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目的:探讨开放ATP敏感性钾通道(K_(ATP)通道)能否抑制Toll样受体4(TLR4)/核因子-κB(NF-κB)通路对抗高糖(HG)引起的H9c2心肌细胞损伤和炎症。方法:应用Western blot测定TLR4和NF-κB p65的蛋白水平;应用ELISA法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;采用细胞计数试剂盒8(CCK-8)测定心肌细胞存活率;罗丹明123染色荧光显微镜照相法测定线粒体膜电位(MMP);双氯荧光素染色荧光显微镜照相法测定细胞内活性氧簇(ROS)水平;Hoechst 33258核染色荧光显微镜照相法检测凋亡细胞数量。结果:H9c2心肌细胞经HG(35 mmol/L葡萄糖)处理24 h,胞内TLR4和磷酸化NF-κB p65(p-NF-κB p65)的蛋白水平明显增加,100μmol/L K_(ATP)通道开放剂二氮嗪(DZ)预处理30 min可抑制HG对TLR4和p-NF-κB p65蛋白水平的上调作用;此外,30μmol/L TAK-242(TLR4抑制剂)和HG共处理心肌细胞24 h也可减轻HG对p-NF-κB p65的上调作用。另一方面,100μmol/L DZ预处理有明确的心肌保护作用,可抑制HG引起的细胞毒性、炎症反应、线粒体损伤、氧化应激和细胞凋亡,使细胞存活率升高,并减少IL-1β和TNF-α分泌水平、MMP丢失、ROS生成及凋亡细胞数量;而30μmol/L TAK-242或100μmol/L PDTC(NF-κB抑制剂)共处理心肌细胞24 h也可发挥和DZ相类似的作用,能抑制HG引起的上述损伤和炎症反应。结论:开放K_(ATP)通道可通过抑制TLR4/NF-κB通路对抗HG引起的H9c2肌细胞损伤和炎症。
OBJECTIVE: To investigate whether opening the ATP-sensitive potassium channel (K-ATP channel) can inhibit the injury of H9c2 cardiomyocytes induced by high glucose (HG) by TLR4 / NF-κB pathway And inflammation. Methods: The protein levels of TLR4 and NF-κB p65 were detected by Western blot. The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell culture medium were detected by ELISA. The viability of cardiomyocytes was measured by counting kit 8 (CCK-8), the mitochondrial membrane potential (MMP) was measured by rhodamine 123 staining fluorescence microscope, the level of intracellular reactive oxygen species (ROS) was measured by fluorocarbon staining The number of apoptotic cells was detected by Hoechst 33258 nuclear staining fluorescence microscopy. RESULTS: The H9c2 cardiomyocytes were treated with HG (35 mmol / L glucose) for 24 h. The protein levels of TLR4 and phosphorylated NF-κB p65 in H9c2 cells were significantly increased, while those of 100 μmol / L K ATP channels The preconditioning with diazoxide (DZ) for 30 min inhibited the upregulation of TLR4 and p-NF-κB p65 protein by HG. In addition, 30 μmol / L TAK-242 (TLR4 inhibitor) and HG co-treated cardiomyocytes 24 h can also reduce the HG p-NF-κB p65 upregulation. On the other hand, pretreatment with 100 μmol / L DZ had definite myocardial protective effect, which could inhibit the cytotoxicity, inflammation, mitochondrial damage, oxidative stress and apoptosis induced by HG, increased the cell survival rate and decreased the expression of IL- 1βand TNF-α secretion, MMP loss, ROS production and apoptotic cells; while 30μmol / L TAK-242 or 100μmol / L PDTC co-treatment of myocardial cells 24 h also play a role in the DZ phase A similar effect, can inhibit the HG caused by the above injury and inflammatory response. CONCLUSION: Open K ATP channels can prevent HG-induced damage and inflammation of H9c2 myocytes by inhibiting TLR4 / NF-|ÊB pathway.