| Objective: To investigate the regulatory mechanism of mitochondrial autophagy in rats with acute liver failure mediated by PINK1/Parkin pathway in combination with bone marrow mesenchymal stem cells.Methods: The mechanism of mitochondrial autophagy in rats with acute liver failure was investigated by detecting biochemical indices,pathological tissues,mitochondrial structure,mitochondrial membrane potential,PINK1(serine/threonine kinase)/Parkin(E3 ubiquitin ligase)and other related proteins in each group.1.Preparation of bone marrow mesenchymal stem cells(BMSCs):cultured BMSCs were identified by flow cytometry The cultured BMSCs were identified by flow cytometry and then used for the intervention treatment in the group of Yin Chen Si Ling combined with BMSCs and the group of BMSCs.2.Procedure: 60 SD male rats were randomly divided equally into group A(normal group),group B(Yin Chen Si combined with BMSCs group),group C(Yin Chen Si Ling group),group D(model group),group E(compound glycyrrhizin group)and group F(BMSCs group).After intraperitoneal injection of thioacetamide(TAA)in each intervention group,groups A and D were given0.9% sodium chloride solution(0.9% Nacl)by gavage(once/day in the morning and evening);group B was given Inocerin granules solution by gavage(once/day in the morning and evening)and BMSCs suspension by tail vein injection;group C was given Inocerin granules solution by gavage(once/day in the morning and evening);group E was given compound glycyrrhizin tablets solution by gavage(once/day in the morning and evening)(Group F: Gavage with 0.9% Nacl solution(once/day in the morning and evening)and tail vein injection of BMSCs suspension;all groups except E and F were injected with0.9% Nacl solution in tail vein.3.Detection: Rats in each group were anesthetized 72 h after intervention,blood was collected from the abdominal aorta and specimens were collected.The indexes of liver function and coagulation were detected;HE staining was observed for liver histopathological sections;transmission electron microscopy was used to observe the mitochondrial structure and autophagic vesicles in liver tissue: flow cytometry was used to detect the level of mitochondrial membrane potential(△Ψm)in liver tissue;The Western Blot method detected the expression of related autophagic proteins PINK1/Parkin,P62(linkerprotein),LC3(microtubule-related protein 1A/1B-light chain 3)and other proteins.Results: 1.Identification by flow cytometry showed low expression of CD34(0.86%)and CD45(0.14%)and high expression of CD90(99.56%)and CD29(97.95%);2.The results of liver function and coagulation function of rats showed that the indexes of each intervention group were improved compared with those of group D,and the indexes of group B were more obvious(P<0.05);3.The morphological observation of liver pathology of rats showed that the liver tissue of group D was infiltrated by a large number of inflammatory and lymphocytes,and the hepatocytes were diffusely necrotic,and the structure of hepatocytes in the necrotic area was vaguely disintegrated and the structure of liver lobules was disturbed,suggesting In group B,the degree of damage was smaller,and new hepatocytes were seen;4.Transmission electron microscopy results: most mitochondria in the cytoplasm of hepatocytes in group D had swollen,dissolved or even disappeared,and apoptosis had occurred;in group B,the mitochondria were only mildly swollen,and the structure still existed,and the degree of damage was smaller;5.Detection results of liver mitochondrial membrane potential: group D suggested serious damage to membrane potential(P<0.05);The positive percentage of each intervention group did not increase,suggesting that the degree of mitochondrial apoptosis was mild,among which the membrane potential of group B was the most stable,indicating that Yin Chen Si Ling granules combined with BMSCs could stabilize the membrane potential of liver mitochondria and reduce the degree of apoptosis;6.The results of protein blotting assay: PINK1,Parkin,LC3,p62 protein expression levels: compared with group D,the expression levels of PINK1,Parkin,LC3 protein expression was significantly increased in each intervention group,among which the increase was obvious in group B(P<0.05),compared with group D,p62 protein expression was decreased in groups B and C(P<0.05),with the most obvious in group B(P<0.05).Conclusion: 1.The combination of Yin Chen Si Ling granules and BMSCs can effectively reduce liver function and coagulation-related indexes,and reduce the degree of liver histopathological damage;2.The intervention of Yin Chen Si Ling granules and BMSCs can improve mitochondrial function and activity by stabilizing membrane potential,inhibit mitochondrial damage,and then affect mitochondrial autophagy,and may activate the PINK1/ Parkin signaling pathway to upregulate autophagy PINK1/ Parkin signaling pathway may be activated to upregulate autophagy LC3 and downregulate P62,effectively promoting hepatic mitochondrial autophagy,clearing damaged mitochondria,and maintaining normal mitochondrial function,thus protecting liver function and improving liver failure. |
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