Based On PKCα/Nrf2/ROS Pathway To Investigate The Antihepatic Fibrosis Rats Effect Of Ruogan Huaxian Granules

Objective:To observe the intervention effect of Rugan Huaxian Granules on the rat model of hepatic fibrosis induced by carbon tetrachloride（CCl₄）complex factors,and to explore the possible mechanism of Rugan Huaxian Granules against hepatic fibrosis from the perspective of PKCα/NRF2/ROS signaling pathway.Methods:Forty-eight male Wistar rats were randomly divided into normal group,pathological model group,colchicine group and Ruogan Huaxian Granules group,with 12 rats in each group.In addition to the normal group,the rest of the each group of rats by subcutaneous injection of 40%CCl₄（after the first use of 5 ml/kg body weight,every 3 days per 3 ml/kg body weight,2 times a week,a total of 8 weeks）,subcutaneous injection,combined high fat,low protein food（is feed with corn flour,1,2 weeks with 0.5%cholesterol,20%lard）,in order to prevent hepatic fibrosis natural repair the effects on the experimental results,in addition to the normal group,the rest of the group still intraperitoneal injection of 40%CCl₄ oil once a week for 3 ml/kg body weight/every time.The normal group was fed normally,and the same volume of peanut oil was injected subcutaneously for 8 weeks and the same volume of normal saline was given intragastric.The hepatic fibrosis model group was given 0.01mg/100g body weight colchicine intragastric 8 weeks after the model was successfully established.Ruogan Huaxian Granules group were administered 4mg/kg intragastric administration period was 8 weeks.Each group was anesthetized by 1%pentobarbital with fasting for 12h after 8 weeks of administration,and sacrificed after femoral vein blood collection.Livers were taken for open dissection.The liver was observed by HE staining and Masson staining,and the contents of Reactive Oxygen Species（ROS）,Superoxide Dismutase（SOD）,Glutathione peroxidase（GSH-Px）and Malondialdehyde（MDA）were detected by ELLSA.The m RNA and protein expressions ofα-smooth Muscle Actin（α-SMA）,E-cadherin（E-cad）,Collagen I（Col I）,Collagen III（Col III）,Heme Oxygenase-1（HO-1）,Quinone dehydrogenase 1（NQO1）,Protein Kinase Cα（PKCα）and NF-E2-related factor2（Nrf2）were detected by PCR and Western Blot.Results:1.Effect of Ruogan Huaxian Granules on Liver TissueIn the normal group,the structure of hepatic lobules was complete,the contour was clear,the liver tissue was compact,no pseudobular nodules were formed,no bile ducts and fibrous tissue hyperplasia were observed in the portal area and its surrounding area,and the nucleus was located in the center.In the pathological model group,the structure of hepatic lobules was destroyed or even disappeared,nodules were formed,hepatic cord arrangement was disordered,a large number of proliferative fibers in portal area were cord-like,hepatocytes were steatosis,and many vacuoles of different sizes appeared in the cytoplasm.In the colchicine group and Ruogan Huaxian Granules group,the structure of hepatic lobules was not clear,fibrous tissue hyperplasia was observed in the portal area and its surrounding bile ducts,hepatocyte steatosis,and many vacuoles of different sizes appeared in the cytoplasm,but the degree was significantly reduced compared with the pathological model group.2.Effect of Rugan Huaxian Granules on SOD,GSH-Px and MDA contents1Compared with normal group,the content of MDA in pathological model group,colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）;Compared with pathological model group,the content of MDA in colchicine group and Ruogan Huaxian Granules group decreased to different degrees（P<0.05）;2Compared with the normal group,SOD and GSH-Px in pathological model group,colchicine group and Ruogan Huaxian Granules group were significantly decreased（P<0.05）;Compared with model group,the levels in colchicine group and Ruogan Huaxian Granules group were significantly increased（P<0.05）.3.Effect of Rugan Chemical Fiber Granules on Serum ROS1Compared with normal group,ROS content in pathological model group was significantly increased（P<0.05）;2Compared with model group,ROS content in colchicine group and Ruogan Huaxian Granules group was decreased（P<0.05）.4.Effect of Rugan chemical fiber granules on the expressions ofα-SMA m RNA,E-cadherin m RNA,PKCαm RNA,Nrf2 m RNA,HO-1 m RNA,Collagen I m RNA and III m RNA1Compared with the normal group,the relative expression of PKCαm RNA in the pathological model group was decreased（P<0.05）,and the relative expression of PKCαm RNA in the Ruogan Huaxian Granules group was increased（P<0.05）,but there was no significant difference in the colchicine group（P>0.05）.Compared with pathological model group,the m RNA relative expression level of PKCαin colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）.2Compared with the normal group,the relative expression of Nrf2m RNA in pathological model group,colchicine group and Ruogan Huaxian Granules group decreased（P<0.05）;Compared with pathological model group,the m RNA relative expression level of Nrf2 in colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）.3Compared with normal group,the m RNA relative expression level of HO-1 in pathological model group,colchicine group and Ruogan Huaxian Granules group decreased（P<0.05）;Compared with pathological model group,the m RNA relative expression level of HO-1 in colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）.4Compared with the normal group,the relative expression of NQO1m RNA in pathological model group was decreased（P<0.05）.Compared with pathological model group,the m RNA relative expression level of NQO1 in colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）.5Compared with normal group,the m RNA relative expression level ofα-SMA in pathological model group,colchicine group and Ruogan Huaxian Granules group was increased（P<0.05）.Compared with pathological model group,the m RNA relative expression level ofα-SMA in colchicine group and Ruogan Huaxian Granules group was significantly decreased（P<0.05）.6Compared with normal group,the m RNA relative expression level of E-cadherin in pathological model group,colchicine group and Ruogan Huaxian Granules group was increased（P<0.05）;Compared with pathological model group,the m RNA relative expression level of E-cadherin in colchicine group and Ruogan Huaxian Granules group was significantly decreased（P<0.05）.7Compared with the normal group,the relative expression level of Collagen I m RNA in the pathological model group was increased（P<0.05）,and the relative expression level of Collagen I m RNA in the Ruogan Huaxian Granules group was significantly decreased（P<0.05）,but there was no significant difference in the colcoline group（P>0.05）.Compared with pathological model group,the m RNA relative expression level of Collagen I in colchicine group and Ruogan Huaxian Granules group was significantly decreased（P<0.05）.8Compared with the normal group,the m RNA relative expression level of Collagen III in the pathological model group was significantly increased（P<0.05）,and the m RNA relative expression level of Collagen III in the Ruogan Huaxian Granules group was significantly decreased（P<0.05）,but there was no significant difference in the colchicine group（P>0.05）.Compared with the pathological model group,the m RNA relative expression level of Collagen III in the colchicine group and the Ruogan Huaxian Granules group was significantly decreased（P<0.05）.5.Effect of Ruogan Huaxian Granules on the protein expression ofα-SMA,E-cadherin,HO-1,NQO1,PKCα,Nrf2,Collagen I and III1Compared with normal group,the expression of PKCαprotein in pathological model group,colchicine group and Ruogan Huaxian Granules group decreased（P<0.05）;Compared with pathological model group,the expression of PKCαprotein in colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）.2Compared with the normal group,the expression of Nrf2 protein in pathological model group and colchicine group was decreased（P<0.05）,but there was no significant difference in the expression of Nrf2 protein in Ruogan Huaxian Granules group（P>0.05）.Compared with pathological model group,the protein expression of Nrf2 in colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）.3Compared with the normal group,the relative expression level of HO-1 protein in the pathological model group and the Ruogan Huaxian Granules group was decreased（P<0.05）,but there was no significant difference in the relative expression level of HO-1 protein in the colchicine group（P>0.05）.Compared with pathological model group,the relative expression level of HO-1 protein in colchicine group was significantly increased（P<0.05）,but the increase of Ruogan Huaxian Granules group was not significant（P>0.05）.4Compared with the normal group,the relative expression of NQO1protein in pathological model group,colchicine group and Ruogan Huaxian Granules group decreased（P<0.05）;Compared with the pathological model group,the relative expression level of NQO1 protein in colchicine group and Ruogan Huaxian Granules group was significantly increased（P<0.05）.5Compared with the normal group,the relative expression level ofα-SMA protein in the pathological model group was increased（P<0.05）,but there was no significant difference in the relative expression level ofα-SMA protein between the colchicine group and the Ruogan Huaxian Granules group（P>0.05）.Compared with pathological model group,the relative expression ofα-SMA protein in colchicine group and Ruogan Huaxian Granules group was significantly decreased（P<0.05）.6Compared with the normal group,the relative expression level of E-cadherin protein in the pathological model group was increased（P<0.05）,but there was no significant difference in the relative expression level of E-cadherin protein between the colchicine group and the Ruogan Huaxian Granules group（P>0.05）.Compared with pathological model group,the relative expression of E-cadherin protein in Ruogan Huaxian Granules group was significantly decreased（P<0.05）.7Compared with the normal group,the relative expression level of Collagen I protein in the pathological model group was increased（P<0.05）,the relative expression level of Collagen I protein in the Ruogan Huaxian Granules group was decreased（P<0.05）,and the relative expression level of Collagen I protein in the colchicine group was not significantly different（P>0.05）.Compared with pathological model group,the relative expression level of Collagen I protein in colchicine group and Ruogan Huaxian Granules group was significantly decreased（P<0.05）.8Compared with the normal group,the relative expression level of Collagen III protein in the pathological model group was increased（P<0.05）,the relative expression level of Collagen I protein in the Ruogan Huaxian Granules group was decreased（P<0.05）,and the relative expression level of Collagen I protein in the colcoline group was not significantly different（P>0.05）.Compared with the pathological model group,the relative protein expression of Collagen III in the colchicine group and the Ruogan Huaxian Granules group was significantly decreased（P<0.05）.Conclusion:Rugan Huaxian Granules can improve the degree of hepatic fibrosis in rats induced by CCl₄.Rougan Huaxian Granules can reduce ROS,SOD,GSH-Px,MDA content,and the changes of ROS,suggesting that Rugan Huaxian Granules has a certain antioxidant stress effect on hepatic fibrosis rats.By activating PKCα/Nrf2 signaling pathway to up-regulate HO-1 and NQO1 expression and down-regulateα-SMA,Collagen I and III expression,Rugan Huaxian Granules played an anti-hepatic fibrosis effect.