IL37 Overexpression Inhibits Autophagy And Apoptosis Induced By Hepatic Ischemia Reperfusion Injury Via Modulating AMPK/mTOR/ULLK1 Signaling Pathways

Purpose:Liver ischemia/reperfusion(ischemia reperfusion,IR)injury is a key problem in hepatobiliary surgery.Autophagy and apoptosis have been shown to be related to liver IR damage.IL37 is an effective anti-inflammatory cytokine,which has a regulatory role in a variety of inflammatory immune responses.It is known that IL37 has a protective effect against ischemiareperfusion injury in the liver.However,the link between autophagy and IL37 in liver IR remains unknown.Therefore,in this study,the aim was to investigate the effect of IL37 on autophagy during hepatic ischemiareperfusion injury and hepatocyte hypoxia-reoxygenation injury and its possible protective mechanism through in vivo and in vitro experiments.Methods:1.Construct a 70% warm ischemia-reperfusion injury model in C57BL/6 mice,each group is ischemic for 1 hour,and then reperfused for 6,12 and 24 hours respectively,Western blot detection of autophagy-related protein LC3 B Ⅱ,Beclin1 expression,immunohistochemistry Detect the level of LC3 Ⅱ protein,H&E staining to observe the degree of tissue damage,and immunofluorescence to observe the apoptosis level of Cleaved-Caspase3.Then according to the results,the time point with the highest autophagy level was selected for follow-up tests.2.According to the above time points,AAV8 overexpressing IL37 was injected through the tail vein 14 days before surgery,and IR surgery was performed 14 days later.The experimental groups were: control group,sham group,ischemia-reperfusion(ischemia reperfusion,IR)group,adenoassociated virus negative control(AAV8-NC+IR)+ IR group,and overexpression of IL37 adeno-associated virus(AAV-IL37+IR)group.RTq PCR,Western blot and laser confocal detection of AAV transfection efficiency;Western blot detection of autophagy-related protein LC3 B Ⅱ and Beclin1 expression;microplate method to detect ALT and AST profit levels;transmission electron microscope observation of autophagosome formation;H&E staining Observe the level of tissue damage;TUNEL method to detect apoptosis.To evaluate the protective effect of IL37 on liver ischemiareperfusion injury in vivo.3.Construct a hypoxia reoxygenation(HR)model of HL7702 cells in vitro.First,the cells were hypoxic for 0,1,2,4,6,and 8 hours.The levels of autophagy proteins LC3 B Ⅱ/Ⅰ,Beclin1 and p62 were observed by Western blot,and the highest autophagy time point was used as the subsequent test point.Then the cells were given 0,1,2,4,6,and 8 hours of reoxygenation at different times.Western blot technology was used to detect the expression of autophagy proteins LC3 B Ⅱ/Ⅰ,Beclin1 and p62,and the changes in apoptosis-related proteins Cleaved Caspase3,Bcl2 and Bax.The phosphorylation activation levels of pathway proteins AMPK,mTOR and ULK1;TUNEL method was used to detect cell apoptosis.4.Treatment of HL7702 cells with different concentrations of recombinant IL37 protein,Western blot technology to detect the expression of LC3 B Ⅱ/Ⅰ and Beclin1 protein,Celltiter kit to detect cell viability.Then combined with Rap and 3-MA to treat the cells together,Western blot detection of LC3 B Ⅱ/Ⅰ and Beclin1,GFP-mRFP-LC3 lentivirus to observe the cell autophagy flow.5.Overexpress IL37 lentivirus to infect HL7702 cells,RT-q PCR,Western blot technology to detect IL37 expression,laser confocal observation of green fluorescence intensity.Then,by applying a stable strain of lentivirus,performing HR,Western blot technology to detect the expression of autophagy proteins LC3 B Ⅱ/Ⅰ,Bcelin1 and p62,transmission electron microscopy to observe the formation of autophagosomes,GFPmRFP-LC3 fluorescence double label to observe the intensity of autophagy;Western blot technology was used to detect the changes of apoptosis-related proteins Cleaved Caspase3,Bcl2 and Bax,and TUNEL staining to observe cell apoptosis.6.Use AMPK inhibitor Compound C to treat HL7702 cells,Western blot technology to detect AMPK and Cleaved Caspase3 protein expression;Compound C combined with lentiviral stable strain to treat HR