Necrostatin-1对急性高眼压大鼠模型中视网膜神经节细胞程序性坏死的抑制作用

Inhibitory effect of necrostatin-1 on necroptosis of retinal ganglion cells in rats with acute ocular hypertension

目的探讨程序性坏死特异性抑制剂necrostatin-1(Nec-1)对急性高眼压大鼠模型中视网膜神经节细胞(RGCs)程序性坏死的抑制作用。方法采用随机数表法将24只雄性SD大鼠随机分为正常对照组、模型对照组、Nec-1处理组和阴性对照组,每组6只。其中,正常对照组大鼠不做任何处理;其余3个组大鼠左眼采用前房灌注质量分数0.9%氯化钠溶液的方法将眼压升至110 mmHg(1 mmHg=0.133 kPa),持续60 min,制备急性高眼压缺血-再灌注模型;Nec-1处理组和阴性对照组大鼠分别于造模后给予玻璃体腔内注射4 mmol/L Nec-1和二甲基亚砜各2μl。造模后7 d,摘取各组大鼠实验眼制备视网膜石蜡切片,采用苏木精-伊红染色法观察视网膜结构;采用免疫组织化学染色法检测胸腺细胞抗原1(Thy-1)和胶质纤维酸性蛋白(GFAP)的表达。结果造模后7 d,与正常对照组大鼠比较,模型对照组和阴性对照组大鼠视网膜神经纤维层均变薄,RGCs层细胞排列疏松,内核层细胞减少、排列紊乱,外核层细胞正常或变大;与模型对照组和阴性对照组比较,Nec-1处理组大鼠神经纤维层厚度及RGCs数量均明显增多。免疫组织化学染色显示,模型对照组、阴性对照组和Nec-1处理组大鼠Thy-1染色阳性RGCs数量均较正常对照组减少,Nec-1处理组大鼠Thy-1染色阳性RGCs数量均较模型对照组和阴性对照组增多。正常对照组、模型对照组、阴性对照组和Nec-1处理组GFAP平均积分吸光度(A)值分别为47.209±15.311、116.220±18.194、116.382±19.020和92.818±10.236,总体比较差异有统计学意义(F=24.675,P<0.001),其中与正常对照组比较,模型对照组、阴性对照组和Nec-1处理组GFAP平均积分A值均明显升高;Nec-1处理组GFAP平均积分A值较模型对照组和阴性对照组明显降低,差异均有统计学意义(均P<0.05)。结论Nec-1对急性高眼压大鼠RGCs的程序性坏死有抑制作用,可促进RGCs存活。

Objective To investigate the inhibitory effect of specific inhibitor of necroptosis necrostatin-1(Nec-1)on necroptosis of retinal ganglion cells(RGCs)in rats with acute ocular hypertension.Methods Twenty-four adult male Sprague Dawley rats were randomly divided into normal control group,model control group,Nec-1 treatment group and negative control group by random number table method,with 6 rats in each group.High intraocular pressure(IOP)-induced ischemia and reperfusion model was established through anterior chamber irrigation of 0.9%sodium chloride solution in left eyes of the rats,raising the IOP to 110 mmHg(1 mmHg=0.133 kPa)for 60 minutes.Nec-1(4 mmol/L,2μl)or dimethyl sulfoxide(2μl)was intravitreally injected immediately in Nec-1 treatment group and negative control group following modeling,respectively,according to grouping.No intervention was administered to the normal control group.Paraffin sections of rat retinas of the left eyes in different groups were prepared seven days after modeling.The retinal structure was observed by hematoxylin-eosin staining,and the expression levels of thymocyte antigen-1(Thy-1)and glial fibrillary acidic protein(GFAP)were detected via immunohistochemical staining.All animal experiments were approved by an Ethics Committee of Tianjin Union Medical Center(No.2017 Quick audit C01).Results Seven days after modeling,compared with normal control group,the retinal nerve fiber layer was thinner in model control group and negative control group,and the RGCs were arranged loosely,and cells in the inner nuclear layer were reduced and arranged disorderly,and cells in the outer nuclear layer were normal or enlarged.Compared with model control group and negative control group,the nerve fiber layer was thickened and the number of RGCs was significantly increased in Nec-1 treatment group.The number of Thy-1-positive RGCs was decreased in model control group,negative control group and Nec-1 treatment group than normal control group,and there were more Thy-1-positive RGCs in Nec-1 treatment group than model control group and negative control group.The integrated absorbance(A)value of GFAP protein in normal control group,model control group,negative control group and Nec-1 treatment group was 47.209±15.311,116.220±18.194,116.382±19.020,92.818±10.236,respectively,showing statistically significant differences among them(F=24.675,P<0.001).The integrated A value of GFAP protein was significantly increased in model control group,negative control group and Nec-1 treatment group than normal control group,and the integrated A value of GFAP protein in Nec-1 treatment group was lower than that in model control group and negative control group,with statistically significant differences(all at P<0.05).Conclusions Nec-1 can promote RGCs survival by inhibiting the necroptosis of RGCs in rats with acute intraocular hypertension.