| Background:It is widely acknowledged that heart disease and stroke are the leading causes of death and disability in high per capita income countries(HIC).What is less appreciated is that this holds true for the low-and middle-income countries(LMIC)as well.CVD is responsible for about 30%of all deaths worldwide each year.Of note,nearly 80%of these deaths occur in LMIC,and half occur in women.CVD is the leading cause of mortality in china too,responsible for over 40%of all deaths,It is 42.61%in the urban area and 45.0%in the rural area,which is far more than the average level of international,indicating that the ability to cure cardiovascular diseases needs to be improved urgently.There are about 290 million patients with cardiovascular disease in China,of which 1 1 million are patients with coronary heart disease.With the rapid development of economic,the living conditions of people has obviously improved,the diet structure has changed(cholesterol,fat content increased),the lifestyle has changed,such as the decrease of sports,and the speed of population aging and urbanization has been quickened.Risk factors of cardiovascular disease increased,causing an increase in the number of cardiovascular disease.The prevalence and mortality of cardiovascular diseases are still rising in China.The increasing burden of cardiovascular diseases has become a major public health problem that needs to be solved.For patients with acute ST-segment elevation myocardial infarction(STEMI),timely myocardial reperfusion is the first option for treatment to reduce the area impacted by the acute myocardial infarction(AMI)and improve prognosis.However,the return of blood flow to the ischemic myocardium can also induce injury;this is referred to as myocardial ischemia/reperfusion(I/R)injury and associated with poorer functional recovery and adverse outcomes.Myocardial ischemia reperfusion injury offset the effect of reperfusion therapy in varying degrees.Therefore,how to reduce myocardial ischemia reperfusion injury is an important subject of medical research.The mechanism of myocardial ischemia-reperfusion injury has not been fully ullustrated,Several main reasons as follows:1.Free oxygen radical damage:under physiological conditions the formation of a small amount of free oxygen radicals can be quickly eliminated.During cell ischemia and hypoxia,cell metabolism is disturbed and free oxygen radical scavenging capacity is insufficient.When ischemia tissue suddenly returns to blood supply,a large amount of free oxygen radicals are produced and cannot be cleared in time.Which can hurt the cell containing them and cells surrounding them,thereby causing myocardial damage.Free radicals damage the structure of cell membrane,make cell membrane to lose its normal barrier function,make substances flow in and out of the cell freely,and finally make the cell lose its normal function.Free radicals can destroy the body protein by oxidation,change the surface structure of protease and make it lose its function.Free radicals can break the genetic material DNA,RNA,make nucleic acid lose its normal function,leading to the cell death finally.2.calcium overload:When the blood supply to an organ is reduced,the cells in the affected region switch to anaerobic glycolysis to generate ATP.This leads to lactate,H+,and NADH+ accumulation and cytosolic pH decreases.In an attempt to reestablish acid-base balance,the plasmolemmal Na+/H+exchangerexchanger(NHE)NHE is activated and transports H+ ions out of the cytosol in exchange for Na+.The influx of Na+ ions in turn activates the plasmolemmal Na+/Ca2+ exchanger which exchanges Na+ for Ca2+.Upon reperfusion,the activity of the NHE is accelerated by the washout of the extracellular H+ ions that accumulated during ischemia,which increases the proton gradient across the plasmolemma and further increases cytosolic Ca2+.In addition,Ca2+ handling by the endoplasmic/sarcoplasmic reticulum(ER/SR)SERCA ATPase is impaired by I/R,which limits Ca2+ reuptake from the cytosol.The massive increases in intracellular Ca+ concentrations activate a variety of processes,which contribute to cell death following I/R.3.Mitochondria play a critical role in the pathogenesis of myocardial IRI.They occupy 30-50%of the cardiomyocyte cytoplasmic volume and are critical in cardiac energy balance since energy supply for cardiomyocytes is mostly derived from mitochondrial oxidative phosphorylation.On the other hand,they are a favoured target of intracellular damage.These cell organelles are the major contributors of ROS as well as the major target for ROS-caused damage.Mitochondrial dysfunction,reflected in the structure,function,and number of mitochondria within the cardiomyocyte,leads to diminished energy production,loss of myocyte contractility,altered electrical properties,and eventual cardiomyocyte cell death.Apoptosis is the major pathogenic mechanism of myocardial I/R injury.Since myocardial cells are nonregenerative,apoptosis may cause myocardial dysfunction,which surfaces during ischemia and then is aggravated after reperfusion.The mechanism through which IR injury increases myocardial cell apoptosis is not clear.The Bcl-2 gene family is recognized as containing the most-important set of genes