Henan Key Laboratory for Modern Research on Zhongjing's Herbal Formulae, Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou, 450046, PR China.
Henan Key Laboratory for Modern Research on Zhongjing's Herbal Formulae, Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou, 450046, PR China; College of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, 450046, PR China.
J Ethnopharmacol. 2023 Oct 28;315:116659. doi: 10.1016/j.jep.2023.116659. Epub 2023 May 30.
ETHNOPHARMACOLOGICAL RELEVANCE: Modified Xiaoyao San (MXYS), a clinical empirical modified formula based on famous traditional Chinese herbal prescription Xiaoyao San, according to the "yu syndrome" theory of traditional Chinese medicine. MXYS has been shown to be an excellent effective therapy for depression patients in clinic, but the antidepressant mechanisms remain unclear. AIM OF THE STUDY: A growing body of evidence indicates the microglia autophagy and M1 polarized microglia (proinflammatory phenotype)-mediated neuroinflammation act critical roles in the pathogenesis of depression. This study aimed to investigate whether MXYS exerts antidepressant efficacy through inhibition of M1 polarized microglia-mediated neuroinflammation and modulation of autophagy involved in PI3K/Akt/mTOR pathway. MATERIALS AND METHODS: In present research, the lipopolysaccharide (LPS)-induced depressive mice and LPS-stimulated N9 microglia cell line were utilized. Behavioral tests (sucrose preference, tail suspension and open field tests) were carried out to evaluate the antidepressant effect of MXYS. The neuronal damage was measured by Nissl's staining in LPS-treated mice. The proinflammatory cytokine levels, the autophagic markers, microglia M1 polarization as well as the PI3K/Akt/mTOR pathway related proteins of MXYS treatment were analyzed by ELISA kits, Western blot and immunofluorescence staining in vivo and vitro. Finally, the influence of autophagy antagonist (3-MA) on the protective effect of MXYS-containing serum in the LPS-stimulated N9 microglia was investigated. RESULTS: Treatment of LPS-induced depressive mice with MXYS significantly reversed depression-like behaviors, accompanied by reduction of proinflammatory cytokine levels (TNF-α, IL-1β) and amelioration of neuronal damage in prefrontal cortex. MXYS suppressed microglia M1 polarization and promoted autophagy in prefrontal cortex and LPS-stimulated N9 cells. Importantly, the remarkable inhibitory effect of the MXYS-medicated serum on microglia M1 polarization was blocked by autophagy antagonist 3-MA in LPS-stimulated N9 cells. Meanwhile, the MXYS treatment exhibited an excellent inhibition effect of PI3K/Akt/mTOR pathway in vivo and vitro. CONCLUSION: Our research suggests that the antidepressant effect of MXYS in LPS-induced depressive mice may be related to alleviate neuroinflammation through suppression of microglia M1 polarization via enhancing autophagy involved in inactivation of the PI3K/Akt/mTOR pathway.
民族药理学相关性：改良逍遥散（MXYS）是一种基于著名中药逍遥散的临床经验改良配方，根据中医的“郁证”理论。MXYS 已被证明是一种治疗抑郁症患者的有效疗法，但抗抑郁机制仍不清楚。
研究目的：越来越多的证据表明，小胶质细胞自噬和 M1 极化小胶质细胞（促炎表型）介导的神经炎症在抑郁症的发病机制中起着关键作用。本研究旨在探讨 MXYS 是否通过抑制 M1 极化小胶质细胞介导的神经炎症和调节参与 PI3K/Akt/mTOR 通路的自噬来发挥抗抑郁作用。
材料和方法：本研究采用脂多糖（LPS）诱导的抑郁小鼠和 LPS 刺激的 N9 小胶质细胞系。通过蔗糖偏好、悬尾和旷场试验进行行为测试，以评估 MXYS 的抗抑郁作用。通过尼氏染色测量 LPS 处理小鼠的神经元损伤。通过 ELISA 试剂盒、Western blot 和免疫荧光染色，在体内和体外分析 MXYS 治疗后的促炎细胞因子水平、自噬标志物、小胶质细胞 M1 极化以及 PI3K/Akt/mTOR 通路相关蛋白。最后，研究了自噬抑制剂（3-MA）对 LPS 刺激的 N9 小胶质细胞中含 MXYS 血清的保护作用的影响。
结果：用 MXYS 治疗 LPS 诱导的抑郁小鼠可显著逆转抑郁样行为，同时降低促炎细胞因子水平（TNF-α、IL-1β）和改善前额叶皮质的神经元损伤。MXYS 抑制小胶质细胞 M1 极化并促进前额叶皮质和 LPS 刺激的 N9 细胞中的自噬。重要的是，自噬抑制剂 3-MA 阻断了 LPS 刺激的 N9 细胞中 MXYS 介导的血清对小胶质细胞 M1 极化的显著抑制作用。同时，MXYS 治疗在体内和体外对 PI3K/Akt/mTOR 通路表现出极好的抑制作用。
结论：我们的研究表明，MXYS 在 LPS 诱导的抑郁小鼠中的抗抑郁作用可能与通过抑制小胶质细胞 M1 极化来减轻神经炎症有关，这种抑制作用是通过增强自噬来实现的，自噬涉及 PI3K/Akt/mTOR 通路的失活。
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