Objective

To investigate the impact and the mechanism of Gadd45α mediating p38MAPK pathway on the retinal ganglion cells (RGCs) injury in chronic ocular hypertension (COH) rats.

Methods

COH model in rats were established and intraocular pressure (IOP) was tested. Retrograde labeling was used for counting RGCs and TUNEL staining was performed for RGCs apoptosis. Western Blotting was conducted to examine the expression of Gadd45α and p38MAPK pathway. Besides, RGC-5 cells cultured in vitro were treated with H₂O₂. Cell viability was detected by CCK-8, ROS level tested by DCFH-DA assay, and cell apoptosis examined by flow cytometry.

Results

COH rats had increased expression of Gadd45α and p-p38/p38 protein 1–4 weeks after surgery. Rats in COH group enhanced obviously in IOP, RGC apoptosis rate and the protein expression of Gadd45α, p-p38/p38, Bax/Bcl-2 and cleaved caspase-3, but declined appreciably in RGC counting. However, the above indicators of COH rats were effectively improved by Gadd45α shRNA treatment. Additionally, RGC-5 cells in H₂O₂ group reduced in cell viability and went up in ROS level and apoptosis rate. The H₂O₂-induced RGC-5 cells treated with Gadd45α shRNA were improved apparently in those indicators, and cells treated with pcDNA Gadd45α showed an opposite trend. Moreover, p38 MAPK inhibitor SB203580 can effectively reverse the damage of pcDNA Gadd45α from H₂O₂-induced RGC-5 cells.

Conclusion

Silencing Gadd45α can reduce the RGC damage in COH rats by inhibiting p38MAPK pathway and such a protective role may be associated with the suppression of RGC apoptosis and the mitigation of oxidative stress.

中文翻译：

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Gadd45α通过调节p38MAPK途径影响慢性高眼压大鼠的视网膜神经节细胞损伤。

目的

探讨Gadd45α介导p38MAPK通路对慢性高眼压（COH）大鼠视网膜神经节细胞（RGCs）损伤的影响及其机制。

方法

建立大鼠COH模型并测试眼压（IOP）。逆行标记用于计数RGC，TUNEL染色用于RGC的凋亡。进行了Western Blotting检测Gadd45α和p38MAPK通路的表达。此外，将体外培养的RGC-5细胞用H ₂ O _2处理。通过CCK-8检测细胞活力，通过DCFH-DA测定法检测ROS水平，并且通过流式细胞术检查细胞凋亡。

结果

术后1-4周，COH大鼠的Gadd45α和p-p38 / p38蛋白表达增加。COH组大鼠眼内压，RGC凋亡率，Gadd45α，p-p38 / p38，Bax / Bcl-2和裂解的caspase-3蛋白表达均明显升高，但RGC计数却明显下降。然而，Gadd45αshRNA处理有效改善了COH大鼠的上述指标。另外，H ₂ O ₂组的RGC-5细胞活力降低，ROS水平和凋亡率升高。H ₂ O ₂用Gadd45αshRNA处理的RGC-5诱导的RGC-5细胞在这些指标上有明显改善，而用pcDNAGadd45α处理的细胞则显示出相反的趋势。此外，p38 MAPK抑制剂SB203580可有效逆转H ₂ O ₂诱导的RGC-5细胞对pcDNAGadd45α的损伤。

结论

沉默Gadd45α可以通过抑制p38MAPK途径减少COH大鼠RGC的损伤，这种保护作用可能与抑制RGC的凋亡和减轻氧化应激有关。