siRNA沉默FoxO1对STZ诱导的糖尿病大鼠视网膜IL-1β的影响及其作用机制

摘要

目的:探讨抑制叉头状转录因子O1(FoxO1)表达对糖尿病大鼠视网膜IL-1β的影响及其作用机制.方法:将人视网膜微血管内皮细胞(HRCECs)用不同浓度葡萄糖处理后,采用RT-PCR和Western blot检测细胞FoxO1、IL-1β表达.HRCECs按照处理方式分为空白对照组(Mock组)、阴性对照组(NC-FoxO1组)和si-FoxO1组(si-FoxO1组),RT-PCR检测各组细胞FoxO1、IL-1β mRNA表达,Western blot检测各组细胞FoxO1、IL-1β、p-P38、p-JNK、p-ERK蛋白表达.60只大鼠随机分为对照组(Control组)、DM组、si-FoxO1转染组(LV-si-FoxO1组)和空病毒转染组(LV-NC组),DM组、LV-si-FoxO1组、LV-NC组建立糖尿病大鼠模型,造模成功后向大鼠玻璃体腔注射LV-si-FoxO1或LV-NC慢病毒载体,注射12周后分离视网膜组织,分别采用免疫组织化学染色、RT-PCR和Western blot检测大鼠视网膜组织FoxO1、IL-1β表达.结果:与5 mmol·L-1葡萄糖浓度组比较,15 mmol·L-1浓度组FoxO1、IL-1β mRNA和蛋白表达差异无统计学意义(P＞0.05),25 mmol·L-1浓度组FoxO1、IL-1β mRNA和蛋白表达明显高于5 mmol·L-1浓度组,而35 mmol·L-1浓度组FoxO1、IL-1β mRNA和蛋白表达增加的更明显,组间比较差异具有统计学意义(P＜0.05).si-FoxO1组IL-1β mRNA和蛋白表达明显低于NC-FoxO1组和Mock组(P＜0.05),NC-FoxO1组和Mock组IL-1β mRNA和蛋白表达比较差异无统计学意义(P＞0.05).DM组和LV-NC组FoxO1、IL-1β mRNA和蛋白表达明显高于Control组和LV-si-FoxO1组(P＜0.05),LV-NC组与Control组FoxO1、IL-1β表达差异无统计学意义(P＞0.05),DM组和LV-NC组FoxO1、IL-1β表达差异无统计学意义(P＞0.05).si-FoxO1组p-P38、p-JNK、p-ERK蛋白表达明显低于NC-FoxO1组和Mock组(P＜0.05),NC-FoxO1组和Mock组p-P38、p-JNK、p-ERK蛋白表达比较差异无统计学意义(P＞0.05).结论:高糖能够诱导HRCECs FoxO1、IL-1β表达增加,抑制FoxO1表达可能是通过降低MAPK磷酸化下调HRCECs细胞IL-1β表达实现的.%Objective:To explore the effect of FoxO1 inhibition in the expressions of IL-1β in the diabetic rats retina induced by STZ and its mechanism.Methods:The human retinal capillary endothelial cells (HRCECs) were treated by different concentration of glucose.The expressions of FoxO1,IL-1β were measured by RT-PCR and Western blot.The HRCECs were divided into Mock group,NC-FoxO1 group and si-FoxO1 group,the expressions of FoxO1,IL-1β mRNA were measured by RT-PCR,the expressions of FoxO1,IL-1β,p-P38,p-JNK,p-ERK protein were measured by Western blot.60 rats were randamly divided into Control group,DM group,LV-si-FoxO1 group and LV-NC group,the diabetic rats models were established in DM group,LV-si-FoxO1 group and LV-NC group.After models were estabilished sucessfully,the LV-si-FoxO1 or LV-NC lentiviral vector was injected in the vitreous cavity of diabetic rats,the retinal tissue was completely separated at the end of 12 weeks,the expressions of FoxO1,IL-1β in retinal tissue were measured by immunohistochemical staining,RT-PCR and Western blot.Results:Compared to the 5 mmol · L-1 glucose group,the expressions of FoxO1,IL-1 β mRNA and protein in 15 mmol · L-1 glucose group had no significant differences (P ＞0.05),the expressions of FoxO1,IL-1β mRNA and protein in 25 mmol · L-1 glucose group were significantly higher than those in 5 mmol · L-1 glucose group,and FoxO1,IL-1 β mRNA and protein in 35 mmol · L-1 glucose group increased more obviously,the difference between groups was statistically significant (P ＜ 0.05).The expressions of IL-1β mRNA and protein in si-FoxO1 group were significantly lower than those in NC-FoxO1 group and Mock group (P ＜ 0.05),there were no significant differences of IL-1β mRNA and protein between NC-FoxO1 group and Mock group (P ＞0.05).The expressions of FoxO1,IL-1β mRNA and protein in DM group and LV-NC group were significantly higher than those in Control group and LV-si-FoxO1 group (P ＜ 0.05),there were no significant differences of FoxO1,IL-1β mRNA and protein between LV-NC group and Control group (P ＞ 0.05),there were also no significant differences between DM group and LV-NC group (P ＞0.05).The expressions of p-P38,p-JNK,p-ERK protein in si-FoxO1 group were significantly lower than those in NC-FoxO1 group and Mock group (P ＜ 0.05),and the expressions of p-P38,p-JNK,p-ERK protein between NC-FoxO1 group and Mock group had no significant differences (P ＞ 0.05).Conclusion:High glucose can promote the expressions of FoxO1,IL-1 β in HRCECs,inhibition the expression of FoxO1 may be done by down-reuglation of IL-1 β expression through decreasing the MAPK phosphorylation.