烟酸对糖尿病大鼠血视网膜屏障的影响及其机制研究

摘要

Objective To investigate the protective effect of Niacin on blood-retina barrier (BRB) in diabetic rats and related mechanism.Methods The male Wistar rats (60) were divided into control (CON) group,diabetes (DM) group and Niacin-treated (NA) group,20 rats in each group.Rats diabetes models were induced with streptozotocin injection.Niacin (40 mg/kg · d) was administrated orally everyday in Niacin-treated group until sacrificed after 3 months.Pathological outcomes,total cholesterol (TC) and highdensity lipoprotein (HDL) were evaluated at month 3.Optical microscopy was used to observe the retinal structure.The integrity of BRB and the vascular permeability was quantified by analyzing albumin leakage using Evans blue (EB) method.The relative expressions of Claudin-5,Occludin,zonula occluden (ZO)-1 and GPR109A mRNA in rat retinas were detected by reverse transcription PCR (RT-PCR) and relative expression of GPR109A,tumor necrosis factor (TNF)-α and interleukin (IL)-6 by Western blot.Results Compared to CON group,the TC content was increased and HDL content was decreased in DM group (t=4.034,5.831;P＜0.05).Compared to DM group,the TC content was decreased and HDL content was increased in NA group (t=6.868,3.369;P＜0.05).The retinal structure of CON group was normal.Pathological changes were found in the DM group,such as tumescent nuclei and disorganized structures.The retinal structure of NA group was similar to the control group.Evans blue dye that the microvascular leakage in DM group was increased compared with CON group (t=24.712,P＜0.05),while in NA group was decreased compared with DM group (t =16.414,P＜ 0.05).The mRNA expression of Occludin,Claudin-5,ZO-1 in DM group were decreased compared with CON group (t=11.422,12.638,12.060;P＜ 0.05),while in NA group were increased compared with DM group (t=5.278,3.952,8.030;P＜0.05).The mRNA expression of GPR109A in NA group were increased compared with DM group (t=5.053,P＜ 0.05).The protein expression of GPR109A,IL-6,TNF-αin DM group were increased compared with CON group (t=4.915,11.106,6.582;P＜0.05).Compared to DM group,the protein expression of GPR109A was increased (t=5.806,P＜0.05),while the protein expression of IL-6 and TNF-α were decreased (t=10.131,5.017;P＜0.05).Conclusion Niacin has the protective effect for BRB by up-regulating GPR109A expression which may suppress inflammation.%目的 观察探讨烟酸对糖尿病大鼠血视网膜屏障(BRB)的影响及机制.方法 采用随机数字表法将60只雄性Wistar大鼠随机分为正常对照组(CON组)、糖尿病模型对照组(DM组)及烟酸处理组(NA组),每组20只.DM组、NA组大鼠尾静脉注射链脲佐菌素溶液建立糖尿病模型.建模成功后第7天,NA组大鼠以40 mg/kg的剂量灌胃给予烟酸,1次/d,持续灌胃3个月,CON组、DM组大鼠正常喂食水.于NA组大鼠灌胃3个月后,各组大鼠检测血清总胆固醇(TC)以及高密度脂蛋白(HDL)含量;分别作视网膜切片苏木精-伊红染色,观察视网膜组织病理学改变;作视网膜铺片,行伊凡思蓝(EB)灌注观察大鼠视网膜EB渗漏量;定量逆转录聚合酶链反应(RT-PCR)检测大鼠视网膜中闭合蛋白(Occludin)、封闭蛋白(Claudin)-5、紧密连接蛋白(ZO)-1、烟酸受体G蛋白偶联受体109A(GPR109A)的mRNA相对表达量;蛋白免疫印迹法(Western blot)检测大鼠视网膜中GPR109A、白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α蛋白相对表达量.结果 与CON组比较,DM组大鼠血清TC含量明显升高,HDL含量明显降低,差异均有统计学意义(t=4.034、5.831,P＜0.05).与DM组比较,NA组大鼠血清TC含量明显降低,HDL含量明显升高,差异均有统计学意义(t=6.868、3.369,P＜0.05).光学显微镜观察发现,CON组大鼠视网膜各层结构清晰,细胞排列整齐、形态正常;DM组大鼠视网膜各层结构变薄,细胞排列紊乱,细胞核肿胀,部分血管明显扩张;NA组大鼠视网膜结构整齐,细胞排列渐规则.DM组EB平均渗漏量较CON组明显增高,差异有统计学意义(t=24.712,P＜0.05);NA组EB平均渗漏量较DM组明显减少,差异有统计学意义(t=16.414,P＜0.05).DM组大鼠视网膜Occludin、Claudin-5、ZO-1相对表达量较CON组明显降低,差异有统计学意义(t=11.422、12.638、12.060,P＜0.05);NA组大鼠视网膜Occludin、Claudin-5、ZO-1mRNA相对表达量较DM组明显增高,差异有统计学意义(t=5.278、3.952、8.030,P＜0.05).NA组大鼠视网膜GPR109A mRNA相对表达量较DM组明显增高,差异有统计学意义(t=5.053,P＜0.05).DM组大鼠视网膜GPR109A、IL-6、TNF-α蛋白相对表达量较CON组明显增高,差异有统计学意义(t=4.915、11.106、6.582,P＜0.05);NA组大鼠视网膜GPR109A蛋白相对表达量较DM组明显增高,差异有统计学意义(t=5.806,P＜0.05);IL-6、TNF-α蛋白相对表达量较DM组明显降低,差异有统计学意义(t=10.131、5.017,P＜0.05).结论 烟酸对糖尿病大鼠BRB有保护作用,其机制可能与上调GPR109A表达,抑制炎症反应有关.