Two previously undescribed cholestanol saponins from the rhizomes of Paris fargesii var. petiolata.

Two previously undescribed cholestanol saponins, parpetiosides F - G (1-2), and six known analogs (3-8) were isolated from the rhizomes of Paris fargesii var. petiolata. Their structures were elucidated by extensive spectroscopic data analysis and chemical methods. Compound 1 was a rare 6/6/6/5/5 fused-rings cholestanol saponin with disaccharide moiety linked at C-26 of aglycone which was hardly seen in genus Paris. All of these compounds were discovered in this plant for the first time. In addition, the cytotoxicities of saponins (1-8) against three human cancer cell lines (U87, HepG2 and SGC-7901) were evaluated by CCK-8 method, and saponins 5-8 displayed certain cytotoxicities. The strong interactions between saponins 5-8 and SCUBE3, an oncogene for glioma cells, were displayed by molecular docking.

Virtual screening-molecular docking-activity evaluation of Ailanthus altissima (Mill.) swingle bark in the treatment of ulcerative colitis.

BACKGROUND: The dried bark of Ailanthus altissima (Mill.) Swingle is widely used in traditional Chinese medicine for the treatment of ulcerative colitis. The objective of this study was to explore the therapeutic basis of the dried bark of Ailanthus altissima (Mill.) Swingle for the treatment of ulcerative colitis based on Virtual Screening-Molecular Docking-Activity Evaluation technology. METHODS: By searching the Traditional Chinese Medicine Systems Pharmacology TCMSP Database and Analysis Platform, 89 compounds were obtained from the chemical components of the dried bark of Ailanthus altissima (Mill.) Swingle. Then, after preliminarily screening the compounds based on Lipinski's rule of five and other relevant conditions, the AutoDock Vina molecular docking software was used to evaluate the affinity of the compounds to ulcerative colitis-related target proteins and their binding modes through use of the scoring function to identify the best candidate compounds. Further verification of the compound's properties was achieved through in vitro experiments. RESULTS: Twenty-two compounds obtained from the secondary screening were molecularly docked with ulcerative colitis-related target proteins (IL-1R, TLR, EGFR, TGFR, and Wnt) using AutoDock Vina. The free energies of the highest scoring compounds binding to the active cavity of human IL-1R, TLR, EGFR, TGFR, and Wnt proteins were - 8.7, - 8.0, - 9.2, - 7.7, and - 8.5 kcal/mol, respectively. The potential compounds, dehydrocrebanine, ailanthone, and kaempferol, were obtained through scoring function and docking mode analysis. Furthermore, the potential compound ailanthone (1, 3, and 10 µM) was found to have no significant effect on cell proliferation, though at 10 µM it reduced the level of pro-inflammatory factors caused by lipopolysaccharide. CONCLUSION: Among the active components of the dried bark of Ailanthus altissima (Mill.) Swingle, ailanthone plays a major role in its anti-inflammatory properties. The present study shows that ailanthone has advantages in cell proliferation and in inhibiting of inflammation, but further animal research is needed to confirm its pharmaceutical potential.

Ilex rotunda Thunb Protects Against Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice by Restoring the Intestinal Mucosal Barrier and Modulating the Oncostatin M/Oncostatin M Receptor Pathway.

Ilex rotunda Thunb (IR) is a traditional Chinese medicine used for the clinical treatment of gastric ulcers and duodenal ulcers; however, the effect of IR on ulcerative colitis (UC) and its underlying mechanism remains unclear. This study investigated the therapeutic effect of IR on UC mice induced by dextran sulfate sodium (DSS) as well as the potential underlying mechanism. The main components of IR were analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Then we established a model of UC mice by administering 2.0% DSS for 7 days followed by 2 weeks of tap water for three cycles and administered IR. On day 56, the disease activity index (DAI), colon length, pathological changes, and inflammatory response of the colon tissue of mice were assessed. The oxidative stress and apoptosis of colon tissue were detected, and the integrity of the intestinal mucosal barrier was evaluated to assess the effect of IR. Furthermore, the relationship between oncostatin M (OSM) and its receptor (OSMR) in addition to the IR treatment of UC were evaluated using a mouse model and Caco2 cell model. The results showed that IR significantly alleviated the symptoms of UC including rescuing the shortened colon length; reducing DAI scores, serum myeloperoxidase and lipopolysaccharide levels, pathological damage, inflammatory cell infiltration and mRNA levels of interleukin one beta, tumor necrosis factor alpha, and interleukin six in colon tissue; alleviating oxidative stress and apoptosis by decreasing kelch-like ECH-associated protein 1 expression and increasing nuclear factor-erythroid factor 2-related factor 2 and heme oxygenase-1 protein expression; and promoting the regeneration of epithelial cells. IR also promoted the restoration of the intestinal mucosal barrier and modulated the OSM/OSMR pathway to alleviate UC. It was found that IR exerted therapeutic effects on UC by restoring the intestinal mucosal barrier and regulating the OSM/OSMR pathway.

