Human lens epithelium of patients with age-related cataract (ARC), both SRA01/04 cells and whole mice lens stimulated by H2O2 were employed. VEGF in human aqueous humor of ARC-patients and the supernatant of SRA01/04 cells was determined by ELISA. The expressions of VEFG in human lens epithelium were detected by immunofluorescence staining. Multiple linear regression analysis and spearman rank-order correlation were used to determine the associations between VEGF and parameters of ARC individuals. In H2O2-induced SRA01/04 cells, Catalase (CAT), PP1 (inhibitor of c-Src kinase) and Avastin (VEGF antibody) were used to inhibit the effects of H2O2, activation of c-Src kinase and VEGF, which were detected by Western blot. The alterations of ZO-1 and N-cadherin were tested by immunofluorescence staining and Western blot. In H2O2-induced whole lens, the changes of opacification area in different treatment of inhibitors were observed.
The secretion of VEGF in aqueous humor and expression of VEGF in the lens epithelium of ARC patients increased significantly with age. In H2O2-induced SRA01/04 cells, the VEGF in the supernatant was increased with the culture duration and the dose of H2O2. The expressions of p-Src418 and VEGF were also up-regulated, whereas the expressions of ZO-1 and N-cadherin were down-regulated. CAT effectively prevented these changes induced by H2O2, while PP1 inhibited not only p-Src418 but also up-regulation of VEGF, Avastin partially inhibited VEGF up-regulation. Both PP1 and Avastin prevented down-regulation of ZO-1 and N-cadherin, respectively, but Avastin combined with PP1 had no significant synergistic effects. In H2O2-induced cataract, CAT prevented development of opacification area effectively, and PP1 and Avastin did partially.
Oxidative stress disrupts connections of lens epithelial cells by activating c-Src/VEGF, inhibiting which may prevent cataract.
■年龄相关性白内障(ARC)患者的人晶状体上皮,使用SRA01/04细胞和H2O2刺激的整个小鼠晶状体。通过ELISA测定ARC患者的人房水和SRA01/04细胞的上清液中的VEGF。免疫荧光染色检测人晶状体上皮中VEFG的表达。使用多元线性回归分析和spearman等级相关来确定VEGF与ARC个体参数之间的关联。在H2O2诱导的SRA01/04细胞中,过氧化氢酶(CAT),PP1(c-Src激酶抑制剂)和Avastin(VEGF抗体)用于抑制H2O2的作用,通过Westernblot检测。通过免疫荧光染色和Westernblot检测ZO-1和N-cadherin的变化。在H2O2诱导的整个晶状体中,观察不同抑制剂处理后混浊面积的变化。
■随着年龄的增长,ARC患者房水VEGF的分泌和晶状体上皮VEGF的表达明显增加。在H2O2诱导的SRA01/04细胞中,上清液中的VEGF随培养时间和H2O2剂量的增加而增加。p-Src418和VEGF的表达也上调,而ZO-1和N-cadherin的表达下调。CAT有效地阻止了H2O2诱导的这些变化,而PP1不仅抑制了p-Src418,还抑制了VEGF的上调,阿瓦斯汀部分抑制VEGF上调。PP1和阿瓦斯汀均可防止ZO-1和N-钙黏着蛋白的下调,分别,但阿瓦斯丁联合PP1无明显协同作用。在H2O2诱导的白内障中,CAT有效阻止了混浊区的发展,PP1和阿瓦斯汀部分出现了。
■氧化应激通过激活c-Src/VEGF破坏晶状体上皮细胞的连接,抑制可以预防白内障。