The Effect Of Safftower Yellow On Inflammatory Signaling Pathways Of Mouse Microglia

Activated microglia, especially the polarized M1 cells, produces pro-inflammatory cytokines and free radicals, thereby contributing directly to neuroinflammation and various brain disorders. Excessive or chronic neuroinflammation within the CNS exacerbates neuronal damages. Therefore, the research of the molecular mechanism about regulation of nerve inflammation can provide targets for neuroprotection. Safflower yellow(SY), as the main active ingredients of the traditional Chinese medicine safflower, has been applied to the clinic, and its anti-inflammatory mechanism may become an important molecular mechanism in drug effect. Based on the preliminary research, this study, taking BV2 cells as the research object, adopts such methods as molecular biological technique and flow-cytometric analysis to evaluate the effect of SY on the expression of microglia and that on inflammatory signaling pathways to partially explain the action mechanism of SY in clinical treatment, which could provide theoretical basis for adopting SY to treat nervous system diseases.PartⅠ Safflower Yellow inhibits the inflammatory response and regulates microglial polarization in LPS-stimulated BV2 cellsObjective:The purpose of this study is to discover the induction effect of LPS on activating microglia and discuss the influence of SY on microglia phenotype as well as related molecular mechanism for restraining the inflammatory reaction of microglia.Methods:We routinely cultured and subcultured BV2 cells. After the cells affixed to the dish, we started the experiments. The study was designed into three groups: PBS control group, LPS stimulation group and LPS+SY intervention group. Firstly, appropriate dose range was determined with MTT method. Secondly, the expression of TLR2, TLR4 m RNA and protein of BV2 cells were detected by fluorescent quantitative PCR method and western blotting. The expression of Myd88, p-NF-κB(p65), p-P38, p-JNK, p-ERK, COX-2, i NOS and Arg-1 were detected by western blot. We detected the expression of TLR2, TLR4, CD206, CD16/32, IL-10, IL-12, i NOS, and Arg-1 with flow cytometry, and the expression of CD206, CD16/32, IL-10, IL-12, i NOS, and Arg-1 with immumofluorescence method at the same time. Thirdly, we detected the expression of TNF-α, IL-1β, IL-6, IL-10 and NO with ELISA or Griess methods.Results:In this study, we provide evidences that SY modulates neuroinflammation by acting directly on BV2 microglia. LPS-stimulated BV2 cells upregulated the expression of TLR4-Myd88 and NF-κB-MAPK signaling pathways to produce IL-1β, IL-6, TNF-α and COX-2, and mediated the downregulation of i NOS, CD16/32 and IL-12 and the upregulation of CD206 and IL-10, indicating the polarization of inflammatory M1 BV2 cells induced with LPS. However, SY treatment inhibited the TLR4-Myd88 and NF-κB-p-38/p-JNK, declined the production of inflammatory cytokines, and converted inflammatory M1 BV2 cells toward anti-inflammatory M2 microglia.Conclusion: SY exhibited anti-inflammatory effect on BV2 microglia possibly through TLR-4/NF-κB/MAPK signaling pathways and the conversion of M1 to M2microglia.PartⅡ The effect of safflower yellow on microglia inflammatory pathways after Oxygen Glucose DeprivationObjective: The purpose of this study is to investigate the anti-inflammatory effect of Safflower yellow(SY) on BV2 microglia with oxygen glucose deprivation/reoxygenation(OGD/R) and possible mechanism.Methods: We routinely cultured and subcultured BV2 cells, build OGD/R model after the cell grew up to about 90%. The study was designed into three groups: Normal control group, OGD/R group and OGD/R plus SY. The expression of TLR4 and p-NF-κB(p65) protein were detected by Western blot, and the production of IL-6, IL-1β and TNF-α were detected by ELISA kits.ResμLts: After being treated with OGD/R, the expression of TLR4 and p-NF-κB(p65) protein was increased compared with normal control group. However, SY had the ability to restrain the expression of TLR4 and p-NF-κB(p65) protein, as well as the secretion of inflammatory factors IL-6, IL-1β and TNF-α on BV2 microglia.Conclusion:OGD/R may induce the activation of the TLR4/NF-κB signaling pathway. SY can reverse OGD/R-induced inflammatory response on BV2 microglia by inhibiting TLR4/NF-κB signaling pathway.