多重酪氨酸激酶受体抑制剂decorin对高糖低氧条件下血-视网膜内屏障的保护作用及机制研究

目的 研究核心蛋白多糖(decorin)对高糖低氧条件下血-视网膜内屏障的保护作用及机制.方法 培养人脐静脉内皮细胞,采用细胞计数试剂盒检测不同浓度decorin对人脐静脉内皮细胞存活率的影响;高糖低氧条件下(25 mmol·L-1右旋葡萄糖+100 μmol·L-1 CoCl2)孵育人脐静脉内皮细胞,通过ELISA检测不同浓度decorin处理后,不同时间点人脐静脉内皮细胞分泌血管内皮生长因子水平.将细胞分为正常对照组(5.5 mmol·L-1右旋葡萄糖)、高糖低氧组(25 mmol·L-1右旋葡萄糖+100 μmol·L-1 CoCl2)、甘露醇对照组(5.5 mmol·L-右旋葡萄糖+ 19.5 mmol·L-1甘露醇)及decorin处理组(25 mmol·L-1右旋葡萄糖+ 100 μmol·L-CoCl2+100 nmol · L-decorin),通过检测跨内皮细胞电阻(transendothelial electrical resistance,TER)、异硫氰酸荧光素标记的右旋糖酐(fluorescein isothiocyanate,FITC-dextran)的渗漏率来评估单层人脐静脉内皮细胞的屏障功能;Western blotting检测内皮细胞间紧密连接蛋白(claudin-5、occludin、ZO-1)及p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)的磷酸化水平.结果 细胞计数试剂盒检测发现,10 nmol·L-1、50 nmol·L-、100 nmol·L-1、200nmol·L-1 decorin处理人脐静脉内皮细胞24h后,各组人脐静脉内皮细胞的存活率均大于90％,差异无统计学意义(均为P＞0.05).在接种后14 d,单层人脐静脉内皮细胞的TER达到最大值且趋于稳定为(170.67±9.07)Ω.与正常对照组比较,高糖低氧处理48 h后,高糖低氧组的TER显著降低至(97.33±6.11)Ω,而decorin组的TER能够维持在(157.67±11.72)Ω,与高糖低氧组相比,差异有统计学意义(P＜0.05).高糖低氧处理48 h后,高糖低氧组FITC-dextran的渗透性明显增加,488nm处的吸光度值为正常对照组的(2.12±0.07)倍(P＜0.05).加入100 nmol·L-1的decorin处理后,FITC-dextran的渗透性明显减少,吸光度值是正常对照组的(1.16±0.03)倍,与高糖低氧组的差异有统计学意义(P＜0.05).高糖低氧处理48 h后,高糖低氧组紧密连接蛋白claudin-5表达量为0.38±0.05、occludin为0.43±0.02及ZO-1为0.25±0.02与正常对照组表达量0.72±0.05、0.90±0.01和0.75±0.02相比,差异均有统计学意义(均为P＜0.05).decorin组claudin-5、occludin及ZO-1表达量分别为0.65 ±0.08、0.87±0.03和0.60±0.01,与高糖低氧组相比,差异均有统计学意义(均为P＜0.05).高糖低氧组p-p38 MAPK/p38 MAPK的比值增加,为0.88±0.02,而decorin组p-p38 MAPK/p38 MAPK的比值(0.58±0.04)接近正常对照组水平(0.56±0.02),与高糖低氧组比较,差异有统计学意义(P＜0.05).结论 decorin能够保护高糖低氧条件下单层人脐静脉内皮细胞的屏障功能并下调p38 MAPK的表达水平,是治疗糖尿病视网膜病变的可能药物.

Protective effects of decorin on blood-retinal barrier function under high-glucose plus hypoxia conditions

Objeetive To evaluate the protective effects of decorin on the inner blood-retinal barrier function under high-glucose plus hypoxia conditions,and explore its potential mechanism.Methods The human umbilical vein endothelial cells (HUVEC) were cultured,and the effect of decorin with different concentrations on HUVEC viability was determined by using the cell counting kit-8 assay (CCK-8).At different hours after incubation under high-glucose plus hypoxia conditions (25 mmol · L-1 D-glucose + 100 μmol · L-1 CoCl2),the suppression effects of various concentrations of decorin on the vascular endothelial growth factor (VEGF) expression of HUVEC was detected by enzyme-linked immunosorbent assay (ELISA).HUVEC were divided into normal control group (5.5 mmol · L-1 D-glucose),high-glucose plus hypoxia group (25 mmol · L-1 D-glucose + 100 μmol · L-1 CoCl2),mannitol control group(5.5 mmol· L-1 D-glucose +19.5 mmol · L-1 mannitol) and decorin treatment group (25 mmol · L-1 D-glucose+ 100 μmol · L-1 CoCl2 + 100 nmol · L-1 decorin).HUVEC barrier function was evaluated by detecting transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate-dextran (FITC-dextran).The content of tight junction proteins (claudin-5,occludin,and ZO-1) and p38 mitogen-activated protein kinase (MAPK) phosphorylation were examined by Western blotting.Results According to the results of CCK-8,the survival rates of HUVEC in all groups were more than 90％,different concentrations of decorin showed no significant effect on HUVEC survival (all P ＞ 0.05).According to the results of CCK-8 and ELISA,the stimulation of hypoxia for 48 hours and 100 nmol · L-1 decorin was taken as the condition of further experiment.At 14 days,TER of HUVEC reached its peak of (170.67 ±9.07) Ω.TER of high-glucose plus hypoxia group (97.33 ±6.11)Ω was significantly lower than that of decorin treatment group (157.67 ± 11.72)Ω (P ＜0.05).The FITC-dextran permeability of high-glucose plus hypoxia group increased to (2.12 ±0.07) times of normal control group(P ＜0.05).Decorin reversed this effect to (1.16 ± 0.03) times of normal control group (P ＜ 0.05).The expression of claudin-5,occludin and ZO-1 in high-glucose plns hypoxia group were 0.38 ±0.05,0.43 ±0.02,0.25 ± 0.02,compared to the normal control group (0.72 ±0.05,0.90 ±0.01,0.75 ±0.02),there were statistical differences (all P ＜0.05).The expression of claudin-5,occludin and ZO-1 m decorin treatment group were 0.65 ±0.08,0.87 ±0.03,0.60 ±0.01,there were statistical differences compared with high-glucose plns hypoxia group (all P ＜0.05).The ration of p-p38 MAPK/p38 MAPK in high-glucose plus hypoxia group (0.88 ± 0.02) was increased,while the decorin treatment group (0.58 ± 0.04) reached the level of the normal control group (0.56 ±0.02),there was statistical difference compared with high-glucose plns hypoxia group (P ＜0.05).Conclusion Decorin can protect the HUVEC barrier function under high-glucose plus hypoxia conditions and inhibit the activation of p38 MAPK signaling pathway.So it may be used to treat diabetic retinopathy.