糖基化低密度脂蛋白诱导的大鼠视网膜微血管内皮细胞凋亡以及根皮苷保护作用的研究

Benifical effects of phlorizin on the apoptosis of retinal microvascular endothelial cells induced by glycated low density lipoproteins in rats

目的观察糖基化低密度脂蛋白(gly-LDL)与根皮苷(PHL)培养对于大鼠视网膜微血管内皮细胞(RMVEC)凋亡的影响,探讨PHL保护糖尿病视网膜损伤的作用。方法体外培养大鼠RMVEC。(1)不同浓度gly-LDL(12.5μg/ml、25μg/ml、50μg/ml与100μg/ml)以及联合PHL(gly-LDL 100μg/ml、gly-LDL 100μg/ml+PHL200μmol/L、gly-LDL100μg/ml+PHL400μmol/L、gly-LDL 100μg/ml+PHL800μmol/L)分别培养RMVEC,于48 h时间点利用四甲基偶氮唑盐(MTT)方法检测细胞活性。(2)将RMVEC分为3组,对照组(CK)、CK+glyL DL100μg/ml组以及CK+glyL DL100μg/ml+PHL800μmol/L组,采用TUNEL分析各组RMVEC的细胞凋亡情况。结果 (1)与对照组比较,glyLDL各浓度组细胞生存率明显降低,且存在浓度依赖性关系[分别为(90.135±0.092)%,(71.802±0.170)%,(67.130±0.308)%,(43.003±0.526)%,P<0.001];联合不同浓度的PHL与RMVEC预先孵育后,能够明显改善gly-LDL诱导的RMVEC细胞活性,随着PHL浓度的增加,细胞生存率逐渐升高[(42.961±0.011)%,(48.249±0.004)%,(53.793±0.007)%,P<0.001];(2)与对照组比较[细胞凋亡率(1.24±0.16)%],gly-LDL显著增加RMVEC的细胞凋亡[gly-LDL组细胞凋亡率(36.52±0.90)%,P<0.001],联合PHL干预后能够明显降低gly-LDL诱导的RMVEC细胞凋亡[gly-LDL+PHL组细胞凋亡率(20.45±1.08)%,P<0.001]。结论 gly-LDL呈浓度依耐性刺激RMVEC可抑制细胞活性、诱导细胞凋亡,经PHL预孵育后能够显著增加细胞生存率、减少细胞凋亡从而抑制细胞损伤,起到保护RMVEC作用。

Objective To investigate the effects of glycated low density lipoproteins（ gly-LDL） on the apoptosis of retinal microvascular endothelial cells（ RMVEC） in rats in the presence or absence of phlorizin,and to explore the protective effects of phlorizin on the diabetic retinal lesions. Methods RMVEC in rats were cultured in vitro.（1）The effects of gly-LDL（ 12. 5 μg/ml,25 μg/ml,50 μg/ml and 100 μg/ml） and different groups of phlorizin（ gly-LDL 100 μg/ml,gly-LDL 100 μg/ml ＋ PHL 200 μmol/L,gly-LDL100 μg/ml ＋ PHL 400 μmol/L,gly-LDL 100 μg/ml ＋ PHL 800 μmol/L） on RMVEC viability were analyzed with MTT at 48 h following by incubation.（2）The apoptosis of RMVEC in 3 groups（ CK,gly-LDL and gly-LDL ＋ PHL） were analyzed using TUNEL. Results（1）The cells viability was significantly decreased in gly-LDL groups than that in control cells（ P〈0. 01）. Moreover,the decreasing viability was concentration-dependently[（ 90. 135 ± 0. 092） %,（ 71. 802 ± 0. 170） %,（ 67. 130 ± 0. 308） % and（ 43. 003 ± 0. 526） %,respectively,P〈0. 01]. The pretreatment of RMVEC with different concentration of phlorizin significantly improved the gly-LDL stimulated cells viability in a dose dependent manner at 48 hours [（ 42. 961± 0. 011） %,（ 48. 249 ± 0. 004） % and（ 53. 793 ± 0. 007） %,respectively,P〈0. 01].（2）gly-LDL-stimulated RMVEC significantly increased the percentage of apoptotic cells compared with control cells [（ 36. 52 ± 0. 90） % vs.（ 1. 24 ± 0. 16） %,P〈0. 01 ],while phlorizin（ 800 μmol/L） significantly attenuated the cell apoptosis for 48 hours [（ 36. 52 ± 0. 90） %,P〈0. 01]. Conclusion Gly-LDL may inhibit the viability of RMVEC and induce the apoptosis of RMVEC in rats,while pretreatment of RMVEC with phlorizin shows the opposite effects,thus,phlorizin could protect diabetic retinal lesions in the early stage.