Fluvastatin Inhibits Cardiomyocyte Apoptosis After Myocardial Infarction Through TLR₄ Pathway

BackgroundAcute myocardial infarction(AMI)is mainly caused by coronary artery stenosis caused by atherosclerosis,leading to myocardial ischemia,injury,cardiogenic shock or cardiac arrest,which is the main cause of sudden death in clinic.In the United States,there are about 620,000 new cases of AMI each year,and 15%of them die from the disease.Compared with the general population,survivors of AMI are more likely to fall ill and die of heart disease or other causes.In addition,patients with a history of AMI had a 79%increased risk of infection.Therefore,the prevention and treatment of AMI is also one of the problems that need to be solved immediately.Statins are competitive inhibitors of 3-hydroxy-3-methylglutaric coenzyme A(HMG-CoA)reductase,which is the rate-limiting enzyme in the cholesterol biosynthesis pathway.Therefore,statins can reduce cholesterol levels in LDL by reducing endogenous cholesterol synthesis.Statins are widely used in patients with hypercholesterolemia and coronary heart disease.The experimental evidence shows that they are very effective in the primary and secondary prevention of atherosclerotic cardiovascular disease,as well as in reducing mortality and the incidence of non-fatal cardiovascular events,and can improve the clinical outcomes of AMI.Among statins,fluvastatin is the most widely used lipid modification drug in clinic.Its side effects and adverse reactions are relatively small,and it plays an important role in the treatment of atherosclerosis.Fluvastatin has the advantages of high selectivity,simple structure and not easy to cause adverse reactions.Fluvastatin has good pharmacological activity without metabolic transformation.Besides its lipid-lowering effect,fluvastatin can also stabilize plaque,inhibit inflammation and regulate coagulation.Since fluvastatin is almost completely metabolized by the liver and only less than 6%of the drug enters the urine,there is no need to adjust the dose for patients with mild to moderate renal insufficiency,but it is prohibited for patients with severe renal insufficiency,active liver disease,persistent unexplained elevated transaminase or heavy drinkers.Cell necrosis was previously considered to be the only cause of cell death during myocardial infarction.Current studies have shown that apoptosis also plays an irreplaceable role in the process of tissue injury after myocardial infarction.Especially during cardiac remodeling after myocardial infarction,apoptosis of myocardial cells in infarcted and boundary regions directly affects cardiac function and is associated with higher mortality.Therefore,inhibiting cardiomyocyte death is of great significance in alleviating cardiac injury after ischemia.In recent years,studies have found that hypoxia,persistent ischemia and reperfusion are the main factors causing apoptosis.Animal experiments showed that myocardial cell apoptosis occurred at different time points after AMI in rats.Myocardial cell apoptosis was mediated by multiple death signals and regulated by different genes.However,the mechanism of different genes on cardiomyocyte apoptosis is not fully understood.Toll-like receptors(TLRs)are important receptor proteins involved in nonspecific immunity.By identifying pathogen-related molecular patterns,cytokines can be induced to play an anti-infective role.TLR4 is expressed in myocardial cells and vascular endothelial cells,which can participate in the inflammatory response of myocardial ischemia-reperfusion injury,promote the formation of reactive oxygen species free radicals,and activate a variety of cytokines.After AMI,TLR4 signaling pathway is activated,and a large number of inflammatory cytokines are expressed and released,which can induce cardiomyocyte apoptosis.Objectives1.To establish a MI model in rats and investigate the expression of TNF-alpha and IL-1 beta and the effect of fluvastatin.2.To observe the effects of fluvastatin on myocardial structure and function in AMI rats 14 days later.3.To study the effects of fluvastatin on myocardial cell apoptosis after AMI.4.To study the effect of fluvastatin on cardiomyocyte apoptosis after myocardial infarction through Toll pathway.MethodsOne hundred and thirty-one rats(6-8 weeks old)were randomly divided into four groups:rats with anterior descending coronary artery ligation were in the AMI group;rats with fluvastatin after ligation were in the treatment group;rats without ligation or suture were in the sham operation group;and rats without ligation were in the normal control group.The AMI group,the sham operation group and the normal control group were all given the same dose of normal saline by gavage.In this experiment,we first collected blood from the inner canthus of rats on the fifth day of the model,detected