The Protective Effect And Mechanism Of Carvedilol On Hypoxia/Reoxygenation Injury Induced Apoptosis In H9C2 Cardiomyocytes

Background and ObjectiveIschemia/reperfusion(I/R)injury is a common occurrence during thrombotic disease.The effects of reperfusion are typically beneficial but often lead to an inflammatory response that causes further damage to viable tissue around the infarct area,a result thought to be associated with increased apoptosis,especially in cardiomyocytes.This form of damage has been demonstrated to lead to cardiomyocytes loss and infarct size increased.Therefore,there have important clinical significance of research the relation of apoptosis and I/R.There are a number of signal pathways that participate in cardiomyocyte apoptosis;however,the toll-like receptor 4(TLR4)/nuclear factor ?-light-chain-enhancer of activated B cells(NF-?B)signal pathway is known to play one of the key roles in I/R injury.TLR4 is one of Toll like receptors(TLRs)and could be activated by danger signals such as stress,injury and necrosis,etc.TLR4 is mainly expressed in heart.TLR4 activation can transduce signal into intra-cellular and activate NF-?B with the help of a series of enzymes,then,NF-?B enters nucleus and induces definitive gene experssions which were responsible for the production of inflammatory cytokines.?-arrestin is a multifunctional adapter protein well known for its role in G-protein-coupled receptor(GPCR)desensitization.?-arrestin take a role of regulate the NF-?B-mediated gene regulation through binding I?B?,the inhibitor of NF-?B.Therefore,?-arrestin is regarded as the negative factor of TLR4-NF-?B signal pathway.Carvedilol is a non-selective ?-adrenoceptor blocker initially used in the treatment of hypertension and angina.It has been shown to reduce the risk of hospitalization and the mortality rate in patients with severe chronic heart failure.In a number of clinical trials carvedilol has been demonstrated to exert a beneficial effect on ventricular remodeling and in preserving the ejection fraction,as compared with other ?-blockers.These effects are considered to be mediated through multiple mechanisms,including the inhibition of cardiomyocyte apoptosis.However,the association between the effect of carvedilol on anti-apoptosis and the TLR4 signaling pathway is not yet known.But there is unknown about the relevance of carvedilol and TLR4-NF-?B mediated apoptosis.It is seldomly reported that the effect of carvedilol on ?-arrestin.Therefore,this study was designed to observe the effect of myocardial injury induced by I/R,to explore the relationship between TLR4/NF-?B signal pathway and myocardial injury.The changes of ?-arrestin level was evaluated at the same time.Base on these findings,we expected to illuminate the anti-apoptosis effect of carvedilol on inhibited TLR4/NF-?B signal pathway and affect the expression of ?-arrestin.Materials and methods The first part:The culture of H9C2 cardiomyocytes and the model of Ischemia/reperfusion.We culture H9C2 cardiomyocytes in vitro in first step.then develop ischemia/reperfusion model by hypoxia/reoxygenation.after the hypoxia 4 hours,experiments were divided into 5 groups according with the difference of reoxygenation time: control group,reoxygenation group 4h,reoxygenation 6h group,reoxygenation the 10 h group.After hypoxia/reoxygenation,the groups were observed under inverted microscope,cell growth,cell activity was detected by MTT.other index include LDH activity and myocardial cell MDA and the content of SOD.ResultsH9C2 cells were cultured in vitro and developed hypoxia / reoxygenation successfully.The morphology changes of H9C2 cells showed increased floating detected by inverted microscope.Compared with the control group,the activity of H9C2 cells detected by MTT in I/R groups significantly decreased,and the content of changes is more significant following the longer I/R time.In this part,the myocardial cell LDH activity and MDA were significantly increased while the SOD value decreased after hypoxia/reoxygenation compared with control group.Conclusion:The H9C2 cardiomyocytes hypoxia / reoxygenation injury model was constructed successfully in this part study.H9C2 myocardial cells showed significantly damage induced by hypoxia reoxygenation,hypoxia for 4h and reoxygenation for 6h.The second part: The effect of carvedilol on the expressions of TLR4-NF?B signal pathway and ?-arrestin in I/R H9C2 myocardium Materials and methods:H9C2 cardiomyocytes were pretreated with carvedilol(1 um,5 um,10 umol/L),TLR4 blocking antibody(20 ug/ml)or NF-?B inhibitor PDTC(100 umol/L)for 1 h,then incubated by hypoxia / reoxygenation for 24 h.The experiment were divided into 7 groups: Control group(Control group),hypoxia / reoxygenation group(I/R group),low-dose carvedilol group(Car1,carvedilol 1umol/L),medium-dose carvedilol group(Car5,Carvedilol 5 umol/L),high-dose carvedilol group(Car10,carvedilol 10 umol/L),TLR4 blocking antibody group(TLR4-Ab group,TLR4 blocking antibody 20 ug/ml)and NF-?B inhibitor PDTC group(PDTC group,PDTC 100 umol/L).MTT detected the cell activity.the apoptosis content was envaluated by TUNNEL assay.BAX and Bcl-2,apoptosis related protein,were detected by Western Blotting.the expressions of TLR4?NF-?B and ?-arrestin were detected by RT-PCR.Results:Compared with the control group,the activity of H9C2 cells detected by MTT in I/R groups significantly decreased,and the content of apoptosis detected by TUNNEL significantly increased.Expression of apoptosis related factor BAX increased and Bcl-2 decreased detected by West-Blotting.At the same time,we found the expression of TLR4 and NF-?B significantly increased in I/R groups,and ?-arrestin decreased through RT-PCR.In carvedilol pretreated H9C2 cardiomyocytes,compared with the I/R group,carvedilol protected the cardiomyocytes from I/R injury,as evidenced by increased cell activity and the level of Bcl-2,decreased apoptosis content and the level of BAX.Through RT-PCR examined,we detected TLR4 and NF-?B decreased in carvedilol pretreated groups,the expression of ?-arrestin increased.the above-mentioned indexes have in moderate and high dose carvedilol pretreated groups.Pretreated with TLR4-antibody or NF-?B inhibitor PDTC,the expression of ?-arrestin of H9C2 cardiomyocyte have significantly difference between I/R and carvedilol pretreated groups.Conclusion: 1.The experiment proved that TLR4-NF-?B signaling pathway were related with the apoptosis of cardiomyocytes.2.Carvedilol could anti-apoptosis in H9C2 cardiomyocytes I/R model through block expression of TLR4 and NF-?B.the effect were significantly in moderate and high dose groups.3.The specific role of carvedilol might be to negative regulated TLR4-NF-?B by activing ?-arrestin2.