Gentiopicroside ameliorates ethanol-induced gastritis via regulating MMP-10 and pERK1/2 signaling.

BACKGROUND: Excessive ethanol consumption results in gastric mucosa damage, which could further develop into chronic gastritis, peptic ulcer, and gastric cancer in humans. Gentiopicroside (GPS), a major active component of Gentianae Macrophyllae radix, was reported to play a critical role in anti-inflammation. In the study, we aimed to investigate the functional role and underlying mechanism of GPS in ethanol-induced gastritis. METHODS: A model of gastritis was created by ethanol in C57BL/6 mice. Enzyme-linked immunosorbent assay was used to determine the concentration of TNF-α, IL-1ß, IL-8, and IL-10. RESULTS: We found that GPS treatment significantly ameliorated ethanol-induced gastritis in mice, with lower production of pro-inflammatory cytokine TNF-α, IL-1ß, and IL-8 and higher levels of anti-inflammatory cytokine IL-10. The anti-inflammatory effect of GPS was further confirmed in vitro in ethanol-treated human gastric mucosal GES cells. Mechanistically, we demonstrated that GPS regulated matrix metallopeptidase expression and pERK1/2 signaling. Knockdown of matrix metallopeptidase 10 (MMP-10) greatly improved cell survival and suppressed inflammatory response in ethanol-treated GES cells. Moreover, inhibition of pERK1/2 signaling using U0126 decreased the expression of MMP-10 in ethanol-induced gastritis. U0126 treatment also suppressed the expression of TNF-α, IL-1ß, and IL-8, and enhanced IL-10 expression in mice gastric mucosa. CONCLUSIONS: Taken together, our findings suggest that GPS ameliorates ethanol-induced gastritis via regulating MMP-10 and pERK1/2 signaling, which might provide a promising therapeutic drug for ethanol-induced gastritis.

Gastroprotective effect of gallic acid against ethanol-induced gastric ulcer in rats: Involvement of the Nrf2/HO-1 signaling and anti-apoptosis role.

Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a phenolic compound found in many medicinal plants traditionally used in China or patent medicine such as Feiyangchangweiyan capsule (FY capsule) for the treatment of gastrointestinal diseases for decades. However, the evidence for the gastroprotective effect of GA is deficient and the pharmacological mechanisms remain limited. The present investigation was initiated to demonstrate the gastroprotective effect and to understand potential underlying mechanism of GA on ethanol-induced gastric ulcer in rats. Gastric ulcers were induced by absolute ethanol (5 mL/kg, i.g.) in male Sprague-Dawley rats, GA (10, 30, and 50 mg/kg), FY capsule (0.4 g/kg) and 30 mg/kg Lansoprazole was administered orally. Physiological saline and lansoprazole were used as negative and positive control, respectively. Induction of rats with ethanol resulted in a significant rise in ulcer index, serum levels of inflammatory cytokines markers (IL-1ß, IL-6 and TNF-α), TBARS, protein expression of Bax and Caspase-3 and a significant reduction in the activities or levels of endogenous antioxidants (SOD, CAT and GSH), gastric mucosal protective factors (PGE2 and NO) and protein expression of Bcl-2. Pretreatment with GA showed a remarkable decrease in ulcer index, inflammatory cytokines markers, TBARS, protein expression of Bax and Caspase-3 and a significant increase in the activities of endogenous antioxidants, levels of PGE2 and NO, and protein expression of Bcl-2, Nrf2 and HO-1 when compared with ethanol treated groups. This study demonstrated the gastroprotective effect of Gallic acid and FY capsule on ethanol-induced gastric ulcer in rats. The underlying mechanism of GA and FY capsule against gastric ulcer in rats caused by ethanol might be involved in Nrf2/HO-1 anti-oxidative pathway and ultimately played an anti-apoptotic role through regulating Bax, Bcl-2 and Caspase-3.

