血管紧张素-(1-7)/M as 受体轴通过调控 NF-κB 通路保护心肌细胞对抗高糖诱导的损伤

摘要

AIM:Tostudywhe ther theangiotens in-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardio-myocytes against high glucose (HG)-induced injury by inhibiting nuclear factor-κB (NF-κB) pathway.METHODS:The cell viability was measured by CCK-8 assay.The intracellular levels of reactive oxygen species ( ROS) were detected by DCFH-DA staining .The number of apoptotic cells was tested by Hoechst 33258 nuclear staining .Mitochondrial membrane potential ( MMP) was examined by JC-1 staining.The levels of NF-κB p65 subunit and cleaved caspase-3 protein were de-termined by Western blotting.RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose (HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated ( p) NF-κB p65, peaking at 60 min.Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30μmol/L and HG for 24 h inhibited HG-induced cy-totoxicity, evidenced by an increase in cell viability .On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage , indicated by decreases in the number of apoptotic cells , cleaved caspase-3 level, ROS generation and MMP loss .However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor (Mas receptor).Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC ( an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG.CONCLUSION:Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway .%目的：探讨血管紧张素－（1－7）[Ang－（1－7）]／Mas受体轴能否通过抑制核因子－κB （NF－κB）通路对抗高糖（ HG）引起的心肌细胞损伤。方法：应用细胞计数检测试剂盒（ CCK－8）检测心肌细胞存活率；双氯荧光素（DCFH－DA）染色荧光显微镜照相法检测胞内活性氧（ROS）水平；Hoechst 33258核染色荧光显微镜照相测定凋亡细胞的形态及数量的变化；JC－1染色法测定线粒体膜电位（ MMP）；应用免疫蛋白印迹法测定NF－κB p65和cleaved caspase－3蛋白的表达水平。结果：应用35 mmol／L葡萄糖分别处理H9c2心肌细胞30、60、90、120和150 min均能明显增加磷酸化（p） NF－κB p65的水平，其中60 min时，表达水平增加最明显；1μmol／L Ang－（1－7）与HG共同处理心肌细胞60 min能抑制HG对p－NF－κB p65表达的上调作用；0．1～30μmol／L的Ang－（1－7）与HG分别共处理心肌细胞24 h均能抑制HG的细胞毒性，使细胞存活率增多；另一方面，1μmol／L Ang－（1－7）能抑制HG引起的细胞凋亡、氧化应激、线粒体损伤等，使凋亡细胞数量、cleaved caspase－3表达、ROS生成水平及MMP丢失减少；但是10μmol／L Ang－（1－7）／Mas受体拮抗剂A－779能明显阻断上述的Ang－（1－7）的心肌细胞保护作用；与Ang－（1－7）的作用相似，100μmol／L PDTC （ NF－κB抑制剂）与HG共处理心肌细胞24 h也能显著抑制上述的HG损伤作用。结论：Ang－（1－7）／Mas受体轴可通过抑制NF－κB通路对抗HG诱导的心肌细胞损伤。