Caloric Restriction Relieves Myocardial Ischemia/Reperfusion Injury By Regulating HIF-1?-mediated Autophagy And Inflammatory Pathways

Objective:Ischemic heart disease ranks first among the world's fatal diseases,and the incidence rate is increasing year by year.The worldwide attention has focused on the prevention and treatment of ischemic heart disease.At present,for the treatment of ischemic heart disease,early-stage revascularization is mostly used,but this method will lead to irreversible fatal myocardial injury—myocardial ischemia/reperfusion injury(MI/R).Therefore,the reduction of myocardial damage caused by MI/R has become the first problem to be solved urgently in clinical treatment.Many studies have found that up-regulation of hypoxia inducible factor-1?(HIF-1?)is an effective measure to protect MI/R,but HIF-1? has extremely high oxygen sensitivity and is very unstable.It is easily degraded and inactivated under normoxic conditions.In recent years,animal and clinical studies have found that caloric restriction(CR)plays a important role in extending lifespan by regulating the body's metabolism and immune function,and has protective effects on important organs such as liver,kidney,brain and ovary.Other studies have reported that CR can up-regulate the expression of HIF-1?,so it is speculated that the protective effect of CR may be related to HIF-1?.Moderate dieting can effectively activate autophagy,and the HIF-1?/Bnip3 pathway can mediate autophagy.However,the relationship between CR and HIF-1?/Bnip3 pathwayhas not been clearly reported.In addition,inflammatory response is an important pathological feature of myocardial ischemia/reperfusion injury.Previous studies in this laboratory confirmed that HIF-1? participates in the regulation of the TLR4/NF-?B pathway.Inhibition of the TLR4/NF-?B pathway may be another protection of the myocardium effective measures.However,the relationship between CR and TLR4/NF-?B pathway is not clear..Therefore,this article aims to explore the effects and possible mechanisms of caloric restriction on myocardial ischemia/reperfusion injury in mice from the perspective of autophagy and inflammatory pathways.Methods:1.Experimental grouping: 8-month-old male C57BL/6 mice were randomly divided into 5 groups:(1)Ad Libitum+Sham group(AL+Sham);(2)Ad Libitum+Myocardial Ischemia/Reperfusion group(AL+I/R);(3)Calorie Restriction+Sham group(CR+Sham);(4)Calorie Restriction+Myocardial Ischemia/Reperfusion group(CR+I/R);(5)Calorie Restriction +Myocardial Ischemia/Reperfusion + YC-1 group(CR+YC-1+I/R),n = 10 in each group.The mice in the Ad Libitum(AL)group were given free daily food intake,and the caloric restriction(CR)group mice were given 90% of the normal food intake,decreased by 10% every two weeks for 8 weeks,and then ligated the coronary arteries The left anterior descending branch was used to construct a mouse model of myocardial ischemia/reperfusion(I/R),and the inhibitor group was injected intravenously with inhibitor YC-1 before surgery.2.During the period of AL and CR,observed the living conditions of the mice,monitored the body weight,and recorded the physiological indicators of the mice.3.Used Even's blue/TTC double staining method to detect the area of myocardial infarction after operation.4.The pathological changes of myocardial tissue were observed with HE staining.5.Corresponding kit was used to measure the indicators of oxidative stress injury in the heart tissue,like superoxide dismutase(SOD)activity,glutathione peroxidase(GSH-Px)activity and malondialdehyde(MDA)content.6.Western blot was used to detect the protein expression levels of HIF-1?,Bnip3,Beclin-1,LC3,P62,TLR4,I???,and P65 in myocardial tissue.Results:1.Calaoric restriction can improve basic physiological indicators of mice: During the experiment,the basic state of the mice was continuously observed.It was found that the mice in the CR group had brighter hair,more flexible movements,and thinner bodies than the AL group.The body weight of the CR group mice showed that gradually decreased,while the weight of the AL group increased steadily.After caloric restriction for8 weeks,the CR group had significantly lower body weight,liver weight,and heart weight than the AL group(P <0.01).However,there was no statistically significant change in tibial length and blood glucose(Figure 1).2.CR reduced myocardial infarction area in mice: Staining results show that the white area in the CR+I/R group is significantly reduced compared to the AL+I/R group.Statistics show that the myocardial infarction area in the CR+I/R group was significantly reduced(P <0.01)(Figure 2).3.CR alleviated the pathological changes of myocardial tissue in mice after I/R:Myocardial tissue sections and HE staining results show that myocardial fibers in the AL+I/R group are disordered,with significant cell degeneration and necrosis.After CR intervention,the myocardium was relatively aligned and the pathological changes were slight(Figure 3).4.CR improved myocardial oxidative stress injury induced by I/R in mice: The test results showed that compared with the AL+I/R group,the SOD and GSH-Px activities of the CR+I/R treatment group increased by 34% and 30%,and the MDA content decreased by 37%(P <0.01)(Figure 4).5.CR promoted the expression of autophagy related protein in myocardial tissue after I/R:Compared with the AL+I/R group,the protein expression levels of Beclin-1 and LC3 II increased by 28% and 38%(P <0.01),while the expression level of P62 decreased by 57%(P <0.01)(Figure 5).6.CR promoted the activation of HIF-1? / Bnip3 pathway in myocardial tissue after I/R: Compared with the AL+I/R group,the expression of HIF-1? protein increased significantly after CR intervention(P <0.05),while the expression of Bnip3 protein increased significantly(P <0.05)(Figure 6).7.Inhibition of HIF-1? can reversed CR upregulation of autophagy in I/R myocardium:To determine whether HIF-1? is involved in the regulation of autophagy,we used its inhibitor YC-1 for further verification.It was found that YC-1 inhibited the expression of HIF-1?(P <0.05),followed by the expression of autophagy-related proteinsBeclin-1(P <0.05),Bnip3(P <0.01),and LC3II(P <0.01)were decreased,while the expression level of P62 increased significantly(P <0.01)(Figure 7).8.CR inhibited TLR4/NF-?B signaling pathway activation after myocardial I/R:TLR4 activates I??? to allow NF-?B to enter the nucleus and thus mediate inflammation.Western blot results showed that myocardial TLR4,I???,and NF-?B protein levels in the AL+I/R group were significantly increased,which were approximately 2.1times,2.65 times,and 1.63 times in the AL+Sham group(P <0.01).After CR-based ascending MI/R surgery,compared with the AL+I/R group,the protein levels of TLR4,I???,and NF-?B were reduced by 20%(P <0.05),21%(P <0.05),31%(P <0.01).(Figure8).9.CR activation of TLR4/NF-?B signaling pathway can be blocked by HIF-1?inhibitor:Compared with CR+I/R group,the expression levels of TLR4/NF-?B signaling pathway related proteins TLR4(P <0.01),NF-?B(P <0.01)and I???(P <0.05)were increased significantly after use YC-1(Figure 9).Conclusion:1.In addition to the eight-week caloric limit,in addition to not causing significant damage to the mice,it can also optimize the basic physiological indicators of the mice,reduce the area of myocardial infarction after I/R,reduce the pathological changes of myocardial tissue,and make myocardial ischemia.The oxidative stress injury caused by ischemia/reperfusion was significantly improved.2.Caloric restriction plays a role in relieving myocardial ischemia/reperfusion injury by up-regulating HIF-1?/Bnip3-mediated autophagy.3.The protective effect of caloric restriction on myocardial/ischemia-reperfusion injury is related to up-regulation of HIF-1? and inhibition of TLR4/NF-?B pathway activation.