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.

Wang XL ，Chen F ，Shi H ，Zhang M ，Yan L ，Pei XY ，Peng XD ... -  《-》

Curcumin inhibits LPS-induced neuroinflammation by promoting microglial M2 polarization via TREM2/ TLR4/ NF-κB pathways in BV2 cells.

Microglia mediate multiple facets of neuroinflammation, which plays a double-edged role in various brain diseases via distinct microglial phenotypes (deleterious M1 and neuroprotective M2). Therefore, the inhibition of overactivated inflammatory M1 microglia by switching to the protective M2 phenotype appears to be a potential therapeutic strategy in neuroinflammatory disorders. Curcumin has been shown to exhibit anti-inflammatory and neuroprotective activities. The present study investigated the potential effects of curcumin on microglial M1/M2 polarization and elucidated the possible molecular mechanisms of action in vitro. In this study, the BV2 microglial cell line was pretreated with different curcumin concentrations in the presence or absence of lipopolysaccharide (LPS) to assess the anti-inflammatory efficacy of curcumin based on the morphological and inflammatory changes. The cytotoxicity of curcumin for BV2 cells was evaluated using the CCK-8 assay. Further, the effect of curcumin concentrations on LPS-induced BV2 cells was studied. The morphological changes were observed using an optical microscope and immunofluorescent staining. Nitric oxide (NO) expression was determined using the Griess reagent. The expression of cytokines and inflammatory mediators was also measured by ELISA, qRT-PCR, flow cytometry, and immunofluorescence. Western blot analysis was used to determine the levels of triggering receptor expressed on myeloid cells 2 (TREM2), toll-like receptor 4 (TLR4), nuclear factor-kappa B (NF-κB) p65, p-NF-κB p65, IκB, and p-IκB expression. Results showed that curcumin concentrations less than 10 μM did not induce any detectable cytotoxicity but decreased BV2 cell viability up to 20 μM. Curcumin inhibited LPS-induced microglial activation. Curcumin treatment switched the M1 pro-inflammatory phenotype to the M2 anti-inflammatory phenotype by decreasing the expression of M1 markers (i.e., iNOS, IL-1β, IL-6, and CD16/32) and elevating the expression of M2 markers (i.e., arginase 1, IL-4, IL-10, and CD206). Interestingly, curcumin attenuated the activation of TLR4/NF-κB pathways and the downregulation of TREM2 expression in LPS-activated BV2 cells. Collectively, these results suggest that curcumin significantly alleviates LPS-induced inflammation by regulating microglial (M1/M2) polarization by reducing the imbalance of TREM2 and TLR4 and balancing the downstream NF-κB activation.

Zhang J ，Zheng Y ，Luo Y ，Du Y ，Zhang X ，Fu J ... -  《-》

Candesartan modulates microglia activation and polarization via NF-κB signaling pathway.

Qie S ，Ran Y ，Lu X ，Su W ，Li W ，Xi J ，Gong W ，Liu Z ... -  《-》

Effects of Betaine on LPS-Stimulated Activation of Microglial M1/M2 Phenotypes by Suppressing TLR4/NF-κB Pathways in N9 Cells.

Shi H ，Wang XL ，Quan HF ，Yan L ，Pei XY ，Wang R ，Peng XD ... -  《MOLECULES》

Oxymatrine inhibits lipopolysaccharide-induced inflammation by down-regulating Toll-like receptor 4/nuclear factor-kappa B in macrophages.

Zhang Y ，Yan R ，Hu Y 《-》