Exploring The Role And Mechanism Of Mitochondrial Autophagy In The Inhibition Of Hepatic Fibrosis By Soft Liver Fibrotic Particles Based On MiR-135a/FOXO1/PINK1 Pathway

Objective:To verify the effect and mechanism of Rougan Huaxian particles in regulating mitochondrial autophagy on carbon tetrachloride（CCl₄）combined with peanut oil induced liver fibrosis in rats and hepatic stellate cells based on the miR-135a/FOXO1/PINK1 pathway through in vivo and in vitro experiments.Methods:1.In the in vivo experimental research:（1）Rats were randomly divided into normal control group,pathological model group,low,medium and high dose group of Rougan Huaxian Granules,etc.The pathological model group and the low,medium and high dose group of Rougan Huaxian Granules were injected subcutaneously with 40%CCl₄oil in combination with peanut oil suspension to establish an animal model of liver fibrosis in rats,and Rougan Huaxian Granules were given to the rats by gavage starting from the 5th week,and the granules were given to the rats by gavage three times per week,for 8 weeks consecutively.After 8 consecutive weeks,the pathological and morphological changes of the liver tissue of rats in each group were compared and analyzed by HE staining,Masson staining and immunohistochemistry,and the expression ofα-smooth muscle actin（α-SMA）and E-Cadherin was detected by Real-time PCR,Western Blot and other methods.（2）The rats were randomly and equally divided into normal control group,pathological model group,saline control group,soft liver fibrotic granule low,medium and high dose group,soft liver fibrotic granule medium dose+3-MA group,and the liver fibrosis model was prepared by subcutaneous injection of CCl₄oil combined with peanut oil suspension,and gavage of soft liver fibrotic granule low,medium and high dose（2g/kg,4g/kg,8g/kg）and 3-MA（15mg/kg）.Real-time PCR,Western Blot and ELISA were used to detect miR-135a,FOXO1,PINK1,Parkin,LC3Ⅱ,Smad2,p-Smad2,TGF-β1,NF-κB p65,p-NF-κB p65,α-SMA,CollagenⅠ,CollagenⅢexpression and ROS,CollagenⅢexpression and ROS content.2.In the in vitro experimental research:The experiment was divided into two parts:（1）HSC-T6 cells were divided into 6 groups:control,H₂O₂,miR-135a-NC,miR-135a-mimic,H₂O₂+miR-135a-inhibitor-NC,H₂O₂+miR-135a-inhibitor;（2）HSC-T6 cells were divided into 6 groups:blank group HSC-T6 cells were divided into 6groups:blank group,blank serum group,H2O2+blank serum group,H₂O₂+drug-containingserumgroup,H₂O₂+drug-containing serum+miR-135a-NC group,H₂O₂+drug-containing serum+miR-135a-mimic group.The miR-135a,FOXO1,PINK1,Parkin,LC3Ⅱ,Smad2,p-Smad2,TGF-β1,NF-κB p65,p-NF-κB p65,andα-SMA were detected by molecular biotechnology techniques,such as Real-time PCR,Western Blot,ELISA with flow cytometry,respectively,CollagenⅠ,CollagenⅢ,TNF-αexpression and mitochondrial membrane potential,ROS generation.Results:1.In the in vivo experimental research:（1）the lobular structure in the liver tissue of rats in the liver fibrosis pathological model group has been mutilated,and there are many abnormal proliferation of liver fibrous tissues,and the collagen fibers formed by the proliferation converge to form a dense fibrous spacer,which turns into a pseudo-lobular structure,and a large number of steatotic hepatocytes with different shapes and sizes appear inside the pseudo-lobular structure,and there is a significant diffuse infiltration of inflammatory cells.Compared with the pathological model group,the degree of destruction of the liver lobular structure was significantly reduced in the low,medium and high dose groups of Soft Liver Fibroblast Granules,with different degrees of proliferation of collagen fibrous tissue concentrated in the central vein to the confluence area,the fibrous intervals were relatively loose,and there was a small amount of inflammatory cell infiltration,with a lesser degree of infiltration than that in the pathological model group.