枸杞多糖对高糖所致RF/6As通透性和Rho激酶的影响

Effects of lycium bararum polysaccharides on changes of monolayer permeability and ROCK1 of RF/6As in high glucose environment

目的探讨高糖条件下猴脉络膜-视网膜内皮细胞(RF/6As)损伤时的单层通透性、Rho激酶1(ROCK1)的变化及它们之间的关系,和枸杞多糖(LBP)对上述变化的影响。方法体外培养RF/6As,分为等渗葡萄糖处理组(对照组)、40mmol/L葡萄糖处理组(高糖组)及LBP+40mmol/L葡萄糖处理组(LBP+高糖组)。各组分别培养1、3、5、7d,应用倒置显微镜观察培养的RF/6As形态,以辣根过氧化物酶(HRP)作为示踪剂用二室弥散系统检测RF/6As单层通透性的变化,应用免疫荧光法观察各组不同时间点细胞骨架的变化,采用Westernblot法检测ROCK1在5d、7d各组细胞中的表达量。结果第3、5、7天,高糖组RF/6As随时间延长逐渐变形,正常细胞数量明显减少,单层通透性逐渐增加;3个时间点HRP的A值分别为0.074±0.011、0.135±0.022、0.253±0.047,明显高于对照组,差异均有统计学意义(P<0.01)。LBP+高糖组仅于7d时细胞形态略有改变,5d及7d时HRP的A值分别为0.069±0.004和0.114±0.009,较对照组明显增加,差异均有统计学意义(P<0.01);但明显低于高糖组,差异均有统计学意义(P<0.01)。Westernblot检测显示,7d时LBP+高糖组ROCK1灰度值为35849.47±1032.36,高糖组为21356.10±1566.66,差异有统计学意义(P<0.01)。结论高糖可引起RF/6As单层通透性增加和细胞骨架的重排以及ROCK1的高表达,而LBP可通过下调ROCK1的表达减轻高糖对RF/6As的损伤。

BackgroundThe destruction of the blood-retina barrier is the primary mechanism of diabetic retinopathy（DR）,and F-actin is verified to involve in this procedure.Rho/ROCK is one of signal pathways of regulating the cytoskeleton.However,seldom researches are published about whether this pathway participate in the damage of retinal vascular endothelial cells in high glucose environment.ObjectiveThe present study was to explore the changes of monolayer permeability and ROCK1 of macaque choroids-retinal endothelial cells（RF/6As）in high glucose environment and to study the effects of lycium bararum polysaccharides（LBP）on above changes.MethodsRF/6A cells were culture and passaged in MEM.The third generation of cells were treated with 5.5mmol/L isoosmia glucose,40mmol/L glucose and 40mmol/L glucose＋1g/L LBP respectively.Morphologic changes of RF/6As in different groups were observed under the inverted microscope at the 1st,3rd,5th and 7th day after culture.Horseradish peroxidase（HRP）was used as a tracer agent to detect the permeability of monolaye RF/6As in transwall chamber.Cytoskeleton distribution in different groups was determined by using immumofluorescence.Western blot was used to determine the expression of ROCK1 on the 3rd and 7th day.ResultsDeformed RF/6As were gradually increased with the prolong of time in 40mmol/L glucose group,but the cells shape was near normal in LBP ＋ 40mmol/L glucose group.The A values of HRP were significantly enhanced in 1,3,5,7 days after treated in 40mmol/L glucose group compared with isoosmia glucose group（P0.01）,and those in 40mmol/L glucose＋LBP group were considerably declined in comparison with 40mmol/L glucose group in various time points（P0.01）.The rupture of stress fibers constructed by F-actin,deformation and digitation of RF/6As were exhibited in 40mmol/L glucose group,but no similar findings were seen in isoosmia glucose and 40mmol/L glucose＋LBP group under the fluorescence microscope.Western blot assay revealed that the expression of ROCK1 was gradually increased in 3 and 7 days after treated by 40mmol/L glucose group in comparison with isoosmia glucose group（P0.01）,and that in 40mmol/L glucose＋LBP group was significantly lower than 40mmol/L glucose group（P0.05）.ConclusionHigh-concentration glucose increases monolayer permeability of vascular endothelial cells.The beneficial effects of LBP limit this damage and may be an approach for the prevention of RF/6As cytoskeleton rearrangement by down-regulating the expression of ROCK1.