Comparative Pharmacokinetics of Gallic Acid After Oral Administration of Gallic Acid Monohydrate in Normal and Isoproterenol-Induced Myocardial Infarcted Rats.

Gallic acid (GA) is a polyphenolic natural product widely distributed in food, beverage, and traditional Chinese herbs with beneficial effects on the cardiovascular system. In this research, a comparative study was conducted to investigate the possible difference of pharmacokinetic process in normal and isoproterenol-induced myocardial infarcted rats after oral administration of GA monohydrate with the dose of 50 and 100 mg/kg, respectively. Quantification of GA in rat plasma was achieved by using a simple and rapid high-performance liquid chromatographic method. The results revealed that pharmacokinetics of GA were greatly different between normal and pathological state. GA exhibited slower absorption into the bloodstream, and yielded 1.7-fold (50 mg/kg GA) and 1.3-fold (100 mg/kg GA) less values of area under concentration-time curve as well as 2.5-fold lower of maximum blood concentration (Cmax) in MI rats than those in normal rats. In addition, significant prolonged T1/2 and MRT as well as decreased CL were also registered in MI rats. Our findings suggest that myocardial infarction could alter the pharmacokinetic process of GA, and thus the potential pharmacokinetic differences of herbal preparations (or dietary nutrition) containing GA between normal and pathological conditions should be brought to the forefront seriously in clinical practice.

Submicron emulsion of cinnamaldehyde ameliorates bleomycin-induced idiopathic pulmonary fibrosis via inhibition of inflammation, oxidative stress and epithelial-mesenchymal transition.

AIMS: Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe form of idiopathic interstitial pneumonias. The pathogenesis is associated with inflammation and oxidative stress and epithelial-mesenchymal transition (EMT). Cinnamaldehyde exhibits antiinflammatory and antioxidant properties, but its effect on IPF is unknown. The present study is to investigate the anti-fibrotic effect and action mechanism of cinnamaldehyde on IPF. MATERIALS AND METHODS: IPF was induced by intratracheal bleomycin in mice. Submicron emulsion of cinnamaldehyde was given by intraperitoneal injection once everyday for 7 or 21 continuous days after bleomycin administration. Lung histological and injury indexes were analyzed. The protein expressions of inflammation and oxidative stress as well as EMT markers alpha-smooth muscle actin (α-SMA) and E-cadherin in mice and cultured A549 cells were measured. RESULTS: Cinnamaldehyde attenuated the bleomycin-induced histological injury, reduced hydroxyproline level and improved pulmonary function by the inhibiting inflammatory cytokines and reactive oxygen species production as well as enhancing total superoxide dismutase activity in bleomycin-induced mice. Cinnamaldehyde also inhibited EMT in both bleomycin-induced mice and TGF-ß1-stimulated A549 cells. CONCLUSIONS: Cinnamaldehyde ameliorated bleomycin-induced IPF via inhibition of inflammation and oxidative stress and EMT.

Wound-healing Activity of Zanthoxylum bungeanum Maxim Seed Oil on Experimentally Burned Rats.