with respect to mediation of apoptosis.Bcl-2 is a protein that inhibits cell apoptosis,while Bax is a protein that promotes cell apoptosis.Accordingly,increases in the Bcl-2/Bax ratio decrease the level of apoptosis.Caspase-3 is a major factor in the activity of the apoptosis pathway and is involved in the final step of the apoptosis process;it is also responsible for the cleavage of many other apoptosis proteins.The important role of caspases in the apoptosis process makes them a potential target for anti-apoptotic treatments.The PI3K/Akt/eNOS pathway is the most important pathway for apoptosis regulation.Activation of Akt restrains GSK-3β,which inhibits mitochondrial permeability transition pore opening,a primary step for apoptotic and necrotic cell death.On the other hand,activation of eNOS by Akt generates NO and activates mitochondrial KATP channels in a cGMP-dependent manner,leading to acute cardioprotection.Considerable effort has gone toward finding ways to reduce the degree of injury from reperfusion;however,few effective treatments exist,and further research is needed to seek new approaches.The sigma-1 receptors are a relatively novel receptor group and are a ubiquitously expressed,unique binding site in the central nervous system and other peripheral tissues.These receptors are currently expected to be a potential target for drugs that treat neurodegenerative disorders,depression,idiopathic pain,and cancer.Recent studies have suggested that sigma-1 receptors agonists have potent cardioprotective effects in mice.Intracerebroventricular infusion of the sigma-1 receptors sigma-1 receptors PRE-084 for 1 month improved both mental and cardiac functioning in mice in which MI was induced.Some selective serotonin reuptake inhibitors(antidepressant drugs),such as sertraline and fluvoxamine,are potent agonists of sigma-1 receptors.Fluvoxamine attenuated cardiac hypertrophy and restored contractility in transverse aortic constriction mice after being delivered for a month by stimulating sigma-1 receptors;co-administation of the sigma-1 receptors antagonist NE-100 eliminated the cardioprotective effect.However,these studies assessed sigma-1 receptors sigma-1 receptors over a relatively long time;whether PRE-084 is effective right after administration(that is,in acute treatment directly after MI is unknown.To date,no studies have been performed regarding the effects of sigma-1 receptors agonists on myocardial I/R injury.Our aim was to explore the mechanism of action and assess the effect of PRE-084 on myocardial I/R injury in rats.Methods:The male Sprague-Dawley rats were injected intraperitoneally with PRE-084 or saline.One hour later,ischemia was induced by ligation at the left anterior descending(LAD)coronary artery.Rats were subjected to 30 minutes of myocardial ischemia and 24 hours of reperfusion.The first part was to observe the effect of PRE084 on cardiac function and myocardial infarction size,and to explore the relationship between PRE084 and oxidative stress in I/R injury rats.The second part was to investigate the effect of PRE084 on myocardial apoptosis and its pathogenesis.Effect on Bax,Bcl-2,Caspase-3 of apoptosis-related protein;PART ONE:Rats were randomized into three groups:(1)the sham group(n=16),in which rats underwent identical surgical procedures but without occlusion of the coronary artery;(2)the I/R group(n=19),ischemia was induced in the rats for a period of 30 minutes;and(3)the PRE-084 group(I/R+ PRE-084 lmg/kg n=19).The maximal velocity of left ventricular pressure development(LV±dp/dt),left ventricular systolic pressure(LVSP),and left ventricular end-diastolic pressure(LVEDP)were measured through a polyethylene-50 tube that was advanced into the left ventricle through the right carotid artery and connected to a multi-channel physiological monitoring system(Biopac MP150)24 h after reperfusion.Blood samples were drawn after 24 hour of reperfusion(8 rats in each group).Serum CK-MB and LDH levels were determined spectrophotometrically according to the manufacturer’s instructions.The MDA level and activities of antioxidant SOD in heart homogenates were determined spectrophotometrically.hematoxylin and eosin(H&E)restaining were performed.Following 24 h reperfusion,myocardial infarct size was evaluated using Evans blue/TTC dye(8 rats in each operation group).The area of infarct size(INF)and area-at-risk(AAR)were measured digitally using Image software.The INF and AAR were expressed as percentages of the left ventricular area(INF/LV and AAR/LV,respectively).PART TWO:Five rats were selected randomized fromeach groups:(1)the sham group,in which rats underwent identical surgical procedures but without occlusion of the coronary artery;(2)the I/R group,ischemia was induced in the rats for a period of 30 minutes;and(3)the PRE-084 group(I/R+ PRE-084 lmg/kg).The tenninal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)assay was used to analyze the degree of myocardial apoptosis,Western blot analyses were performed to assess Akt,eNOS,cleaved caspase-3,Bax and Bcl-2 protein expression,PCR were performed to assess mRNA of sigma-1 receptor.Statistical analysisThe statistical analysis was performed using SPSS 19.0 software.Data were described as