Inhibitory effect of small interfering RNA-TLR9 on high glucose-induced retinal ganglion cell apoptosis

Authors: Yang Huihui,  Kan Quane,  Yu Lu,  Li Xuqing,  Huang Aiguo,  Yuan Huijuan

DOI: 10.3760/cma.j.issn.2095-0160.2019.10.008
Published 2020-01-10
Cite as Chin J Exp Ophthalmol, 2020,38(01): 38-44.

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.

Key words:

Toll-like receptor 9; Small interfering RNA; Retinal ganglion cells; High glucose; Apoptosis; Cell culture

Contributor Information

Yang Huihui
Department of Endocrinology, Henan Provincial People’s Hospital, Zhengzhou 450003, China
Kan Quane
Department of Endocrinology, Henan Provincial People’s Hospital, Zhengzhou 450003, China
Yu Lu
Department of Endocrinology, Henan Provincial People’s Hospital, Zhengzhou 450003, China
Li Xuqing
Department of Endocrinology, Henan Provincial People’s Hospital, Zhengzhou 450003, China
Huang Aiguo
Henan Provincial People’s Hospital, Henan Eye Hospital, Henan Eye Institute, Henan Key Laboratory of Ophthalmology and Visual Sciences, People’s Hospital of Zhengzhou University, People’s Hospital of Henan University, Zhengzhou 450003, China
Yuan Huijuan
Department of Endocrinology, Henan Provincial People’s Hospital, Zhengzhou 450003, China
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Updated: March 17, 2020 — 8:41 am