BACKGROUND: The seed oil of Zanthoxylum bungeanum Maxim (ZBSO) is considered to be rich source of fatty acids, mainly oleic and linoleic acids, and has been used for the treatment of burns in Chinese medicine. OBJECTIVE: We evaluated the healing efficacy of ZBSO and explored its possible mechanism on scalded rats. MATERIALS AND METHODS: Sprague-Dawley rat models with deep second-degree burns were set up, and ZBSO (500 and 1000 µl/wound) was topically applied twice daily for 7 days and then once daily until wound healing. The therapeutic effects of ZBSO were evaluated by observing wound closure time, decrustation time, wound-healing ratio, and pathological changes. Collagen type-III, matrix metalloproteinase-2 (MMP-2), MMP-9, phospho-nuclear factor-κB (p-NF-κB) p65, inhibitor of NF-κB subunit α p-IκBα, and inhibitor of NF-κB subunit α (IκBα) expression were determined using Western blotting. RESULTS: The ZBSO-treated group showed a higher wound-healing ratio and shorter decrustation and wound closure times than the untreated group. The topical application of ZBSO increased collagen synthesis as evidenced by an increase in hydroxyproline level and upregulated expression of collagen type-III on days 7, 14, and 21 posttreatment. A reduction in MMP-2 and MMP-9 expressions also confirmed the collagen formation efficacy of ZBSO. Furthermore, there was a significant increase in superoxide dismutase levels and a decrease in malondialdehyde levels in ZBSO-treated wounds. ZBSO also decreased tumor necrosis factor alpha, interleukin-1 (IL-1) ß, and IL-6 levels in serum, upregulated IκBα, and downregulated p-NF-κB p65 and p-IκBα expression in vivo, indicating the anti-inflammatory action of ZBSO. CONCLUSION: ZBSO has significant potential to treat burn wounds by accelerating collagen synthesis and the anti-inflammatory cascade of the healing process. SUMMARY: The seed oil of Zanthoxylum bungeanum Maxim (ZBSO) is rich of fatty acidsThe healing efficacy of ZBSO on experimentally scalded rats was evaluatedZBSO has significant potential to treat deep second-degree burn woundsZBSO could accelerate collagen synthesis and inhibit the inflammatory signaling. Abbreviations used: ECL: Enhanced chemiluminescence; ECM: Extracellular matrix; ELISA: Enzyme-linked immunosorbent assay; GC-MS: Gas chromatography-mass spectrometry; HRP: Horseradish peroxidase; HYP: Hydroxyproline; IκBα: Inhibitor of NF-κB subunit α; IL: Interleukin; MDA: Malondialdehyde; MMP: Matrix metalloproteinase-2; NF-κB: Nuclear factor-κB; SFE: Supercritical fluid extraction; SOD: Superoxide dismutase; SSD: Silver sulfadiazine; TCM: Traditional Chinese medicine; TNF: Tumor necrosis factor.

Protective Effects of Amarogentin against Carbon Tetrachloride-Induced Liver Fibrosis in Mice.

Amarogentin, a secoiridoid glycoside that is mainly extracted from Swertia and Gentiana roots, has been suggested to exhibit many biological effects, including anti-oxidative, anti-tumour, and anti-diabetic activities. The present study was designed to evaluate the protective effects of amarogentin on carbon tetrachloride-induced liver fibrosis in vivo and the underlying mechanism. Fibrosis was induced by subcutaneous injections of 6 mL/kg of 20% carbon tetrachloride (dissolved in olive oil) twice per week for seven weeks. Mice were orally treated with 25, 50, and 100 mg/kg amarogentin and with colchicine as a positive control. Biochemical assays and histopathological investigations showed that amarogentin delayed the formation of liver fibrosis; decreased alanine aminotransferase, aspartate aminotransferase, malondialdehyde and hydroxyproline levels; and increased albumin, cyclic guanosine monophosphate, glutathione peroxidase, and superoxide dismutase levels. Moreover, amarogentin exhibited downregulation of α-smooth muscle actin and transforming growth factor-ß1 levels in immunohistochemical and Western blot analyses. The levels of phosphorylated extracellular regulated protein kinases, c-Jun N-terminal kinase, and p38 were also significantly reduced in all amarogentin-treated groups in a dose-dependent manner. These findings demonstrated that amarogentin exerted significant hepatoprotective effects against carbon tetrachloride-induced liver fibrosis in mice and suggested that the effect of amarogentin against liver fibrosis may be by anti-oxidative properties and suppressing the mitogen-activated protein kinase signalling pathway.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside protects murine hearts against ischemia/reperfusion injury by activating Notch1/Hes1 signaling and attenuating endoplasmic reticulum stress.