means±standard deviations.The basis of homogeneity of variance was evaluated by a one-way analysis of variance(ANOVA);multiple comparisons between the groups were performed using the Student-Newman-Keuls method,with P<0.05 considered statistically significant.ResultsThe first part:1.Histopathologic observation of the myocardium in rats:Pathologic changes of myocardial tissue were observed under hematoxylin-eosine staining(HE).In Sham group there Was no detectable histopathological change.Rats in the I/R control group displayed a serious degree ofmyocardial neutrophilic infiltrate,necrosis, hemorrhage,and spindle—shaped interstitial cells as compared with mts in the sham group.pathologic changes of cardiac tissue in the PRE-084 group were significantly milder than that of the I/R.2.The effect of PRE-084 on cardiac functionTo assess the effect of PRE-084 on cardiac function,hemodynamic parameters such as LVEDP,LVSP,and LV±dp/dt were measured.Compared with those in I/R group,LV±dp/dt(+dp/dt 2279.26±189.04 versus 1845.4±250.54 mmHg/s,P<0.01)(-dp/dt 2284.87±195.00 versus 1774.24±225.96 mmHg/s,P<0.01)and LVSP(103.23±9.52 vs 87.19±13.68 mmHg,P<0.01)significantly increased in the PRE-084 group.LVEDP(5.05±1.71 vs 6.75±2.17 mmHg,P>0.05)was not significantly changed between the two groups.3.The effects of PRE-084 on the levels of serum LDH、CK-MB in rats:Compared with Sham group,levels of serum LDH and CK-MB were significantly increased in PRE-084 group and I/R group(p<0.0 1).Compared with I/R group,PRE-084 group markedly decreased the activity of serum LDH、CK.-MB.4.The effects of PRE-084 on the levels of SOD and MDA in myocardial ischemia-reperfusion rats:Compared with Sham group,the content of MDA in myocardium were significantly increased,but SOD in myocardium was significantly decreased in PRE-084 group and I/R group(p<0.01).Compared with I/R group,PRE-084 group markedly decreased the content of MDA in myocardium,but significantly increased the activity of SOD in myocardium(p<0.01).5.To examine the effect of PRE-084 on the infarct size following myocardial I/R,the myocardial infarct sizes of rats in each group were determined by TTC/Evans blue dye 24 h after reperfusion.The representative mid-ventricular cross-sections are shown in Figure 1A(n=8 in each group).rats pre-treated with PRE-084 displayed much smaller INF/LV(10.1%±2.1%)compared to I/R model mice(25.7%±3.9%,P<0.01,),suggesting that treatment of PRE-084 significantly reduced the infarct size.Moreover,both groups displayed similar AAR/LV percentages(28.3%±4.3%in the PRE-084group vs 31.4%±5.2%in the I/R group,P=0.347).The second part1.myocardial cell apoptosis:The TUNEL assay was used to assess the effect of PRE-084 on myocardial apoptosis during I/R injury.PRE-084 treatment significantly decreased the degree of myocardial apoptosis in the PRE-084 group(as measured by percentage of apoptotic cells)compared with that of the I/R group(22.1%±2.8%versus 51.1%±5.8%,P<0.01,n=5 in each group).2.The effect of PRE-084 on mRNA expression of sigma-1 receptor:The sigma-1 receptor mRNA expression were significantly higher in myocardial cells in PRE-084 group than in I/R group(P<0.01).3.The effect of PRE-084 on Akt and eNOS:The expression of phosphorylated protein kinase B(p-Akt)(Ser473),and endothelial nitric oxide synthase(p-eNOS)(Ser1177)was assessed to explore the mechanisms underlying the cardioprotective effects of PRE-084.Phosphorylation levels of Akt and eNOS decreased significantly in I/R group compared with the sham group(0.35±0.09 versus 0.96±0.02,0.30±0.07 versus 0.95±0.02,respectively,P<0.01).PRE-084 treatment led to significantly increased expression of p-Akt(0.73±0.07 versus 0.35±0.09,P<0.01)and p-eNOS(0.79±0.06 versus 0.30±0.07,P<0.01)in the PRE-084 group compared with those in the I/R group.4.Effect of PRE-084 on cleaved caspase-3 and Bax and Bcl-2 protein expression:As caspase-3 and Bcl-2,Bax are the most important factors in the regulation of apoptosis.we assessed their expression levels to determine the potential mechanism.Compared with the sham group,Bcl-2 protein expression was significantly decreased(0.62±0.06 versus 0.32±0.03,P<0.01)and Bax protein expression was significantly increased(0.16±0.05 versus 0.54±0.07,P<0.01),with the Bcl-2/Bax ratio significantly reduced in the I/R group(4.46±1.08 versus 0.57±0.09,P<0.01);PRE-084 pretreatment restored most of the changes as illustrated in(P<0.01).Compared with the sham group,the expression of cleaved caspase-3 significantly increased in the I/R group(0.52±0.10 versus 0.14±0.05,P<0.01),and decreased in the PRE-084 group(0.52±0.10 versus 0.33±0.06,P<0.01).Conclusion1.Our study showed that myocardial isehemia/reperfusion could lead to myocardial injury in rats,which can be reduced by Pretreatment with PRE-084,it can reduce concentration of LDH、CK-MB and increase concentration of MDA,decrease infarct size and improve cardiac function.2.The heart protective effect of PRE-084 was probably achieved through decreasing myocardium apoptosis,and modulating expression of Bcl-2 and Bax, increasing the ratios of Bcl-2/Bax and decreasing the activity of caspase-3.All these effects maybe following activating of Akt and eNOS. |
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