Inhibitory effect of small interfering RNA-TLR9 on high glucose-induced retinal ganglion cell apoptosis / 中华实验眼科杂志

ABSTRACT

Objective To investigate the effect of Toll-like receptor 9 (TLR9) on high glucose-induced retinal ganglion cells-5 (RGC-5) apoptosis and the inhibitory effects of small interfering RNA-TLR9 (si-TLR9) on apoptosis.Methods RGC-5 cells were divided into normal control group,high glucose group,high glucose+ negative control group and high glucose + si-TLR9 group which cells were respectively dealt with normal culture medium,high glucose medium,transfection of non-specific siRNA under high glucose and transfection of siRNA-TLR9 under high glucose.The expression of TLR9 mRNA was detected by real time PCR;the survival rate of the cells was evaluated by MTT assay;the apoptotic rate of the cells was detected by flow cytometry;the caspase-3 activity in the cells was detected by related kit,and the expressions of TLR9,B cell lymphoma (bcl-2),bcl-2 associated X protein (bax),p38 mitogen-activated protein kinases (p38MAPK) and phosphorylated (p)-p38MAPK proteins were detected by Western blot.Results The expressions of TLR9 mRNA and protein in the high glucose+si-TLR9 group were significantly decreased in comparison with the high glucose+negative control group (both at P＜0.05).The cell survival rate of the high glucose group and high glucose+negative control group was (78.36±5.13)％ and (75.12± 4.25) ％,respectively,which was significantly lower than (95.48± 7.25) ％ in the normal control group,and that in the high glucose+si-TLR9 group was (86.58±5.32)％,which was significantly reduced in comparison with the high glucose+negative control group (all at P＜0.05).The apoptotic rate of the cells in the high glucose group and high glucose+negative control group was (13.23 ± 1.22) ％ and (12.52± 1.38) ％,respectively,which was significantly higher than (2.26±0.15)％ of the normal control group,and apoptotic rate in the high glucose+si-TLR9 group was (7.15±0.24) ％,which was significantly lower than that in high glucose+negative control group (all at P＜0.05).The expression of bax in the cells of the high glucose+si-TLR9 group was significantly decreased,and the expression of bcl-2 in the cells of the high glucose+si-TLR9 group was significantly increased in comparison with the high glucose+ negative control group (both at P＜ 0.05).Caspase-3 activity in the cells was significantly decreased in the high glucose+si-TLR9 group compared with the high glucose+negative control group (P＜0.05).The relative expression of p-p38MAPK in the cells was significantly decreased in the high glucose+ si-TLR9 group compared with the high glucose+negative control group (P＜0.05).Conclusions Down-regulation of TLR9 expression in the RGC-5 cells can inhibit the apoptosis induced by high glucose probablely by suppressing p38MAPK signaling pathway.