Dichotomine B Attenuates Neuroinflammatory Responses by Regulating TLR4/MyD88-mTOR Signaling Pathway in BV2 Cells.

Abstract

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.