Oxymatrine inhibits neuroinflammation byRegulating M1/M2 polarization in N9 microglia through the TLR4/NF-κB pathway

Microglia are the primary immune cells involved in the immune response, inflammation, and injury repair in the central nervous system. Under different stimuli, the dual polarization of classically-activated M1 microglia and anti-inflammatory selectively-activated M2 microglia is observed. Oxymatrine (OMT) exerts various anti-inflammatory and neuroprotective effects, but the mechanism underlying its action remains unclear. In the present study, we investigated the effects of OMT on the polarization of M1/M2 microglia in a lipopolysaccharide (LPS)-induced inflammation model in order to elucidate the potential molecular mechanism of action of OMT in vitro. We first used a Cell Counting Kit-8 (CCK-8) to evaluate the effects of different concentrations OMT on the viability of N9 microglia to determine the appropriate concentration for follow-up experiments. Next, Griess reagent and enzyme-linked immunosorbent assay (ELISA) kits were used to detect the expression of the inflammation-related factors nitric oxide (NO), tumour necrosis factor-alpha (TNF-α), and interleukin (IL)-6, -1β, and -10. To evaluate the protective effects of OMT, the ultrastructure of the cells was observed using electron microscopy. Immunofluorescence, flow cytometry, and western blotting were performed to evaluate the effects of OMT on the following markers of M1 and M2 microglia: CD16/32, CD206, Arginase-10 (Arg-1), and inducible nitric oxide synthase (iNOS). Lastly, western blotting and quantitative polymerase chain reaction (qPCR) were used to detect factors associated with the Toll-like Receptor 4/nuclear factor-κB (TLR4/NF-κB) signalling pathway in order to explore the potential mechanism by which OMT regulates microglial polarization. The viability of N9 cells did not decrease when treated with a concentration of 1000 μg/mL OMT. Electron microscopy revealed that a concentration of 100 μg/mL OMT exerted a protective effect on N9 cells stimulated by LPS. The results of the present study indicated that OMT inhibited the over-activation of microglia, increased the levels of the M2 marker IL-10, decreased the levels of the M1 markers NO, TNF-α, IL-6, and IL-1β, promoted the polarization of N9 microglia to the M2 phenotype, and regulated M1/M2 polarization in the microglia by inhibiting TLR4/NF-κB signalling, which effectively attenuated the LPS-induced inflammatory response.