The Study Of The Effect And Mechanism Of Huagan-tongluo Prescription On Hepatic Stellate Cells Of Rat

Objective:To study treatment of primary biliary cirrhosis (PBC) in vitro and investigate Huagan-Tongluo prescription’s effect on hepatic stellate cell proliferation of rats and the mechanism.Method:1. Preparation of serum containing drug:40 SD rats were randomized into 4 groups: Huagan-Tongluo high-dose group(73.7g/kg/d), Huagan-Tongluo middle-dose group(36.8g/kg/d), Huagan-Tongluo low-dose group(18.4g/kg/d) and untreated group(0.9%NaCl). Rats were intragastric administrated 2ml every day for 7 days. Jejunitas for 24h before the last administration. Collect blood from aorta abdominalis 1h after the last administration, let stand for centrifugal, inactivate complement, filter bacteria and cryopreserve in -80℃.2. Composition Determination in drug. Examine composition and compare with standard substance by means of mass spectrometry, then confirm the composition in drug.3. Investigate Huagan-Tongluo prescription’s effect on hepatic stellate cell proliferation of rats:Use MTT method to measure OD value change between different groups after serum containing drug treatment for 24h to determine the cell proliferation affected by drug.4. Investigate the mechemism of Huagan-Tongluo formulation on hepatic stellate cell proliferation:cell treated by different concentration of serum containing drug for 24h, examine the expression of TGF-β1、smad2/3、smad7 with WB method after extracting protein from the cell and determine the role of the formulation in TGF-β/smad pathway.Results:1. Preparation of serum containing drug:Effective constituent was confirmed in Huagan-Tongluo formulation through comparision with standard substance via mass spectrometry.2. Investigate Huagan-Tongluo prescription’s effect on hepatic stellate cell proliferation of rats:OD value decreased significantly after treated with serum containing drug, indicates that this formulation was capable of inhibiting proliferation of hepatic stellate cell. There is statistical difference in high-dose group (*P<0.05) and middle-dose group, except low-dose group (P> 0.05)3. Investigate the mechemism of Huagan-Tongluo prescription on hepatic stellate cell proliferation:WB results indicate that there is no significant difference between low-dose group and untreated group in terms of TGF-β1、smad2/3 expression (P> 0.05), expression in middle-dose group and high-dose group was down regulated (*P<0.05).Expression of smad7 was up regulated in high-dose group and middle-dose group (*P<0.05), but not that remarkable in low-dose group. (P> 0.05)Conclusion:1. There is objective constituentin Huagan-Tongluo prescription containing drug.2. Huagan-Tongluo prescription serum containing drug is able to inhibit proliferation of hepatic stellate cell.3. Treatment of PBC by Huagan-Tongluo prescription is partly based on TGF-β/smad pathway.