Research Of The Phenolic Components Extracted From Gastrodia Elata Blume On Inflammation Model Of BV-2 Cells Stimulated By LPS

Objective:The present study was designed to establish an inflammatory model of BV-2 cells stimulated by LPS, illustrate the mechanism on reducing the neuro-inflammation by component B,C,D and H.Methods:1. Effects of component B, C, D and H on the cytokines and protein expression of M1/M2 microglia phenotypesBV-2 microglia was stimulated by LPS to establish an inflammatory model.ELISA kits were used to determine the cytokines of M1 microglia phenotype (TNF-α,IL-1β, IL-6, PGE2) and cytokines of M2 microglia phenotype (IL-10, TGF-β).CD 16/32 and CD206 were determined by flow cytometry. Arginase-1 and iNOS were determined by western blot.2. Effects of component B, C, D and H on MAPK and NF-κB signaling pathway following microglia activationThe protein expression of MAPK and NF-κB signaling pathway (MyD88, p38 MAPK, p-p38 MAPK, JNK, p-JNK, ERK1/2, p-ERK1/2, NF-κB p65, p-NF-κB p65)were determined by western blot.Results:1. Effects of component B, C, D and H on cytokines of M1 microglia phenotypeThe result showed that BV-2 cells were induced by LPS in the concentration of 1μg·mL-1 for 24h, the release of specific cytokines of Ml phenotype microglia significantly increased. Minocycline could significantly reduce the levels of NO,TNF-a, IL-6 and PGE2. Component B, C, D, and H could all significantly reduce the release of TNF-a, but there was some difference on the effect among these four components about other cytokines.2. Effects of phenolic component B, C, D and H on cytokines of M2 microglia phenotypeThe result showed that component B, C, D, H, minocycline and IL-4 could significantly increase the release of IL-10, TGF-β by BV-2 cells without inducing.When BV-2 cells were induced by LPS, the effects of different components were selective.3. Effects of component B, C, D and H on protein expression of M1/M2 microglia phenotypesThe result showed that BV-2 cells were induced by LPS, expression of protein of CD 16/32 and CD206 had little changed, component B, C, D, H, minocycline and IL-4 could all reduce the protein expression of CD 16/32 and increase the protein expression of CD206. The protein expression of iNOS was significantly increased,component H could reduce the protein expression of iNOS.4. Effects of component B, C, D and H on MAPK and NF-κB signaling pathway following microglia activationThe result showed that BV-2 cells were induced by LPS, the protein phosphorylated expression of p3 8 MAPK,JNK and NF-κB p65 was significantly increased,the protein expression of MyD88 and ERK1/2 did not show any changing.Minocycline could reduce the protein phosphorylated expression of p38 MAPK, JNK and NF-κB p65. Component B,C,D and H could all reduce the protein phosphorylated expression of JNK and NF-κB p65. The effects of different components on the expression of other proteins were selective.Conclusions:Component B, C, D, and H have the effect on reducing neuro inflammation with the mechanism of inhibiting the activation of microglia to M1 phenotype and promoting the transformation of M2 phenotype. They played a role in reducing neuro inflammation after cerebral ischemia by down-regulation of p-p38 MAPK and p-NF-κB p65 signaling pathway. The mechanism of effect concretely manifested as that component B, C, D, and H could all significantly reduce the release of TNF-a of BV-2 cells induced by LPS and increase the release of IL-10 and TGF-p of BV-2 cells without inducing. They could all decrease the protein expression of CD 16/32 and increase the protein expression of CD206. And they could all regulate the protein phosphorylated expression of JNK and NF-κB p65. The effects of different components on the release of other cytokines and protein expression were selective.