A Study Of MiR-101 Regulating MTOR Signaling Pathway Mediated Autophagy In Hepatic Ischemia/Reperfusion Injury

Objective:Liver ischemia/reperfusion injury(LIRI)is a common complication in liver transplantation and liver surgery,which has a significant influence on the effect of liver surgery and the recovery of liver function of postoperative patients.For many years,how to reduce liver damage,to protect the liver as much as possible and to improve the prognosis of patients has been a hot topic and difficulty in liver surgery.In recent years,the relevant research on the specific mechanism of LIRI has been increasing,but it has not been fully elucidated.The aim of this study was to explore the role of miR-101 mediated autophagy in mouse LIRI by investigating the expression of miR-101 in LIRI and the changes of cell autophagy activity,and to reveal the potential function and mechanism of miR-101 mediated autophagy regulation in LIRI,in order to provide a new idea for clinical treatment and research of LIRI.Methods:The model of hepatic segmental(70%hepatic lobe clipping)ischemia and reperfusion injury was established.The serum biochemical parameters were used to detect the changes of serum transaminase(ALT,AST)content.HE staining to observe the mice liver tissue pathology damage change.TUNEL staining to observe the mouse liver cell apoptosis.The proliferation and apoptosis of liver cells were observed by immunohistochemistry.Changes of miR-101,AMPK mRNA and mTOR mRNA were detected by qRT-PCR.Western Blot was used to detect the expression of AMPK,mTOR,p-mTOR,P62,Caspase-3 and LC3.Mice were pre-administered with miR-101 agomir/antagomir and autophagy inhibitor 3-MA to further validate the role of miR-101 in LIRI.AML12 cells were transfected with miR-101 mimics/inhibitor,and a hypoxia/reoxygenation(H/R)model was established to simulate LIRI in vitro.The autophagy in AML 12 cells was observed by confocal microscopy and immunofluorescence.The changes of miR-101,AMPK mRNA and mTOR mRNA were detected by qRT-PCR.The expressions of AMPK,mTOR,p-mTOR,P62,Caspase-3 and LC3 were detected by Western Blot.The model of H/R was established by pre-treatment of AML12 cells with autophagy activator rapamycin and autophagy inhibitor 3-MA to further clarify the effect of autophagy on the viability of AML 12 cells induced by H/R treatment and the effect of miR-101 on LIRI by affecting autophagy expression.Results:Serum AST and ALT levels were increased after hepatic ischemia/reperfusion in mice,and reached the peak at 12 h after reperfusion.Pathological examination revealed that edema swelling、steatosis、vacuolization、flaky necrosis、neutrophil infiltration and hepatic sinus congestion were observed in the liver tissue after reperfusion.These pathological changes were gradually aggravated with the extension of reperfusion time,and the damage was most serious at 12h after reperfusion.Immunohistochemistry and TUNEL detection of liver cell proliferation and apoptosis after LIRI showed that apoptosis increased significantly after reperfusion and cell proliferation decreased significantly.The results of qRT-PCR showed that the expression of miR-101 and mTOR decreased gradually after reperfusion,while the expression of AMPK increased continuously.Western Blot showed that the levels of AMPK,Caspase-3 and LC3 protein gradually increased with the time of ischemia/reperfusion.In contrast,the expression of mTOR,p-mTOR,and P62 decreased with the prolongation of ischemia/reperfusion time.After overexpression of miR-101 in vivo,the pathological damage and apoptosis caused by ischemia/reperfusion were significantly reduced,and the mTOR gene and protein levels increased,while the AMPK gene and protein levels decreased significantly.This reduction of liver damage by miR-101 was more pronounced after the addition of the autophagy inhibitor 3-MA.Normal mouse hepatocytes AML 12 were cultured in vitro and a hypoxia/reoxygenation model was established to simulate LIRI in vitro.The study found that the increase in autophagy level was positively correlated with the degree of hepatocyte injury.When miR-101 agomir was used,autophagy level was inhibited and hepatocyte injury was significantly reduced.Confocal results showed that after overexpression of miR-101 the number of phagosomes and autophagic lysosomes was significantly reduced,and immunofluorescence suggested that P62 protein accumulation could not be degraded;MTT results showed that cell survival rate decreased during hypoxia/reoxygenation.The cell survival rate was significantly reduced when treated with rapamycin and the combination of rapamycin and miR-101 inhibitors.In contra’st,the survival rate of cells treated with autophagy inhibitor 3-MA was significantly improved.The results of qRT-PCR showed that overexpression of miR-101 promoted the expression of mTOR and inhibited the expression of AMPK after hypoxia/reoxygenation.Western Blot results showed that the expression levels of AMPK,Caspase-3 and LC3 were significantly decreased after overexpression of miR-101,and the level of mTOR protein was significantly increased.When rapamycin and miR-101 inhibitor were used,the expression levels of mTOR,p-mTOR and P62 decreased,Caspase-3 and LC3 expression increased,and autophagy increased.C onclusion:The expression of miR-101 was significantly decreased in the liver after ischemia/reperfusioin injury,the-expression of AMPK was significantly increased,and the expression of mTOR was inhibited,and the expression of autophagy was up-regulated,whieh aggravated liver injury.Therefore,miR-101 can inhibit the expression of autophagy and thus reduce the LIRI of mice and promote the recovery of damaged liver tissue,In mouse LIRI,miR-101 can attenuate the damage of autophagy to liver tissue by regulating the AMPK-mTOR signaling pathway.