特异性阻断剂Nec-1对视网膜缺血再灌注损伤模型小鼠坏死性凋亡的影响及作用

摘要

目的 探讨特异性阻断剂Nec-1对视网膜缺血再灌注损伤(retinal ischemia reperfusion injury,RIRI)模型中坏死性凋亡的影响及作用.方法 取60只野生型C57小鼠随机分为实验组、对照组及空白组.每组20只.在进行Nec-1对坏死性凋亡影响研究中,空白组15只小鼠不做任何处理,实验组15只小鼠给予玻璃体内注射2 μL Nec-1 (2 mol·L-1)预处理,对照组15只小鼠无预处理;预处理4h后实验组及对照组通过前房灌注法建立RIRI模型;再灌注损伤后3d,每种检测方法各取5只小鼠分别于取材后行Western blot、免疫荧光定量PCR、免疫荧光染色,检测以下基因mRNA和蛋白的表达变化:IL-13、IL-6、TNF-α、RIP3、RIP1及Caspase-8.在进行Nec-1对视网膜神经节细胞(retinal ganglion cell,RGC)的影响研究中,实验组、对照组及空白组各5只小鼠纳入荧光金标记.空白组小鼠荧光金标记后不做任何处理.荧光金标记7d后,实验组小鼠给予玻璃体内注射2μL Nec-1(2 mol·L-1)预处理,对照组无预处理.预处理4h后实验组及对照组通过前房灌注法建立RIRI模型.再灌注损伤后3d,收集视网膜组织行铺片RGC计数研究.结果 与对照组相比,实验组RIP3、RIPI的mRNA表达明显降低,差异均有统计学意义(均为P ＜0.001);IL-1β、IL-6、TNF-α的mRNA表达亦均明显降低,差异均有统计学意义(均为P＜0.001);Caspase-8的表达变化不明显,与对照组相比差异无统计学意义(P =0.654 8).通过Western blot检测发现,实验组RIP3蛋白表达较对照组明显降低,其变化趋势与RIP3 mRNA水平的变化基本一致.通过视网膜切片免疫荧光染色检测亦发现,实验组RIP3蛋白表达较对照组明显降低.实验组全视网膜铺片中每个视野下荧光金逆行标记RGC数(197.3±3.6)较对照组(107.5±6.1)明显增多,差异有统计学意义(P＜0.001).结论 实验性RIRI模型中,Nec-1能阻断坏死性凋亡,并显著增加RGC数.%Objective To observe the influence of necrostatin-1 (Nec-1) on necroptosis and retinal ganglion cells (RGC) in mice with retinal ischemia reperfusion injury (RIRI).Methods Together 60 wild-type C57 mice were randomly divided into three groups (n =20),and they were control group,experimental group and blank group.Firstly,as for investigation the effect of Nec-I on necroptosis,15 mice in the blank group left untreated,15 mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,and 15 in the control group without pretreatment.After 4 h,the RIRI model was established by anterior chamber perfusion in the latter two groups.Then retinas in 5 mice in each group were harvested 3 days after ischemia reperfusion injury for Western blot,immunofluorescence quantitative PCR and immunofluorescence staining to detect the expression of mRNA and protein of IL-1β,IL-6,TNF-α,RIP3,RIP1 and Caspase-8.Secondly,the rest of 5 mice in 20 of each group were collected and retrogradely labeled with fluoro-gold (FG) to explored the influence of Nec-1 on RGC.Mice in the blank group left untreated.Mice in the experimental group were injected with 2 μL Nec-1 (2 mol · L-1) intravitreally,while the control group was not treated anything 7 days after fluorescence labeling;after 4 h,the RIRI model was established by anterior chamber perfusion in the two groups.The retinal tissue was harvested and RGC counting was performed 3 days after ischemia reperfusion injury.Results When compared with control group,mRNA expression levels of IL-1β,IL-6,TNF-α,RIP3 and RIP1 in the experimental group were decreased significantly (all P ＜ 0.001),but Caspase-8 mRNA did not change obviously (P =0.654 8).Western blot and immunofluorescence staining showed that RIP3 expression decreased dramatically in the experimental group when compared with the control group.FG labeled RGC counting presented that RGC number of each field in experimental group was significantly larger than that in the control group (P ＜ 0.001).Couclusion Nec-1 can block necroptosis and significantly increase RGC number in mice model of experimental RIRI.