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目的研究细胞凋亡信号调节激酶1(apoptosis signal-regulating kinase1,ASK1)对高糖诱导的视网膜神经节细胞(retinal ganglion cell,RGC)损伤的保护作用及其可能的机制。方法建立大鼠RGC细胞的分离培养和体外高糖(50 mmol/L葡萄糖)损伤模型。转染ASK1 siRNA沉默ASK1的表达。实时荧光定量PCR(qRT-PCR)检测ASK1 mRNA表达水平;Western blot检测ASK1、JNK蛋白表达量和c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)磷酸化水平;MTT和CCK-8方法、Ki67染色分别检测RGC生长活力和增殖情况;Annexin-V FITC/PI方法和TUNEL检测RGC凋亡情况;试剂盒方法检测Caspase-3活性。结果 RGC高糖环境下培养4d,细胞生长活力降低明显(P<0.05);ASK1表达水平在高糖损伤的RGC中明显上升(P<0.05);ASK1 siRNA的转染实验结果显示:ASK1的表达水平明显下降(P<0.05),说明沉默表达实验成功;ASK1表达被抑制后RGC的生长活力(P<0.05)和增殖能力(P<0.05)明显上升,RGC的凋亡(P<0.01)和Caspase-3的活力(P<0.01)明显降低。沉默ASK1的表达有效地降低JNK蛋白的磷酸化水平。结论抑制ASK1的表达对RGC的高糖损伤有保护作用,其机制可能与抑制JNK通路有关。
Objective To investigate the protective effect of apoptosis signal-regulating kinase 1 (ASK1) on high glucose-induced retinal ganglion cell (RGC) injury and its possible mechanism. Methods The rat RGC cells were isolated and cultured in vitro and the injury model of high glucose (50 mmol / L glucose) in vitro was established. ASK1 siRNA transfection silence ASK1 expression. The expression of ASK1 and JNK protein and the phosphorylation of c-Jun N-terminal kinase (JNK) were detected by Western blot. The expressions of MTT and CCK-1 were detected by qRT-PCR. 8 and Ki67 staining respectively. The apoptosis of RGCs was detected by Annexin-V FITC / PI assay and TUNEL assay. Caspase-3 activity was detected by kit assay. The results showed that the expression of ASK1 was significantly increased in RGC induced by high glucose (P <0.05). The results of ASK1 siRNA transfection showed that the expression of ASK1 (P <0.05), and the level of RGC decreased significantly (P <0.05), indicating that the silencing expression was successful; the growth vigor and proliferation ability of RGC were significantly increased (P <0.01) Caspase-3 activity (P <0.01) was significantly lower. Silencing ASK1 expression effectively reduces the phosphorylation of JNK protein. Conclusion Inhibition of ASK1 expression has a protective effect on RGC induced by high glucose, and its mechanism may be related to the inhibition of JNK pathway.