HL7702 cells,Western blot technology to detect AMPK,mTOR,ULK1 phosphorylation activation levels,Autophagy-related proteins LC3 B Ⅱ/Ⅰ,Beclin1 and p62 protein expression,Cleaved Caspase3,Bax and Bcl2 protein expression.Results:1.Compared with the sham group,the protein expression levels of LC3 B Ⅱ/Ⅰ and Beclin1 in the I1R6,I1R12 and I1R24 groups increased most significantly when I1R12,and the difference was statistically significant(p<0.01);LC3 immunohistochemistry and Western blot The results were consistent(p<0.01);meanwhile,liver tissue damage gradually aggravated with the reperfusion time(p<0.01);the expression of ALT and AST at each time point also gradually aggravated with the reperfusion time(p<0.01,p<0.001);liver tissue apoptosis gradually increases with the prolongation of reperfusion time.2.Compared with the control group,the protein and mRNA expression levels of IL37 in the AAV-IL37 group were significantly higher than those in the control group and the AAV-NC group(p<0.001);compared with the sham group,the IR group and the AAV-NC+IR group The expression levels of LC3 B Ⅱ/Ⅰ(p<0.01)and Beclin1(p<0.01)were significantly increased,while AAV-IL37+IR could effectively inhibit the expression of autophagy protein,and the difference was statistically significant(p<0.01).H&E staining showed that IR(p<0.001 The degree of liver damage in the)group and AAV-NC+IR(p<0.001)group was significantly higher than that of the control group and sham group.AAV-IL37(p<0.01)can significantly improve the degree of tissue damage.The highest rate of hepatocyte apoptosis was seen in the IR(p<0.001)group and the AAV-NC+IR(p<0.001)group.AAVIL37(p<0.01)can effectively reduce the rate of hepatocyte apoptosis.3.Compared with the control group,LC3 B Ⅱ/Ⅰ(p<0.01)and Beclin1(p<0.01)protein expression levels of HL7702 were highest when hypoxia for 2 h,and then declined,p62(p<0.01)expression levels were the lowest,and then Increase,so take this as the research time point.According to the selected hypoxia time points,and then given different time of reoxygenation treatment,compared with the control group,the expression level of LC3BⅡ/Ⅰ(p<0.01)and Beclin1(p<0.01)was the highest in H2R6,and then decreased,p62(p<0.01)The expression level is lowest in H2R6.At each time point,the apoptotic proteins Cleaved Caspase3 and Bax gradually increased(p<0.01),and the expression of Bcl2 gradually decreased(p<0.05).The TUNEL results are consistent with the Western blot results(p<0.01).At each time point,the expression of signaling pathway proteins p-AMPK and p-ULK1(s317)gradually increased and reached the highest at H2R6(p<0.01),while pmTOR gradually decreased,and the lowest at H2R6(p<0.01).4.Compared with the control group,different concentrations of rh IL37(0,50,100,200,300 ng/ml)had a dose-dependent inhibitory effect on autophagy-related proteins LC3 B Ⅱ/Ⅰ and Beclin1(p<0.05,p<0.01),the protein expression of p62 gradually increased(p<0.01).At the same time,compared with the control group,300ng/ml recombinant IL37 can promote cell proliferation most at the same time(p<0.01).Compared with the control group,the expression of LC3 B Ⅱ/Ⅰ(p<0.05)and Beclin1(p<0.05)in the HR group was significantly increased.rh IL37 can effectively inhibit the expression of LC3 B Ⅱ/Ⅰ(p<0.05)and Beclin1(p<0.05).,Rap has the effect of synergistically promoting autophagy caused by HR.IL37 can partially weaken the expression of LC3 B Ⅱ/Ⅰ(p<0.05)and Beclin1(p<0.05)caused by Rap.3MA-can partially weaken the expression of LC3 B Ⅱ/Ⅰ caused by HR.