Baicalin,Geniposide And Their Combination In Regulating M1/M2 Polarization Of Microglia Against Cerebral Ischemic Inflammatory Injury Through 5-LOX/LTB4 Pathway

ObjectiveTo study the mechanism of 5-lipoxygenase(5-LOX)/leukotriene B4(LTB4)pathway regulating M1/M2 polarization of microglia,and baicalin(BC),geniposide(GP)and their compatibility(BC/GP,7:3)regulating M1/M2 polarization of microglia by inhibiting 5-LOX/LTB4 pathway.Methods1.The M1 microglia model was established by stimulating BV2 microglia with lipopolysaccharide(LPS)in vitro to explore the mechanism of BC,GP and BC/GP(7:3)regulating M1/M2 polarization of microglia through 5-LOX/LTB4 pathway.After administration of BC,GP,BC/GP(7:3),LTB4 inhibitor ARM1,LTB4 receptor(BLT1 and BLT2)inhibitors LY223982 and LY255283,the contents of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-10(IL-10)and transforming growth factor-β(TGF-β)in M1 microglia were detected by ELISA,and the content of NO in cell supernatant was detected by Griess.q PCR and Western blotting were used to detect the expression of inducible nitric oxide synthase(i NOS),TNF-α and M2 markers arginase-1(Arg-1),IL-10,STAT6 in microglia;q PCR and WB were used to detect the expression of 5-LOX/LTB4,TLR4/My D88 and MAPK pathways related proteins;immunofluorescence double staining was used to detect the distribution of M1 marker CD86 and M2 marker CD206;immunofluorescence single staining was used to detect the distribution of nuclear factor kappa B(NF-κB).2.BV2 microglia were stimulated in vitro with interleukin-4(IL-4)to establish a model of type M2 microglia,and the effects of BC,GP and BC/GP(7:3)on M2 microglia through 5-LOX/LTB4 pathway were investigated.Microglia were co-cultured with different concentrations(1 n M,10 n M,100 n M and 500 n M)LTB4 and IL-4,and phagocytic ability was detected after 24 h,48 h and 72 h;cell proliferation ability was detected by CCK8 assay;NO content in cell supernatant was detected by Griess method;NF-κB DNA binding activity was detected by EMSA;The expressions of M1 marker i NOS,TNF-α and M2 marker Arg-1,IL-10,STAT6 in microglia were detected by q PCR and Western blotting;The protein expression of BLT1 and BLT2 was detected by Western blotting.Result1.BV2 microglia were stimulated by LPS(100 ng/m L)to change to M1 phenotype.After administration of ARM1,LY223982,LY255283,BC,GP and BC/GP,the expression of pro-inflammatory cytokines TNF-α,IL-6 and M1 markers i NOS,NO decreased significantly(p<0.01),while the expression of anti-inflammatory cytokines IL-10,TGF-β1 and M2 markers Arg-1,STAT6 increased significantly by ELISA(p<0.01),q PCR and Western blotting.Immunofluorescence showed that the expression of M1 marker CD86 was significantly decreased(p<0.01)and that of M2 marker CD206 was significantly increased(p<0.01)after administration.q PCR and Western blotting detection showed that the expression of 5-LOX/LTB4,TLR4/My D88 and MAPK pathway related proteins decreased significantly after administration(p<0.01).Immunofluorescence detection showed that NF-κB transferred from the nucleus to the cytoplasm and inhibited the expression of NF-κB.2.Co-culture of microglia with different concentrations of LTB4(1 n M,10 n M,100 n M and 500 n M)could significantly increase the phagocytic ability and cell viability(p<0.01)of microglia in a time-dependent manner,and 100 n M LTB4 had the strongest effect on proliferation and phagocytosis.Griess and q PCR assay showed that the contents of M1 markers i NOS,NO and TNF-α increased significantly(p<0.01),while M2 markers Arg-1,STAT6 and IL-10 decreased significantly(p<0.01).The protein expression of BLT1 and BLT2 increased significantly(p<0.01)after exogenous LTB4 was given in a dosedependent manner.EMSA detection showed that the DNA binding activity of NF-κ B increased with the increase of LTB4 concentration.3.Microglia were co-cultured with LTB4 and IL-4 at 100 n M.After administration of BC,GP and BC/GP,the phagocytosis of microglia was significantly increased(p<0.01),the expression of M1 marker i NOS,NO and TNF-α was significantly decreased(p<0.01),and the expression of M2 marker Arg-1,STAT6 and IL-10 was increased.In addition,Western blotting data showed that BLT1 and BLT2 proteins expression were significantly decreased(p<0.01)after administration,and the effect of BC/GP was better than that of BC and GP alone.Conclusion5-LOX/LTB4 pathway can regulate M1/M2 polarization of microglia.BC,GP and BC/GP(7:3)inhibited the expression of TLR4/My D88 and MAPK pathways by downregulating the expression of 5-LOX/LTB4 pathway,and promote microglia from M1 to M2.