目的 研究根皮苷(PHL)对糖基化低密度脂蛋白(gly-LDL)诱导大鼠视网膜微血管内皮细胞(RMVEC)表达乳脂肪球表皮生长因子8(MFG-E8)的影响,探讨PHL治疗糖尿病视网膜病变(DR)的作用及其机制.方法 体外培养大鼠RMVEC,分别进行不同浓度的gly-LDL与联合PHL+gly-LDL的处理.采用TUNEL方法观察不同处理条件下大鼠RMVEC的细胞凋亡.利用蛋白质免疫印迹方法(Western blotting)检测大鼠RMVEC 表达MFG-E8的变化.结果 与对照组相比,gly-LDL诱导大鼠RMVEC发生明显的细胞凋亡形态学变化,凋亡细胞的比例显著增多达到36.52%(P<0.01),预先经PHL(800 μmmol/L)孵育后,细胞凋亡明显减少至20.45%(P<0.01);与对照组(蛋白表达灰度值0.290±0.008)比较,gly-LDL诱导组蛋白MFG-E8表达明显降低,并呈现浓度依赖性(蛋白表达灰度值分别为0.215±0.009、0.165±0.002、0.117±0.014与0.092±0.004,P<0.01);经PHL预处理后,gly-LDL诱导的MFG-E8表达明显升高,伴随PHL给药浓度的增加,MFG-E8的表达逐渐上调,差异有统计学意义(蛋白表达灰度值分别为0.343±0.008、0.373±0.002与0.404±0.006,P<0.01).结论 PHL可能通过显著改善蛋白MFG-E8的表达,从而抑制gly-LDL诱导的大鼠RMVEC的细胞凋亡,实现对DR的保护作用.%Objective To study the effects of phlorizin on the expression of MFG-E8 in rats retinal microvascular endothelial cells (RMVEC) in response to glycated low density lipoproteins (gly-LDL).Methods The primary RMVEC in rats were cultured and identified in vitro.The apoptosis of RMVEC induced gly-LDL was estimated using TUNEL.The expressions of MFG-E8 in RMVEC in the absence or presence of phlorizin were determined by Western blotting.Results Gly-LDL caused an obvious apoptosis of RMVEC compared with the control group (36.52% VS.1.24%,P<0.01),while pretreatment with phlorizin attenuated gly-LDL mediated cells apoptosis significantly (20.45% VS.36.52%,P<0.01).Moreover,stimulation of RMVEC with different concentrations of gly-LDL(0,12.50,25.00,50.00,100.00 μg/mL) resulted in a significant decrease in the expression of MFG-E8 (P<0.01),while the pretreatment of RMVEC with different concentrations of phlorizin (200,400,800 μmmol/L) significantly restored the gly-LDL induced decreased MFG-E8 levels in a dose dependent manner for 48h (P<0.01).Conclusions Phlorizin could inhibit RMVEC apoptosis in response to gly-LDL,possibly by the up-regulation of the protein MFG-E8 expression.
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