| Objective: Mechanism of Shenren Huoxue granule on Hepatic Fibrosis in Rats based on TGF-? 1/Smad Pathway.Methods:1.Modeling and intervention:Fifty 6-week-old male SD rats of clean grade were randomly divided into five groups: blank control group,model control group,low dose of Chinese medicine,medium dose of Chinese medicine,high dose of traditional Chinese medicine,and 10 rats in each group,are fed with general feed.The blank control group was given normal saline 1ml/kg,intraperitoneal injection twice a week,a total of 8 weeks;the model control group was given 40% carbon tetrachloride(CCL4)0.2ml/100 g intraperitoneal injection twice a week for 8 weeks;in the low dose group of traditional Chinese medicine,0.5times of equivalent dose of Shenren Huoxue granule was given to the stomach from the 4th week of model making,and the rats were killed at the 8th week;in the middle dose group of traditional Chinese medicine,the rats were given the equivalent dose of Shenren Huoxue granule at the 4th week,and the rats were killed at the 8th week;in the high dose group of traditional Chinese medicine,2 times of equivalent dose ofShenren Huoxue granule was given to the stomach from the 4th week of model making,and the rats were killed at the 8th week.2.Sprague-Dawley rats in each group were killed in the 8th week,and then liver tissue was taken for detection:Pathological examination:HE stain,Msson staining;immunohistochemical:collagen I,collagen?,TGF-?1;RT-PCR:TGF-?1,Smad3,Smad7;Western-blot:TGF-?1,Smad3,Smad7.Results:1.HE and Msson staining of liver pathological tissue:(1)HE stain:compared with the blank control group,the model group showed liver lobular structure destruction,cellular cord structure disorder,inflammatory cell infiltration,cytoplasmic loosening,hepatocyte degeneration,severe necrosis and pseudolobules formation(P<0.05);compared with the model group,the degree of hepatocyte necrosis and inflammatory cell infiltration in the traditional Chinese medicine group was obviously reduced,the proliferation of fibrous connective tissue was decreased,the high dosage group of traditional Chinese medicine was the most significant,followed by the middle dose group of traditional Chinese medicine(P< 0.05);(2)Masson staining:compared with the blank control group,the fibers in the liver tissue of the model group were obviously increased,the hepatic lobule structure was destroyed,and a large number of fibrous tissues were partitioned around the liver tissue to form pseudolobules of different sizes(P<0.05);compared with the model group,the collagenous fibers in eachgroup were thinned,decreased,pseudoblobule formation was significantly decreased,and the degree of hepatic fibrosis was significantly improved,the high dosage group of traditional Chinese medicine was the most significant,followed by the middle dose group of traditional Chinese medicine(P< 0.05).2.Immunohistochemical results showed:the positive expression of Collagen ? and Collagen ? protein could be observed in the interstitial cells of the hepatocytes in each group.In the blank control group,the portal area was small and light brown,which was a small amount of fibrous tissue in normal liver tissue and had no positive expression.The staining of model group was thick and deep brown,the staining of model group was the deepest,lowest,middle and high dose groups were lighter than the model group,and showed a positive correlation with the dose;the positive expression of TGF-?1 protein could be found in the cell membrane,cytoplasm and interstitial of hepatocytes in each group,mainly in the cytoplasm.The brown-yellow staining was light in the blank control group,while the model group was the deepest,the highest,the middle and low dose groups were lighter than the model group,and showed a positive correlation with the dose.3.Realtime-PCR test results show:compared with the normal group,the expression of Smad7 mRNA in liver fibrosis tissues of the model group decreased and the expression of TGF-?1mRNA and Smad3 mRNA increased(P<0.05);compared with the modelgroup,the expression of Smad7 mRNA was significantly increased in the liver fibrosis tissues of rats in each Chinese medicine group,and the effect was the best in the high dose group,followed by the middle dose group in the traditional Chinese medicine group(P<0.05);compared with the model group,the expression of TGF-beta 1mRNA and Smad3 mRNA decreased obviously,the effect of high dose group of Chinese medicine was the most obvious,the effect of middle dose group of Chinese medicine was the second one(P<0.05);4.Western-blot test results show:compared with the normal group,the expression of Smad7 protein in the liver fibrosis tissue of the model group decreased significantly,and the expression of TGF-? 1 and Smad3 protein increased significantly in the model group(P<0.05);compared with the model group,the expression of Smad7 protein in the liver fibrosis tissue of rats in each Chinese medicine group was significantly increased,the effect of high dose Chinese medicine group was the best,and the middle dose Chinese medicine group was the second(P<0.05);compared with the model group,the expression of TGF-?1 and Smad3 protein in the liver fibrosis tissue of each Chinese medicine group was significantly decreased,and the effect of the high dose group was the most obvious(P<0.05).Conclusion: 1.40% CCL4 0.2 ml/100 g could successfully induce hepatocyte damage in SD rats,under microscope,HE staining and Masson staining in liver tissue showed the proliferation of fibrousconnective tissue,the formation of fibrous septum,and the formation of pseudolobules in some liver tissues,the results showed that the SD rat model of hepatic fibrosis was successful;2.Shenren Huoxue granule can obviously improve the pathological structure of liver tissue of SD rats with hepatic fibrosis,alleviate the degree of liver fibrosis and liver inflammation in HF rats;3.Shenren Huoxue granule may decrease the synthesis of Collagen ?and Collagen ? by inhibiting the activation of TGF-?1 and Smad3 and upregulating the expression of Smad7. |
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