大花红景天乙酸乙酯提取物的化学成分及其对阿尔茨海默病模型细胞神经保护作用的研究

Study on the Chemical Components of the Ethyl Acetate Extract of Rhodiola crenulata and Its Neuroprotective Effects on Alzheimer’s Disease Model Cells

    摘要
  • 摘要:
    目的 研究大花红景天乙酸乙酯提取物（Ethyl acetate extract of Rhodiola crenulata， EAERC）的主要化学成分及其对阿尔茨海默病（Alzheimer’s disease， AD）模型细胞的神经保护作用。
    方法 采用液液萃取法制备EAERC，通过UPLC-Q-Exactive Orbitrap MS技术对EAERC的化学成分进行定性分析，并采用HPLC法同时测定没食子酸、红景天苷等6种成分的含量。随后，采用Aβ25-35诱导PC12细胞建立AD细胞模型，运用MTT、ELISA和荧光染色等方法评估EAERC对细胞活力，炎症因子（TNF-α、IL-1β、IL-6），氧化应激指标（ROS、MDA、GSH-Px）及凋亡的影响，并通过Western blot检测凋亡（Bax/Bcl-2、Caspase-3）、NF-κB通路（IκBα、p65）及MAPK通路（ERK、p38、JNK、GSK-3β、Tau）相关蛋白的磷酸化水平。
    结果 共鉴定出56种化合物（包括黄酮类、有机酸等），成功对没食子酸、红景天苷等6种成分进行定量分析，线性关系良好（r≥0.999 7）。细胞实验结果表明，EAERC（25 μg·mL-1）可显著提高AD模型细胞活力（P0.01），降低炎症因子TNF-α、IL-1β和IL-6水平（P0.000 1），降低氧化应激指标ROS、MDA水平，提高GSH-Px活性（P0.000 1），并抑制细胞凋亡（P0.000 1），Bax/Bcl-2比值降低（P0.000 1），Cleaved caspase-3/Caspase-3下调（P0.01）。此外，EAERC可抑制NF-κB信号通路中p-IκBα/IκBα及p-p65/p65的磷酸化（P0.000 1），抑制p38、Tau及JNK蛋白的磷酸化（P0.000 1），同时激活MAPK信号通路中ERK、GSK-3β的磷酸化（P0.000 1），提示其可能通过调节NF-κB/MAPK信号通路缓解Aβ25-35诱导的神经毒性。
    结论 EAERC可通过抑制炎症反应、调节氧化应激及凋亡过程，调控NF-κB/MAPK通路，有效改善Aβ25-35诱导的AD细胞损伤。

     

    Abstract:
    OBJECTIVE To investigate the main chemical components of the ethyl acetate extract of Rhodiola crenulata (EAERC) and its neuroprotective effects on Alzheimer's disease (AD) model cells.
    METHODS EAERC was prepared using liquid-liquid extraction. Qualitative analysis of its chemical components was performed via UPLC-Q-Exactive Orbitrap MS, and the contents of six components, including gallic acid and salidroside, were simultaneously determined using HPLC. Subsequently, an AD cell model was established by inducing PC12 cells with Aβ25-35. Methods such as MTT, ELISA, and fluorescence staining were employed to evaluate the effects of EAERC on cell viability, inflammatory factors (TNF-α, IL-1β, IL-6), oxidative stress markers (ROS, MDA, GSH-Px), and apoptosis. Additionally, Western blot analysis was used to detect the expression or phosphorylation levels of proteins related to apoptosis (Bax/Bcl-2, Caspase-3), the NF-κB pathway (IκBα, p65), and the MAPK pathway (ERK, p38, JNK, GSK-3β, Tau).
    RESULTS A total of 56 compounds (including flavonoids, organic acids, etc.) were identified. Six components, including gallic acid and salidroside, were successfully quantified, and the method showed good linearity (r≥0.999 7). Cellular experiment results demonstrated that EAERC (25 μg·mL⁻¹) significantly increased the viability of AD model cells (P0.01), reduced the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 (P0.000 1), decreased levels of oxidative stress markers ROS and MDA while increasing GSH-Px activity (P0.000 1), inhibited apoptosis (P0.000 1), reduced Bax/Bcl-2 ratio (P0.000 1), and downregulated Cleaved caspase-3/Caspase-3 (P0.01). Furthermore, EAERC inhibited the phosphorylation of p-IκBα/IκBα and p-p65/p65 in the NF-κB signaling pathway (P0.000 1) and the phosphorylation of p38, Tau, and JNK proteins (P0.000 1), while activating the phosphorylation of ERK and GSK-3β in the MAPK signaling pathway (P0.000 1), suggesting that it might alleviate Aβ25-35-induced neurotoxicity by modulating the NF-κB/MAPK signaling pathway.
    CONCLUSION EAERC effectively ameliorates Aβ25-35-induced AD-like cellular damage by inhibiting inflammatory responses, regulating oxidative stress and apoptotic processes, and modulating the NF-κB/MAPK pathway.

     

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