| 1.Backgroud and objectivesMyocardial infarction(MI)is one of the major diseases that endanger human health.Inflammatory response plays an important role in the pathological process of MI.Orderly and the moderate inflammatory response is one of the preconditions for myocardial repair after MI.However,excessive inflammatory response will also lead to the additional loss of functional myocardium,aggravate myocardial injury and ventricular remodeling.NLRP3 inflammasome is the key mediator to promote aseptic inflammatory response.Some studies have shown that NLRP3 inflammasome mediates ischemic injury and aggravates myocardial injury by expanding inflammatory response.Mitochondria are the main place of energy metabolism.The content of mitochondria in cardiomyocytes is more than 40%of its volume,which provides more than 90%of energy.In the process of MI,prolonged ischemia and hypoxia lead to mitochondrial damage,and then damaged mitochondria release a lot of mitochondrial ROS(mtROS)and oxidized mitochondrial DNA(ox-mtDNA)into the cytoplasm,which promotes the assembly and activation of NLRP3 inflammasome and then aggravate the myocardial injury.Mitophagy is an important factor that negatively regulates the activation of NLRP3 inflammasome.It can inhibit the activation of NLRP3 inflammasome by eliminating damaged mitochondria to reduce the release of mtROS and mtDNA.Therefore,we establish the MI model of rat in vivo and the ischemia and hypoxia model of H9c2 cells in vitro to investigate the effect of SL extract on myocardial infarction and oxidative stress,and further explored the intervention effect of SL extract on the activation of NLRP3 inflammasome based on mitophagy.2.Methods2.1 Cardioprotective effects of SL extract on myocardial infarctionThe rat model of MI was established using coronary artery ligation method.Then the rats were randomly divided into seven groups:sham group,MI group,three SL extract groups(The dose is 62.16,124.32,248.64 mg/kg/d),compound Danshen Tablet(259.20 mg/kg/d)group and diltiazem hydrochloride(21.60 mg/kg/d)group.21 days after administration:(1)Echocardiography combined with hemodynamics was used to detect the changes of cardiac structure and function;(2)TTC staining was used to detect the size of myocardial infarction;(3)enzyme and ELISA were used to detect the levels of serum myocardial injury markers,including CK-MB,cTnT and oxidative stress indicators including SOD,GSH-Px and MDA;(4)HE staining was used to detect the pathological changes of myocardial tissue;(5)Masson staining was used to detect the changes of cardiac fiber tissue.To observe the protective effect of SL extract on MI.2.2 Effects of SL extract on NLRP3 inflammasome by regulating mitophagy in MI ratsOn the basis of "2.1",by:(1)TUNEL staining was used to detect myocardial apoptosis;(2)ELISA was used to detect the levels of inflammatory cytokines IL-1βand IL-18 downstream of NLRP3 inflammasome;(3)Immunohistochemistry was used to detect the expression of mitophagy protein PINK1,parkin and inflammasome component NLRP3;(4)Western blot was used to detect the expression of mitophagy related proteins LC3-Ⅱ/LC3-I,p62,PINK1,Parkin,and NLRP3 inflammasome related proteins NLRP3,caspase-1,IL-1 β,IL-18.To further explore the effects of SL extract on NLRP3 inflammasome by regulating mitophagy.2.3 Effects of SL extract on the ischemia and hypoxia model of H9c2 cellsH9c2 cells were cultured in glucose and serum-free medium for 24 hours in a 94%N2,1%O2 and 5%CO2 incubator to establish the ischemia and hypoxia model of H9c2 cell in vitro.Then:(1)MTT assay was used to detect cell activity;(2)Annexin-V/PI double staining method was used to detect cell apoptosis;(3)Enzyme method was used to detect the levels of LDH,SOD and MDA in cell culture supernatant.Objective to observe the protective effect of SL extract on H9c2 cells against ischemia and hypoxia injury.2.4 Effects of SL extract on NLRP3 inflammasome by regulating mitophagy in H9c2 cells model of ischemia and hypoxiaBased on the "2.3",through:(1)The levels of reactive oxygen species(ROS)were detected by fluorescent probe DCFH-DA;(2)Mitochondrial membrane potential was detected by JC-1 fluorescent probe;(3)Cell ultrastructure and autophagy were observed by transmission electron microscope;(5)The secretion of IL-1 β downstream of NLRP3 inflammasome was detected by ELISA;(6)Western blot was used to detect the expression of mitophagy related proteins LC3-II/LC3-I,p62,PINK1,Parkin,and NLRP3 inflammasome related proteins NLRP3,caspase-1,IL-1 β and IL-18.To investigate the mechanism of SL extract regulating mitophagy to intervene NLRP3 inflammasome and reduce myocardial cell injury.3.Results3.1 Cardioprotective effects of SL extract on myocardial infarction(1)Cardiac structure and function:echocardiography results showed that the left ventricular anterior wall thickness(LVAWd/s)of model group was significantly decreased(P<0.01),left ventricular posterior wall thickness(LVPWd/s)did not change significantly(P>0.05),left ventricular inner diameter(LVIDd/s)was significantly expanded(P<0.01),while ejection fraction(EF)and short axis shortening rate(FS)of cardiac function parameters were significantly reduced in model group(P<0.01)The results showed that the level of EF and FS were significantly improved compared with those in model group(P<0.01),and LVAWd/s,LVPWd/s and LVIDd/s were improved compared with the model group,among which the high dose of SL extract could significantly reduce LVIDs(P<0.01).The hemodynamic results showed that compared with the model group,SL extract could significantly improve LVSP,+dp/dtmax,-dp/dtmin,reduce LVEDP(P<0.01),and improve the systolic and diastolic function of the heart.