番茄红素对高糖诱导的足细胞自噬以及凋亡的影响

摘要

Objective To investigate the effects of lycopene (Lyc) on high glucose-induced autophagy and apoptosis of podocytes in order to provide a reliable theoretical basis for the clinical treatment of diabetic nephropathy.Methods The immortalized mouse podocyte line was used as the study subjects,and the high glucose-induced podocytes were intervened by the addition of different doses of lycopene.The experimental cells were divided into 6 groups:control group (5.5 mmol/L glucose),hyperosmolar group (5.5 mmol/L glucose + 19.5 mmol/L mannitol),high glucose group (25 mmol/L glucose),high glucose + low dose Lyc group (25 mmol/L glucose + 3.125 μmol/L Lyc),high glucose + middle dose Lyc group (25 mmol/L glucose + 6.25 μmol/L Lyc) and high glucose + high dose Lyc group (25 mmol/L glucose + 12.5 μmol/L Lyc).After culture for 48 h,the autophagic changes of the cells were observed by acridine orange staining.The flow cytometry was used to detect the apoptosis rate of each group,and the expression levels of LC3-Ⅱ,beclin-1,p62/SQSTMI mRNA and protein were detected by Real-Time PCR and Western blotting,respectively.Results The number of autophagosomes in the high glucose group was significantly greater than that in the normal glucose group and high permeability group.The apoptosis rate of the cells in high glucose group was significantly higher (P＜0.01).The expression of autophagy-related genes LC3-Ⅱ and beclin-1 was significantly increased (P＜0.05),while that of p62/SQSTMI gene was significantly decreased (P ＜ 0.05).As compared with the high glucose group,the number of autophagosomes in different doses of lycopene group was significantly increased;the apoptosis rate of the cells was significantly reduced (P＜0.05),and the apoptosis rate of the cells in high glucose + high dose lycopene group was extremely significantly decreased (P＜0.01).The expression of autophagy-related genes LC3-Ⅱ and beclin-1 was significantly increased (P＜0.05).The expression of p62/SQSTMI gene was significantly reduced (P＜ 0.05),and that in high glucose + high dose lycopene group was decreased extremely significantly (P＜0.01).The results of Western blotting on the protein level were consistent with those of RT-PCR.Conclusions Lycopene can further enhance the autophagy of podocytes induced by high glucose,but it still inhibits the apoptosis of podocytes,and the effect of high-dose lycopene is more obvious.%目的 探讨番茄红素(Lyc)对高糖诱导的足细胞自噬及凋亡的影响,旨在为临床治疗糖尿病肾病提供可靠的理论依据.方法 采用条件永生化小鼠足细胞系为研究对象,通过添加不同剂量的Lyc对高糖诱导的足细胞进行干预.实验细胞分6组,分别为正常对照组(5.5mmol/L葡萄糖)、高渗对照组(5.5 mmol/L葡萄糖+19.5 mmol/L甘露醇)、高糖组(25 mmol/L葡萄糖)、低剂量Lyc组(25 mmol/L葡萄糖+3.125 μmol/L Lyc)、中剂量Lyc组(25 mmol/L葡萄糖+ 6.25 μmol/L Lyc)和高剂量Lyc组(25 mmol/L葡萄糖+12.5 μmol/L Lyc).在细胞培养48 h后,用吖啶橙染色法观察各组细胞的自噬体变化情况;流式细胞技术检测各组细胞的凋亡率变化;采用实时定量荧光PCR和蛋白质印迹法分别检测自噬相关基因LC3、beclin-1、p62/SQSTMI mRNA和蛋白的表达变化情况.结果 与正常对照组、高渗对照组相比,高糖组细胞中的自噬体数目明显增多,细胞的凋亡率极显著增加(P＜0.01),自噬相关基因LC3-Ⅱ、beclin-1的表达量显著增加(P＜0.05),而p62/SQSTMI基因的表达量则显著降低(P＜0.05).与高糖组比较,不同剂量Lyc组中的足细胞自噬体数目更多;细胞的凋亡率显著降低(P＜0.05),且高剂量Lyc组细胞的凋亡率降低极显著(P＜0.01);自噬相关基因LC3-Ⅱ、beclin-1表达量显著增加(P＜0.05);而p62/SQSTMI基因表达量则显著降低(P＜0.05);且高剂量Lyc组基因表达量变化极显著(P＜0.01);蛋白水平上的检测结果与mRNA水平基本一致.结论 Lyc能进一步增强高糖诱导引起的足细胞的自噬,但对足细胞的凋亡却起到了一定的抑制作用,且高剂量的Lyc作用效果更加明显.