Inhibitory effects of oxymatrine on hepatic stellate cells activation through TGF-ß/miR-195/Smad signaling pathway.

BACKGROUND: Oxymatrine (OM), a quinolizidine alkaloid extracted from a herb Sophorae Flavescentis Radix, has been used to treat liver fibrotic diseases. However, the mechanism of its anti-fibrosis effects is still unclear. TGF-ß/Smad signaling and miR-195 have been proved to paly an important role in hepatic stellate cells (HSCs) activation and liver fibrosis. In this study, we investigated whether OM could inhibit HSCs activation through TGF-ß1/miR-195/Smads signaling or not. METHODS: First, the effects of OM on HSC-T6 in different concentrations and time points were tested by MTT assay. We choose three appropriate concentrations of OM as treatment concentrations in following experiment. By Quantitative Real-time PCR and Western Blot, then we investigated the effect of OM on miR-195, Smad7 and α-SMA's expressions to prove the correlation between OM and the TGF-ß1/miR-195/Smads signaling. Last, miR-195 mimic and INF-γ were used to investigate the relation between miR-195 and OM in HSC activation. RESULTS: Our results showed that the proliferation of HSC was significantly inhibited when OM concentration was higher than 200 µg/mL after 24 h, 100 µg/mL after 48 h and 10 µg/mL after 72 h. The IC50 of OM after 24, 48 and 72 h were 539, 454, 387 µg/mL respectively. OM could down-regulate miR-195 and α-SMA (P < 0.01), while up-regulate Smad7 (P < 0.05). In HSC-T6 cells transfected with miR-195 mimic and pretreated with OM, miR-195 and α-SMA were up-regulated (P < 0.05), and Smad7 was down-regulated (P < 0.05) . CONCLUSIONS: Given these results, OM could inhibit TGF-ß1 induced activation of HSC-T6 proliferation in a dose-dependent and time-dependent manner to some extent. We proved that OM inhibited HSC activation through down-regulating the expression of miR-195 and up-regulating Smad7.

[UPLC-MS/MS determination of tanshinone â ¡A, salvianolic acid B and ginsenoside Rg1 in Fufang Danshen preparation in rat plasma and brain tissues].

A sensitive and specific ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed for analysis of tanshinone ⅡA(TSⅡA), salvianolic acid B(SAB) and ginsenoside Rg1 (GRg1) in rat plasma and brain tissues. Male healthy Sprague-Dawley(SD) rats were orally given single dose of Fufang Danshen preparation (TS ⅡA 60 mg•kg⁻¹, SAB 300 mg•kg⁻¹, GRg1 150 mg•kg⁻¹, borneol 300 mg•kg⁻¹), and their blood samples and brain tissues were collected at different time points. The drug plasma and brain tissue concentrations of the three analytes were determined by UPLC-MS/MS method. Subsequently, the main pharmacokinetics parameters of plasma and brain tissues were calculated by using Phoenix WinNolin 6.1 software. The methodological test showed that all of analytes in both plasma and brain homogenate exhibited a good linearity within the concentration range(r>0.992 2). Their mean recoveries were between 58.86% and 112.1%. Intra-day and inter-day precisions of the investigated components exhibited RSD≤9.7%, and the accuracy(RE) ranged from -9.68% to 8.20% at all quality control levels. The results of accuracy and stability meet the requirements for biopharmaceutical analysis. For TSⅡA, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.58±0.081) h, (725.4±88.20) µg•L⁻¹, (2 101.3±124.85) µg•h•L⁻¹ and (3.66±0.05) h, respectively. For SAB, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.29±0.21) h, (307.9±46.75) µg•L⁻¹, (537.4±88.24) µg•h•L⁻¹ and (2.08±0.11) h, respectively. For GRg1, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.42±0.20) h, (460.38±154.60) µg•L⁻¹, (383.4±88.16) µg•h•L⁻¹ and (1.87±0.046) h, respectively. For TSⅡA, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the brain tissue were (0.75±0.22) h, (1.41±0.42) ng•g⁻¹, (4.34±2.48) ng•h•g⁻¹ and (4.00±1.90) h, respectively. For SAB, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (1.08±0.20) h, (21.09±4.850) ng•g⁻¹, (14.83±3.160) ng•h•g⁻¹ and (0.99±0.08) h, respectively. For GRg1, the pharmacokinetics parameters Tmax, Cmax, AUC0-t, MRTlast in the plasma were (0.50±0.16) h, (130.96±54.220) ng•g⁻¹, (136.24±34.350) ng•h•g⁻¹ and (2.87±0.33) h, respectively. The developed method was successfully applied in pharmacokinetic studies on content of TS ⅡA, SAB and GRg1 in rat plasma and brain tissues.

