表没食子儿茶素没食子酸醋对高糖环境下小鼠足细胞增殖和凋亡作用

Effect of epigallocatechin-3-gallate on cell proliferation and apoptosis of mouse podocytes exposed to high glucose

目的观察作为绿茶主要活性成分的表没食子儿茶素没食子酸醋(EGCG)对高糖环境下足细胞增殖和凋亡的作用并探讨其机制。方法将足细胞分为9组。组1:正常糖(5.6mmol/L)。组2:正常糖(5.6mmol/L)+0.1mmol/LH2O2。组3:DMEM高糖(25mmol/L)。组4:20μmol/LEGCG+DMEM高糖(25mmol/L)。组5:2.0μmol/LEGCG+DMEM高糖(25mmol/L)。组6:0.2μmol/LEGCG+DMEM高糖(25mmol/L)。组7:0.2mmol/L维生素E+DMEM高糖(25mmol/L)。组8:0.1μmol/LEGCG+0.1mmol/L维生素E+DMEM高糖(25mmol/L)。组9:24.4mmol/L甘露醇+5.6mmol/L葡萄糖。采用溴脱氧核苷尿嘧啶(BrdU)酶联免疫吸附试验(ELISA)检测细胞增殖,在Hoechst染色共聚焦激光显微镜下观察不同浓度EGCG作用24h对足细胞凋亡的影响,采用AnnexinV-FITC法检测足细胞凋亡,CM-H2DCFDA荧光探针检测足细胞内活性氧(ROS)生成。结果①足细胞增殖:与组1比较,组2在刺激24h时的足细胞增殖的光密度(D)450值无显著变化(P>0.05),48和72h时D450值均显著降低(P值均<0.05)。与组2比较,组7和组8高糖刺激24h时的D450值显著升高(P值均<0.05),组4、组5和组6无显著变化(P值均>0.05);组4、组7和组8在刺激48、72h时的D450值显著升高(P值均<0.01)。②激光共聚焦显微镜下,凋亡细胞表现为较多的核固缩,DNA浓缩并向核膜靠拢,核质比减小,以及胞膜皱缩等早期凋亡形态学变化。组2、组3的足细胞凋亡较组1增加,组7明显少于组4、组5,组9较组2无明显增加。③不同浓度EGCG作用后,组4、组5、组8的早期凋亡细胞膜联蛋白V(+)PI(-)比例和坏死细胞膜联蛋白V(+)PI(+)V(+)比例均显著低于组2(P值均<0.05);组4、组8的早期凋亡细胞膜联蛋白V(+)PI(-)比例均显著低于组7(P值均<0.05)。④与组2比较,组424h时的ROS显著减少(P<0.05),但组6无显著改变(P>0.05);与组7比较,组424h时的ROS显著减少(P<0.05),6、12h时无显著变化(P值均>0.05)。结论EGCG(20μmol/L)作用72h促进高糖环境下足细胞增殖,EGCG降低高糖刺激下小鼠足细胞凋亡的作用可能是通过减少足细胞ROS的生成实现的。

Objective To investigate the influence of epigallocatechin-3-gallate（EGCG）on apoptosis and cell proliferation of mouse podocytes exposed to high glucose. Methods Mouse podocytes were randomly divided into 9 groups, normal glucose group （5.6 mmol/L, group 1 ）, normal glucose （5.6 mmol/L） ＋ 0.1 mmol/L H2O2 （group 2）, DMEM high glucose （25 mmol/L, group 3）, 20 μmol/L EGCG＋ DMEM high glucose （25 mmol/L, group 4）, 2.0 μmol/L EGCG ＋ DMEM high glucose （25 mmol/L, group 5）, 0.2 μmol/L EGCG ＋ DMEM high glucose （25 mmol/L, group 6）,0.2 mmol/L vitamin E ＋ DMEM high glucose （25 mmol/L）, 0.1 μmol/L EGCG ＋ 0.1 mmol/L vitamin E ＋ DMEM high glucose （25 mmol/L, group 8）, and 24.4 mmol/L mannitol ＋ 5.6 mmol/L glucose （group 9 ）. Cell proliferation was examined by BrdU ELISA assay. The influences of different concentrations of EGCG （24 h treatment） on cell apoptosis were observed by Hoechst 33258 staining and confocal laser scanning microscopy. The apoptosis of cells was examined by annexin fluorescein isothiocyanate （V-FITC） binding by flow cytometry. The intracellular ROS production was measured by fluorescent probe CM-H2DCFDA by fluorescence microscopy. Results ①Compared with group 1, the proliferation of podocyte （D450 value） in group 2 was comparable 24 h after stimulation, and was significantly lower 48 and 72 h after stimulation （P〈0.05）. Compared with group 2, the D450 values in group 7, 8 were significantly higher （P〈0.05） and in group 4, 5, and 6 had no significant changes; those in group 4, 7, and 8 were significantly increased after 48 and 72 h （ P〈0.01 ）.②Cell apoptosis in group 2 and 3 was more severe than that in group 1; that in group 7 was slighter than that in group 4 and 5; and that in group 9 was similar to that in group 2.③EGCG decreased the early apoptosis of cells exposed to high glucose in a time- and dose-dependent manner. EGCG （2.0 Um,20 μmol/L）, a-Tocopherol （0.2 μmol/L） with EGCG （0.1μmol/L）and a-Tocopherol （0.2 μmol/L） decreased the percentage of apoptosis after 24 hr of incubation in high glucose （P〈0.05）. Compared with a-Tocopherol （0.2 μmol/L） or EGCG （20μmol/L）, a-Tocopherol （0.2 μmoi/L） combined with EGCG （0.1 μmol/L） significantly decreased the percentage of apoptosis after 24 h exposure to high glucose （P〈0.05）.④Compared with group 2, group 4 had significantly lower ROS production after 24 h （P〈0.05）, group 6 had a similar ROS production to group 2. Compared with group 7, group 4 had significantly decreased ROS production at 24 h （P〈0.05）, but had no significant changes after 6, and 12 h. Conclusion EGCG （20 μmol/L） can stimulate podocyte proliferation after 72 h incubation under high glucose condition. EGCG can also inhibit high glucose-induced podocyte apoptosis through suppressing the production of ROS.