Abstract：
OBJECTIVE: To investigate the effect of emodin on high glucose (HG)-induced podocyte apoptosis and whether the potential anti-apoptotic mechanism of emodin is related to induction of adenosine-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)-mediated autophagy in podocytes (MPC5 cells) in vitro.
METHODS: MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.
RESULTS: HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation.
CONCLUSIONS: Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.
METHODS: MPC5 cells were treated with different concentrations of HG (2.5, 5, 10, 20, 40, 80 and 160 mmol/L), emodin (2, 4, 8 µ mol/L), or HG (40 mmol/L) and emodin (4 µ mol/L) with or without rapamycin (Rap, 100 nmol/L) and compound C (10 µ mol/L). The viability and apoptosis of MPC5 cells were detected using cell counting kit-8 (CCK-8) assay and flow cytometry analysis, respectively. The expression levels of cleaved caspase-3, autophagy marker light chain 3 (LC3) I/II, and AMPK/mTOR signaling pathway-related proteins were determined by Western blot. The changes of morphology and RFP-LC3 fluorescence were observed under microscopy.
RESULTS: HG at 20, 40, 80 and 160 mmol/L dose-dependently induced cell apoptosis in MPC5 cells, whereas emodin (4 µ mol/L) significantly ameliorated HG-induced cell apoptosis and caspase-3 cleavage (P<0.01). Emodin (4 µ mol/L) significantly increased LC3-II protein expression levels and induced RFP-LC3-containing punctate structures in MPC5 cells (P<0.01). Furthermore, the protective effects of emodin were mimicked by rapamycin (100 nmol/L). Moreover, emodin increased the phosphorylation of AMPK and suppressed the phosphorylation of mTOR. The AMPK inhibitor compound C (10 µ mol/L) reversed emodin-induced autophagy activation.
CONCLUSIONS: Emodin ameliorated HG-induced apoptosis of MPC5 cells in vitro that involved induction of autophagy through the AMPK/mTOR signaling pathway, which might provide a potential therapeutic option for diabetic nephropathy.
摘要：
目的：探讨大黄素对高糖(HG)诱导的足细胞凋亡的影响及其潜在的抗凋亡机制是否与体外诱导单磷酸腺苷活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)介导的自噬有关。
方法：用不同浓度的HG（2.5、5、10、20、40、80和160mmol/L）处理MPC5细胞，大黄素（2，4，8µmol/L），或HG（40mmol/L）和大黄素（4µmol/L），有或没有雷帕霉素（Rap，100nmol/L)和化合物C(10µmol/L)。采用细胞计数试剂盒-8(CCK-8)和流式细胞术分析检测MPC5细胞的活力和凋亡,分别。裂解的caspase-3，自噬标记轻链3（LC3）I/II的表达水平，Westernblot检测AMPK/mTOR信号通路相关蛋白。显微镜下观察形态和RFP-LC3荧光的变化。
结果：20、40、80和160mmol/L的HG剂量依赖性地诱导MPC5细胞凋亡，而大黄素(4µmol/L)显著改善HG诱导的细胞凋亡和caspase-3裂解(P<0.01)。大黄素(4µmol/L)可显著提高MPC5细胞中LC3-II蛋白的表达水平并诱导含RFP-LC3的点状结构(P<0.01)。此外，雷帕霉素（100nmol/L）模拟了大黄素的保护作用。此外，大黄素增加AMPK的磷酸化,抑制mTOR的磷酸化。AMPK抑制剂化合物C(10µmol/L)逆转大黄素诱导的自噬激活。
结论：大黄素通过AMPK/mTOR信号通路诱导自噬，可改善HG诱导的MPC5细胞凋亡，这可能为糖尿病肾病提供潜在的治疗选择。
方法：用不同浓度的HG（2.5、5、10、20、40、80和160mmol/L）处理MPC5细胞，大黄素（2，4，8µmol/L），或HG（40mmol/L）和大黄素（4µmol/L），有或没有雷帕霉素（Rap，100nmol/L)和化合物C(10µmol/L)。采用细胞计数试剂盒-8(CCK-8)和流式细胞术分析检测MPC5细胞的活力和凋亡,分别。裂解的caspase-3，自噬标记轻链3（LC3）I/II的表达水平，Westernblot检测AMPK/mTOR信号通路相关蛋白。显微镜下观察形态和RFP-LC3荧光的变化。
结果：20、40、80和160mmol/L的HG剂量依赖性地诱导MPC5细胞凋亡，而大黄素(4µmol/L)显著改善HG诱导的细胞凋亡和caspase-3裂解(P<0.01)。大黄素(4µmol/L)可显著提高MPC5细胞中LC3-II蛋白的表达水平并诱导含RFP-LC3的点状结构(P<0.01)。此外，雷帕霉素（100nmol/L）模拟了大黄素的保护作用。此外，大黄素增加AMPK的磷酸化,抑制mTOR的磷酸化。AMPK抑制剂化合物C(10µmol/L)逆转大黄素诱导的自噬激活。
结论：大黄素通过AMPK/mTOR信号通路诱导自噬，可改善HG诱导的MPC5细胞凋亡，这可能为糖尿病肾病提供潜在的治疗选择。