(2)Myocardial infarction size:the results showed that after 21 days of treatment with SL extract,the myocardial infarction size of rats was significantly lower than that of model group(P<0.01).(3)Serum myocardial injury markers and oxidative stress indexes:compared with model group,SL extract can significantly reduce CK-MB and cTnT levels in rats serum(P<0.01),improve SOD,GSH-PX activity and reduce MDA level(P<0.05 or P<0.01).(4)HE staining:The results showed that the cardiomyocytes in sham group were arranged in order,the structure was complete and normal,the myofibers were arranged closely,the texture was clear,the color was uniform,the size of the nucleus was consistent,and no inflammatory cell infiltration was found;The model group had swelling of cardiomyocytes,myofibrils vacuolation and arrangement disordered,and the nucleus of the heart was scattered and stained with cytoplasm,accompanied by a large number of inflammatory cell infiltration;The degree of myocardial tissue lesion was significantly reduced after SL extract intervention,and the necrosis of cardiomyocytes was decreased,and the arrangement of myocardial fibers was relatively neat,and only a few inflammatory cells were infiltrated.(5)Masson staining of collagen fiber:The results showed that the myocardial tissue structure of rats in sham group was normal,there were a small amount of collagen fibers in the gap between cells,and the cardiac cells were arranged in order and closely;the cardiomyocytes in model group were necrosis and replaced by collagen fibers,and the myocardial fibrosis was serious;After intervention by SL extract,the myocardial tissue structure of rats was relatively complete and the myocardial was fine,the number of remaining cardiomyocytes increased and only a few were replaced by collagen fibers,and the degree of myocardial fibrosis was improved significantly.3.2 Effects of SL extract on NLRP33 inflammasome by regulating mitophagy in MI rats(1)Apoptosis of cardiomyocytes:TUNEL staining showed that there were only a few apoptosis cells in the sham group,but a large number of apoptosis cells were observed in the model group,and the number of apoptosis cells decreased after SL intervention.The results of apoptosis index calculation also showed that SL extract could significantly reduce the apoptosis index of cardiomyocytes compared with the model group.(2)Mitophagy:compared with model group,SL extract can improve mitophagy level,up regulate LC3-II/LC3-I,PINK1,Parkin expression,and down regulate P62 protein expression(P<0.01).(3)Activation of NLRP3 inflammatory corpuscles:compared with model group,SL extract can significantly inhibit the activation of NLRP3 inflammasome,inhibit the expression of NLRP3 and caspase-1 protein,and reduce the expression and secretion of downstream inflammatory factors IL-1β and IL-18(P<0.05 or P<0.01).3.3 Effect of SL extract on the ischemia and hypoxia model of H9c2 cellsThe results showed that compared with the model group,SL extract could significantly improve the activity of H9c2 cells and reduce apoptosis(P<0.01),reduce LDH and MDA levels,improve SOD activity(P<0.01),and alleviate the H9c2 cell damage caused by ischemia and hypoxia.3.4 Effect of SL extract on NLRP3 inflammasome by regulating mitophagy in H9c2 cells model of ischemia and hypoxiaThe results showed that:(1)Compared with the model group,SL extract can significantly reduce the ROS level in cells(P<0.05),improve the mitochondrial membrane potential(P<0.05 or P<0.01);(2)The results of transmission electron microscope showed that mitochondria swelled,mitochondria were broken,part of mitochondria membrane was broken,and autophagosomes were found in the model group.After SL extract intervention,the damage of mitochondria was alleviated,the accumulation of damaged mitochondria was reduced,and a large number of autophagosomes were observed.(3)Compared with the model group,the level of mitophagy in SL extract group was significantly increased,and the expression of LC3-Ⅱ/LC3-Ⅰ,PINK1 and Parkin was significantly increased,while the expression of P62 protein was significantly decreased(P<0.05 or P<0.01);(4)Compared with the model group,SL extract could significantly inhibit the secretion of IL-1β(P<0.01),and reduce the expression of NLRP3,caspase-1,IL-1 β and IL-18(P<0.01).4.Conclusion(1)In this study,the MI model of rat in vivo was established by ligating the left anterior descending branch of coronary artery.The myocardial tissue of model group had obvious infarct site,the level of CK-MB and cTnT were increased significantly.The cardiac function of rats was significantly decreased by echocardiography and hemodynamic test;The pathological examination showed obvious myocardial damage,indicating the model replication.(2)SL extract can reduce the myocardial infarction size,reduce oxidative stress injury,inhibit inflammation,improve cardiac function,and thus fight myocardial ischemia injury.(3)SL extract can significantly improve cell activity and antioxidant capacity,increase ROS clearance,reverse mitochondrial membrane potential decline,reduce apoptosis,and improve H9c2 cells ischemia and hypoxia injury(4)SL extract may inhibit the activation of NLRP3 inflammatory bodies by improving the level of PINK 1/parkin-mediated mitophagy,thus playing a protective role in myocardium. |
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