Role of Toll-like receptor 4 in cardiomyocyte apoptosis induced by hypoxia-reoxygenation in newborn rats / 中华麻醉学杂志

ABSTRACT

Objective To evaluate the role of Toll-like receptor 4 (TLR4) in cardiomyocyte apoptosis induced by hypoxia-reoxygenation (H/R) in newborn rats.Methods Cardiomyocytes obtained from newborn Sprague-Dawley rats,aged 1-3 days,were primarily cultured in DMEM culture medium supplemented with 20％ fetal bovine serum and divided into 4 groups (n=10 each) using a random number tablecontrol group (group C),H/R group,negative control group (con-siRNA group) and TLR4-siRNA group.Cardiomyocytes were routinely cultured for 24 h in group C.Cardiomyocytes were subjected to hypoxia for 2 h followed by 4 h of reoxygenation in group H/R.Cardiomyocytes were transfected with siRNA and TLR4-siRNA at the final concentration of 80 nmol/L in con-siRNA group and TLR4-siRNA group,respectively.After successful establishment of H/R injury model,the cell survival rate was measured by MTT assay,flow cytometry was used to detect apoptosis in cardiomyocytes,the expression of TLR4 mRNA was determined by real-time polymerase chain reaction,and the expression of TLR4,Bcl-2,Bax and caspase-3 was detected by Western blot.The apoptosis rate was calculated.Results Compared with group C,the cell survival rate was significantly decreased,the expression of TLR4 protein and mRNA,caspase-3 and Bax was up-regulated,the apoptosis rate was increased and the expression of Bcl-2 was down-regulated in the other three groups (P＜0.01).Compared with H/R and con-siRNA groups,the cell survival rate was significantly increased,the expression of TLR4 protein and mRNA,caspase-3 and Bax was down-regulated,the apoptosis rate was decreased and the expression of Bcl-2 was up-regulated in group TLR4-siRNA (P＜0.01).Conclusion TLR4 is involved in cardiomyocyte apoptosis induced by H/R in newborn rats.