| Objective:Liver fibrosis is the pathological basis for the progression of chronic liver disease to cirrhosis,cancer of liver and even liver failure,which seriously endangers people’s health.Hepatocyte injury is the core factor in the occurrence and development of liver fibrosis.Fuzheng Huayu Decoction(FZHY)has been proven to be effective in the treatment of liver fibrosis,and F03 is an effective active ingredient in FZHY,which has the effect of protecting liver and lowering enzyme.Combined with our previous research,based on the role of hepatocyte in the occurrence and development of liver fibrosis,explore the partial effect mechanism of F03 against liver fibrosis.Methods:(1).Observe the effects of different doses of F03 on different animal models of liver fibrosis:① The 0.3%DMN solution was injected intraperitoneally three times a week for 5 weeks,to prepare a mouse model of liver fibrosis.From the 3rd week,three doses of F03 0.9,2.7,8.1 mg/kg and FZHY(4g/kg)were given intragastric intervention for 3 weeks,and the animals were sacrificed at the end of the 6th week.②A 15%CC14 olive oil mixed solution was injected intraperitoneally three times a week for 6 weeks to prepare a mouse model of liver fibrosis.From the 4rd week,three doses of F03 0.9,2.7,and 8.1 mg/kg were given intragastric intervention for 3 weeks.Sorafenib(7.2 mg/kg)was used as the positive control,and the samples were sacrificed at the end of the 6th week.③Mdr2-/-mice grow freely to the age of 9 weeks and are randomly divided into model group and F03 group according to their body weight.Mdr2+/+mice were used as the normal group.The F03 group was given F03(2.7mg/kg)intragastric intervention for 3 weeks,and samples were collected after treatment.Serum liver function indexes(AST,ALT,ALB,ALP,TBIL,DBIL)were detected after samples were collected from the above animals.H&E and SR staining were used to observe the pathological changes of liver tissue.The content of HYP was determined by kits to evaluate its efficacy.The expression of α-SMA,Col-Ⅰ,Col-Ⅳ in liver tissue were detected by qRT-PCR,Western blot and IHC.(2).Preliminary exploration of the partial effect mechanism of F03 intervention in different animal models of liver fibrosis:the model-building method of administration is the same as the above part.The expression of TNF-α,NLRP3,CD163,BAX/BCl-2,ECM1,TGF-β1,DDAH1,p-eNOS/eNOS,CD31,CD44,VEGF in liver tissue were detected by qRT-PCR and Western blot.The expressions of F4/80,CD163,ECM1,CD31 and CD44 in liver tissue were detected by immunohistochemistry.Apoptosis was detected by Tunel method.ADM A expression was detected by HPLC.The expression of NO was detected by kit.(3).①To study the protective effect of F03 on injured L02 cells and its partial mechanism:H2O2(100 μM)was used to stimulate the L02 cell injury model.Different doses of F03 were used to intervene normal L02 cells and injured L02 cells(stimulated by 100 μM H2O2 for 6 h).After 24 h,CCK8 was used to detect cell proliferation,EdU was used to detect cell proliferation,and flow cytometry was used to detect cell apoptosis.The mRNA and protein expression levels of DDAH1 were detected by qRT-PCR and Western blot.②To silence DDAH1 expression,use DDAH1 gene to interfere with lentivirus transfection of L02 cells.After F03 treatment,the EdU incorporation method was used to detect the number of cell proliferation,and qRT-PCR was used to detect the mRNA expression level of DDAH1.Results:①In the CCl4 liver fibrosis model,DMN liver fibrosis model and Mdr2-/spontaneous primary sclerosis cholangitis mouse model,compared with the normal group,the serum biochemical,pathological staining and Hyp content of the model group were significantly changed,which was consistent with the pathological characteristics of liver fibrosis.Compared with model group,serum biochemical changes(AST,ALT,ALB,ALP,TBIL,DBIL),histomathology and HYP content in F03 groups with different doses were improved to varying degrees.In the CCl4 and DMN liver fibrosis mouse models and Mdr2-/-spontaneous primary sclerosis cholangitis mouse model,after F03 intervention,F03 reduced the expressions of α-SMA,Col-Ⅰ,Col-Ⅳ.②In the CCl4 and DMN liver fibrosis mouse models,after F03 intervention,the three dose groups of F03 reduced the expressions of liver inflammation indexes F4/80,TNF-α,and NLRP3;increasing the expression of ECM1 and DDAH1,reduces the ratio of liver tissue cell apoptosis indicators BAX/BCl-2,reduces the expression of ADMA,and increases the content of NO,decrease the expression of liver angiogenesis index CD31 and increase the expression of CD44 in the liver tissues of model mice to different degrees.③In the L02 injury model induced by H2O2,F03 could promote cell proliferation to varying degrees in the range of 0.01-10 μM,inhibit hepatocyte apoptosis to varying degrees in the range of 0.01-10 μM,and reduce the expression of IL-6 gene in the range of 0.01-1 μM,increase the expression of DDAH1 gene at a dose of 0.01μM,and increase the expression of DDAH1 protein to varying degrees within the range of 0.01-1 μM.Increasing DDAH1 gene expression at 0.01μM dose and increasing DDAH1 protein expression in the range of 0.01 to 1μM could increase DDAH1 protein expression in different degrees.After the loss of DDAH1 in L02 cells,the proliferation ability of hepatocytes decreases,and the proliferation ability of liver cells increased after F03 treatment.However,the ability of F03 to promote the proliferation of hepatocytes is significantly weaker than the ability of F03 to promote the proliferation of L02 cells transfected with negative viruses.This part of the results suggests that F03 can promote the proliferation of hepatocytes by upregulating the expression of DDAH1,but F03 may have other targets in promoting the proliferation of damaged hepatocytes.Conclusion:① F03 could play a good role in the anti-hepatic fibrosis by improving liver function,reducing collagen deposition,alleviating inflammatory response,resisting liver cell injury,inhibiting sinusoidal capillarization,etc.②F03 may degrade ADMA in liver tissue by increasing the expression of DDAH1 in liver cells,thus exerting the effects of anti-liver cell injury and promoting liver cell proliferation;F03 increased the expression of DDAH1 in liver cells and promoted the synthesis of NO after degrading ADMA in liver tissue,thereby inhibiting sinusoidal capillarization. |
PDF Full Text Request