血管紧张素-(1-7)通过抑制TLR4激活和坏死性凋亡的相互作用对抗高糖引起的H9c2心肌细胞损伤

目的:研究血管紧张素-(1-7)Ang-(1-7)]能否通过抑制Toll样受体4(TLR4)激活和坏死性凋亡的相互作用对抗高糖(HG)引起的H9c2心肌细胞损伤。方法:应用Western blot检测心肌细胞受体相互作用蛋白3(RIP3;反映坏死性凋亡的指标)和TLR4的表达水平;CCK-8法测定心肌细胞存活率;用试剂盒检测细胞培养液中乳酸脱氢酶(LDH)的活性;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)的分泌水平;双氯荧光素染色法测定细胞内活性氧簇(ROS)水平;罗丹明123染色法测定线粒体膜电位(MMP)。结果:HG(35 mmol/L葡萄糖)作用H9c2心肌细胞24 h可使RIP3的表达水平明显升高,应用30μmol/L TAK-242(TLR4抑制剂)和HG共处理心肌细胞24 h可抑制HG对RIP3的上调;另一方面,HG可上调TLR4的表达,100μmol/L坏死性凋亡的特异性抑制剂necrostatin-1(Nec-1)和HG共处理心肌细胞24 h可抑制HG对TLR4的上调;而1μmol/L Ang-(1-7)和HG共处理心肌细胞24 h能同时抑制HG对RIP3和TLR4的上调。此外,1μmol/L Ang-(1-7)、30μmol/L TAK-242或100μmol/L Nec-1与HG共处理心肌细胞均能对抗HG引起的心肌细胞损伤,细胞存活率升高,LDH活性降低,ROS生成和MMP丢失减少,同时IL-1β和TNF-α的分泌减少。结论:Ang-(1-7)通过抑制TLR4激活和坏死性凋亡的相互作用对抗高糖引起的H9c2心肌细胞损伤。

Angiotensin-(1-7) protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis

AIM: To investigate whether angiotensin-(1-7)Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) activation and necroptosis. METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and TLR4 were determined by Western blot. Cell viability was measured by CCK-8 assay. The activity of lactate dehydrogenase (LDH) in the culture medium was measured with a commercial kit. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was analyzed by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) stating followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased. Co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG. Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100 μmol/L necrostatin-1 (Nec-1; a specific inhibitor of necroptosis) and HG for 24 h attenuated the up-regulation of TLR4 expression induced by HG. Moreover, 1 μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG. On the other hand, co-treatment of the cells with 1 μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response, leading to the increase in the cell viability, and the decreases in the activity of LDH, ROS generation, MMP loss as well as the releases of IL-1β and TNF-α. CONCLUSION: Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis.