Dexmedetomidine Preconditioning Attenuates Hypoxia/Reoxygenation Injury By Suppression Of TLR4/NF-?B Signal Pathway In Rat Cardiomyocytes

Part One: Dexmedetomidinepreconditioning attenuates inflammatoryresponse induced by hypoxia/reoxygenation injury of cardiomyocytes.Objective To observe the effect of the dexmetomidine(Dex)preconditioning on the inflammatory response of cardiomyocytes exposed tohypoxia/reoxygenation injury.Methods Rat cardiomyocytes were cultured in vitro(H9C2 cell line and primary neonatal rat cardiomyocytes).Cardiomyocytes were divided into five groups: control group(C);H/R group(H/R);Dex(0.1?M,1?M,10?M)preconditioning group(D+H/R).Cell viability,lactate dehydrogenasel(LDH)activity,m RNA expression of TNF-?,IL-6 and IL-1? were detected,the morphology of cardiomyocytes was observed by inverted microscope.Results After reperfusion,the survival rate of cardiomyocytes was markedly decreased and higher LDH levels in the H/R group compared with that in the control group(p<0.01).Dex preconditioning improved the cell survival rate,and the concentration of 1 ?M resulted in the most significant improvement compared with the H/R group(p<0.05).Similarly,the most significant reduction was detected in LDH levels when 1 ?M of DEX was applied(p<0.01).The results of quantitative RT-PCR showed that the levels of IL-1?,TNF-? and IL-6 m RNA expression were significantly increased in the H/R group compared with the control group(p<0.01).Dex preconditioning partly blocked the elevation of these inflammatory factors(p<0.05).The condition of cell necrosis,irregular cell arrangement,and the decline of cell membrane refractive index caused by H/R injury were partly reversed by Dex.The same effects of Dex were found in H9C2 cell line and primary neonatal rat cardiomyocytes.Conclusion These results suggest that DEX preconditioning provides protective effects for cardiomyocytes following H/R injury by alleviate inflammation.Part Two: The effect of dexmedetomidine preconditioningon TLR4/NF-?Bsignaling pathway of cardiomyocytes induced by hypoxia/reoxygenationinjuryObjective To observe the effect of Dex preconditioning on TLR4 / NF-?B pathway of cardiomyocyts exposed to hypoxia / reoxygenation injury.Methods Rat cardiomyocytes were cultured in vitro(H9C2 cell line and primary neonatal rat cardiomyocytes).The effect of Dex on TLR4 / NF-?B signaling pathway in cardiomyocytes hypoxia / reoxygenation injury was investigated by knockdown and overexpression of TLR4 gene.Cardiomyocytes were divided into four groupsrespectively.Control group(C);H/R group(H/R);DEX(1?M)preconditioning group(D+H/R);TLR4 si RNA group(TLR4 si RNA +H/R).H/R group(H/R);DEX(1?M)preconditioning group(D+H/R);TLR4 DNA group(TLR4 DNA+D+H/R);Control DNA group(Control DNA +D+H/R).Cell viability,lactate dehydrogenase(LDH)activity,m RNA expression of TNF-?? IL-6 and IL-1?,protein levels of TLR4 and nuclear NF-?B p65 were detected.Results Compared with the H/R group,Dex preconditioning and TLR4 knockdown resulted in significantly higher cell survival rate(p<0.05)and lower LDH levels(p<0.05);The expressions of IL-1?,TNF-?,and IL-6 m RNA were suppressed by Dex preconditioning or TLR4knockdown(p<0.05).TLR4 knockdownwas also found to significantly inhibit the levels of TLR4 and nuclear NF-?Bp65 protein(p<0.05).TLR4 overexpression reversed the effects of Dex preconditioning on cell survival rate,LDH,the levels of IL-1?,TNF-?,IL-6 and TLR4 and nuclear NF-?Bp65protein compared and with the D group(p<0.05),while con DNA transfection didn't affect the results of Dex.The same effects of Dex were found in H9C2 cell line and primary neonatal rat cardiomyocytes.Conclusion These results suggest that Dex preconditioning provides protective effects for cardiomyocytes following H/R injury by down-regulationof the TLR4-NF-?B signaling pathway.