Role of Toll-like receptor 4 in cardiomyocyte apoptosis induced by hypoxia-reoxygenation in newborn rats

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 table: control 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. Key words: Toll-like receptor 4; RNA, small interfering; Myocytes, cardiac; Anoxia; Apoptosis