Wang Yan-Jun, Dai Guo-Liang, Chen Pei-Yao, Hang Hua-Xi, Bian Xin-Fang, Chen Yu-Jie, Ju Wen-Zheng
Affiliated Hospital of Nanjing University of Chinese Medicine Nanjing 210029, China.
Zhongguo Zhong Yao Za Zhi. 2025 Jun;50(11):3117-3126. doi: 10.19540/j.cnki.cjcmm.20250310.402.
Based on the α7 nicotinic acetylcholine receptor(α7nAChR), this study examined how tetrahydropalmatine(THP) affected BV2 microglia exposed to lipopolysaccharide(LPS), aiming to clarify the possible mechanism underlying the anti-depression effect of THP from the perspectives of preventing inflammation and regulating polarization. First, after molecular docking and determination of the content of Corydalis saxicola Bunting total alkaloids, THP was initially identified as a possible anti-depression component. The BV2 microglia model of inflammation was established with LPS. BV2 microglia were allocated into a normal group, a model group, low-and high-dose(20 and 40 μmol·L(-1), respectively) THP groups, and a THP(20 μmol·L(-1))+α7nAChR-specific antagonist MLA(1 μmol·L(-1)) group. The CCK-8 assay was used to screen the safe concentration of THP. A light microscope was used to examine the morphology of the cells. Western blot and immunofluorescence were used to determine the expression of α7nAChR. qRT-PCR was performed to determine the mRNA levels of inducible nitric oxide synthase(iNOS), cluster of differentiation 86(CD86), suppressor of cytokine signaling 3(SOCS3), arginase-1(Arg-1), cluster of differentiation 206(CD206), tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1β. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of TNF-α, IL-6, and IL-1β in the cell supernatant. The experimental results showed that THP at concentrations of 40 μmol·L(-1) and below had no effect on BV2 microglia. THP improved the morphology of BV2 microglia, significantly up-regulated the protein level of α7nAChR, significantly down-regulated the mRNA levels of iNOS, CD86, SOCS3, TNF-α, IL-6, and IL-1β, significantly up-regulated the mRNA levels of Arg-1 and CD206, and dramatically lowered the levels of TNF-α, IL-6, and IL-1β in the cell supernatant. However, the antagonist MLA abolished the above-mentioned ameliorative effects of THP on LPS-treated BV2 microglia. As demonstrated by the aforementioned findings, THP protected LPS-treated BV2 microglia by regulating the M1/M2 polarization and preventing inflammation, which might be connected to the regulation of α7nAChR on BV2 microglia.
基于α7烟碱型乙酰胆碱受体（α7nAChR），本研究探讨了延胡索乙素（THP）对暴露于脂多糖（LPS）的BV2小胶质细胞的影响，旨在从预防炎症和调节极化的角度阐明THP抗抑郁作用的可能机制。首先，通过分子对接和测定岩黄连总生物碱含量，初步确定THP为一种可能的抗抑郁成分。用LPS建立BV2小胶质细胞炎症模型。将BV2小胶质细胞分为正常组、模型组、低剂量和高剂量（分别为20和40 μmol·L⁻¹）THP组以及THP（20 μmol·L⁻¹）+α7nAChR特异性拮抗剂MLA（1 μmol·L⁻¹）组。采用CCK-8法筛选THP的安全浓度。用光学显微镜观察细胞形态。采用蛋白质免疫印迹法和免疫荧光法检测α7nAChR的表达。采用qRT-PCR法检测诱导型一氧化氮合酶（iNOS）、分化簇86（CD86）、细胞因子信号转导抑制因子3（SOCS3）、精氨酸酶-1（Arg-1）、分化簇206（CD206）、肿瘤坏死因子（TNF）-α、白细胞介素（IL）-6和IL-1β的mRNA水平。采用酶联免疫吸附测定（ELISA）法检测细胞上清液中TNF-α、IL-6和IL-1β的水平。实验结果表明，浓度为40 μmol·L⁻¹及以下的THP对BV2小胶质细胞无影响。THP改善了BV2小胶质细胞的形态，显著上调了α7nAChR的蛋白水平，显著下调了iNOS、CD86、SOCS3、TNF-α、IL-6和IL-1β的mRNA水平，显著上调了Arg-1和CD206的mRNA水平，并显著降低了细胞上清液中TNF-α、IL-6和IL-1β的水平。然而，拮抗剂MLA消除了THP对LPS处理的BV2小胶质细胞的上述改善作用。上述结果表明，THP通过调节M1/M2极化和预防炎症来保护LPS处理的BV2小胶质细胞，这可能与α7nAChR对BV2小胶质细胞的调节有关。
