| Objective:Through the observation of "nourishing kidney to produce marrow and form liver" rule of treatment in Hubei Provincial Traditional Chinese Medical Hospital hospital preparations to "Diwuyanggan capsule"(Hubei pharmaceutical word Z20120221) intervention effect on CC14-induced liver fibrosis in rats. To explore the mechanism of " DiwuYanggan capsule " regulation of EMT/MET imbalance, provide new treatment ideas and direction for the clinical treatment of liver fibrosis,reveals the EMT/MET imbalances caused by"marrow producing liver abnormally" pathogenesis and "nourishing kidney to produce marrow and form liver" regulation of EMT/MET imbalance in the scientific connotation.Methods:Rat hepatic fibrosis model induced by50%CCL4/oil solution was subcutaneous multi-point injection method.20SPF Wistar male rats were randomly divided into three groups:blank control group, hepatic fibrosis model group (hereinafter referred to as the "model group"),"Diwuyanggan capsule" treatment group (hereinafter referred to as the "treatment group"). The model group and treatment group rats were given50%CCL4/soybean oil was established by subcutaneous injection for six weeks,2times per week, a dose of1ml/kg. At the same time, the blank control group received doses of soybean oil by subcutaneous injection for6weeks. In modeling at the same time, the treatment group rats were given "DiwuYanggan capsule" solution gavage,1times/day, a dose of360mg/kg, a total of6weeks. And the control group and model group rats were given the same amount of saline lavage in comparison. Modeling six weeks, the rats were sacrificed, blank control group, model group and treatment group were taken liver tissue, determination of liver index and hydroxyproline content, and liver tissue haematin-eosin (HE) staining and Masson staining, in order to observe the degree of liver damage and the proliferation of collagen fibers extent. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting observed mRNA and protein expression in liver tissue of E-cadherin and vimentin. The image was processed by Gel-pro analyzer software, the selected analysis area, obtaining the average value of the integrated optical density IOD value. All data were processed by statistical software SPSS17.0, the result is in x±s. Group of multiple samples were compared using analysis of variance United SNK test, P<0.05was considered statistically significance. Draw a diagram according to the statistical results.Results:The hydroxyproline content results showed that the hydroxyproline content of liver tissue in model group rats (207.39±31.66) than the control group rats (135.12±17.74) was significantly increased, Statistically, the difference was significant, p<0.05. After "Diwuyanggan capsule" treatment, the content of hydroxyproline in liver tissue of rats treated group (155.16±25.22) was significantly lower than that of model group, statistically, significant difference between the two, p<0.05. HE staining and Masson staining showed that "Diwuyanggan capsule" therapy could alleviate liver cell injury, and reduce the degree of liver fibrosis. PCR results showed that:the epithelial cell marker E-cadherin mRNA levels of the model group (0.364±0.12) was significantly lower than the blank control group (0.593±0.12), and mesothelial cell marker Vimentin mRNA levels (0.456±0.08) was significantly higher than the blank control group (0.310±0.06), after statistics processing, the difference significant, p<0.05. Model group, the decrease in markers of epithelial cells, mesenchymal cell markers increased, indicating that CCL4-induced rat liver fibrosis occurs EMT. After "Diwuyanggan capsule" treatment, the treatment group compared with the model group and blank control group, the content of mRNA in liver tissue of rats with E-cadherin (0.476±0.17) was significantly higher than the model group (0.364±0.12) and lower than that of control group (0.593±0.12), after the statistical treatment, the difference was significant, p<0.05. Treatment of liver tissue Vimentin mRNA levels (0.348±0.12) was significantly lower than that in the model group (0.456±0.08), both Statistically, the significant difference, p<0.05. Western blotting results showed that:the model group compared to the control rats, the model group, the epithelial cell marker E-cadherin protein expression (0.631±0.57) was significantly lower than the control group (3.666±2.38), mesothelial cells Vimentin protein markers (2.745±1.85) than the control group (1.441±1.37) statistically, the difference significant, p<0.05. After "Diwuyanggan capsule" treatment, the treatment group compared with the model group, the expression of E-cadherin protein in the treatment group (1.723±1.47) was higher than that in the model group (0.631±0.57), p<0.05. While the expression of Vimentin protein (2.274±0.93) lower than that of model group (2.745±1.85), p>0.05. Found that after the treatment of "Diwuyanggan capsule" in the rat liver f ibrosis in the process of EMT is a certain degree of inhibitiont, promote the occurrence of MET, thus reducing the progress of liver fibrosis disease.Conclusion:CC14-induced hepatic fibrosis in rats exist EMT/MET imbalance caused by etiology and pathogenesis of"marrow producing liver abnormally", the liver tissue epithelial cell marker expression levels down, and mesenchymal cell marker expression levels increase."Diwuyanggan capsule" can delay or reduce the development of liver fibrosis, regulating EMT/MET imbalance (inhibition of EMT and promote MET), to improve the"marrow producing liver abnormally "may be one of the mechanisms. |
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