Hubei Key Laboratory of Diabetes and Angiopathy, Hubei University of Science and Technology, Xianning, 437100, China.
Institute of Preventative Medicine, Zhejiang Provincial Key Laboratory of Pathological and Physiological Technology, School of Medicine, Ningbo University, Ningbo, 315211, China.
J Ethnopharmacol. 2021 Mar 25;268:113579. doi: 10.1016/j.jep.2020.113579. Epub 2020 Nov 12.
Gnaphalium affine D. Don is an important Traditional Chinese herbal Medicine (TCM) used to treat hyperuricemia, asthma, rheumatic arthritis, antitussive, expectorant and cardiovascular in folk medicine because of anti-inflammatory and anti-oxidant activity. The aim of this study was to investigate the potential beneficial effect of G. affine extract (GAE) on hydrogen peroxide (HO)-induced apoptosis and explore the possible underlying mechanism in cardiomyocyte.
The ingredients of GAE were isolated and tentatively identified by HPLC-ESI-Q-Qribatrip-MS/MS. The cardioprotective and anti-oxidant effects of GAE were evaluated in the experimental model with HO induced apoptosis in H9c2 cells. H9c2 cells were pretreated for 3 h with or without GAE or with GAE plus PX866 (PI3K inhibitor), then exposed to HO for 6 h, H9c2 cells viability were detected by CCK8 kit, the content of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) and intracellular superoxide dismutase (SOD) activity were measured by the commercial biochemical kits, western blotting, immunohistochemical (IHC), immunofluorescence (IF) and reverse transcription-polymerase chain reaction (RT-PCR) assays were performed to evaluate the proteins and mRNA expression, propidium iodide (PI) staining was adopted to indicate H9c2 cells apoptosis.
Firstly, seventeen polyphenols and flavonoids compounds with the characteristics of anti-inflammatory and anti-oxidant in GAE were tentatively identified by HPLC-ESI-Q-Qribatrip-MS/MS. In the experimental model, GAE not only significantly improved cells viability, but also showed anti-oxidant effects through improving SOD activity, up-regulating nuclear factor E2-related factor 2 (Nrf2), and decreasing intracellular concentration of ROS and MDA and the proteins expression of p47, p67 and gp91. On the other hand, GAE revealed anti-apoptotic effect through up-regulating the expression of B-cell lymphoma-2 (Bcl-2), down-regulating Bcl2-associated X (BAX) and cleaved-caspase 3. Furthermore, GAE significantly facilitated phosphorylation of AKT and glycogen synthase kinase-3 beta (GSK-3β) but not AMPK, while the effects were blocked by PX866 (PI3K inhibitor).
Our data suggested that GAE showed strong anti-oxidant effect to ameliorate oxidative stress and attenuate apoptosis induced by HO in H9c2 cells by targeting PI3K/AKT/GSK-3β signaling pathway.
茵陈蒿 D. 唐是一种重要的中药，用于治疗高尿酸血症、哮喘、风湿性关节炎、镇咳、祛痰和心血管疾病，因为其具有抗炎和抗氧化活性。本研究旨在探讨茵陈蒿提取物（GAE）对过氧化氢（HO）诱导的细胞凋亡的潜在有益作用，并探讨其在心肌细胞中的可能作用机制。
采用高效液相色谱-电喷雾串联四极杆飞行时间质谱（HPLC-ESI-Q-Qribatrip-MS/MS）对 GAE 的成分进行分离和初步鉴定。采用 HO 诱导 H9c2 细胞凋亡的实验模型评价 GAE 的心脏保护和抗氧化作用。H9c2 细胞先用或不用 GAE 或 GAE 加 PX866（PI3K 抑制剂）预处理 3 h，然后暴露于 HO 6 h，用 CCK8 试剂盒检测 H9c2 细胞活力，用商业生化试剂盒检测细胞内活性氧（ROS）和丙二醛（MDA）含量及细胞内超氧化物歧化酶（SOD）活性，采用 Western blot、免疫组化（IHC）、免疫荧光（IF）和逆转录-聚合酶链反应（RT-PCR）检测蛋白和 mRNA 表达，碘化丙啶（PI）染色检测 H9c2 细胞凋亡。
首先，采用 HPLC-ESI-Q-Qribatrip-MS/MS 对 GAE 中的十七种多酚和类黄酮化合物进行了初步鉴定，这些化合物具有抗炎和抗氧化作用。在实验模型中，GAE 不仅显著提高了细胞活力，还通过提高 SOD 活性、上调核因子 E2 相关因子 2（Nrf2）、降低细胞内 ROS 和 MDA 浓度以及 p47、p67 和 gp91 蛋白表达来发挥抗氧化作用。另一方面，GAE 通过上调 B 细胞淋巴瘤-2（Bcl-2）的表达，下调 Bcl2 相关 X（BAX）和 cleaved-caspase 3 的表达，显示出抗凋亡作用。此外，GAE 显著促进 AKT 和糖原合成酶激酶-3β（GSK-3β）的磷酸化，但不影响 AMPK，而这些作用被 PX866（PI3K 抑制剂）阻断。
我们的数据表明，GAE 通过靶向 PI3K/AKT/GSK-3β 信号通路，显示出强大的抗氧化作用，可改善 H9c2 细胞中由 HO 引起的氧化应激和细胞凋亡。
