School of Pharmacy, Ningxia Medical University, Yinchuan, 750004, China.
Hainan Health Vocational College, Haikou, 813099, China.
Neurochem Res. 2023 Aug;48(8):2451-2462. doi: 10.1007/s11064-023-03920-0. Epub 2023 Apr 3.
Dichotomine B is a [Formula: see text]-Carboline alkaloid isolated from Stellariae Radix. Stellariae Radix, also known as Yin Chai Hu, is a common Chinese medicine in clinical practice. This herb has been demonstrated to have anti-inflammatory activity. This study aimed to investigate the effects and mechanisms of Dichotomine B on neuroinflammation by BV2 microglia induced by lipopolysaccharide (LPS) and adenosine triphosphate (ATP). The experiment was divided into a control group, a model group (10 µg/mL LPS + 5 mM ATP), a model+ TLR4 inhibitor (TAK-242, 10 µmol/L) group, model+ Dichotomine B (20, 40 and 80 µmol/L) groups and Dichotomine B (80 µmol/L) group. The BV2 cell viability was detected by MTT assay, the morphology of BV2 cells was observed by inverted microscope, and the levels of IL-6, IL-1[Formula: see text] and TNF-[Formula: see text] in BV2 cells were determined by ELISA. The expression levels of TLR4, MyD88, p-mTOR/mTOR, p62, p-RPS6/RPS6, LC3II/LC3I and Beclin-1 proteins were detected by western blot assay. The expression levels of TLR4, MyD88, mTOR, p62, RPS6, LC3B and Beclin-1 mRNA were detected by PCR assay. Finally, molecular docking was performed to predict the affinity of Dichotomine B with TLR4, MyD88 and mTOR by LibDock of Discovery Studio and MOE. The results showed that compared with the model group, the survival rates of damaged cells were significantly increased by TAK-242 and Dichotomine B, and the morphology of these BV2 cells improved. The levels of IL-6, IL-1[Formula: see text] and TNF-[Formula: see text] were significantly decreased by TAK-242 and Dichotomine B in LPS/ATP-induced BV2 cells. 80 µmol/L Dichotomine B has no effect on normal BV2 cells. Further mechanism investigation showed that TAK-242 and Dichotomine B significantly inhibited the protein and mRNA expression levels of TLR4, MyD88, p-mTOR/mTOR (mTOR), p62, p-RPS6/RPS6 (RPS6) and increased the protein and mRNA expression levels of LC3II/LC3I (LC3B), Beclin-1. Docking study showed the LibDock scores of Dichotomine B with TLR4, MyD88 and mTOR were all higher than those of positive drugs (Diazepam). These findings indicated that Dichotomine B attenuated neuroinflammatory responses in LPS/ATP-induced BV2 microglia, and its mechanism may be related to TLR4/MyD88-mTOR signaling pathway and autophagy.
B 型二氢血根碱是从丹参中分离得到的一种[化学式：见正文] - 咔啉生物碱。丹参，又称银柴胡，是临床常用的中药。该草药已被证明具有抗炎活性。本研究旨在通过脂多糖（LPS）和三磷酸腺苷（ATP）诱导的 BV2 小胶质细胞探讨 B 型二氢血根碱对神经炎症的影响及其机制。实验分为对照组、模型组（10 µg/mL LPS + 5 mM ATP）、模型+TLR4 抑制剂（TAK-242，10 µmol/L）组、模型+B 型二氢血根碱（20、40 和 80 µmol/L）组和 B 型二氢血根碱（80 µmol/L）组。采用 MTT 法检测 BV2 细胞活力，倒置显微镜观察 BV2 细胞形态，ELISA 法检测 BV2 细胞中 IL-6、IL-1β 和 TNF-α 的水平。Western blot 法检测 TLR4、MyD88、p-mTOR/mTOR、p62、p-RPS6/RPS6、LC3II/LC3I 和 Beclin-1 蛋白的表达水平。PCR 法检测 TLR4、MyD88、mTOR、p62、RPS6、LC3B 和 Beclin-1mRNA 的表达水平。最后，通过 Discovery Studio 的 LibDock 和 MOE 对分子对接进行了 TLR4、MyD88 和 mTOR 与 B 型二氢血根碱亲和力的预测。结果表明，与模型组相比，TAK-242 和 B 型二氢血根碱显著提高了受损细胞的存活率，改善了这些 BV2 细胞的形态。TAK-242 和 B 型二氢血根碱显著降低了 LPS/ATP 诱导的 BV2 细胞中 IL-6、IL-1β 和 TNF-α 的水平。80 µmol/L 的 B 型二氢血根碱对正常 BV2 细胞没有影响。进一步的机制研究表明，TAK-242 和 B 型二氢血根碱显著抑制 TLR4、MyD88、p-mTOR/mTOR（mTOR）、p62、p-RPS6/RPS6（RPS6）的蛋白和 mRNA 表达水平，并增加 LC3II/LC3I（LC3B）、Beclin-1 的蛋白和 mRNA 表达水平。对接研究表明，B 型二氢血根碱与 TLR4、MyD88 和 mTOR 的 LibDock 评分均高于阳性药物（地西泮）。这些发现表明，B 型二氢血根碱可减轻 LPS/ATP 诱导的 BV2 小胶质细胞中的神经炎症反应，其机制可能与 TLR4/MyD88-mTOR 信号通路和自噬有关。