[Effect of notoginseng radix on expression quantity of TGF-beta1/ Smads and CTGF mRNA in rats with alcoholic liver disease].

OBJECTIVE: To evaluate the effect of Notoginseng Radix on hepatic expression of transforming growth factor beta1 (TGF-beta1) and connective tissue growth factor (CTGF) in rats with alcoholic liver disease (ALD), in order to discuss its protective effect on alcoholic cirrhosis. METHOD: Fifty SD male rats were divided into the normal control group, the model group, the high-dose and low-dose Notoginseng Radix groups (3.0, 12.0 g x kg(-1)) and the magnesium isoglycyrrhizinate group (24 mg x kg(-1)), with 10 rats in each group. Apart from the control group, other groups were administered with ethanol-cornoil-pyrazole for 14 weeks to establish the alcoholic liver disease model. During the establishment of the model, the high-dose and low-dose Notoginseng Radix groups were administered with 12 g x kg(-1) x d(-1) Notoginseng Radix for 14 weeks, once everyday. Efforts were made to detect liver function, pathology with Masson staining, and the expressions of TGF-beta1, Smad3, Smad7 and CTGF mRNA. RESULT: Compared with the rats in model group, rats in Notoginseng Radix groups showed significant reduction in liver ALT, AST, collagen fiber deposition, and TGF-beta1, Smad3 and CTGF mRNA expressions in liver tissues, with the increase in the expression quantity of Smad7 mRNA. There were differences between the Notoginseng Radix groups. No significant difference was observed between the high-dose Notoginseng Radix group and the magnesium isoglycyrrhizinate group. CONCLUSION: Notoginseng Radix can affect TGF-beta1/Smads signaling pathway and reduce the expression of CTGF.

Effects of Ginkgo biloba extracts on diazepam metabolism: a pharmacokinetic study in healthy Chinese male subjects.

AIM: It has been reported that Ginkgo biloba extract (GBE) is an inducer or inhibitor of microsomal cytochrome P450 (CYP) 2C19, and diazepam is a substrate of CYP2C19. Thus, it could be expected that GBE may alter the metabolism of diazepam. METHODS: The pharmacokinetic parameters of diazepam and one of its metabolites, N-demethyldiazepam, were compared after oral administration of diazepam (10 mg) in the absence or presence of oral GBE (120 mg bid, for 28 days) in 12 healthy volunteers. The pharmacokinetic analysis was performed using a noncompartmental method. RESULTS: The 90% confidence intervals (CIs) of the ratios of mean pharmacokinetic parameters of diazepam presence and absence of GBE were well within the 80-125% bioequivalence range, indicating no pharmacokinetic interaction. The ratio of AUC(0-408) with GBE to AUC(0-408) without GBE was 95.2 (90%CI: 91.6-98.8) and 101.8 (90%CI: 99.4-104.1) for diazepam and N-desmethyldiazepam, respectively. The two drugs were well tolerated, and no drug-related serious adverse events were reported. CONCLUSION: The above data suggest that GBE, when taken in normally recommended doses over a 4-week time period, may not affect the pharmacokinetics of diazepam via CYP2C19 and the excretion of N-desmethyldiazepam in healthy volunteers. No drug-drug interaction was observed between GBE and diazepam.