阻断DLK/MKK4/JNK/c-JUN通路对ITON小鼠视网膜神经节细胞存活的影响

Effect of blocking the DLK/MKK4/JNK/c-JUN pathway on survival of retinal ganglion cells in ITON mice

目的研究阻断DLK/MKK4/JNK/c-JUN通路的表达能否促进间接性创伤性视神经病变(ITON)小鼠视网膜神经节细胞(RGCs)的存活并比较阻断不同位点产生的阻断效果。方法使用能够产生创伤性轴突损伤(TAI)的撞击加速(IA)模型对6~8周龄的小鼠进行处理模拟间接性创伤性视神经病变,筛选出符合条件的小鼠分为4组:空白对照组,IA组,IA+sunitinib组,IA+SP600125组。在实验要求的各个时间点留取标本,进行γ-synuclein或p-c-JUN免疫荧光染色、TUNEL染色和Western blot检测,并进行统计学分析。结果免疫荧光染色结果显示,与IA组相比,IA+sunitinib组和IA+SP600125组存活的RGCs密度显著升高、p-c-JUN(+)RGCs密度显著降低(P<0.05);TUNEL检测结果显示,与IA组相比,IA+sunitinib组和IA+SP600125组凋亡的RGCs密度显著降低(P<0.05);Western blot检测结果显示,与IA组相比,IA+sunitinib组和IA+SP600125组DLK/MKK4/JNK/c-JUN通路中的相应下游蛋白表达水平均降低(P<0.05)。结论DLK/MKK4/JNK/c-JUN通路的激活与ITON小鼠中RGCs的存活率相关,应用DLK抑制剂或JNK抑制剂可有效阻断该通路的蛋白表达,并促进RGC的存活,且后者效果更佳。

Objective To investigate whether blocking the DLK/MKK4/JNK/c-JUN pathway can promote the survival of retinal ganglion cells(RGCs)in mice with indirect traumatic optic neuropathy(ITON)and compare the blocking effects of different sites.Methods 6-8 weeks old mice were treated with the impact acceleration(IA)model capable of generating traumatic axonal injury(TAI)to simulate indirect traumatic optic neuropathy(ITON),then eligible mice were screened out and divided into four groups:a shamoperation group,an IA group,an IA+sunitinib group and an IA+SP600125 group.Samples collected at different times of the experiment were detected byγ-synuclein or p-c-JUN immunofluorescence staining,TUNEL staining and Western blot,and the results were statistically analyzed.Results Immunofluorescence staining results showed that compared with IA group,the survival RGCs density of IA+sunitinib group and IA+SP600125 group increased significantly,while the density of p-c-JUN(+)RGCs decreased remarkably(P<0.05).TUNEL test results showed that compared with IA group,the apoptosis RGCs density in both IA+sunitinib group and IA+SP600125 group reduced dramatically(P<0.05).Western blot results showed that compared with the IA group,the expression of corresponding downstream proteins in the DLK/MKK4/JNK/cJUN pathway in IA+sunitinib group and IA+SP600125 group decreased noticeably(P<0.05).Conclusions The activation of the DLK/MKK4/JNK/C-JUN pathway is related to the survival rate of RGCs in ITON mice.The application of DLK inhibitors or JNK inhibitors can effectively block the expression of the pathway and promote the survival of RGC,with the latter blocking method having a better effect.