补肾活血中药方血清对离体状态下血-视网膜内屏障模型的保护作用

Protective effect of serum of Bushenhuoxue decoction on the inner blood-retinal barrier model in vitro

目的探讨补肾活血中药方血清对离体状态下血-视网膜内屏障(iBRB)模型的保护作用。方法取新生7 d SD大鼠用于取材及细胞原代培养。将大鼠视网膜微血管内皮细胞(RMEC)和视网膜Müller胶质细胞(RMGC)分离及传代培养,利用Transwell小室将RMEC与RMGC共培养以构建正常条件下的体外iBRB模型,制备补肾活血中药方血清,并分别标记为中药含药血清和空白血清。本实验分8组。选取构建成功的体外iBRB细胞共培养模型,按不同培养条件进行分组。其中正常对照组用含体积分数20%空白血清的DMEM液培养;中药干预正常组用含体积分数20%中药含药血清的DMEM液培养;低糖基化终末产物(AGEs)组用含体积分数20%空白血清的DMEM液及终浓度50 mg·L^(-1)AGEs培养;中药干预低AGEs组用含体积分数20%中药含药血清的DMEM液及终浓度50 mg·L^(-1)AGEs培养;高AGEs组用含体积分数20%空白血清的DMEM液及终浓度100 mg·L^(-1)AGEs培养;中药干预高AGEs组用含体积分数20%中药含药血清的DMEM液及终浓度100 mg·L^(-1)AGEs培养;缺氧组用含体积分数20%空白血清的DMEM液及终浓度1.0 mmol·L^(-1)连二亚硫酸钠培养;中药干预缺氧组用含体积分数20%中药含药血清的DMEM液及终浓度1.0 mmol·L^(-1)连二亚硫酸钠培养。利用细胞电阻仪检测各组RMEC层的跨内皮细胞电阻(TEER),采用免疫荧光双标法检测各组Occludin、ZO-1蛋白在RMEC层的表达。结果在AGEs或缺氧条件下,低AGEs组、高AGEs组及缺氧组RMEC层TEER在细胞培养24 h、48 h和72 h后均低于正常对照组,差异均有统计学意义(均为P<0.05)。在细胞培养24 h后,中药干预正常组与正常对照组RMEC层TEER相比,差异无统计学意义(P>0.05),在细胞培养48 h后中药干预正常组RMEC层TEER高于正常对照组,差异有统计学意义(P<0.05),在细胞培养72 h后中药干预正常组RMEC层TEER低于正常对照组,差异有统计学意义(P<0.05);中药干预低AGEs组、中药干预高AGEs组、中药干预缺氧组RMEC层TEER在细胞培养24 h、48 h和72 h后均高于同一时段相对应的非中药干预组,差异均有统计学意义(均为P<0.05)。低AGEs组、高AGEs组及缺氧组Occludin、ZO-1蛋白在RMEC层的阳性表达在细胞培养24 h、48 h和72 h后均弱于正常对照组。在细胞培养24 h、48 h和72 h后,中药干预正常组、中药干预低AGEs组、中药干预高AGEs组、中药干预缺氧组Occludin、ZO-1蛋白在RMEC层的阳性表达均强于同一时段相对应的非中药干预组。结论AGEs及缺氧均可导致体外iBRB模型RMEC层TEER降低及Occludin、ZO-1蛋白表达减弱,补肾活血中药方血清能有效提高Occludin和ZO-1蛋白的表达,抑制iBRB模型通透性增加,从而起到对体外iBRB模型的保护作用。

Objective To explore the protective effect of serum of Bushenhuoxue decoction on the inner blood-retinal barrier(iBRB)model in vitro.Methods The 7-day-old SD rats were used for material collection and primary cell culture.Retinal microvascular endothelial cells(RMEC)and retinal Müller glial cells(RMGC)of rats were isolated and subcultured.RMEC and RMGC were co-cultured in Transwell chamber to construct the in vitro iBRB model under normal conditions.The serum of Bushenhuoxue decoction was prepared and labeled as Chinese medicine serum and blank serum respectively.The experiment was divided into 8 groups.The successful co-culture model of iBRB in vitro was selected and divided into groups according to different culture conditions.The normal control group was cultured with DMEM containing 20%blank serum;while the Chinese medicine intervention normal group was cultured with DMEM containing 20%Chinese medicine serum;low advanced glycosylation end products(AGEs)group was cultured with DMEM containing 20%blank serum and final concentration of 50 mg·L^(-1) AGEs;the Chinese medicine intervention low AGEs group was cultured with DMEM containing 20%Chinese medicine serum and final concentration of 50 mg·L^(-1) AGEs;high AGEs group was cultured with DMEM containing 20%blank serum and final concentration of 100 mg·L^(-1) AGEs;the Chinese medicine intervention high AGEs group was cultured with DMEM containing 20%Chinese medicine serum and final concentration of 100 mg·L^(-1) AGEs;hypoxia group was cultured with DMEM containing 20%blank serum and final concentration of 1.0 mmol·L^(-1) sodium disulfite;the Chinese medicine intervention hypoxia group was cultured with DMEM containing 20%Chinese medicine serum and final concentration of 1.0 mmol·L^(-1) sodium disulfite.The transendothelial electronic resistance(TEER)of RMEC cell layer in each group was detected by cell resistance meter,and the expression of Occludin and ZO-1 protein in RMEC cell layer was detected by immunofluorescence double labeling method.Results Under the condition of AGEs or hypoxia,the TEER of RMEC cell layer in low AGEs group,high AGEs group and hypoxia group was lower than that in normal control group at 24 hours,48 hours and 72 hours after cell culture,and the differences were statistically significant(all P<0.05).After 24 hours of cell culture,there was no significant difference in TEER between the Chinese medicine intervention normal group and the normal control group(P>0.05).After 48 hours of cell culture,the TEER of RMEC cell layer of the normal group treated with traditional Chinese medicine was higher than that of the normal control group,and the difference was statistically significant(P<0.05).After 72 hours of cell culture,the TEER of RMEC cell layer of the normal group treated with traditional Chinese medicine was lower than that of the normal control group,and the difference was statistically significant(P<0.05).The TEER of RMEC cell layer in Chinese medicine intervention low AGEs group,the Chinese medicine intervention high AGEs group and the Chinese medicine intervention hypoxia group were higher than those in the corresponding non-Chinese medicine intervention group at 24 hours,48 hours and 72 hours after cell culture,and the differences were statistically significant(all P<0.05).The positive expression of Occludin and ZO-1 protein of RMEC cell layer in low AGEs group,high AGEs group and hypoxia group were weaker than that of normal control group at 24 hours,48 hours and 72 hours after cell culture.After 24 hours,48 hours and 72 hours of cell culture,the positive expressions of Occludin and ZO-1 protein of RMEC cell layer in the Chinese medicine intervention normal group,the Chinese medicine intervention low AGEs group,the Chinese medicine intervention high AGEs group and the Chinese medicine intervention hypoxia group were stronger than those of non-Chinese medicine intervention group at the same time.Conclusion Both AGEs and hypoxia can lead to the decrease of TEER and the expression levels of Occludin and ZO-1 protein in iBRB cell model in vitro.The serum of Bushenhuoxue decoction can effectively increase the expression levels of Occludin and ZO-1 protein and inhibit the increase of permeability,thus playing a protective role in iBRB model in vitro.