Research On Biejiajian Pills Molecular Mechanism Of Anti-Liver Fibrosis

Research source:Traditional Chinese Medicine Agency-funded projects of Guangdong Province (307012).Purpose and significance:To investigate the Biejiajian Pills molecular mechanism of anti-liver fibrosis,provide a more adequate theoretical basis for clinical use of the Biejiajian Pills in curing liver fibrosis and the development of antifibrosis Chinese medicineMethods:We observed the Effects of Biejiajian Pills on symptoms, signs and liver morphology, liver function and serum HA, Pc III and TGF in liver tissue-β1 and its signal transduction pathway in hepatic fibrosis model rats induced by multiple factors.60 Wistar rats were randomly assigned to normal control group (A group), model group (B group), colchicine group (C group),Biejiajian Pills high-dose group (D group)and BiejiajianPills medium-dose group (E group). All rats were subcutaneously injected 0.5ml·100g-lof 40% (volume fraction) CC14 peanut oil in day 1 follwed by 0.3ml·100g-1 every 3 days and feed with mixed diet(79.5% pure Corn flour,20% lard and 0.5% cholesterol) and 15% ethanol except group A.While rats of group A were injected peanut oil of same amount,and feed with normal diet and water.Each group get appropriate medication daily by intragastric administration from day 2,0.011mg·100g-lcolchicine for group C,2.2g·100g-1,1.1 g·100g-1 Biejiajian Pills respectively for group D and E,distilled water for group A and B for control.At the end of 6th week, rats were killed, blood and liver tissue reserved for further use. Part of the liver tissue were fixed in 10% neutral formalin solution. After conventional dehydration and paraffin embedation, liver tissue was sliced by thickness of 5μm for light microscopy and immunohistochemical purposes.we also measured serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity by Lai and colorimetry respectively and serum HA, Pc HI level by radioimmunoassay according to kit instruction.we Observed pathological changes in liver tissue under light microscope and graded the liver fibrosis;measured TGF-β1, CTGF and other expression by immunohistochemical technology. Statistical analysis:measurement data with the mean±standard deviation (x±s), data were processed using SPSS11.0, ANOVA (one-way ANOVA). Ranked data were analysis by Ridit.Research content and processes:1.observe the effect of Biejiajian Pills on model rats symptoms, signs and liver morphologyModel group rats were lackadaisical with weight loss, anorexia, decreased drinking, loose stool, yellow and less urine; compared with control group,their liver surface were less smooth and colored,more blunt in edge and hard, uneven surface of some specimens showed a small nodule shape. Mental status, hair color,diet,drink,defecation and other general conditions of each drug group of each drug group were improved significantly,especially in those two Biejiajian pills groups. Biejiajian pills of high and medium-dose group was superior to colchicine group (P all<0.05)in improving water consumption,while Biejiajian pills of high-dose group was superior to colchicine Group (P all<0.05)in improving the appetite and body weight.Light microscopy showed that normal hepatic lobule structure damage, hepatic cord disorder, scattered spotty necrotic liver cells, liver cells vacuolation, marked interstitial hyperplasia,fibrous septa and complete pseudolobules in model group rats. Hepatic lobules were significantly improved with less collagen interval and liver cell necrosis, rare inflammatory cell infiltration in drug groups, but incomplete pseudolobules still exist in very few rats. High and medium-dose of Biejiajian pills could significantly reduce liver fibrosis in rats (P<0.05), but there was no significant difference between colchicine and Biejiajian pills(P> 0.05).2. the effect of Biejiajian Pills on rats liver function and serum HA, PcⅢlevel model group rats were significantly higher than those in control group (P all<0.01); compared with model group, ALT, AST activity, HA, Pc III levels were significantly decreased (P all<0.01) in Biejiajian Pills high and medium-dose groups,as well as AST/ALT ratio(P<0.01, P<0.05); Biejiajian Pills high and medium-dose groups were superior to colchicine group(P all<0.05)in lowering ALT activity; Biejiajian Pills high-dose group was superior to lower AST activity compared with colchicine (P <0.05); but there was no significant difference (P> 0.05)among the drug group on AST/ALT ratio and the HA, Pc III level.3.effect of Biejiajian Pills on TGF-β1 in fibrotic liver tissue of model ratsThe expression of TGF-β1:there was almost no expression in normal liver cells,little expression in sinusoidal space, matrix and interstitial cells with portal area. There was a significantly increase in model group, especially in stromal cells, inflammatory cells and injured liver cells, and weak positive in fibrotic septum. Positive staining of each drug group significantly reduced compared with the model group, expression mainly concentrated in the cytoplasm of interstitial cells and inflammatory cells.Statistics show that the expression of TGF-β1 in liver tissue of model group increased significantly compared with control group (P<0.01). TGF-β1 expression in Biejiajian Pills high and medium-dose groups decreased significantly(P all<0.01) compared with the model group.But there was no significant difference between those two groups and colchicine group(P all> 0.05).4.effect of Biejiajian Pills on CTGF in fibrotic liver tissue of model rats There was almost no CTGF expression in normal liver cells,while a significantly increased expression occured in Model group, showing brownish yellow cytoplastic distribution,especially in the spindle-like interstitial cells spaced along septa within the portal area and fiber interval instead of liver cells. The degree of positive staining in the cytopalsm of interstitial cells within fibrous septa and inflammatory cells and the positive cells number of drug groups decreased significantly compared with model group.CTGF expression of Model group increased significantly(P<0.01) compared with normal group;CTGF expression of Biejiajian Pills high, medium-dose groups reduced significantly(P all<0.05) compared with model group;there were no significant difference(P> 0.05) between Biejiajian Pills high-dose group and colchicine group, but the Biejiajian Pills medium-dose group was significantly weaker than the colchicine group (P<0.05) in lowering CTGF expression.Conclusion:Biejiajian Pills is effective in curing hepatic fibrosis in model rats, mechanism as following:1. Biejiajian Pills can improve general conditions such as the mental state body weight, appetite, water consumption and other symptoms to improve life quality of the liver fibrosis model rats.2. Biejiajian Pills can significantly reduced ALT, AST, HA, Pc III content, suggesting that Biejiajian pills can effectively reduce inflammation as well as serum fibrosis markers level and protect liver cells,thereby inhibit the initiation of early hepatic fibrosis;3. light microscope observation showed that Biejiajian Pills liver cell loss could apparently improve liver structure and reduce the proliferation of fibrous tissue;4. Biejiajian pills can restrict the TGF-β1 expression,suggesting that Biejiajian Pills maybe be able to block the TGF-β1 signal transduction;5. Biejiajian Pills liver tissue can significantly inhibit the expression of CTGF, which is one of the targets of Biejiajian Pills to cure liver fibrosis. In summary, Biejiajian Pills realize its integrated anti-liver fibrosis effects through multi-channels and targets. Inhibition of TGF-β1 and its signal transduction together with CTGF expression may be one of the molecular mechanisms,Which is closely related to inflammation inhibition,liver cells protection,serum fibrosis markers and cytokine cascade reduction,and thus inhibit the initiation of liver fibrosis.