of IALP in a time-dependent manner, which might strengthen the intestinal detoxification system. A duality of YPFS in modulating the expression of iNOS and COX-2 was determined here. The expression of iNOS and COX-2 in macrophages was induced by YPFS, and this activation was partially blocked by the NF-κB-specific inhibitor BAY 11-7082, indicating a role of NF-κB signaling. These YPFS-induced changes in gene regulation strongly suggest that the anti-inflammatory effects of YPFS are mediated through the regulation of inflammatory enzymes.
PLoS ONE 06/2014; 9(6):e100382. DOI:10.1371/journal.pone.0100382 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Danggui Buxue Tang, an ancient formula composed of Astragali Radix and Angelicae Sinensis Radix, has been used for treating menopausal irregularity in women for more than 800 years in China. In Danggui Buxue Tang, the complete functions of Astragali Radix require the assistance of Angelicae Sinensis Radix, and both herbs have to work harmoniously in order to achieve the maximal therapeutic purposes. In order to analyze the relationship of the two herbs, the role of ferulic acid, a major chemical within Angelicae Sinensis Radix, in chemical and biological properties of Astragali Radix was determined. Using ferulic acid in the extraction of Astragali Radix, the amounts of astragaloside IV, calycosin, and formononetin were increased in the final extract; however, the Astragali Radix polysaccharide showed a minor increase. The chemical-enriched Astragali Radix extract showed robust induction in osteogenic and estrogenic activities in cultured osteosarcoma MG-63 and breast MCF-7 cells. However, ferulic acid itself did not show such biological responses. The current results strongly suggest that Angelicae Sinensis Radix-derived ferulic acid is a positive regulator for Danggui Buxue Tang, which enhanced the solubilities of active ingredients derived from Astragali Radix, and which therefore increased the biological efficacies of Danggui Buxue Tang.
[Show abstract][Hide abstract] ABSTRACT: Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3' end produces different isoforms, including tailed (AChET), read-through (AChER), and hydrophobic (AChEH). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChET transcript per 100 ng of total RNA were 5.7 × 10(4) in PC12 (rat neuronal cell), 1.3 × 10(4) in Caco-2 (human intestinal cell), 0.67 × 10(4) in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChEH in these cell types were 0.3 × 10(4), 3.3 × 10(4), 2.7 × 10(4), 133.3, and 46.7, respectively, and AChER were 0.07 × 10(4), 0.13 × 10(4), 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChET mRNA but not AChER or AChEH mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChET.
[Show abstract][Hide abstract] ABSTRACT: Flavonoids, a family of phenolic compounds, are widely present in our daily diet and exist in traditional Chinese medicines, in which they act as the major active functional ingredients. Different lines of evidence indicate that flavonoids have positive impacts on human health. Here, different subclasses of flavonoids were analyzed for their inductive roles in promoting the expression of synaptic proteins, synaptotagmin, and post-synaptic density protein-95 in cultured rat cortical neurons. Among the screened 65 flavonoids, (-)-catechin, luteolin, and isorhamnetin, in micromolar concentration, were found to induce the expression of synaptic proteins in a dose-dependent manner: the induction values were from 2- to 8-fold that of the control. Similar results were revealed in the flavonoid-treated hippocampal neurons. The identification of these synapse-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer's disease and depression.
[Show abstract][Hide abstract] ABSTRACT: Yu Ping Feng San (YPFS), a Chinese herbal decoction, is composed of Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu) and Saposhnikoviae Radix (SR; Fangfeng) in a weight ratio of 1∶2∶1. Clinically, YPFS has been widely used to regulate immune functions; however, the action mechanism of it is not known. Here, we addressed this issue by providing detail analyses of chemical and biological properties of YPFS. By using rapid resolution liquid chromatography coupled with mass spectrometry, fifteen chemicals deriving from different herbs of YPFS were determined, and which served as a control for the standardization of the herbal extract of YPFS. In general, the amounts of chosen chemical markers were higher in a preparation of YPFS as compared to that of single herb or two-herb compositions. In order to reveal the immune functions of YPFS, the standardized extract was applied onto cultured murine macrophages. The treatment of YPFS stimulated the mRNA and protein expressions of pro-inflammatory cytokines via activation of NF-κB by enhancing IκBα degradation. In contrast, the application of YPFS suppressed the expressions of pro-inflammatory cytokines significantly in the lipopolysaccharide (LPS)-induced chronic inflammation model. In addition, YPFS could up regulate the phagocytic activity in cultured macrophages. These results therefore supported the bi-directional immune-modulatory roles of YPFS in regulating the releases of cytokines from macrophages.
