Department of Ophthalmology, Affiliated Hospital of Inner Mongolia University for the Nationalities, No 1742, HuoLinHe Street, Neimenggu, Tongliao, 028050, China.
Department of Ophthalmology, Tongliao Hospital, No.668, KeErQin Street, Neimenggu, Tongliao, 028000, China.
Biomed Pharmacother. 2018 May;101:494-500. doi: 10.1016/j.biopha.2018.02.107. Epub 2018 Mar 22.
To determine the effectiveness of GRGM-13 on oxidative stress induced apoptosis of retinal ganglion cells (RGCs) and revealed its possible mechanism.
Caspase-3 activity, MDA level, and glutathione peroxidase level were detected by Caspase-3 assay kit, Lipid Peroxidation MDA Assay Kit, and Total Glutathione Peroxidase Assay Kit, respectively. Protein levels of Bax, Bcl-2, p-p38 and p38 were observed by Western Blot. Reactive oxygen species assay kit was used to determine intracellular ROS level. Apoptotic cells were measured by flow cytometry.
GRGM-13 inhibited apoptosis of RGCs and ROS level in rat retinal tissue and RGC-5 cells, and the decrease degree strengthened with the increase of GRGM-13 concentration. In addition, ROS upregulated p-p38 expression, while GRGM-13 reversed this effect. We also found that p38 inhibitor SB202190 did not change L-glutamate (Glu) or HO-induced ROS level, while SB202190 inhibited apoptosis of RGC-5 cells. Finally, we observed that P2 × 7R agonist BzATP reversed the inhibition effect of GRGM-13 on RGC-5 cell apoptosis, ROS level and p-p38 expression, while si-P2 × 7R inhibited oxidative stress-induced phosphorylation of p38.
GRGM-13 could inhibit oxidative stress-induced RGCs apoptosis via inhibiting P2RX7/p38 MAPK pathway, which revealed the possible mechanism of GRGM-13 on stress-induced RGCs apoptosis and provided new Chinese medicine for the treatment of glaucoma.
探讨 GRGM-13 对氧化应激诱导的视网膜神经节细胞（RGCs）凋亡的作用，并揭示其可能的作用机制。
采用 Caspase-3 活性检测试剂盒、脂质过氧化物 MDA 检测试剂盒和总谷胱甘肽过氧化物酶检测试剂盒分别检测 Caspase-3 活性、MDA 水平和谷胱甘肽过氧化物酶水平，Western blot 法观察 Bax、Bcl-2、p-p38 和 p38 蛋白水平，活性氧（ROS）检测试剂盒测定细胞内 ROS 水平，流式细胞术检测细胞凋亡。
GRGM-13 抑制大鼠视网膜组织和 RGC-5 细胞中 RGCs 的凋亡和 ROS 水平，且随着 GRGM-13 浓度的增加，抑制程度增强。此外，ROS 上调 p-p38 的表达，而 GRGM-13 逆转了这一作用。我们还发现，p38 抑制剂 SB202190 不改变 L-谷氨酸（Glu）或 H2O2 诱导的 ROS 水平，但 SB202190 抑制 RGC-5 细胞凋亡。最后，我们观察到 P2X7R 激动剂 BzATP 逆转了 GRGM-13 对 RGC-5 细胞凋亡、ROS 水平和 p-p38 表达的抑制作用，而 si-P2X7R 抑制氧化应激诱导的 p38 磷酸化。
GRGM-13 可通过抑制 P2RX7/p38 MAPK 通路抑制氧化应激诱导的 RGCs 凋亡，揭示了 GRGM-13 抑制应激诱导的 RGCs 凋亡的可能机制，为治疗青光眼提供了新的中药。
