Dichotomine B attenuates neuroinflammatory responses via regulating TLR4/MyD88-mTOR signaling pathway in BV2 cells

Abstract Dichotomine B is a β-Carboline alkaloid isolated from Stellariae Radix. Stellariae Radix is a common Chinese medicine in clinical practice. This herb has been proved to have anti-inflammatory activity. The aim of this study was 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 control group, model group (10 µg/mL LPS + 5mM ATP), 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, the levels of IL-6, IL-1β and TNF-α 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, the molecular docking was performed to predict the affinity of Dichotomine B with TLR4, MyD88 and mTOR by LibDock of Discovery Studio. 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-1β and TNF-α were significantly decreased by TAK-242 and Dichotomine B in LPS/ATP-induced BV2 cells. 80 µmol/L Dichotomine B have 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.