摘要
目的观察过表达S100A4蛋白对视网膜缺血再灌注损伤(RIRI)后视网膜毛细血管细胞和视网膜神经节细胞(RGC)的影响。方法采用随机数字表法将100只健康雄性成年C57BL/6小鼠分为正常对照组(C组)、RIRI组、玻璃体腔注射腺相关病毒(AAV2)-S100A4-绿色荧光蛋白(GFP)组(S组)、RIRI+玻璃体腔注射AAV2-GFP组(GIR组)、RIRI+玻璃体腔注射AAV2-S100A4-GFP组(SIR组),每组各20只。RIRI组、GIR组、SIR组小鼠采用前房高眼压法建立RIRI模型。建模后3 d过量麻醉处死摘除眼球。视网膜胰蛋白酶消化铺片和苏木精-伊红、高碘酸-雪夫染色观察各组小鼠视网膜毛细血管内皮细胞、周细胞数量;免疫荧光染色观察内皮细胞、周细胞覆盖率和RGC存活率;蛋白质免疫印迹法检测小鼠视网膜组织中Toll-样受体4(TLR4)、p38蛋白、核因子E2相关因子2(NRF2)蛋白相对表达量。多组间数据比较采用单因素方差分析。结果建模后3 d,内皮细胞与周细胞比值:C组与S组、SIR组比较,差异均无统计学意义(F=106.30,P>0.05);SIR组与RIRI组、GIR组比较,差异均有统计学意义(F=106.30,P<0.0001)。内皮细胞覆盖率:各组比较,差异无统计学意义(F=3.44,P>0.05)。周细胞覆盖率:与C组比较,RIRI组、GIR组显著降低,差异均有统计学意义(F=62.69,P<0.001)。RGC存活率:与C组比较,RIRI组、GIR组明显降低,差异有统计学意义(F=171.60,P<0.0001);与RIRI组、GIR组比较,SIR组明显增高,差异有统计学意义(F=171.60,P<0.0001)。TLR4、p38、NRF2蛋白相对表达量:各组比较,差异均有统计学意义(F=42.65、20.78、11.55,P<0.05)。结论RIRI后,周细胞较内皮细胞对缺血更敏感,早期毛细血管细胞变性以周细胞丢失为主,而不是内皮细胞。S100A4蛋白过表达通过抑制TLR4/p38/NRF2信号通路保护了RIRI后周细胞和RGC的丢失。
Objective To observe the effects of overexpression of S100A4 protein on retinal capillary cells and retinal ganglion cells(RGC)after retinal ischemia-reperfusion injury(RIRI).Methods One hundred healthy adult male C57BL/6 mice were randomly divided into normal control group(group C),RIRI group,adeno-associated virus(AAV2)-S100A4 green fluorescent protein(GFP)intravitreal injection group(group S),RIRI+AAV2-GFP intravitreal injection group(group GIR),and RIRI+AAV2-S100A4-GFP intravitreal injection group(group SIR),with 20 mice in each group.The RIRI model was established using the high intraocular pressure anterior chamber method in the RIRI,GIR and SIR groups of mice.Eyes were enucleated 3 days after modelling by over anaesthesia.The number of retinal capillary endothelial cells and pericytes in the retinal capillaries of mice in each group was observed by retinal trypsinised sections and hematoxylin-eosin and periodic acid-Schiff staining;immunofluorescence staining was used to observe endothelial cell,pericyte coverage and RGC survival;The relative expression of Toll-like receptor 4(TLR4),p38 MAPK and nuclear factor erythroid 2-related factor 2(NRF2)in retinal tissues was measured by Western blot.One-way analysis of variance was used to compare data between groups.Results Three days after modeling,the endothelial cell to pericyte ratio in group C was compared with group S and SIR,and the difference was not statistically significant(F=106.30,P>0.05);the SIR group was compared with group RIRI and GIR,and the difference was statistically significant(F=106.30,P<0.0001).Comparison of endothelial cell coverage in each group,the difference was not statistically significant(F=3.44,P>0.05);compared with the pericyte coverage in group C,the RIRI group and the GIR group were significantly lower,and the difference was statistically significant(F=62.69,P<0.001).Compared with the RGC survival rate in group C,it was significantly lower in RIRI and GIR groups,and the difference was statistically significant(F=171.60,P<0.0001);compared with RIRI and GIR groups,the RGC survival rate in SIR group was significantly higher,and the difference was statistically significant(F=171.60,P<0.0001).The relative expression levels of TLR4,p38 and NRF2 proteins were statistically significant among all groups(F=42.65,20.78,11.55;P<0.05).Conclusions Pericytes are more sensitive to ischemia than endothelial cells after retinal RIRI in mice,and early vascular cell loss is dominated by pericytes rather than endothelial cells.The overexpression of S100A4 protein protects against loss of pericytes and RGC after RIRI by inhibiting the TLR4/p38/NRF2 signaling pathway.
作者
潘奕吉
杨家翼
邢怡桥
贺涛
Pan Yiji;Yang Jiayi;Xing Yiqiao;He Tao(Eye Center,Renmin Hospital of Wuhan University,Wuhan 430060,China)
出处
《中华眼底病杂志》
CAS
CSCD
北大核心
2024年第4期296-302,共7页
Chinese Journal of Ocular Fundus Diseases