ATF3在视神经损伤后大鼠视网膜的表达及作用

Expression of ATF3 and its role in retina of rats after optic nerve injury

目的探讨转录激活因子3(ATF3)表达对视神经损伤后大鼠视网膜神经节细胞存活的影响。方法成年雌性SD大鼠65只,随机选取10只大鼠作为对照组,剩余55只建立大鼠视神经损伤模型作为视神经损伤组。采用实时荧光定量PCR检测两组视网膜中ATF3 mRNA的表达;对照组大鼠、视神经损伤组于感染后3,7,14,35 d采用免疫组织化学检测ATF3的表达,免疫荧光组织化学检测胶质原纤维酸性蛋白(GFAP)的表达。取40只视神经损伤大鼠随机分为4组:NC组、ATF3过表达质粒(pIRES2/ATF3)组、阴性对照(shNC)组和ATF3-shRNA组,各组大鼠分别腹腔注射生理盐水、ATF3过表达质粒转染的复合物、shNC的复合物或ATF3-shRNA表达质粒的复合物,然后感染后0,7,14,35 d,采用OCT扫描测量视网膜神经纤维层(RNFL)厚度,CCK-8法检测视网膜神经节细胞存活情况,流式细胞术检测视网膜神经节细胞凋亡情况,免疫印迹法检测Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)蛋白的表达。结果视神经损伤组ATF3 mRNA明显高于对照组(P<0.05)。在感染后3,7,14,35 d,视神经损伤组大鼠ATF3和GFAP的表达逐渐增加,并且明显高于对照组(P<0.05)。与NC组比较,在感染后14,35 d,pIRES2/ATF3组RNFL平均厚度显著减少(P<0.05),视网膜神经节细胞存活率降低(P<0.05),pIRES2/ATF3组细胞凋亡率、Bax蛋白水平升高,Bcl-2蛋白水平降低(P<0.05)。与shNC组比较,在感染后14,35 d,ATF3-shRNA组RNFL厚度明显增加,视网膜神经节细胞存活率升高(P<0.05),细胞凋亡率、Bax蛋白水平降低,Bcl-2蛋白水平升高(均P<0.05)。结论ATF3在视神经损伤大鼠视网膜中的表达升高,ATF3能够调控视神经损伤后大鼠视网膜神经节细胞存活和凋亡能力。

Objective To investigate the effect of activating transcription factor 3(ATF3)expression on the survival of retinal ganglion cells in rats after optic nerve injury.Methods Ten adult female SD rats were randomly selected as control group,and the remaining 55 rats were chosen to establish the optic nerve injury models in optic nerve injury group.The expression of ATF3 mRNA in the retina in the two groups was detected by real-time fluorescence quantitative PCR.The expression of ATF3 in control group and optic nerve injury group at days 3,7,14 and 35 after infection was detected by immunohistochemistry,and the expression of glial fibrillary acidic protein(GFAP)was detected by immunofluorescence histochemistry.Forty optic nerve injury rats were randomly divided into four groups:NC group,ATF3 overexpression plasmid(pIRES2/ATF3)group,shNC group and ATF3-shRNA group,and the rats were intraperitoneally injected with normal saline,ATF3 overexpression plasmid transfected complex,shNC complex,and ATF3-shRNA expression plasmid complex in four groups,respectively.At days 0,7,14 and 35 after infection,the retinal nerve fiber layer(RNFL)thickness was measured by OCT scanning,the survival of retinal ganglion cells was detected by CCK-8 method,the apoptosis of retinal ganglion cells was detected by flow cytometry,and the expression levels of Bcl-2 related X protein(Bax)and B lymphocytoma-2(Bcl-2)were detected by Western blotting.Results ATF3 mRNA in optic nerve injury group was significantly higher than that in control group(P<0.05).The expression of ATF3 and GFAP in rats with optic nerve injury increased gradually at days 3,7,14,35 after infection,and it was significantly higher than that in control group(P<0.05).Compared with NC group,the average thickness of RNFL in pIRES2/ATF3 group was decreased significantly at days 14,35 after infection(P<0.05),and the survival rate of retinal ganglion cells was decreased(P<0.05).The apoptosis rate and Bax protein level in pIRES2/ATF3 group were higher than those in NC group,while Bcl-2 protein level was lower than that in NC group(P<0.05).Compared with shNC group,the thickness of RNFL and the survival rate of retinal ganglion cells were increased significantly in ATF3-shRNA group at days 14,35 after infection(P<0.05).The apoptosis rate and Bax protein level in ATF3-shRNA group were lower than those in shNC group,while Bcl-2 protein level was higher than that in shNC group(P<0.05).Conclusion The expression of ATF3 is increased in the retina of rats with optic nerve injury,and ATF3 could regulate the survival and the apoptosis of retinal ganglion cells after optic nerve injury.