(p<0.05),Beclin1(p<0.05)expression increased,and compared with the3-MA group alone,rh IL37 combined with 3-MA can inhibit LC3BⅡ/Ⅰ(p<0.05),Beclin1(p<0.05)Expression,the difference was statistically significant(p<0.05).The results of GFP-mRFP-LC3 showed that: Compared with the control group,the induction of autophagosomes(yellow dots)in the HR group was increased.Compared with the HR group,the induction of the rapamycin group was further enhanced.This enhancement effect can be suppressed in the rapamycin group.In addition,only compared with the HR group,the use of 3-MA can reduce the number of autophagosomes formed(yellow dots),IL37 enhanced the inhibitory effect of 3-MA,all results are statistically significant(p<0.05).5.Compared with the LV-NC group,the IL37 mRNA and protein expression levels in the LV-IL37 group were significantly increased(p<0.01,p<0.001).Compared with the NC group,the expression of LC3 B Ⅱ/Ⅰ and Beclin1 in the NC+HR group was significantly increased,while the expression of p62 was decreased(p<0.05,p<0.01).In the LV-IL37+HR group,LC3 B Ⅱ/Ⅰ and Beclin1 protein levels were significantly suppressed,while p62 protein expression was increased(p<0.05).Compared with the NC group,the number of autophagosomes in the NC+HR group was significantly increased(p<0.001),and the number of autophagosomes in the LV-IL37+HR group was significantly reduced(p<0.01).The results of GFPmRFP-LC3 showed that in LV-NC+HR,the green and red dots were significantly more than those in the LV-NC(p<0.001)group,indicating that autophagosomes were formed,and autophagolysosomes were formed,and autophagy Smooth circulation;IL37 can effectively inhibit the formation of autophagosomes and autophagolysosomes caused by HR(p<0.01),and the autophagy flow is partially unobstructed,which is consistent with the expected results.6.Compared with the LV-NC+HR group,LV-IL37 can effectively increase the cell proliferation rate(p<0.01).In the LV-NC+HR group,the expression of apoptosis protein Cleaved Caspase3 and Bax was significantly higher than that of the LV-NC group(p<0.05,p<0.01),while the expression of Bcl2 was reduced(p<0.05),and LV-IL37 could be significantly Decrease Cleaved Caspase3,Bax,and increase the protein expression of Bcl2d(p<0.05,p<0.01).TUNEL results showed that in LV-NC+HR,the apoptosis rate was significantly increased(p<0.01),and IL37 could effectively inhibit hepatocyte apoptosis(p<0.05).7.Different concentrations of Compound C(0,5,10,20,40μM)can effectively inhibit the expression of p-AMPK(p<0.01,p<0.001)and Cleaved Caspase3(p<0.05).Compared with the LV-NC+HR group,in the LV-IL37+HR group and the LV-NC+HR+Compound C group,the expression of LC3BⅡ/Ⅰ,Beclin1,Cleaved Caspase3,and Bax protein was significantly inhibited(p<0.05,p <0.01,p<0.001),the expression of p62 and Bcl2 increased(p<0.05).In the LV-IL37+HR+Compound C group,we observed that the protein expression of autophagy-related proteins LC3 B Ⅱ/Ⅰ,Beclin1,Cleaved Caspase3 was lower than that of the experimental group using IL37 and Compound C alone,while Bax,p62 and Bcl2 The expression of the protein has not been statistically significant.In addition,Western blot results showed that,compared with the LV-NC+HR group,in the LVIL37+HR group and LV-NC+HR+Compound C group,the protein expression of p-AMPK and p-ULK1(s317)was significantly affected.Inhibition(p<0.05,p<0.01),the expression of p-mTOR,p-ULK1(s757)increased(p<0.05,p<0.01).In the LV-IL37+HR+Compound C group,we observed that the protein expression of p-AMPK and p-ULK1(s317)was lower than that of the experimental group that only applied IL37 and Compound C(p<0.05,p<0.01),and The protein expressions of p-mTOR and p-ULK1(s757)were higher than those of the experimental group using IL37 and Compound C alone(p<0.05,p<0.01).IL37 can inhibit autophagy and apoptosis through this pathway and play a role in reducing liver ischemiareperfusion injury.Conclusion:1.The expression of autophagy and apoptosis-related proteins gradually increases during liver ischemia-reperfusion injury,and is positively correlated with many indicators such as ALT and AST.2.Successfully constructed an animal model of AAV8 overexpressing IL37,and conducted a preliminary study on the IR model to confirm that IL37 can inhibit IR-induced autophagy and apoptosis in vivo and reduce liver IR damage.3.Both recombinant human IL37 and IL37 overexpression lentiviral stable strains can effectively inhibit the autophagy of HL7702 hepatocytes caused by HR,and LV-IL37 can effectively inhibit autophagy while reducing apoptosis and promoting cell proliferation.4.IL37 reduces the effects of hypoxia-reoxygenation injury of liver cells and liver ischemia-reperfusion injury,which may be achieved by regulating AMPK/mTOR/ULK1 signaling pathway.