Li Ruolan, Wang Lingyu, Zhang Qing, Duan Huxinyue, Qian Die, Yang Fei, Xia Jun
School of Pharmacy, School of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, China.
Front Pharmacol. 2022 Aug 25;13:966348. doi: 10.3389/fphar.2022.966348. eCollection 2022.
Alzheimer's disease (AD) is a typical neurodegenerative disease, which occurs in the elderly population. Fructus (AOF) is a traditional Chinese medicine that has potential therapeutic effect on AD, but the mechanism behind it is unclear. Firstly, the main chemical components of AOF were identified by LC-MS, while the main active ingredients and targets were screened by TCMSP database. At the same time, AD-related target proteins were obtained using Genecards and OMIM databases. PPI was constructed by cross-linking AOF and AD targets, and GO enrichment analysis and KEGG pathway enrichment analysis were performed to identify the relevant biological processes and signaling pathways. Finally, based on the HO-stimulated PC12 cell, flow cytometry, WB and immunofluorescence experiments were performed to verify the protective effect of AOF on AD. We identified 38 active ingredients with 662 non-repetitive targets in AOF, of which 49 were potential therapeutic AD targets of AOF. According to the GO and KEGG analysis, these potential targets are mainly related to oxidative stress and apoptosis. The role of AOF in the treatment of AD is mainly related to the PI3K/AKT signaling pathway. Protocatechuic acid and nootkatone might be the main active ingredients of AOF. In subsequent experiments, the results of CCK-8 showed that AOF mitigated PC12 cell damage induced by HO. Kits, flow cytometry, and laser confocal microscopy indicated that AOF could decrease ROS and increase the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), while AOF could also increase mitochondrial membrane potential (MMP), thereby inhibiting apoptosis. Finally, immunofluorescence and WB results showed that AOF inhibited the expression of BAX and caspase-3 in PC12 cells, and promoted the expression of Bcl-2. At the same time, the phosphorylation levels of PI3K and Akt proteins were also significantly increased. This study suggests that AOF had the potential to treat AD by suppressing apoptosis induced by oxidative stress via the PI3K/Akt pathway.
阿尔茨海默病（AD）是一种典型的神经退行性疾病，多见于老年人群。山茱萸果实（AOF）是一种对AD具有潜在治疗作用的传统中药，但其背后的机制尚不清楚。首先，通过液相色谱-质谱联用（LC-MS）鉴定了AOF的主要化学成分，同时利用中药系统药理学数据库（TCMSP）筛选了主要活性成分和靶点。与此同时，使用Genecards和在线孟德尔人类遗传数据库（OMIM）获得AD相关靶蛋白。通过交联AOF和AD靶点构建蛋白质-蛋白质相互作用（PPI）网络，并进行基因本体（GO）富集分析和京都基因与基因组百科全书（KEGG）通路富集分析，以确定相关的生物学过程和信号通路。最后，基于过氧化氢（HO）刺激的PC12细胞，进行了流式细胞术、蛋白质免疫印迹（WB）和免疫荧光实验，以验证AOF对AD的保护作用。我们在AOF中鉴定出38种活性成分，具有662个非重复靶点，其中49个是AOF潜在的治疗AD靶点。根据GO和KEGG分析，这些潜在靶点主要与氧化应激和细胞凋亡有关。AOF在治疗AD中的作用主要与磷脂酰肌醇-3激酶/蛋白激酶B（PI3K/AKT）信号通路有关。原儿茶酸和诺卡酮可能是AOF的主要活性成分。在后续实验中，细胞计数试剂盒（CCK-8）结果显示，AOF减轻了HO诱导的PC12细胞损伤。试剂盒、流式细胞术和激光共聚焦显微镜检查表明，AOF可降低活性氧（ROS）水平，提高超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和谷胱甘肽过氧化物酶（GSH-Px）活性，同时AOF还可增加线粒体膜电位（MMP），从而抑制细胞凋亡。最后，免疫荧光和WB结果显示，AOF抑制了PC12细胞中促凋亡蛋白BAX和半胱天冬酶-3（caspase-3）的表达，并促进了抗凋亡蛋白Bcl-2的表达。同时，PI3K和Akt蛋白的磷酸化水平也显著升高。本研究表明，AOF具有通过PI3K/Akt通路抑制氧化应激诱导的细胞凋亡来治疗AD的潜力。
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