Background Oxidative stress-induced apoptosis plays an important role in the development of heart failure. 3,5-Dicaffeoylquinic acid (3,5-diCQA), a phenolic compound, has shown protective effects against oxidative stress in many diseases. Objective The objective of this study was to investigate the anti-apoptosis potential of 3,5-diCQA in cardiomyocyte cells under oxidative stress and explore its underlying mechanisms. Design A model of tert-butyl hydroperoxide (TBHP)-induced apoptosis in a cardiomyocyte cell line (H9C2) was established. Cell viabilities on cell lines were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. The apoptosis was measured by hoechst33342 and propidium iodide (PI) fluorescent staining. PI (in red) stained the regions of cell apoptosis; Hoechet33342 (in blue) stained the nuclei. The Western blot was used to determine the expressions of related proteins such as p-PI3K: phosphorylated phosphatidylinositol-3-kinase (p-PI3K), phosphorylated Serine and Threonine kinase AKT (p-AKT), p-PTEN, Bcl-2, Bax, and caspase-3. Afterward, a PI3K inhibitor, LY294002, was applied to confirm the influence of the PI3K/Akt pathway on TBHP-treated cells of 3,5-diCQA. Then, H9C2 cells were pre-incubated with 3,5-diCQA alone to determine if the expression of activated PI3K/Akt signaling was mediated by 3,5-diCQA in H9C2 cells. Results The results showed that TBHP resulted in an increase in cardiomyocyte apoptosis, whereas 3,5-diCQA treatment protected cells from TBHP-induced apoptosis in a dose-dependent manner. Moreover, 3,5-diCQA decreased expressions of Bax and caspase-3 but increased the phosphorylation levels of PI3K and Akt in TBHP-treated cells, which are the key molecules mediating cell survival, whereas phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphorylation was unchanged. Importantly, pre-incubation with a PI3K inhibitor (LY294002) partly abolished the anti-apoptosis effects of 3,5-diCQA. Further, 3,5-diCQA enhanced the phosphorylation levels of PI3K and Akt in H9C2 cells directly, while LY294002 attenuated the effects of 3,5-diCQA on PI3K and Akt. Conclusion This study suggested that 3,5-diCQA rescued myocardium from apoptosis by increasing the activation of the PI3K/Akt signaling pathway.

中文翻译：

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3,5-二咖啡酰奎尼酸通过激活 PI3K/Akt 信号通路保护 H9C2 细胞免受氧化应激诱导的细胞凋亡

背景 氧化应激诱导的细胞凋亡在心力衰竭的发展中起重要作用。3,5-二咖啡酰奎尼酸 (3,5-diCQA) 是一种酚类化合物，在许多疾病中显示出对氧化应激的保护作用。目的本研究旨在探讨3,5-diCQA在氧化应激下心肌细胞的抗凋亡作用，并探讨其潜在机制。设计 建立了叔丁基过氧化氢 (TBHP) 诱导的心肌细胞系 (H9C2) 细胞凋亡模型。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑 (MTT) 测定法测定细胞系上的细胞活力。通过 hoechst33342 和碘化丙啶 (PI) 荧光染色测量细胞凋亡。PI（红色）染色细胞凋亡区域；Hoechet33342（蓝色）对细胞核进行染色。Western blot检测p-PI3K：磷酸化磷脂酰肌醇-3-激酶（p-PI3K）、磷酸化丝氨酸和苏氨酸激酶AKT（p-AKT）、p-PTEN、Bcl-2、 Bax 和 caspase-3。之后，应用 PI3K 抑制剂 LY294002 来确认 PI3K/Akt 通路对 TBHP 处理的 3,5-diCQA 细胞的影响。然后，将 H9C2 细胞与单独的 3,5-diCQA 预孵育以确定活化的 PI3K/Akt 信号传导的表达是否由 H9C2 细胞中的 3,5-diCQA 介导。结果 结果表明，TBHP 导致心肌细胞凋亡增加，而 3,5-diCQA 处理以剂量依赖性方式保护细胞免受 TBHP 诱导的细胞凋亡。此外，3，5-diCQA 降低了 Bax 和 caspase-3 的表达，但增加了 TBHP 处理的细胞中 PI3K 和 Akt 的磷酸化水平，这是介导细胞存活的关键分子，而 10 号染色体上缺失的磷酸酶和张力蛋白同源物 (PTEN) 磷酸化没有变化. 重要的是，与 PI3K 抑制剂 (LY294002) 预孵育部分消除了 3,5-diCQA 的抗细胞凋亡作用。此外，3,5-diCQA 直接增强了 H9C2 细胞中 PI3K 和 Akt 的磷酸化水平，而 LY294002 减弱了 3,5-diCQA 对 PI3K 和 Akt 的影响。结论 本研究表明，3,5-diCQA 通过增加 PI3K/Akt 信号通路的激活来挽救心肌细胞凋亡。而10号染色体上缺失的磷酸酶和张力蛋白同源物（PTEN）磷酸化没有改变。重要的是，与 PI3K 抑制剂 (LY294002) 预孵育部分消除了 3,5-diCQA 的抗细胞凋亡作用。此外，3,5-diCQA 直接增强了 H9C2 细胞中 PI3K 和 Akt 的磷酸化水平，而 LY294002 减弱了 3,5-diCQA 对 PI3K 和 Akt 的影响。结论 本研究表明，3,5-diCQA 通过增加 PI3K/Akt 信号通路的激活来挽救心肌细胞凋亡。而10号染色体上缺失的磷酸酶和张力蛋白同源物（PTEN）磷酸化没有改变。重要的是，与 PI3K 抑制剂 (LY294002) 预孵育部分消除了 3,5-diCQA 的抗细胞凋亡作用。此外，3,5-diCQA 直接增强了 H9C2 细胞中 PI3K 和 Akt 的磷酸化水平，而 LY294002 减弱了 3,5-diCQA 对 PI3K 和 Akt 的影响。结论 本研究表明，3,5-diCQA 通过增加 PI3K/Akt 信号通路的激活来挽救心肌细胞凋亡。