雷公藤红素上调Nrf2/SIRT3信号通路缓解H2O2诱导心肌细胞氧化应激损伤

    Celastrol alleviates H2O2-induced oxidative stress injury in cardiomyocytes by up-regulating Nrf2/SIRT3 signaling pathway

      摘要
    • 摘要:
      目的 探究雷公藤红素（CEL）激活核因子E2相关因子2（Nrf2）/沉默信息调节因子3（SIRT3）信号通路改善过氧化氢（H2O2）诱导心肌细胞氧化应激损伤。
      方法 用浓度为（0.5～3.0）μmol/L的雷公藤红素与H2O2共同处理AC16细胞，CCK-8法测细胞活性，筛选雷公藤红素最佳浓度；将AC16细胞分为Control组、H2O2组、雷公藤红素低、中、高浓度组（CEL-L+H2O2组、CEL-M+H2O2组、CEL-H+H2O2组）、雷公藤红素高浓度+Nrf2抑制剂组（CEL-H+ML385+H2O2组）；用流式细胞仪检测细胞凋亡；用酶标仪检测细胞丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性、乳酸脱氢酶（LDH）活性；用免疫荧光检测AC16细胞活性氧（ROS）含量；免疫荧光法检测细胞线粒体膜电位（MMP）；Western blot检测B细胞淋巴瘤-2相关X蛋白（Bax）、促凋亡蛋白（Caspase-3）、Nrf2、SIRT3蛋白表达。
      结果 浓度为（1.5～2.5）μmol/L的雷公藤红素可促进H2O2诱导的AC16细胞增殖，选择浓度为1.5 μmol/L、2.0 μmol/L、2.5 μmol/L进行后续实验。与Control组相比，H2O2组细胞凋亡率、活化的Caspase-3（Cleaved-Caspase-3）、Bax蛋白表达、ROS、LDH、MDA水平明显升高，MMP、SOD水平、Nrf2、SIRT3蛋白表达降低（P<0.01）；与H2O2组比较，CEL-L+H2O2、CEL-M+H2O2、CEL-H+H2O2组AC16细胞凋亡率、C-Caspase-3、Bax蛋白表达水平、ROS、LDH、MDA水平依次降低，MMP、SOD水平、Nrf2、SIRT3蛋白表达升高（P<0.05，P<0.01）；与CEL-H+H2O2组比较，CEL-H+ML385+H2O2组AC16细胞凋亡率、C-Caspase-3、Bax蛋白表达水平、ROS、LDH、MDA水平显著上升，MMP、SOD水平、Nrf2、SIRT3蛋白表达下降（P<0.01）。
      结论 雷公藤红素能够减轻H2O2诱导的心肌细胞氧化应激损伤，其可能是通过激活Nrf2/SIRT3信号通路实现的。

       

      Abstract:
      AIM To investigate the effect of celastrol (CEL) on improving hydrogen peroxide (H2O2)-induced oxidative stress injury in cardiomyocytes by activating nuclear factor E2-related factor 2 (Nrf2)/silent information regulator 3 (SIRT3) signaling pathway.
      METHODS AC16 cells were treated with a concentration of 0.5～3.0 μmol/L of celastrol and H2O2. CCK-8 method was applied to measure the cell activity and screen for the optimal concentration of celastrol. AC16 cells were grouped into control group, H2O2 group, low, medium and high concentration celastrol groups (CEL-L+H2O2 group, CEL-M+H2O2 group, CEL-H+H2O2 group) and high concentration celastrol+Nrf2 inhibitor group (CEL-H+ML385+H2O2 group). Flow cytometry was applied to detect cell apoptosis and enzyme-linked immunosorbent assay (ELISA) was applied to detect cellular malondialdehyde (MDA) content, superoxide dismutase (SOD) activity and lactate dehydrogenase (LDH) activity. Immunofluorescence was applied to detect the content of reactive oxygen species (ROS) in AC16 cells and immunofluorescence assay was applied to detect mitochondrial membrane potential (MMP) in cells. Western blot was applied to detect the expressions of B-cell lymphoma associated X protein (Bax), pro apoptotic protein (Caspase-3), Nrf2 and SIRT3 proteins.
      RESULTS Celastrol at concentrations of 1.5-2.5 μmol/L was able to promote H2O2-induced proliferation of AC16 cells. Subsequent experiments were conducted at concentrations of 1.5 μmol/L, 2.0 μmol/L and 2.5 μmol/L. Compared with those in the control group, the apoptosis rate, C-Caspase-3, Bax protein expression, ROS, LDH and MDA levels in the H2O2 group were obviously increased, while MMP, SOD levels, Nrf2 and SIRT3 protein expression were reduced (P<0.01). Compared with those in the H2O2 group, the apoptosis rate, C-Caspase-3, Bax protein expression level, ROS, LDH and MDA levels of AC16 cells in the CEL-L+H2O2, CEL-M+H2O2 and CEL-H+H2O2 groups decreased sequentially, while MMP, SOD levels, Nrf2 and SIRT3 protein expression increased (P<0.05, P<0.01). Compared with those in the CEL-H+H2O2 group, the apoptosis rate of AC16 cells, C-Caspase-3, Bax protein expression level, ROS, LDH and MDA levels in the CEL-H+ML385+H2O2 group obviously increased, while MMP, SOD levels, Nrf2 and SIRT3 protein expression decreased (P<0.01).
      CONCLUSION Celastrol activates the Nrf2/SIRT3 signaling pathway to alleviate H2O2 induced oxidative stress damage in AC16 cells.

       

      • HTML全文
      • 参考文献(21)
      • 相关文章
      • 施引文献
    • 资源附件(0)

    /

    返回文章
    返回