METHODS: H9c2 cells were incubated with H2O2 (final concentration of 1 mM) and then intervened with donepezil at doses of 2.5 and 10 μM. Subsequently, the necroptosis inhibitor necrostatin-1 (Nec-1) was introduced to treat H9c2 cells. For cell function experiments, cell proliferation; the contents of creatine kinase (CK), lactate dehydrogenase (LDH), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), and malondialdehyde (MDA); the protein and mRNA levels of the necroptosis-related proteins receptor-interacting serine-threonine kinase 3 (RIP3) and mixed lineage kinase-like (MLKL); and calcium ion fluorescence intensity were detected using Cell Counting Kit-8, enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative reverse transcription polymerase chain reaction, and flow cytometry, respectively.
RESULTS: Cell viability was conspicuously decreased; CK and LDH contents, RIP3 and MLKL expression levels, and MDA production were preeminently elevated; and the production of SOD, CAT, and GSH was prominently reduced under H2O2 stimulation, which were dose-dependently countered by donepezil intervention. Nec-1 decreased the cell necroptosis, oxidative stress, and calcium overload caused by H2O2. However, on the premise of donepezil intervention, the addition of Nec-1 failed to further improve the situation, suggesting that donepezil exerts cardioprotective effects partly by inhibiting RIP3 and MLKL levels.
CONCLUSIONS: Donepezil reduced H2O2-inflicted oxidative stress and necroptosis in cardiomyocytes by suppressing RIP3 and MLKL levels and calcium ion overload.
方法:将H9c2细胞与H2O2(终浓度为1mM)一起孵育,然后以2.5和10μM的剂量用多奈哌齐进行干预。随后,引入坏死凋亡抑制剂necrostatin-1(Nec-1)来治疗H9c2细胞。对于细胞功能实验,细胞增殖;肌酸激酶(CK)的含量,乳酸脱氢酶(LDH),超氧化物歧化酶(SOD),过氧化氢酶(CAT),谷胱甘肽(GSH),和丙二醛(MDA);坏死相关蛋白受体相互作用丝氨酸-苏氨酸激酶3(RIP3)和混合谱系激酶样(MLKL)的蛋白和mRNA水平;使用CellCountingKit-8,酶联免疫吸附试验(ELISA)检测钙离子荧光强度,西方印迹,定量逆转录聚合酶链反应,和流式细胞术,分别。
结果:细胞活力明显下降;CK和LDH含量,RIP3和MLKL表达水平,MDA产量显著升高;SOD的产量,CAT,GSH在H2O2刺激下显著降低,多奈哌齐干预是剂量依赖性的。Nec-1降低了细胞坏死,氧化应激,和H2O2引起的钙超载。然而,在多奈哌齐干预的前提下,Nec-1的加入未能进一步改善这种情况,提示多奈哌齐部分通过抑制RIP3和MLKL水平发挥心脏保护作用。
结论:多奈哌齐通过抑制RIP3和MLKL水平和钙离子超负荷来降低H2O2引起的心肌细胞氧化应激和坏死。