Study On The Mechanism Of Shuganhuazheng Decoction On CCl₄ Induced Hepatic Fibrosis Model Rats Based On MAPK Pathway

ObjectiveTo verify the therapeutic effect of Shugan Huazheng Decoction(SGHZ)on hepatic fibrosis model rats,and to explore its mechanism based on MAPK signaling pathway.MethodsFifty-four male SPF SD rats were selected and fed adaptively for 3 days.They were randomly divided into six groups:blank control group(BG),model group(MG),colchicine group(CG),Shugan Huazheng Decoction High Dose Group(SGHZ-H),Shugan Huazheng Medium Dose Group(SGHZ-M)and Shugan Huazheng Decoction Low Dose Group(SGHZ-L).There were 9 rats in each group,and there was no significant difference in body weight between the groups.BG was not treated.The rats in the other groups were intraperitoneally injected with 40%CCl4 olive oil solution at a dose of 2.0 m L/kg(injection dose/rat body weight),twice a week for 8 weeks.BG without any treatment conventional feeding.CG was intragastrically administered with 0.2 mg/kg(colchicine content/rat body weight)colchicine aqueous solution.SGHZ-H,SGHZ-M and SGHZ-L were intragastrically administered with29.52 g/kg(crude drug content/rat body weight),14.76 g/kg and 7.38 g/kg,respectively.Intragastrically administered once a day for 8 weeks.After that,(1)the general condition of rats in each group was dynamically monitored.(2)On the 56th day after anesthesia,serum was taken to detect liver function by automatic biochemical analyzer.(3)LN and Col IV were detected by ELISA.(4)Observation of hepatic morphology and structural changes in rats in each group by HE.(5)WB was used to detect the expression of p38,p-p38,ERK,p-ERK,JNK and p-JNK protein.(6)Q-PCR was used to detect the expression of p38 mRNA,ERK mRNA and JNK mRNA.(7)The number and proportion of CD4~+and CD8~+cells in mesenteric lymph nodes of rats in each group were detected by flow cytometry.Results1 General observation of rats in each groupDuring the experiment,MG rats had obvious abdominal expansion,poor mental state,less activity,slow response,less diet and drinking water,sparse hair,dark coat color,weight loss,loose stool,yellow urine.The physiological state of SGHZ-H,SGHZ-M and SGHZ-L rats was better than that of MG,and the activity was increased.The diet,drinking water,hair,hair color,feces and activity were improved,especially SGHZ-H rats.2 Effect of SGHZ on hepatic function of rats groupAfter 8 weeks of modeling,there were significant differences in ALT,AST,TP,ALB,GLB and A/G in MG serum compared with BG(P<0.05).ALT,AST,TP,ALB,GLB and A/G in serum of CG,SGHZ-H,SGHZ-M and SGHZ-L were lower than those of MG.Compared with MG,the ALT,AST,TP,ALB,GLB and A/G in CG and SGHZ-H serum decreased significantly(P<0.05).3 Effects of SGHZ on HYP Content in Hepatic Tissue of Rats in Each GroupThe experimental results showed that compared with BG,the HYP content in hepatic tissue of MG rats was significantly increased after CCl4 modeling(P<0.05).Compared with MG,the content of HYP in hepatic tissue of CG,SGHZ-H,SGHZ-M and SGHZ-L decreased significantly(P<0.05),among which,the content of HYP in hepatic tissue of SGHZ-H decreased most obviously.4 Effect of SGHZ on hepatic fibrosis indexesELISA experiments showed that compared with BG,the levels of LN and Col IV in hepatic tissue of MG rats were significantly increased(P<0.05).Compared with MG,the level of LN in CG and SGHZ-H decreased significantly(P<0.05).The levels of Col IV in hepatic tissues of CG,SGHZ-H,SGHZ-M and SGHZ-L rats were significantly decreased(P<0.05).5 Effect of SGHZ on the changes of hepatic tissue structure of each groupThe results of HE showed that the hepatic color of BG was dark red,the texture was soft,the corners were sharp,and there were almost no lipid droplets.The structure of hepatic lobule was complete and clear,and the hepatic cord and hepatic sinus were radially arranged around the central vein.The hepatic tissue of MG was seriously damaged,the color became lighter,the texture became tougher,the corners became blunt,and the lipid droplets increased significantly.The connective tissue in the hepatic lobules proliferated diffusely,and even formed fibrous septa,which destroyed the normal hepatic lobules and formed pseudolobules.Degenerative and necrotic hepatocytes could be seen around the lobules and portal areas.Compared with MG,the proliferation,segmentation and lipid droplets of liver fibrous connective tissue in CG,SGHZ-H,SGHZ-M and SGHZ-L rats were reduced to varying degrees.Among them,the proliferation and segmentation of hepatic fibrous connective tissue in CG and SGHZ-H rats were significantly(P<0.05)reduced,lipid droplets were nearly hours,the outline of hepatic lobules was relatively clear,inflammation and hepatocyte degeneration and necrosis were reduced,and liver morphology,color and texture were close to normal.6 Effect of SGHZ on the expression of p38,p-p38,ERK,p-ERK,JNK and p-JNK protein in each groupWB results showed that compared with BG,the expression of p-p38,p-ERK,JNK and p-JNK protein in MG was significantly increased(P<0.05).Compared with MG,CG,SGHZ-H,SGHZ-M,and SGHZ-L did not significantly affect the expression levels of p38,ERK,and JNK proteins.Compared with MG,the expression of p-p38,p-ERK and p-JNK protein in CG and SGHZ-H was significantly(P<0.05)decreased.7 Effect of SGHZ on the expression of p38 mRNA,ERK mRNA and JNK mRNA in each groupQ-PCR results showed that compared with BG,the levels of p38 mRNA,ERK mRNA and JNK mRNA in hepatic tissue of MG rats were significantly increased(P<0.05).Compared with MG,the level of p38 mRNA in CG and SGHZ-H decreased significantly(P<0.05).The levels of ERK mRNA in CG,SGHZ-H and SGHZ-M were significantly decreased(P<0.05).The levels of JNK mRNA in CG and SGHZ-H decreased significantly(P<0.05).8 Effect of SGHZ on CD4~+and CD8~+cells in intestinal lymph nodes in each groupFlow Cytometry(FCM)results showed that compared with BG,the number and proportion of CD4~+cells and CD8~+cells in MG were significantly decreased(P<0.05).Compared with MG,the number and proportion of CD4~+cells and CD8~+cells in CG,SGHZ-H,SGHZ-M and SGHZ-L increased.Compared with MG,the number of CD4~+cells and CD8~+cells in CG and SGHZ-H was significantly increased,and the proportion of cells was basically consistent with BG group.Conclusion1.The SGHZ-H has a good effect on the treatment of hepatic fibrosis.2.SGHZ can reduce the levels of p38 mRNA,ERK mRNA and JNK mRNA in hepatic fibrosis model rats,and can reduce the expression levels of p-p38,p-ERK and p-JNK.It is proved that MAPK signaling pathway may be one of the signaling pathways of SGHZ in treating hepatic fibrosis.