| Hepatic fibrosis may be viewed as a repair response to hepatocellular injury, a common sequel to a variety of liver diseases of varying causes. Activation of hepatic stellate cells is an invariant feature of liver diseases that ultimately lead to hepatic fibrosis. Meanwhile, many kinds of cytokines participated in hepatic fibrosis, and transforming growth factor-β1 (TGF-β1) is considered to be one of the most important cytokines to hepatic fibrosis. TGF-β1 can activate hepatic stellate cells, promote deposition of extracellular matrix (ECM) and restrain degradation of collagenous fibers.Hepatocyte growth factor (HGF) is a multifunctional cytokine with mitogenic, anti-apoptotic, and anti-inflammatory functions in hepatocytes. Considering the insufficient production of hepatocyte growth factor is responsible for the manifestation of chronic hepatitis, and hepatic fibrosis was formed in the process of chronic hepatitis, supplementation with hepatocyte growth factor represents a new strategy for attenuating hepatic fibrosis.ObjectiveTo observe the changes of hepatic fibrosis in model rats induced by CCl4 and composite factors at different time-points, and seek the optimal time points for administration and ending of experimentation. To observe gene therapeutic effects of hepatocyte growth factor(HGF)on hepatic-fibrosis rats induced by CCl4 and composite factors, and focusing on the mechanisms of Ad-HGF for reversing hepatic fibrosis. Method1. Changes of hepatic fibrosis in model rats induced by CCl4 and composite factors Male Wistar rats were randomly divided into two groups: model group and normal group. Rats in model group were intoxicated continuously with 40% CCl4 in olive oil twice a week and fed on the fatty-rich diet and 5% alcohol, and the ones in normal group were identically treated with olive oil. At the end of the 1st, 4th, 8th week, the rats in normal group and model group were randomly selected to make up of six groups: 1-week model group, 1-week normal group, 4-week model group, 4-week normal group, 8-week model group, 8-week normal group;and were respectively killed at these three different time points. Serum biochemical indicators (GOT, GPT, TP, ALB, TBil), liver index and content of Hyp in the liver tissues were then analyzed. The histopathological changes of HE stained were observed by microscopy. The optimal time points for administration and ending of experimentation were determined by the result.2. Therapeutical effect of Ad-HGF on hepatic fibrosis25 Male wistar rats were randomly divided into five groups: control group, model group, low-dose(1×109 pfu)group, middle-dose(1×109 pfu)group, high-dose(5×109 pfu)group. The rats in treated group and model group were intoxicated continuously with 40% CCl4 in olive oil twice a week and fed on the fatty-rich diet and 5% alcohol, and the ones in normal group were identically treated with olive oil. At the optimal time point for administration, the rats in low-dose, middle-dose and high-dose group were respectively injected 1×109 pfu, 2.5×109 pfu and 5×109 pfu Ad-HGF via the tail vein three times a week for two weeks. At the optimal time point for ending of experimentation, the rats were killed, and then Serum biochemical indicators (GOT, GPT, TP, ALB, TBil), liver index and content of Hyp in the liver tissues were analyzed. The histopathological changes of HE stained were observed by microscopy, and disposition of collagen in liver tissues was detected by Sirius-red staining. In addition, area of collagen in liver tissues was analyzed by American professional software of image analysis (Image—Pro Plus). Expression of HGF and TGF-β1 in liver tissues were also detected by Immunohistochemical staining.Result1. Changes of hepatic fibrosis of model rats in different phases Compared with 1-week normal group, GOT, GPT, TBil in plasma and liver index were markedly(P<0.05)enhanced in 1-week model group. Compared with 4-week normal group, GOT, GPT, TBil in plasma , liver index, and content of Hyp in the liver tissues were markedly(P<0.01)enhanced in 4-week model group, and TP, ALB in plasma were remarkably (P<0.05)declined . Compared with 8-week normal group, GOT, GPT, TBil in plasma , and content of Hyp in the liver tissues were markedly(P<0.05)enhanced in 8-week model group, and TP, ALB in plasma were remarkably (P<0.01)declined. The histopathological changes of HE stained: no fibroplasia was in the liver tissue of 1-week model rats; a lot of fibrous tissue was in the liver tissue of 4-week model rats; notho-lobulus of liver were in the liver tissue of 8-week model rats. The end of 4th week was determined to be the optimal time point for administration, and the end of 8th week to be the optimal time point for ending experimentation.2. Therapeutic Effect of Ad-HGF on anti-hepatic fibrosisGOT,GPT,TP,ALB,TBil in plasma were markedly (P<0.05) improved in the high-dose group compared with the model group. Area of collagen, content of Hpy in liver tissues, degree of hepatocyte damage and hepatic fibrosis were remarkably (P<0.05)declined in middle-dose group and high-dose group compared with the model group. The immunohistochemical result indicated that Ad-HGF could remarkably up-regulate expression of HGF and down-regulate expression of TGF-β1 in liver tissues in low-dose group, middle-dose group and high-dose group.Conclusion1. Changes in different stages of hepatic-fibrosis were detected in model rats induced by CCl4 and composite factors. The optimal time points for administration was 4th week, and the optimal time points for ending of experimentation was 8th week.2. Ad-HGF gene therapy could exert anti-hepatic fibrosis effect in rats and its action mechanism might be related to: 1) up-regulating expression of HGF gene, and making effects of HGF protein on promoting hepatocellular regeneration , refraining hepatocellular apoptosis and lessening the degree of hepatic injury; 2) restraining expression of TGF-β1, promoting collagen degradation, inhibiting deposition of ECM, and ultimately blocking the process of hepatic fibrosis. These suggested that Ad-HGF might be a reasonable strategy to for the gene therapy of hepatic fibrosis. |
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