Eye Center, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan, 430060, Hubei Province, China.
Eye Center, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan, 430060, Hubei Province, China.
Exp Eye Res. 2021 Jun;207:108587. doi: 10.1016/j.exer.2021.108587. Epub 2021 Apr 21.
The purpose of this study is to investigate the effects of Krüppel-like factor 7 (KLF7) on retinal ganglion cells (RGCs) and retinal function after retinal ischemia-reperfusion (RIR) injury in mice.
Male C57BL/6J mice were intravitreally injected with recombinant adeno-associated vectors (rAAV-KLF7-EGFP or rAAV-EGFP), and subsequently used to induce RIR injury. Retinal cryosections were used to access the efficacy of virus transfection, 1, 2, 3, and 4 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rate was observed and quantified by immunofluorescent staining, 7 days after RIR injury. Meanwhile, electroretinogram (ERG) and optomotor response were used to evaluate the electrophysiological functions and visual acuity. Apoptosis was evaluated by TUNEL staining 1 day after RIR injury. Expression of KLF7, Akt, phospho-Akt, Bcl-2, and Bax were further detected by western blot to excavate the underlying mechanism.
The transfection efficiency of rAAV-KLF7-EGFP was increased in a time-dependent manner, and the number of EGFP-positive cells was increased significantly 3 weeks after rAAV-KLF7-EGFP transfer. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were decreased after RIR injury. With the increase of light intensity, the amplitudes of scotopic ERG a- and b-wave were gradually increased while the incubation period was gradually shortened. RGCs survival rates, amplitudes of ERG a-, b-wave, Ops, PhNR, and visual acuity of mice were increased after rAAV-KLF7-EGFP transfer. The protein level of KLF7 was up-regulated after rAAV-KLF7-EGFP transfer. Up-regulation of KLF7 significantly inhibited cells apoptosis, increased phospho-Akt and Bcl-2 expression, and decreased Bax expression. There were no significant changes in Akt expression.
Overexpression of KLF7 can not only prevent the loss of RGCs, but also preserve the electrophysiological function. In addition, overexpression of KLF7 can ameliorate the retinal dysfunction after RIR injury, and ultimately improve the visual acuity of mice. The activation of Akt pathway and the suppression of the mitochondrial apoptotic pathway contribute to the neuroprotection of KLF7.
本研究旨在探讨 Krüppel 样因子 7（KLF7）对小鼠视网膜缺血再灌注（RIR）损伤后视网膜神经节细胞（RGCs）和视网膜功能的影响。
雄性 C57BL/6J 小鼠玻璃体腔注射重组腺相关病毒（rAAV-KLF7-EGFP 或 rAAV-EGFP），随后诱导 RIR 损伤。转染 rAAV-KLF7-EGFP 后 1、2、3、4 周，通过视网膜冰冻切片评估病毒转染的效果。RIR 损伤后 7 天，通过免疫荧光染色观察和定量 RGC 存活率。同时，通过视网膜电图（ERG）和光动反应评估视网膜的电生理功能和视力。RIR 损伤后 1 天，通过 TUNEL 染色评估细胞凋亡。通过 Western blot 进一步检测 KLF7、Akt、磷酸化 Akt、Bcl-2 和 Bax 的表达，以挖掘潜在的机制。
rAAV-KLF7-EGFP 的转染效率呈时间依赖性增加，转染 rAAV-KLF7-EGFP 3 周后，EGFP 阳性细胞数量明显增加。RIR 损伤后，小鼠的 RGCs 存活率、ERG a-和 b-波的振幅、Ops、PhNR 和视力均降低。随着光照强度的增加，暗适应 ERG a-和 b-波的振幅逐渐增加，潜伏期逐渐缩短。rAAV-KLF7-EGFP 转染后，小鼠的 RGCs 存活率、ERG a-和 b-波的振幅、Ops、PhNR 和视力均增加。rAAV-KLF7-EGFP 转染后，KLF7 蛋白水平上调。KLF7 的上调显著抑制细胞凋亡，增加磷酸化 Akt 和 Bcl-2 的表达，减少 Bax 的表达。Akt 表达无明显变化。
KLF7 的过表达不仅可以防止 RGCs 的丢失，还可以保护视网膜的电生理功能。此外，KLF7 的过表达可以改善 RIR 损伤后的视网膜功能障碍，最终提高小鼠的视力。Akt 通路的激活和线粒体凋亡通路的抑制有助于 KLF7 的神经保护作用。