PLoS ONE 11/2013; 8(11):e78622. DOI:10.1371/journal.pone.0078622 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Song Bu Li decoction (SBL) is a traditional Uyghur medicinal herbal preparation, containing Nardostachyos Radix et Rhizoma. Recently, SBL is being used to treat neurological disorders (insomnia and neurasthenia) and heart disorders (arrhythmia and palpitation). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness. Here, we aimed to evaluate the protective and differentiating activities of SBL in cultured PC12 cells. The pretreatment of SBL protected the cell against tBHP-induced cell death in a dose-dependent manner. In parallel, SBL suppressed intracellular reactive oxygen species (ROS) formation. The transcriptional activity of antioxidant response element (ARE), as well as the key antioxidative stress proteins, was induced in dose-dependent manner by SBL in the cultures. In cultured PC12 cells, the expression of neurofilament, a protein marker for neuronal differentiation, was markedly induced by applied herbal extract. Moreover, the nerve growth factor- (NGF-) induced neurite outgrowth in cultured PC12 cells was significantly potentiated by the cotreatment of SBL. In accord, the expression of neurofilament was increased in the treatment of SBL. These results therefore suggested a possible role of SBL by its effect on neuron differentiation and protection against oxidative stress.
Evidence-based Complementary and Alternative Medicine 09/2013; 2013(5134):687958. DOI:10.1155/2013/687958 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite the classical hormonal effect, estrogen has been reported to mediate neuroprotection in the brain, which leads to the searching of estrogen-like substances for treating neurodegenerative diseases. Flavonoids, a group of natural compounds, are well known to possess estrogenic effects and used to substitute estrogen, that is, phytoestrogen. Flavonoid serves as one of the potential targets for the development of natural supplements and therapeutic drugs against different diseases. The neuroprotection activity of flavonoids was chosen for a possible development of anti-Alzheimer's drugs or food supplements. The estrogenic activity of two flavonoids, baicalein and daidzein, were demonstrated by their strong abilities in stimulating estrogen receptor phosphorylation and transcriptional activation of estrogen responsive element in MCF-7 breast cells. The neuroprotection effects of flavonoids against β -amyloid (A β ) were revealed by their inhibition effects on in vitro A β aggregation and A β -induced cytotoxicity in PC12 neuronal cells. More importantly, the estrogenic and neuroprotective activities of individual flavonoid could be further enhanced by the cotreatment in the cultures. Taken together, this synergistic effect of baicalein and daidzein might serve as a method to improve the therapeutic efficacy of different flavonoids against A β , which might be crucial in developing those flavonoidsin treating Alzheimer's disease in the future.
Evidence-based Complementary and Alternative Medicine 08/2013; 2013(1):635694. DOI:10.1155/2013/635694 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chinese date, the fruit of Ziziphus jujuba Mill., has thousands of years cultivation history, and about 700 cultivars of dates in China. Two types of dates are commonly found in the market: (i) fresh immature dates consumed as fruits, and (ii) dried mature dates used as Chinese medicines. Here, chemical and biological properties of these dates were revealed. Different sources of dates showed similar chemical profiles; however, the amounts of identified chemicals showed a great variation. The amount of nucleotides, flavonoids and polysaccharides in dates could be affected by its maturity and drying process. In parallel, the anti-oxidative functions of their extracts were compared. The date extracts protected PC12 cells against tBHP-induced cytotoxicity, and which also stimulated the transcriptional activity of anti-oxidant response element. The anti-oxidative effects were varied amongst different dates. The current results suggested the optimization of sources and specific usage of different maturity dates.
