Neocryptotanshinone protects cardiomyocyte hypoxia/reoxygenation-induced H9C2 cell injury through targeting RxRα

摘要

OBJECTIVE Neocryptotanshinone(NCTS)is a natural product extracted from traditional Chinese herb Salvia miltiorrhiza Bunge.Previous studies have demonstrated the anti-inflammatory of NCTS in lipopolysaccharide(LPS)-stimulated mouse macrophage(RAW 264.7).However,the protective effect and mechanism of NCTS in cardiomy⁃ocytes are still undefined.This study is to investigate whether NCTS exerts its cardioprotective effect against hypoxia/re⁃oxygenation(H/R)-induced H9C2 cell injury.METHODS The model of H/R injury was established through hypoxia for 8 h and reoxygenation for 12 h in H9C2 cardiomyocytes of rats.Cultured cardiomyocytes were randomly divided into four groups,control group,H/R group,H/R+NCTS pretreated group(1,2,5 and 10μmol·L^-1),and H/R+NCTS+HX531(an RXRαantagonist,2μmol·L^-1)co-treated group.The cell viability was measured by Cell Counting Kit-8,Hoechst33258 staining was used to observe the morphology of apoptotic changes.Mitochondrial membrane potential was detected by JC-1 fluorescent probe,and protein expressions of RXRα,Bcl-2,Bax,caspase-3 and cleaved caspase-3 with Western blotting.RESULTS Compared with control group,the cell viability in model group was decreased(P<0.05).After treated with NCTS in different concentrations,the CCK8 results showed that NCTS in 2μmol·L^-1 had protective effects.Result of Hoechst33258 staining suggested that the apoptosis was notably increased in model group(P<0.05),Meanwhile,the JC-1 results showed that the mitochondrial membrane potential of the model group decreased which was consistent with previous study.impressively,NCTS could restore the mitochondrial membrane potential as well as apoptosis.Fur⁃ther western blot experiments showed that NCTS treat could upregulate Bcl-2 protein,and downregulate the levels of Bax and cleaved caspase-3/caspase-3 ratio.Since RXRαis a critical upstreaming proteins which can directly mediate the apoptosis,we then determined the effect of NCTS on it.Intriguingly,RXRαwas notably activated by NCTS,while the HX531,the antagonist of RXRα,could abolished NCTS'effect when co-treated with NCTS.CONCLUSION NCTS in 2μmol·L^-1 was effective to protect H9C2 cell from H/R-induced cell injury through RXRα-mediated mitochondria apop⁃tosis.Current results provide possible drugs for the treatment of ischemic cardiomyopathy.