Protective Effect And Mechanism Of Simvastatin On Hypoxia/reoxygenation-induced Apoptosis In Rats' Cardiomyocytes

Background and Aims:Myocardial ischemia/reperfusion injury (M I/R I)is a consequence of cardiac thrombosis or coronary spasm followed by the restoration of the coronary blood flow. Ischemia/reperfusion can aggravate the injury of myocardial ischemia. Myocardial ischemia/reperfusion may cause cardiomyocyte apoptosis, which may have adverse impact on cardiac structure and function. Therefore, prevention against M I/R I is fairly important in clinic.Toll-like receptor4 recognizes innate immunity and acquire immunity reaction of pathogens. TLR4 are expressed differentially among immune cells. Activation of TLR4 can increase the expression of pro-inflammatory cytokines and active several signaling pathways in different cells. TLR4 plays a major role in inflammation and non-infectious inflammation. Several studies have demonstrated that the myocardial infarct size after MIRI decreased in TLR-4 gene deficient mice(C3H / HeJ mice) compared with that in wild-type mice (C3H/HeN mice). Recent studies have also confirmed that TLR-4 inhibitor eritoran which blocked TLR-4 signaling pathway after neonatal rat cardiomyocytes can protect the heart against MIRI injury.Simvastatin is a semi-synthetic lipid-lowering drugs derived from degradation product of terrecin and have widely used in clinic for hyperlipidemia. A large number of studies have shown that simvastatin have effects independent of lipid-lowering including anti-inflammation, immunomodulation, anti-apoptosis, left ventricular remodeling reversement, etc. However, the mechanisms of simvastatin on cardiovascular disease,especially on myocardial ischemia/reperfusion injury in still not well understood.Our aims was to investigate the protection effects of simvastatin on cardiomyocyte aopotosis induced-by MI/RI and whether the mechanism is associated with TLR4 signaling pathway.MethodsPart I: CMs culture and establishment of H/R model. CMs were isolated from 1-3d Sprague-Dawley neonatal rat ventricle. Ventricular tissue was cut into small pieces in serum-free DMEM solution and digestion in 0.125% trypsin.CMs at passage 3 were cultured in in anaerobic system at 37℃for 2h, then reoxygenation for 4h,8h,12h,24h. The cell survival rate was detected by trypan blue staining. The release of lactate dehydrogenase(LDH) was tested by LDH assay kit. The apoptosis index of CMs was detected by Flow cytometry.Part II: Changement of TLR4 signaling pathway during the injury of cardiomyocyte MI/RI. After hypoxia for 2h and reoxygenation for 4h,8h,12h,24h, the expressions of TLR-4 were analyzed by Western blotting. Part III: Effects of TLR4 signaling pathway inhibition on the protection against cardiomyocytes hypoxia(2h)/ reoxygenation(4h) injury. Two to there passages of cardiomyocytes were administrated with the TLR4 neutralizing antibody MST510 (10ug/mL) and pretreated with simvastatin (0.1μmol/L,1μmol/L, 10μmol/L) for 1h befor hypoxia. The release of LDH was detected by LDH assay kit. The apoptosis of CMs were analyzed by Flow cytometry. The expression of TLR4 was investigated by Western blotting.Results1. CMs were successfully isolated and cultured in vitro.2. The release of LDH was increased in each group ( P<0.05) after hypoxia/reoxygenation injury compared with the control group. The apoptosis index were increased in each reoxygenation group( P<0.05).3. TLR-4 expression were increased after reoxygenation for 4h,8h,12h,24h ( P<0.05) compared to the control group.4. MTS510, a TLR4 signaling pathway neutralizing antibody, blocked the release of LDH and increased the apoptosis index of CMs compared to the control group and the H/R group (P<0.05). Pretreatment with simvastatin (0.1μmol/L, 1μmol/L,10μmol/L) 1h befor hypoxia, reduced the release of LDH and apoptosis index of CMs compared to the control group and the H/R group (P<0.05).Conclusion:1. H/R injury enhanced the release of LDH and increased the apoptosis index of CMs.2. H/R injury actived the expression of TLR4 which lead to the increase of CMs's apoptosis 3. TLR-4 signal pathway blocker (MTS510) decreased the apoptosis index of CMs caused by H/R injury.4. Pretreatment with simvastatin could protect against apoptosis induced by H/R injury.