Journal of Agricultural and Food Chemistry 07/2013; 61(30). DOI:10.1021/jf402379u · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neurotrophic factors are playing vital roles in survival, growth, and function of neurons. Regulation of neurotrophic factors in the brain has been considered as one of the targets in developing drug or therapy against neuronal disorders. Flavonoids, a family of multifunctional natural compounds, are well known for their neuronal beneficial effects. Here, the effects of flavonoids on regulating neurotrophic factors were analyzed in cultured rat astrocytes. Astrocyte is a major secreting source of neurotrophic factors in the brain. Thirty-three flavonoids were screened in the cultures, and calycosin, isorhamnetin, luteolin, and genistein were identified to be highly active in inducing the synthesis and secretion of neurotrophic factors, including nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF). The inductions were in time- and dose-dependent manners. In cultured astrocytes, the phosphorylation of estrogen receptor was triggered by application of flavonoids. The phosphorylation was blocked by an inhibitor of estrogen receptor, which in parallel reduced the flavonoid-induced expression of neurotrophic factors. The results proposed the role of flavonoids in protecting brain diseases, and therefore these flavonoids could be developed for health food supplement for patients suffering from neurodegenerative diseases.
Evidence-based Complementary and Alternative Medicine 06/2013; 2013(4):127075. DOI:10.1155/2013/127075 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adenosine 5'-triphosphate (ATP) functions as an extracellular signaling molecule that is co-stored and co-released with neurotransmitters at central and peripheral neuronal synapses. Stimulation by ATP up-regulates the expression of synaptic genes in muscle - including the genes for nicotine acetylcholine receptor (α-, δ- and ε-subunits) and acetylcholinesterase (AChE) - via the P2Y receptor (P2YR), but the trophic response of neurons to the activation of P2YRs is less well understood. We reported that cultured cortical neurons and the developing rat brain expressed different types of P2YRs, and among these the UTP-sensitive P2Y2R was the most abundant. P2Y2R was found to exist in membrane rafts and it co-localized with the post-synaptic protein PSD-95 in cortical neurons. Notably, agonist-dependent stimulation of P2Y2R elevated the neuronal expression of cholinergic genes encoding AChE, PRiMA (an anchor for the globular form AChE) and choline acetyltransferase, and this induction was mediated by a signaling cascade that involved Ca(2+) mobilization and ERK1/2 activation. The importance of P2Y2R action was further shown by the receptor's synergistic effect with P2Y1R in enhancing cholinergic gene expression via the robust stimulation of Ca(2+) influx. Taken together our results revealed a developmental function of P2Y2R in promoting synaptic gene expression and demonstrate the influence of co-stimulation of P2Y1R and P2Y2R in neurons.
[Show abstract][Hide abstract] ABSTRACT: Immunoglobulin superfamily (IgSF) molecules are actively involved in cell-cell adhesion, neuronal migration, axonal guidance and synapse formation in the nervous system. Kirre, as a member of this family, has been implicated in mammalian neuronal differentiation and development. Although the distribution of rKirre (a rat homologue of Drosophila Kirre) mRNA was previously analyzed in adult rat cerebellum by in situ hybridization, the expression levels of transcript and protein were not well studied. Here, we showed that the expressions of rKirre mRNA and protein significantly increased during postnatal development of rat cerebellum. rKirre mRNA was mainly expressed in the granular layers and Purkinje cell layer in the developing cerebellum, revealing a possible involvement of rKirre in granule cell migration and Purkinje cell development. An essential relationship between rKirre and Purkinje cells was implied by the co-localization of rKirre and NF-200 on the cell bodies of Purkinje cells. These results suggest that rKirre may play a potential role in postnatal developing rat cerebellum.
[Show abstract][Hide abstract] ABSTRACT: Acetylcholinesterase (AChE) activity has been used to evaluate the exposure of mollusk bivalves to organophosphates, carbamate pesticides, and heavy metals. Crassostrea hongkongensis is a Hong Kong endemic oyster, and has a high commercial value along the coastal area of South China. The use of this species as a bio-indicator of the marine environment, and the use of AChE activity measurements in tissues of C. hongkongensis require prior characterization of AChE in this species. Here, we report that gill tissue contains the highest AChE activity in C. hongkongensis, and that the molecular form of AChE is most likely to be a dimeric form. In addition, the effect of the pesticide acephate on AChE activity in the gill of C. hongkongensis was analyzed, and the mean inhibition concentration (IC50) value was determined. This study suggests that AChE activity in the gill tissue of C. hongkongensis might be used as a biomarker in monitoring organophosphate contamination in the marine fauna of South China.
