| ObjectiveBy monitoring the changes of autophagy,the signal regulation mechanism of autophagy was analyzed,and the relationship between autophagy and combined anti-rat liver fibrosis was explored.After treatment with Taurine,EGCG and Genistein in rat hepatic stellate cells and liver fibrosis rats.We elucidate the molecular mechanism of the combination drugs to inhibit autophagy anti-rat liver fibrosis,and lay a certain pharmacological basis for the clinical application of drugs.Finally,providing new ideas for the treatment of liver fibrosis.Methods1.Inhibition of autophagy in rat hepatic stellate cells by combination therapy.(1)HSC-T6 was used as the research object,which was divided into the control group,the combination drug group,the autophagy inhibitor Bafilomycin-A1 group,and the drug autophagy inhibitor group.After the rat hepatic stellate cells were treated with a combination of drugs,the cell viability and survival rate were measured by CCK-8 method,and the cell death wasobserved.(2)Detection of autophagosomes and autophagy lysosomes by transmission electron microscopy and acridine orange fluorescence staining(3)Analysis of expression changes of autophagy marker proteins LC3 and Beclin-1by Western blot(4)mRFP-GFP-LC3 B lentivirus was transfected into HSC-T6 marker to track LC3 protein to monitor the dynamic changes of autophagy.2.Animal level verification study on autophagy induced by combination drug in rat hepatic stellate cells.(1)SD rats with CCl4-induced liver fibrosis model were divided into control group,CCl4 model group,combination drug group,autophagy inhibitor 3-MA group,combined drug +autophagy inhibitor 3-MA group.Blood was taken from the abdominal aorta,serum was separated,and serum ALT and AST activities were detected.HE staining and Masson staining were used to detect the degree of liver fibrosis in each group.(2)The state of autophagosomes in liver tissue cells was examined by transmission electron microscopy.(3)Western blot and IHC techniques were used to analyze the expression of autophagy-related proteins LC3 and Beclin-1in rat liver tissue,and the expression of autophagy induced by combination drug was verified at the animal level.3.Signal regulation mechanism of combined use of drugs to inhibit autophagy in rat hepatic stellate cells.(1)After the combination of hepatic stellate cells,the mRNA,protein expression and phosphorylation levels of key factors such as AMPK and mTOR in the AMPK-mTOR signaling pathway were analyzed by qPCR and Western blot,and cell proliferation and liver fibrosis related factors were detected.The expression of TGF-β1 and COL-I was analyzed to analyze the regulation of AMPK-mTOR signaling pathway in combination therapy against rat liver fibrosis.(2)After interfering with hepatic stellate cells with Compound C,an inhibitor of the AMPK-mTOR pathway,cellswere used to observe cell viability and survival rate,and the expression levels of LC3 and Beclin1 and the phosphorylation levels of AMPK and mTOR in the AMPK pathway were detected.The combination of AMPK-mTOR pathway was used to inhibit the autophagy regulation of rat hepatic stellate cells.Results1.Combined drug inhibits autophagy of rat hepatic stellate cells.(1)Transmission electron microscopy showed that the number of autophagosomes in activated HSCs was significantly higher than that in normal liver cells.After the combination drugs,the number of autophagosomes in HSCs was significantly reduced.(2)The survival rate of HSCs was determined by CCK-8method,and the combination drugs and autophagy inhibitor Bafilomycin-A1 inhibited the proliferation of HSCs.(3)In the acridine orange staining experiment,the red fluorescent region of the HSCs in the control group was exceptionally obvious,and the cell morphology was intact.After the combination drug and the autophagy inhibitor,the cells had only weak red fluorescence.(4)Western blot analysis showed that the expression levels of autophagy marker proteins LC3-II and Beclin1 were significantly decreased after treatment with combination drugs in HSCs.(5)Using the autophagy inhibitor Bafilomycin-A1 as a control,combination drugs and Bafilomycin-A1 intervene cells,the expression of LC3-II in the combination drug + Baf-A1 group was smaller than that in the Bafilomycin-A1 group,indicating that the combination drugs reduced autophagosomes.At the same time,the double fluorescence of mRFP-GFP-LC3 showed that compared with the control group,the other three groups showed obvious green fluorescence,and the combined drug + Baf-A1 group had the strongest green fluorescence,and the Merge image showed that the fluorescence of the combined drug group was biased yellow,which further demonstrated that the combination drugs inhibit the production of autophagosomes in HSCs.2.Combination drugs to reduce autophagy in rats with hepatic fibrosis.(1)After 8 weeks of treatment with CCl4,the AST and ALT levels were significantly increased,and after combination therapy,gradually returned to normal levels.(2)HE and Masson staining images showed that compared with the normal group,hepatocytes in the model group showed turbidity and degeneration,pseudo-lobule formation,connective tissue hyperplasia around the nodules,interlaced complex blue-green reticular streaks,After combination drugs treatment.,the fibrous tissue proliferation in the portal area was reduced,the fiber spacing was narrowed,and the formation of inflammatory fat vacuoles was significantly reduced.(3)Rat liver tissue was collected and observed under transmission electron microscope.Compared with normal rat liver tissue,many autophagosomes appeared in the liver of the liver fibrosis model group.After combination drugs treatment,the number of autophagosomes decreased.(4)Western blot and IHC results showed that the expression of LC3-II and Beclin1 was increased in rats with hepatic fibrosis compared with the normal group,and the expression of LC3-II and Beclin1 was significantly decreased after combined drug treatment.3.Combination drugs on hepatic stellate cell autophagy signaling pathway.(1)Compared with the control group,qPCR assay showed that the combination drug significantly inhibited the expression of LC3,Beclin1,AMPK and mTOR mRNA levels in HSCs.Among them,the expression of the above four genes in the combination drug + Compound C group was significantly lower than that in the Compound C group.(2)Western blot results showed that the expression of p-AMPK was decreased and the expression of p-mTOR wasincreased after the intervention of HSCs.Conclusion1.The anti-rat liver fibrosis effect of the combination drug is closely related to the autophagy of hepatic stellate cells.Many autophagosomes appear in the liver of activated hepatic stellate cells and liver fibrosis models,while the combination drugs reduce the number of autophagosomes and inhibits cell proliferation,and the combination drugs reduces the autophagy marker proteins LC3 and Beclin1.The partial mechanism of the combination drugs against rat liver fibrosis may be to achieve the purpose of anti-fibrosis by regulating the autophagy of the cells and affecting the proliferation of the cells.2.The combination drugs inhibits autophagy of hepatic stellate cells by blocking the AMPK-mTOR signaling pathway of hepatic stellate cells.After combined drugs intervention in hepatic stellate cells,the expression of genes,proteins and phosphorylated proteins of key factors LC3,Beclin1,AMPK and mTOR in the AMPK-mTOR signaling pathway were significantly reduced,liver fibrosis factors TGF-β1 and Col-1 also declined.We hypothesize that one of the molecular mechanisms of the combination of anti-hepatic fibrosis may be that the combination drug can inhibit the AMPK-mTOR signaling pathway in activated HSCs.The combined drug causes the autophagy process in the cell to be blocked,and the activated HSCs can not continue to proliferate without energy supply,thereby achieving anti-rat liver fibrosis. |
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