| Background:The prevalence and mortality of cardiovascular disease remains high all the year around.At present,the treatment of patients with acute ischemic heart disease mainly adopts ischemic regional reperfusion,which is an important measure to improve the success rate of myocardial infarction treatment and the quality of patients’ life.However,a large number of studies have found that simple reperfusion therapy can aggravate the existing ischemic myocardial injury,and even threat the lives of patients,namely myocardial ischemia/reperfusion injury(MI/RI).The mechanism of MI/RI is complex.Studies suggest that excessive oxygen free radical generation,neutrophil infiltration,mitochondrial structure and function damage,myocardial cell necrosis and apoptosis are involved in it.In addition,myocardial ischemia/reperfusion process will trigger a large number of inflammatory reactions,affecting the repair and prognosis of ischemic myocardium.However,there is no effective way to alleviate the injury.Pyroptosis is a programmed form of cell death characterized by activation of NLRP3 inflammasome,activation of caspase-1,and formation of cell membrane pores,leading to the release of IL-1β and IL-18 in the cytoplasm.It has been reported that pyroptosis is involved in the occurrence and development of a variety of cardiovascular diseases,such as atherosclerosis,myocardial infarction,MI/RI and so on.It is known that caloric restriction(CR)is an anti-aging,anti obesity,delayed or reduced aging related diseases diet regulation method,which has multiple protective effects on the cardiovascular system,can prevent vascular atherosclerosis,improve myocardial tolerance and delay cardiac aging.Our previous experiment found that CR can up-regulate HIF-1α and regulate cardiomyocyte autophagy,but whether CR can affect pyroptosis and MI/RI is still uncertain.Therefore,this study intends to explore the effect of CR on MI/RI and its related mechanism from the perspective of pyroptosis.Objective:1.To determine whether caloric restriction improves myocardial ischemia/reperfusion injury in mice by regulating cardiomyocyte pyroptosis;2.To investigate whether the regulation of pyroptosis by caloric restriction is related to the inhibition of TLR4/NLRP3 signaling pathway.Methods:1.Animal experiment:(1)Experimental grouping: male C57BL/6J mice aged 8 months were randomly divided into 4 groups: Ad libitum + Sham operation group(AL+Sham);Ad libitum + myocardial ischemia/reperfusion group(AL+I/R);caloric restriction + Sham operation group(CR+Sham);caloric restriction + myocardial ischemia/reperfusion group(CR+I/R),n=10 in each group.In the Ad libitum group,the mice were fed freely every day.The caloric restriction method was to start with 90% of the normal diet,and then decrease by 10% every two weeks for 8 weeks.MI/R was performed by ligation of the left anterior descending coronary artery for 30 minutes after ischemia and 24 hours after reperfusion;(2)The body weight of mice was monitored during caloric restriction,and the final body weight was collected after 8 weeks;(3)The heart of mice was stained with Evan’s blue and TTC to observe the changes of myocardial infarction area after I/R;(4)HE staining was used to determine the pathological damage of myocardial tissue;(5)The expression of CD45 in myocardium was detected by immunohistochemistry;(6)The contents of LDH,CK-MB,SOD and MDA reflected the myocardial injury;(7)Western blot was used to detect the expression of pyroptosis related proteins NLRP3、GSDMD、ASC、caspase-1 and TLR4 in myocardial tissue;(8)The levels of IL-1β and IL-18 were detected by ELISA.2.Cell experiment:(1)Experimental group: H9c2 cardiomyocytes were divided into the following five groups: control group(Control);caloric restriction group(CR);hypoxia/reoxygenation group(H/R);caloric restriction + hypoxia/reoxygenation group(CR+H/R);TLR4 inhibitor TAK-242 + hypoxia/ reoxygenation group(TAK-242+H/R);(2)CCK-8 method was used to determine the cell survival rate;(3)The expressions of TLR4,NLRP3,GSDMD,ASC and caspase-1 were detected by RT-PCR and Western blot;(4)The expression of NLRP3 and ASC was detected by immunofluorescence.Results:1.Caloric restriction attenuates myocardial ischemia/reperfusion injury in mice by inhibiting pyroptosis1.1 The results of weight monitoring showed that CR made the mice lose weight: during the experiment,the weight of AL group increased steadily,while that of CR group decreased slightly;The weight of CR group was less than that of AL group after 8 weeks(P < 0.05).1.2 The results of Evan’s blue and TTC staining showed that the area of myocardial infarction in AL+I/R group increased,while that of CR+I/R group was 35% lower than that in AL+I/R group(P < 0.01),indicating that CR treatment could reduce the myocardial infarction area of mice.1.3 The results of HE staining showed that after I/R,the myocardial fibers were broken and the gap was enlarged;Compared with AL+I/R group,CR+I/R group was significantly improved,with occasional myocardial fiber rupture.These results indicate that CR