硫化氢通过抑制ROS-TLR4通路对抗高糖诱导的H9c2心肌细胞损伤和炎症反应

Hydrogen Sulfide Protects Against High Glucose-induced H9c2 Cardiomyocyte Injury and Inflammatory Response by Inhibiting ROS-TLR4 Pathway

目的探讨外源性硫化氢(H2S)能否通过抑制活性氧(ROS)-Toll样受体4(TLR4)通路对抗高糖诱导的H9c2心肌细胞损伤和炎症反应。方法应用细胞计数盒(CCK-8)检测细胞存活率,用试剂盒检测细胞培养液中乳酸脱氢酶(LDH)活性,ELISA检测细胞培养液中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)的水平,Hoechst 33258核染色荧光显微镜照相法测定凋亡细胞数量,Western blot测定TLR4和Cleaved Caspase 3蛋白的表达水平,双氯荧光素染色荧光显微镜照相法测定细胞内ROS水平,罗丹明123染色荧光显微镜照相法检测线粒体膜电位(MMP)。结果应用35 mmol/L葡萄糖(高糖)作用H9c2心肌细胞24 h能上调TLR4的表达水平。在高糖作用前,应用1000μmol/L N-乙酰半胱氨酸(NAC)预处理60 min或400μmol/L硫氢化钠(Na HS)预处理30 min能明显减轻高糖对TLR4蛋白表达的上调作用。高糖作用心肌细胞24 h可引起心肌细胞损伤和炎症反应,使细胞存活率降低,LDH活性、凋亡细胞数量、Cleaved Caspase 3蛋白的表达、ROS生成、MMP丢失及IL-1β和TNF-α的分泌增加;400μmol/L Na HS预处理心肌细胞30 min后再予高糖作用24 h或30μmol/L TAK-242(TLR4抑制剂)和高糖共处理心肌细胞24 h能显著拮抗高糖引起的上述损伤和炎症反应。结论外源性H2S可通过抑制ROS-TLR4通路对抗高糖诱导的H9c2心肌细胞损伤和炎症反应。

Aim To investigate whether exogenous hydrogen sulfide（H2S） protects against high glucose（HG）-induced H9c2 cardiomyocyte injury and inflammation response by inhibiting reactive oxygen species（ROS）-Toll-like receptor 4（TLR4） pathway.Methods Cell counter kit-8（CCK-8） assay was used to measure the cell viability,the activity of lactate dehydrogenase（LDH） in the culture medium was measured with commercial kits,the levels of interleukin-1β（IL-1β） and tumor necrosis factor-α（TNF-α） were detected by ELISA,the number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography,the expression levels of TLR4 and Cleaved Caspase 3 were determined by Western blot,the intracellular level of ROS was detected by 2＇,7＇-dichlorfluorescein-diacetate（DCFHDA） staining followed by photofluorography,mitochondrial membrane potential（MMP） was examined by Rhodamine 123 staining followed by photofluorography.Results After H9c2 cardiac cells were exposed to 35 mmol/L glucose（HG）for 24 h,the expression level of TLR4 was significantly increased.Pre-treatment of the cells with 1000 μmol/L N-acetylL-cysteine（NAC） for 60 min or with 400 μmol/L sodium hydrogen sulfide（Na HS） for 30 min before HG exposure considerably attenuated the up-regulation of TLR4 expression level induced by HG.HG induced considerable injuries and inflammatory response,leading to a decrease in cell viability,increases in the activity of LDH,the number of apoptotic cells,the expression of Cleaved Caspase 3,ROS generation,MMP loss as well as the secretion levels of IL-1β and TNF-α.Pretreatment of the cells with 400 μmol/L Na HS for 30 min before HG exposure or co-treatment of the cells with 30 μmol/L TAK-242（an inhibitor of TLR4） and HG for 24 h obviously reduced the above injuries and inflammatory response induced by HG.Conclusion Exogenous H2 S protects against the HG-induced H9c2 cardiomyocyte injury and inflammation response by inhibiting ROS-TLR4 pathway.