（2）Compared with the pathological model group,the expression level ofα-SMA in the low,medium and high dose groups of Soft Liver Fibroblast Granules was significantly lower（p<0.01）,and the expression level of E-Cadherin was significantly higher in all groups（p<0.01）.（3）miR-135a,α-SMA,CollagenⅠ,CollagenⅢ,p-Smad2,TGF-β1,p-NF-κB p65,TNF-αexpression and ROS content were significantly up-regulated in the liver fibrosis pathological model group（p<0.05）;the expression of FOXO1,PINK1,Parkin and LC3Ⅱwas significantly down-regulated（p<0.05）.There was no significant difference in the expression effect between the physiological saline gavage control group and the pathological model group（p>0.05）;the low,medium and high dosage groups of Soft Liver Fibroblast Granules were able to significantly inhibit the expression of miR-135a,α-SMA,CollagenⅠ,CollagenⅢ,p-Smad2,TGF-β1,p-NF-κB p65,TNF-α,and ROS generation;Up-regulation of FOXO1,PINK1,Parkin,LC3Ⅱexpression（p<0.05）.The efficacy of the middle-dose group of Soft Liver Fiber Granules was significantly better than that of the low-dose group（p<0.05）,while there was no significant difference between the efficacy of the middle-and high-dose groups（p>0.05）.The mitochondrial autophagy inhibitor-added Rougan Huaxian Granules medium-dose+3-MA group could significantly inhibit the efficacy of Rougan Huaxian Granules（p<0.05）,and prevent the up-regulation effect of Rougan Huaxian Granules on the expression of FOXO1,PINK1,Parkin and LC3Ⅱ（p<0.05）.2.In terms of in vitro experimental studies:（1）Administration of H₂O₂and overexpression of miR-135a significantly up-regulated HSC-T6 miR-135a,α-SMA,CollagenⅠ,CollagenⅢ,p-Smad2,TGF-β1,p-NF-κB p65,and TNF-αexpression and ROS generation（p<0.01）;down-regulated FOXO1,PINK1,Parkin,LC3 II expression and mitochondrial membrane potential（p<0.01）.（2）Administration of Rougan Huaxian Granules and inhibition of miR-135a expression significantly down-regulated HSC-T6 miR-135a,α-SMA,CollagenⅠ,CollagenⅢ,p-Smad2,TGF-β1,p-NF-κB p65,and TNF-αexpression and ROS generation（p<0.01）;up-regulated FOXO1,PINK1,Parkin,LC3Ⅱexpression and mitochondrial membrane potential（p<0.01）.（3）Administration of soft liver fibroblast particles with simultaneous overexpression of miR-135a inhibited the effect of soft liver fibroblast particles on HSC-T6（p<0.01）.Conclusion（s）:1.Ruogan Huaxian Granules can affect mitochondrial autophagy and improve liver fibrosis by regulating the miR-135a/FOXO1/PINK1 pathway.2.Ruogan Huaxian Granules can inhibit liver fibrosis in rats,and its mechanism may be related to the downregulation of Ruogan Huaxian Granulesα-The expression of SMA protein,upregulation of E-cadherin protein expression,inhibition of epithelial mesenchymal transition（EMT）,regulation of lipid metabolism,and reduction of collagen synthesis in the body are related;At the same time,it inhibits the expression of miR-135a and activates the miR-135a/FOXO1/PINK1 pathway,thereby inducing mitochondrial autophagy,reducing oxidative stress effects,and inhibiting TGF-β1/Smad2 activation is related.3.Ruogan Huaxian Granules can inhibit HSC-T6 activation,and its mechanism may be related to the inhibition of miR-135a expression by Ruogan Huaxian Granules,activation of the miR-135a/FOXO1/PINK1pathway,thereby promoting mitochondrial autophagy and inhibiting ROS generation.