2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-glucoside (TSG) is a water-soluble active component extracted from Polygonum multiflorum Thunb. A number of studies demonstrate that TSG exerts cardioprotective effects. Since endoplasmic reticulum (ER) stress plays a key role in myocardial ischemia/reperfusion (MI/R)-induced cell apoptosis, we sought to determine whether modulation of the ER stress during MI/R injury was involved in the cardioprotective action of TSG. Male mice were treated with TSG (60 mg·kg-1·d-1, ig) for 2 weeks and then were subjected to MI/R surgery. Pre-administration of TSG significantly improved post-operative cardiac function, and suppressed MI/R-induced myocardial apoptosis, evidenced by the reduction in the myocardial apoptotic index, serum levels of LDH and CK after 6 h of reperfusion. TSG (0.1-1000 µmol/L) did not affect the viability of cultured H9c2 cardiomyoblasts in vitro, but pretreatment with TSG dose-dependently decreased simulated ischemia/reperfusion (SIR)-induced cell apoptosis. Furthermore, both in vivo and in vitro studies revealed that TSG treatment activated the Notch1/Hes1 signaling pathway and suppressed ER stress, as evidenced by increasing Notch1, Notch1 intracellular domain (NICD), Hes1, and Bcl-2 expression levels and by decreasing p-PERK/PERK ratio, p-eIF2α/eIF2α ratio, and ATF4, CHOP, Bax, and caspase-3 expression levels. Moreover, the protective effects conferred by TSG on SIR-treated H9c2 cardiomyoblasts were abolished by co-administration of DAPT (the Notch1 signaling inhibitor). In summary, TSG ameliorates MI/R injury in vivo and in vitro by activating the Notch1/Hes1 signaling pathway and attenuating ER stress-induced apoptosis.

Paeonol and danshensu combination attenuates apoptosis in myocardial infarcted rats by inhibiting oxidative stress: Roles of Nrf2/HO-1 and PI3K/Akt pathway.

Paeonol and danshensu is the representative active ingredient of traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizae, respectively. Paeonol and danshensu combination (PDSS) has putative cardioprotective effects in treating ischemic heart disease (IHD). However, the evidence for the protective effect is scarce and the pharmacological mechanisms of the combination remain unclear. The present study was designed to investigate the protective effect of PDSS on isoproterenol (ISO)-induced myocardial infarction in rats and to elucidate the potential mechanism. Assays of creatine kinase-MB, cardiac troponin I and T and histopathological analysis revealed PDSS significantly prevented myocardial injury induced by ISO. The ISO-induced profound elevation of oxidative stress was also suppressed by PDSS. TUNEL and caspase-3 activity assay showed that PDSS significantly inhibited apoptosis in myocardia. In exploring the underlying mechanisms of PDSS, we found PDSS enhanced the nuclear translocation of Nrf2 in myocardial injured rats. Furthermore, PDSS increased phosphorylated PI3K and Akt, which may in turn activate antioxidative and antiapoptotic signaling events in rat. These present findings demonstrated that PDSS exerts significant cardioprotective effects against ISO-induced myocardial infarction in rats. The protective effect is, at least partly, via activation of Nrf2/HO-1 signaling and involvement of the PI3K/Akt cell survival signaling pathway.

Proliferation of rat cardiac stem cells is induced by 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-D-glucoside in vitro.

AIM: To study the effects of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-ß-d-glucoside (THSG) on proliferation of rat cardiac stem cells (CSCs) in vitro. MATERIALS AND METHODS: C-kit(+) cells were isolated from neonatal (1 day old) Sprague-Dawley rats by using flow cytometry. Optimal THSG treatment times and doses for growth of CSCs were analyzed. CSCs were treated with various THSG doses (0, 1, 10, and 100 µM) for 12h. RESULTS: Sorted c-kit(+) cells exhibited self-renewing and clonogenic capabilities. Cell Counting Kit (CCK-8) and Proliferating Cell Nuclear Antigen (PCNA) ELISA test positive cells were significantly increased in THSG-treated groups compared with untreated controls. The percentage of S-phase cells also increased after THSG treatment. Moreover, we show that some c-kit(+) cells spontaneously express vascular endothelial growth factor (VEGF), T-box transcription factor (Tbx5), hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2), hyperpolarization-activated cyclic nucleotide gated 4 (HCN4), alpha myosin heavy chain (αMHC), and beta myosin heavy chain (ßMHC) mRNA, and stem cell antigen 1 (Sca-1), cardiac troponin-I, GATA-4, Nkx2.5, and connexin 43 protein were also assessed in CSCs. However, their expression was significantly increased with THSG treatment when compared to untreated controls. CONCLUSION: THSG can increase proliferation of rat CSCs in vitro and thus, shows promise as a potential treatment strategy for stimulating endogenous stem cells to help repair the injured heart after myocardial infarction in patients.

