石斛多糖对糖尿病大鼠血-视网膜屏障的保护作用及其机制

背景 糖尿病视网膜病变(DR)的发病机制涉及多条通路,近年来炎症因子相关的通路学说在DR发病中的作用越来越受到人们的关注.研究表明,高糖环境下视网膜内肿瘤坏死因子-α(TN F-α)等促炎因子含量增加,从而加速视网膜中紧密连接蛋白的异常,导致血-视网膜屏障(BRB)破坏.研究发现石斛多糖可抑制TNF-α的高表达,但其在DR早期对TNF-α表达的影响尚不清楚. 目的 研究石斛多糖对糖尿病大鼠TNF-α诱导的BRB通透性异常及视网膜内紧密连接蛋白1(ZO-1)、闭合蛋白(occludin)、连接蛋白-5(claudin-5)表达的影响.方法 将50只清洁级成年SD大鼠分为正常对照组、糖尿病模型组及低、中、高剂量石斛多糖组,每组10只大鼠,糖尿病模型组和低、中、高剂量石斛多糖组大鼠均采用链脲佐菌素腹腔内注射法诱导糖尿病模型.低、中、高剂量石斛多糖组于建模成功后6周按照分组分别用100、200及300 mg/(kg·d)石斛多糖灌胃,正常对照组和糖尿病模型组大鼠用生理盐水灌胃.干预后8周各组大鼠心脏灌注伊文思蓝并获取双侧眼球,分别进行相关指标检测,通过测定大鼠视网膜中伊文思蓝质量浓度评估大鼠视网膜渗漏量;采用Western blot法检测大鼠视网膜内ZO-1、occludin、claudin-5蛋白的相对表达量,采用ELISA法检测大鼠视网膜中及血清中TNF-α的质量浓度.结果 正常对照组、糖尿病模型组及低、中、高石斛多糖组大鼠视网膜伊文思蓝渗漏量分别为(12.68±1.30)、(30.45±2.60)、(22.12±1.15)、(17.99±1.00)和(21.49±1.00) μg/μl,糖尿病模型组大鼠视网膜伊文思蓝渗漏量明显高于正常对照组,低、中、高石斛多糖组视网膜伊文思蓝渗漏量明显低于糖尿病模型组,差异均有统计学意义(均P＜0.01).Western blot检测显示,糖尿病模型组大鼠视网膜中TNF-α蛋白相对表达量为1.12±0.10,明显高于正常对照组的0.27±0.03,低、中、高石斛多糖组视网膜中TNF-α蛋白表达量明显低于糖尿病模型组,ZO-1、occludin和claudin-5蛋白表达量明显高于糖尿病模型组,差异均有统计学意义(均P＜0.05);ELISA法检测显示,糖尿病模型组大鼠视网膜和血清中TNF-α质量浓度明显高于正常对照组,低、中、高石斛多糖组大鼠视网膜和血清中TNF-α质量浓度明显低于糖尿病模型组,差异均有统计学意义(均P＜0.05).不同剂量石斛多糖组各指标的比较差异均无统计学意义(均P＞0.05).结论 石斛多糖可能通过下调糖尿病大鼠视网膜中TNF-α的表达和上调紧密连接蛋白的表达而改善糖尿病大鼠BRB通透性的异常.

Protection of polysaccharides of dendrobium candidum on blood-retinal barrier in diabatic rats and its mechanism

Background The pathogenesis of diabetic retinopathy (DR) involves a variety of biological pathways.Recently,inflammation factor has been thought to paly an important role in the pathogenesis of DR.Studies show that the concent of tumor necrosis factor-α (TNF-α) is increased in high-glucose environment,which leads to the abnormality of tight junction protein and follows by blood-retinal barrier (BRB) damage.Polysaccharides of dendrobium candidum (PDC) can inhibit the overexpression of TNF-α,but its effect on TNF-α in early DR procedure has been unelucidated.Objective This study was to investigate the effects of PDC on permeability of BRB and its mechanism in daibetic rats.Methods Fifty clear adult SD rats were divided into normal control group,diabetic model group and low-(100 mg/[kg · d]),moderate-(200 mg/[kg · d]) and high-dose (300 mg/[kg · d]) PDC groups,10 rats for each group.Streptozotocin was intraperitoneally injected to establish diabetic model in 40 rats,expect for normal control group.PDC at the concentrations of 100,200 and 300 mg/(kg · d) was intragastrically administered in the low-,moderate-and high-dose groups respectively at 6 weeks after modeling,and normal saline solution was used at the same way in the normal control group and diabetic model group.Evans blue was perfused via cardic chamber and eyes were obtained.Evants blue leakage was measured to evaluate the BRB permeability.The relative expressions of TNF-α,zonula occludens-1 (ZO-1),occludin and claudin-5 proteins were detected by Western blot;TNF-α contents in the retina and serum of the rats were detected by ELISA.Results The leakage concents of Evans blue in the retinas were (12.68±1.30),(30.45±2.60),(22.12±1.15),(17.99±1.00) and (21.49±1.00) in the normal control group,diabetic model goup and low-,moderate-and high-dose PDC groups,respectively,and the retinal leakage concents in the diabetic model group were significantly higher than those in the normal control group,and the retinal leakage contents in the low-,moderate-and high-dose PDC groups were lower than those in the diabetic model group (all at P ＜ 0.01).Western blot showed that the relative expression level of retinal TNF-α was significantly higher in the diabetic model group compared with the normal control group(1.12±0.10 vs.0.27±0.03),and that in the diabetic model group was significantly higher than that in the normal control group;while the relative expression levels of retinal TNF-α in different doses PDC groups were significantly lower,and the relative expression levels of retinal ZO-1,occludin and claudin-5 were significantly higher than those in the diabetic model group (all at P＜0.05).ELISA showed that the concentrations of retinal and serum TNF-α were higher in the diabetic model group compared with the normal control group,and those in the different doses of PDC groups were lower than those in the diabetic model group (all at P＜0.05).No significant differences were found among various doses of PDC groups (all at P＞0.05).Conclusions PDC can improve the permeability of BRB by down-regulating the expression of TNF-α and up-regulating the expressions of tight junction proteins in the retina of diabetic rats,which is probably related to suppressing the development of early DR.