Abstract：
OBJECTIVE: Necroptosis, as a form of regulated cell necrosis, could participate in myocardial oxidative damage. We investigated whether donepezil attenuates H2O2-induced oxidative stress injury and necroptosis in rat cardiomyocytes.
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.
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.
摘要：
目标：坏死，作为调节细胞坏死的一种形式，参与心肌氧化损伤。我们研究了多奈哌齐是否减轻H2O2诱导的大鼠心肌细胞氧化应激损伤和坏死。
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。
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。
