Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine (BUCM), Beijing, 100700, China; Key Laboratory of Pharmacology Dongzhimen Hospital (BUCM), State Administration of Traditional Chinese Medicine, Beijing, 100700, China.
Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine (BUCM), Beijing, 100700, China; Key Laboratory of Pharmacology Dongzhimen Hospital (BUCM), State Administration of Traditional Chinese Medicine, Beijing, 100700, China; Institute for Brain Disorders, Beijing University of Chinese Medicine (BUCM), Beijing, 100029, China.
J Ethnopharmacol. 2021 Oct 5;278:114264. doi: 10.1016/j.jep.2021.114264. Epub 2021 Jun 1.
Shenzhiling oral liquid (SZL), a traditional Chinese medicine (TCM) compound, is firstly approved by the Chinese Food and Drug Administration (CFDA) for the treatment of mild to moderate Alzheimer's disease (AD). SZL is composed of ten Chinese herbs, and the precise therapy mechanism of its action to AD is far from fully understood.
The purpose of this study was to observe whether SZL is an effective therapy for amyloid-beta (Aβ)-induced myelin sheath and oligodendrocytes impairments. Notably, the primary aim was to elucidate whether and through what underlying mechanism SZL protects the myelin sheath through the PI3K/Akt-mTOR signaling pathway in Aβ-induced OLN-93 oligodendrocytes in vitro.
APP/PS1 mice were treated with SZL or donepezil continuously for three months, and Aβ-induced oligodendrocyte OLN-93 cells mimicking AD pathogenesis of myelin sheath impairments were incubated with SZL-containing serum or with donepezil. LC-MS/MS was used to analysis the active components of SZL and SZL-containing serum. The Y maze test was administered after 3 months of treatment, and the hippocampal tissues of the APP/PS1 mice were then harvested for observation of myelin sheath and oligodendrocyte morphology. Cell viability and toxicity were assessed using CCK-8 and lactate dehydrogenase (LDH) release assays, and flow cytometry was used to measure cell apoptosis. The expression of the myelin proteins MBP, PLP, and MAG and that of Aβ and Aβ in the hippocampi of APP/PS1 mice were examined after SZL treatment. Simultaneously, the expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR were also examined. The expression of proteins, including CNPase, Olig2, NKX2.2, MBP, PLP, MAG, MOG, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR, was determined by immunofluorescence and Western blot, and the corresponding gene expression was evaluated by qPCR in Aβ-induced OLN-93 oligodendrocytes.
LC-MS/MS detected a total of 126 active compounds in SZL-containing serum, including terpenoids, flavones, phenols, phenylpropanoids and phenolic acids. SZL treatment significantly improved memory and cognition in APP/PS1 mice and decreased the G-ratio of myelin sheath, alleviated myelin sheath and oligodendrocyte impairments by decreasing Aβ and Aβ accumulation and increasing the expression of myelin proteins MBP, PLP, MAG, and PI3K/Akt-mTOR signaling pathway associated protein in the hippocampi of APP/PS1 mice. SZL-containing serum also significantly reversed the OLN-93 cell injury induced by Aβ by increasing cell viability and enhanced the expression of MBP, PLP, MAG, and MOG. Meanwhile, SZL-containing serum facilitated the maturation and differentiation of oligodendrocytes in Aβ-induced OLN-93 cells by heightening the expression of CNPase, Olig2 and NKX2.2. SZL-containing serum treatment also fostered the expression of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, and mTOR, indicating an activating PI3K/Akt-mTOR signaling pathway in OLN-93 cells. Furthermore, the effects of SZL on myelin proteins, p-Akt, and p-mTOR were clearly inhibited by LY294002 and/or rapamycin, antagonists of PI3K and m-TOR, respectively.
Our findings indicate that SZL exhibits a neuroprotective effect on the myelin sheath by promoting the expression of myelin proteins during AD, and its mechanism of action is closely related to the activation of the PI3K/Akt-mTOR signaling pathway.
ETHNOPHARMACOLOGICAL 相关性：Shenzhiling 口服液 (SZL)，一种中药 (TCM) 复方，是中国食品药品监督管理局 (CFDA) 首次批准用于治疗轻度至中度阿尔茨海默病 (AD) 的药物。SZL 由十种中药组成，其对 AD 的精确治疗机制还远未完全了解。
本研究的目的是观察 SZL 是否对淀粉样蛋白-β (Aβ) 诱导的髓鞘和少突胶质细胞损伤有治疗作用。值得注意的是，主要目的是阐明 SZL 是否以及通过何种潜在机制通过 PI3K/Akt-mTOR 信号通路在体外 Aβ 诱导的 OLN-93 少突胶质细胞中保护髓鞘。
APP/PS1 小鼠连续用 SZL 或多奈哌齐治疗三个月，并用含 SZL 的血清或多奈哌齐孵育 Aβ 诱导的 OLN-93 少突胶质细胞 OLN-93 细胞，模拟 AD 发病机制中的髓鞘损伤。LC-MS/MS 用于分析 SZL 和含 SZL 的血清中的活性成分。治疗 3 个月后进行 Y 迷宫测试，然后采集 APP/PS1 小鼠的海马组织观察髓鞘和少突胶质细胞形态。使用 CCK-8 和乳酸脱氢酶 (LDH) 释放测定法评估细胞活力和毒性，并用流式细胞术测量细胞凋亡。用 SZL 处理后，检测 APP/PS1 小鼠海马中的髓鞘蛋白 MBP、PLP 和 MAG 以及 Aβ 和 Aβ 的表达。同时，还检测了 p-PI3K、PI3K、p-Akt、Akt、p-mTOR 和 mTOR 的表达。通过免疫荧光和 Western blot 检测 CNPase、Olig2、NKX2.2、MBP、PLP、MAG、MOG、p-PI3K、PI3K、p-Akt、Akt、p-mTOR 和 mTOR 等蛋白质的表达，并通过 qPCR 评估 Aβ 诱导的 OLN-93 少突胶质细胞中的相应基因表达。
LC-MS/MS 检测到 SZL 含血清中的 126 种活性成分，包括萜类、类黄酮、酚类、苯丙素和酚酸。SZL 治疗可显著改善 APP/PS1 小鼠的记忆和认知能力，降低髓鞘 G 比值，通过减少 Aβ 和 Aβ 蓄积，增加 APP/PS1 小鼠海马中的髓鞘蛋白 MBP、PLP、MAG 和 PI3K/Akt-mTOR 信号通路相关蛋白的表达，减轻髓鞘和少突胶质细胞损伤。含 SZL 的血清还通过增加细胞活力，增强 MBP、PLP、MAG 和 MOG 的表达，显著逆转 Aβ 诱导的 OLN-93 细胞损伤。同时，含 SZL 的血清通过提高 CNPase、Olig2 和 NKX2.2 的表达，促进 Aβ 诱导的 OLN-93 细胞中少突胶质细胞的成熟和分化。含 SZL 的血清处理还促进了 OLN-93 细胞中 p-PI3K、PI3K、p-Akt、Akt、p-mTOR 和 mTOR 的表达，表明 PI3K/Akt-mTOR 信号通路在 OLN-93 细胞中被激活。此外，LY294002 和/或 rapamycin（PI3K 和 mTOR 的拮抗剂）明显抑制了 SZL 对髓鞘蛋白、p-Akt 和 p-mTOR 的作用。
我们的研究结果表明，SZL 通过在 AD 期间促进髓鞘蛋白的表达对髓鞘具有神经保护作用，其作用机制与激活 PI3K/Akt-mTOR 信号通路密切相关。