Anticancer effects of bufalin on human hepatocellular carcinoma HepG2 cells: roles of apoptosis and autophagy.

The traditional Chinese medicine bufalin, extracted from toad's skin, has been demonstrated to exert anticancer activities in various kinds of human cancers. The mechanisms of action lie in its capacity to induce apoptosis, or termed type I programmed cell death (PCD). However, type II PCD, or autophagy, participates in cancer proliferation, progression, and relapse, as well. Recent studies on autophagy seem to be controversial because of the dual roles of autophagy in cancer survival and death. In good agreement with previous studies, we found that 100 nM bufalin induced extensive HepG2 cell apoptosis. However, we also noticed bufalin triggered autophagy and enhanced Beclin-1 expression, LC3-I to LC3-II conversion, as well as decreased p62 expression and mTOR signaling activation in HepG2 cells. Blockage of autophagy by selective inhibitor 3-MA decreased apoptotic ratio in bufalin-treated HepG2 cells, suggesting a proapoptotic role of bufalin-induced autophagy. Furthermore, we investigated the underlying mechanisms of bufalin-induced autophagy. Bufalin treatment dose-dependently promoted AMPK phosphorylation while AMPK inhibition by compound C significantly attenuated bufalin-induced autophagy. Taken together, we report for the first time that bufalin induces HepG2 cells PCD, especially for autophagy, and the mechanism of action is, at least in part, AMPK-mTOR dependent.

Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats.

BACKGROUND: Traditional Chinese medicinal herbs Cortex Moutan and Radix Salviae Milthiorrhizaeare are prescribed together for their putative cardioprotective effects in clinical practice. However, the rationale of the combined use remains unclear. The present study was designed to investigate the cardioprotective effects of paeonol and danshensu (representative active ingredient of Cortex Moutan and Radix Salviae Milthiorrhizae, respectively) on isoproterenol-induced myocardial infarction in rats and its underlying mechanisms. METHODOLOGY: Paeonol (80 mg kg(-1)) and danshensu (160 mg kg(-1)) were administered orally to Sprague Dawley rats in individual or in combination for 21 days. At the end of this period, rats were administered isoproterenol (85 mg kg(-1)) subcutaneously to induce myocardial injury. After induction, rats were anaesthetized with pentobarbital sodium (35 mg kg(-1)) to record electrocardiogram, then sacrificed and biochemical assays of the heart tissues were performed. PRINCIPAL FINDINGS: Induction of rats with isoproterenol resulted in a marked (P<0.001) elevation in ST-segment, infarct size, level of serum marker enzymes (CK-MB, LDH, AST and ALT), cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant decrease in the activities of endogenous antioxidants (SOD, CAT, GPx, GR, and GST) and protein expression of Bcl-2. Pretreatment with paeonol and danshensu combination showed a significant (P<0.001) decrease in ST-segment elevation, infarct size, cTnI, TBARS, protein expression of Bax and Caspase-3 and a significant increase in the activities of endogenous antioxidants and protein expression of Bcl-2 and Nrf2 when compared with individual treated groups. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial infarction in rats. The mechanism might be associated with the enhancement of antioxidant defense system through activating of Nrf2 signaling and anti-apoptosis through regulating Bax, Bcl-2 and Caspase-3. It could provide experimental evidence to support the rationality of combinatorial use of traditional Chinese medicine in clinical practice.

[Study on the HPLC fingerprint of toad skin from different regions].