[Show abstract][Hide abstract] ABSTRACT: Roots of (Danggui) have been used in promoting blood circulation as herbal medicine for over 2000 years in China. Another species of roots called is being used in Korea. To reveal the efficiency of different roots, the chemical and biological properties of roots from different cultivated regions were compared. Roots of contained higher levels of ferulic acid, Z-ligustilide, and senkyunolide A, while high amounts of butylphthalide and Z-butylenephthalide were found in roots. The extracts deriving from roots showed better effects in osteogenic and estrogenic properties than that of from China. However, this difference was markedly reduced when the roots were being prepared in a Chinese herbal decoction together with Astragali Radix as Danggui Buxue Tang. In contrast, the herbal decoction prepared from roots showed better responses in cell cultures. In addition, the extracts of roots showed strong cell toxicity both as single herb and as Danggui Buxue Tang. This result revealed the distinct properties of roots from China and Korea suggesting the specific usage of herb in preparing a unique herbal decoction.
Evidence-based Complementary and Alternative Medicine 02/2013; 2013(8):483286. DOI:10.1155/2013/483286 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2∶3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+) levels in HUVECs, and the Ca(2+) chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+) and a reduction of ROS.
PLoS ONE 12/2012; 7(12):e51670. DOI:10.1371/journal.pone.0051670 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1a's synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1a in fast-twitch muscles.
[Show abstract][Hide abstract] ABSTRACT: Acetylcholinesterase (AChE) is well-known for its cholinergic functions in the nervous system; however, this enzyme is also found in other tissues where its function is still not understood. AChE is synthesized through alternative splicing as splicing variants, with isoforms including read-through (AChER), tailed (AChET) and hydrophobic (AChEH). In human erythrocytes, AChEH is a glycophosphatidylinositol-linked dimer on the plasma membrane. Three N-linked glycosylation sites have been identified in the catalytic domain of human AChE. Here, we investigate the roles of glycosylation in assembly and trafficking of human AChEH. In transfected fibroblasts, expression of AChEH was able to mimic the function of the dimeric form of AChE on the erythrocyte membrane. A glycan-depleted form was constructed by site-directed mutagenesis. By comparison with the wild-type AChEH, the mutant had a much lower enzymatic activity and a much higher Km value. In addition, the mutant was dimerized in the endoplasmic reticulum, but was not trafficked to the Golgi apparatus. The results suggest that the glycosylation may affect AChEH enzymatic activity and trafficking, but not dimer formation. The present findings indicate the significance of N-glycosylation in controlling the biosynthesis of the AChEH dimer form.
Structured digital abstract
[Show abstract][Hide abstract] ABSTRACT: Acetylcholinesterase (AChE) is organized into globular tetramers (G(4)) by a structural protein called proline-rich membrane anchor (PRiMA), anchoring it into the cell membrane of neurons in the brain. The assembly of AChE tetramers with PRiMA requires the presence of a C-terminal "t-peptide" in the AChE catalytic subunit (AChE(T)). The glycosylation of AChE(T) is known to be required for its proper assembly and trafficking; however, the role of PRiMA glycosylation in the oligomer assembly has not been revealed. PRiMA is a glycoprotein containing two putative N-linked glycosylation sites. By using site-directed mutagenesis, the asparagine-43 was identified to be the N-linked glycosylation site of PRiMA. Abolishing glycosylation on mouse PRiMA appeared not to affect its assembly with AChE(T), the enzymatic properties of AChE, and the membrane trafficking of PRiMA-linked AChE tetramers. This result is contrary to the reports that glycosylation is essential for conformation and trafficking of membrane glycoproteins.
[Show abstract][Hide abstract] ABSTRACT: Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of
L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimer’s disease and depression.
Evidence-based Complementary and Alternative Medicine 06/2012; 2012(1):278273. DOI:10.1155/2012/278273 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R(2) at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.