摘要
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。
AIM： To study whether the angiotensin-（ 1-7） [Ang-（ 1-7） ]/Mas receptor axis protects cardiomyocytes 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 determined 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 - 30 μmol / L and HG for 24 h inhibited HG-induced cytotoxicity,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.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2015年第2期267-273,共7页
Chinese Journal of Pathophysiology
基金
广东省科技计划(No.2012B031800358)