OBJECTIVE: To study the HPLC fingerprint of toad skin and provide a reliable method for quality control and identification. METHODS: It used HPLC for detection and computer aided similarity evaluation system for processing and analysing HPLC fingerprint. RESULTS: The common pattern of HPLC fingerprint of toad skin was astablished, 29 peaks were identified as the characteristic fingerprints, in which 9 peaks corresponded to 9 bufogenins. (2) Each samples' similarity of relative retention time was all above 0.99, but the similarity of relative peak areas was low. CONCLUSION: (1) The method is accurate and with good reproducibility. The fingerprints can be used for the identification and quality control of toad skin. (2) The toad skin from different regions are stable in composition, but the contents of the components are different.

Effect of combined OX40Ig and CTLA4Ig gene local transfer on allograft rejection and the underlying mechanisms.

BACKGROUND: OX40Ig and CTLA4Ig fusion proteins have been suggested to induce immune tolerance and prevent rejection in allografts. The present study aims to investigate and compare the effects of ex vivo combined OX40Ig and CTLA4Ig lentivirus-mediated gene transfer on the long-term survival of the graft, as well as potential underlying mechanisms. METHODS: We ex vivo transferred Brown Norway rats' superficial groin free flap with lentivirus vectors expressing OX40Ig or CTLA4Ig, or OX40Ig and CTLA4Ig combined, and transplanted the free flaps to Lewis rats. Short-course rapamycin was administered after transfection and transplantation. RT-PCR and Western blot were employed to evaluate expression of OX40Ig and CTLA4Ig. We assessed the survival time of the grafts and the degree of acute graft rejection after indicated treatment. Mixed lymphocyte reaction, flow cytometry, and ELISA were also used to evaluate systemic immune reactions. RESULTS: Ex vivo transfer of OX40Ig or CTLA4Ig lentivirus vectors led to local expression of corresponding mRNA and proteins in the donor flap without affecting other organs of the recipient. The graft survival time was significantly expanded and rejection was markedly attenuated after transfection. Mixed lymphocyte reaction, flow cytometry (CD4(+) and CD8(+) T lymphocyte proportions), and serum ELISA analysis (IL-2, IFN-γ, IL-4, and IL-10) also showed decreased immune response following transfection. Combined OX40Ig and CTLA4Ig transfer exerted superior effect on improving graft survival and preventing graft rejection, inhibiting the immune response and decreasing the production of proinflammatory cytokines, compared with singular transfer of either OX40Ig or CTLA4Ig. CONCLUSION: Combined ex vivo transfer of OX40Ig and CTLA4Ig lentivirus vectors provided superior benefits on long-term survival and restoration of the graft through inhibiting immune response and decreasing the production of proinflammatory cytokines.

The bone-protective effect of genistein in the animal model of bilateral ovariectomy: roles of phytoestrogens and PTH/PTHR1 against post-menopausal osteoporosis.

Genistein, a major phytoestrogen of soy, is considered a potential drug for the prevention and treatment of post-menopausal osteoporosis. Mounting evidence suggested a positive correlation between genistein consumption and bone health both in vivo and in vitro. Earlier studies have revealed that genistein acted as a natural estrogen analogue which activated estrogen receptor and exerted anti-osteoporotic effect. However, it remains unclear whether PTH, the most crucial hormone that regulates mineral homeostasis, participates in the process of genistein-mediated bone protection. In the present study, we compared the therapeutic effects between genistein and nilestriol and investigated whether PTH and its specific receptor PTHR1 altered in response to genistein-containing diet in the animal model of ovariectomy. Our results showed that genistein administration significantly improved femoral mechanical properties and alleviates femoral turnover. Genistein at all doses (4.5 mg/kg, 9.0 mg/kg and 18.0 mg/kg per day, respectively) exerted improved bending strength and b-ALP limiting effects than nilestriol in the present study. However, genistein administration did not exert superior effects on bone protection than nilestriol. We also observed circulating PTH restoration in ovariectomized rats receiving genistein at the dose of 18 mg/kg per day. Meanwhile, PTHR1 abnormalities were attenuated in the presence of genistein as confirmed by RT-PCR, Western blot and immunohistochemistry. These findings strongly support the idea that besides serving as an estrogen, genistein could interact with PTH/PTHR1, causing a superior mineral restoring effect than nilestriol on certain circumstance. In conclusion, our study reported for the first time that the anti-osteoporotic effect of genistein is partly PTH/PTHR1-dependent. Genistein might be a potential option in the prevention and treatment of post-menopausal osteoporosis with good tolerance, more clinical benefits and few undesirable side effects.