and compared the concentration of TNF-alpha and IL-1 beta in the plasma of rats in each group,determined the effect of fluvastatin on the secretion of inflammatory factors,and inferred its control effect on acute inflammatory response induced by AMI in rats.We observed the function and structure of heart by ultrasound in AMI rats 14 days later.Myocardial specimens were collected from rats with myocardial infarction for 7 days.The degree of apoptosis was detected by TUNEL;the expression of apoptosis-related genes Caspase-3,Bcl-2,Bax and HSP70 was detected by immunofluorescence.Quantitative RT-PCR was used to detect the expression of TLR4 in myocardial cells of four groups of rats,and immunoblotting was used to detect the expression of TLR4 protein to prove the potential relationship between Toll-like signaling pathway and the treatment of AMI and fluvastatin.In addition,TUNEL method was used to detect the apoptosis of rat cardiomyocytes.ResultsAfter ligation,white anterior ventricular wall,cyanosis and left atrial swelling were observed,electrocardiogram showed marked elevation of ST segment,echocardiography showed a significant decrease in EF an FS,suggesting that the model was successful.The concentration of IL-lbeta and TNF-alpha in plasma increased significantly 5 days after the establishment of AMI model in rats,compared with normal control group and sham operation group(P<0.001),and decreased after the intervention of fluvastatin(P<0.05).These results suggest that fluvastatin therapy can inhibit acute inflammation induced by AMI to some extent.Cardiac function in the fluvastatin group was improved compared with that in the untreated AMI group in AMI rats 14 days later.After 14 days of AMI modeling,BNP was increased,while it was decreased in the fluvastatin group(P<0.05).TUNEL positive cells were occasionally found in cardiac myocytes of normal control group,and few TUNEL positive cells were scattered in sham operation group,which had no significant difference from that of normal control group(P>0.05).A large number of TUNEL positive cells were found in the AMI group,apoptotic cells accounted for 30.41±3.12%of all cells,which had significant difference compared with the normal control group and sham operation group(P<0.001).Compared with MI group,TUNEL positive cells in fluvastatin group decreased significantly(P<0.05),apoptotic cells accounted for 19.28±3.86%of all cells..Under laser confocal microscopy,Caspase-3 protein expression was not detected in the normal control group,Caspase-3 immunopositive cells were occasionally seen in the sham operation group,while Bax and HSP70 protein were occasionally weak expressed in both groups,while Bcl-2 protein expression was strong,and there was no significant difference between the two groups(P>0.05).After the establishment of AMI model,a large number of Caspase-3,Bax immunopositive cells were distributed,and the expression of Bcl-2,HSP70 was weakened.Compared with normal control group and sham operation group,there were significant differences.The expression of Bcl-2,HSP70 in fluvastatin group was higher than that in AMI group,and the expression of the other two proteins was lower than that in AMI group,with significant difference.These data suggest that fluvastatin can significantly inhibit cardiomyocyte apoptosis during the intervention of AMI in rats.TLR4 was expressed in cardiac myocytes of four groups.The expression of TLR4 in normal control group was 0.902±0.140,while that in sham-operated group was 1.214+0.274.The expression of TLR4 in myocardial cells of AMI group was significantly higher than that of the first two groups,reaching 3.412±0.879,and the difference was statistically significant(P<0.05).It is worth noting that compared with the AMI group,the expression of TLR4 in rat cardiac myocytes after fluvastatin treatment decreased significantly to 1.864±0.467(P<0.05).Similarly,there was no significant difference in TLR4 protein expression between normal control group and sham-operated group(P>0.05).Compared with normal control group and sham-operated group,the expression of TLR4 protein in AMI group was significantly higher(P<0.05).The expression of TLR4 protein in fluvastatin group was significantly lower than that in AMI group(P<0.05).Conclusions1.After myocardial infarction in rats,a series of inflammation appeared,Inflammatory factors IL-1β and TNF-aincreases,cardiac function is damaged to some degree,and myocardial remodeling occurs.Fluvastatin may inhibit myocardial inflammation,reduce myocardial remodeling,improve the heart function.2.In summary,fluvastatin can reduce cardiomyocyte apoptosis and inhibit the occurrence and development of AMI by inhibiting the expression of TLR4.The study will be helpful to understand the therapeutic mechanism of fluvastatin in the treatment of AMI at the molecular and cellular level.