treatment can reduce the pathological damage of myocardial tissue after MI/R in mice.1.4 Caloric restriction reduced the expression of CD45 in myocardial tissue after MI/R in mice: compared with AL+Sham group,CD45 positive granules in AL+I/R group were significantly increased,while CD45 positive granules were reduced after CR treatment,indicating that CR can reduce the inflammatory infiltration after MI/R in mice.1.5 CR can significantly improve the myocardial injury caused by MI/R in mice:compared with AL+Sham group,the contents of LDH,CK-MB and MDA in AL+I/R group were increased(P <0.01),while SOD activity decreased(P <0.05);In MI/R mice treated with CR,the contents of LDH,CK-MB and MDA decreased by 17%,15% and 21%,respectively,while SOD activity was increased by 16%(P <0.05).1.6 CR inhibited the expression of pyroptosis related proteins after MI/R: the expression of NLRP3,GSDMD,ASC and caspase-1 in AL+I/R group were significantly up-regulated(P < 0.01),while the expression of these proteins in CR+I/R group were 32%,36%,26%and 30% lower than that in AL+I/R group(P < 0.05).1.7 CR decreased the levels of IL-1β and IL-18 after MI/R: compared with AL+I/R group,the contents of IL-1β and IL-18 in CR+I/R group decreased by 31% and 33%respectively,indicating that CR can reduce the release of inflammatory cytokines(P < 0.05).1.8 CR inhibited the expression of TLR4 in myocardial tissue after MI/R: compared with AL+Sham group,the expression of TLR4 in AL+I/R group increased significantly(P< 0.01),while that in CR+I/R group decreased by 26%(P < 0.05).2.CR attenuates hypoxia/reoxygenation induced pyroptosis of H9c2 cardiomyocytes by regulating TLR4 / NLRP3 signaling pathway2.1 Establishment of hypoxia/reoxygenation model of H9c2 cardiomyocytes and determination of caloric restriction protocol: the results of CCK-8 method showed that the survival rate of cells was decreased after hypoxia for 12 h and reoxygenation for 3h(P <0.05).H9c2 cardiomyocytes were cultured in the medium containing 1 g/L,2 g/L,3 g/L,4g/L and 4.5 g/L glucose for 12 h.It was found that the survival rate of H9c2 cardiomyocytes after H/R could be improved by treatment with 2 g/L glucose for 12 h.Therefore,the following experimental conditions were hypoxia 12 h / reoxygenation 3h,2 g/L glucose concentration 12 h to simulate caloric restriction.2.2 CR could reduce the m RNA expression of pyroptosis protein in H9c2 cardiomyocytes after H/R: RT-PCR results showed that compared with Control group,the m RNA expression levels of NLRP3,GSDMD,ASC,caspase-1,IL-1β and IL-18 in H/R group were increased(P < 0.01),while the m RNA expression of these proteins in CR+H/R group decreased by 33%,42%,34%,39%,28% and respectively(P < 0.05).2.3 CR could inhibit the expression of pyroptosis related proteins in H9c2 cardiomyocytes after H/R: the results of Western blot detection showed that the protein expressions of NLRP3,GSDMD,ASC and caspase-1 were significantly up-regulated in the H/R group,while the expression of these proteins in the CR+H/R group decreased by 27%,39%,21% and 33%,respectively(P < 0.05).2.4 CR could reduce the expression of TLR4 in H9c2 cardiomyocytes induced by H/R:the protein and m RNA expression of TLR4 in H/R group was significantly higher than that in Control group(P < 0.01).After CR treatment,the expression levels of TLR4 protein and m RNA were decreased by 30% and 31%,respectively(P < 0.05).2.5 TLR4 inhibitor could reduce the m RNA expression of pyroptosis proteins: RT-PCR results showed that compared with Control group,the m RNA expression of TLR4 and pyroptosis related-proteins in H/R group were significantly increased(P < 0.01).After administration of TLR4 inhibitor TAK-242,the m RNA expression levels of TLR4,NLRP3,GSDMD,ASC and caspase-1 were decreased(P < 0.05),and CR treatment had similar effect(P < 0.05).2.6 TLR4 inhibitor attenuates H/R-induced cardiomyocyte pyroptosis in H9c2 cells:compared with control group,the expression of TLR4,NLRP3,GSDMD,ASC and caspase-1 in H/R group were obviously increased.After the cells were pretreated with CR,the protein expression levels of these proteins were decreased(P < 0.05).Compared with H/R group,the expression of TLR4,NLRP3,GSDMD,ASC and caspase-1 in TAK-242+H/R group were also decreased(P < 0.01),which was similar to the effect of CR treatment.At the same time,IF staining showed that NLRP3 showed red fluorescence and ASC showed green fluorescence.Compared with H/R group,the number of NLRP3 and ASC positive fluorescent cells in CR+H/R and TAK-242+H/R group were decreased,and there was no statistical difference between CR+H/R and TAK-242+H/R group.Conclusion:1.Caloric restriction can reduce the size of myocardial infarction,reduce the release of LDH,CK-MB and the level of oxidative stress in myocardial tissue,alleviate the degree of MI / R pathological injury and improve myocardial ischemia/reperfusion injury;2.Caloric restriction improves myocardial ischemia/reperfusion injury by inhibiting NLRP3 mediated pyroptosis,in which TLR4/NLRP3 signaling pathway may be involved. |
PDF Full Text Request