Protective effects of hyperoside against human umbilical vein endothelial cell damage induced by hydrogen peroxide.

ETHNOPHARMACOLOGICAL RELEVANCE: Hyperoside (Hyp) is a flavonoid compound isolated from Rhododendron ponticum L. leaves that elicits vascular protective effects in vitro. Treatment with Hyp has been found to attenuate endothelial cell damage induced by oxidative stress, but its mechanisms of action remain unclear. This study investigated the action of Hyp in an endothelial injury model induced by hydrogen peroxide (H(2)O(2)), as well as its possible mechanisms. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with H(2)O(2) alone or in combination with Hyp. The protective effects of Hyp against H(2)O(2) were evaluated, and the activation of extracellular signal-regulated protein kinase (ERK) in Hyp was assayed in HUVECs. RESULTS: Loss of cell viability as well as excessive cell apoptosis and death were observed in HUVECs after 18 h of challenge with H(2)O(2) (400µM); however, both cell apoptosis and death were attenuated in the Hyp-pretreated cells. Western blot analysis revealed that Hyp increased the expression of Bcl-2 but decreased that of Bax. In addition, Hyp induced the phosphorylation of ERK1/2 in HUVECs. CONCLUSION: These observations provide preliminary evidence that Hyp protects HUVECs against H(2)O(2) damage, at least partially, by activating the ERK signaling pathway.

Burn-induced apoptosis of cardiomyocytes is survivin dependent and regulated by PI3K/Akt, p38 MAPK and ERK pathways.

Survivin belongs to the family of genes known as inhibitors of apoptosis, and although it has been implicated in the prevention of cancer, its potential role in burn-induced cardiac injury is unknown. In this study, we investigated the effects of survivin blockade on burn-induced cardiac apoptosis. Using a standardized Sprague-Dawley rat model of third-degree burn injury over 40% of total body surface area, apoptosis was measured in vivo followed by in vitro assessment of burn serum-stimulated cardiomyocytes. Based on the Western blot analyses, real-time PCR, ELISA, and TUNEL, apoptosis and caspase activation both in vivo and in vitro were significantly increased after severe burn injury, while survivin expression was increased (up to 2.90-fold) during the early stage of burn injury and was almost completely abolished 8 h after the burn. Survivin-deficient cardiomyocytes, as well as hearts from rats treated with the survivin inhibitor YM155, exhibited increased caspase-3 protein and mRNA expression and apoptosis ratio at different times after the burn. Furthermore, inhibition of ERK, phosphoinositol 3-kinase contributed the burn serum-induced increase in apoptosis and caspase-3 protein expression, and decreased survivin expression, whereas burn serum-induced increase in apoptosis was attenuated by P38 mitogen-activated protein kinase inhibition. These data identify survivin as a critical anti-apoptotic regulator of cardiomyocytes after burn injury. ERK, P38 MAPK and PI3K were found to be upstream regulators of survivin.

Madecassoside suppresses LPS-induced TNF-alpha production in cardiomyocytes through inhibition of ERK, p38, and NF-kappaB activity.

Madecassoside (MA) is a major triterpenoid component of Centella asiatica that has a wide range of biological activities, including wound-healing and antioxidative activities. In the present study, we evaluated the therapeutic effect of MA on rat cardiac dysfunction during sepsis induced by lipopolysaccharide (LPS), as well as the possible mechanism. Pretreatment of the neonatal rat cardiomyocytes with MA inhibited LPS-induced TNF-alpha production in a concentration-dependent manner. In addition, pretreatment of the rats with MA (20 mg/kg, i.g.) significantly inhibited the elevation of plasma TNF-alpha, delayed the fall of mean arterial blood pressure, and attenuated the tachycardia induced by LPS. We further observed that MA prevented the LPS-induced nuclear factor-kappa B (NF-kappaB) translocation from the cytoplasm into the nucleus, and inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38. These results suggest that MA inhibits LPS-stimulated TNF-alpha production through the blocking of ERK1/2, p38 and NF-kappaB pathways in cardiomyocytes. MA may have cardioprotective effects in LPS-mediated sepsis.