Baicalin,Geniposide And Their Compatibility On M2 Polarization Mechanism In Microglia And Neuron Protection

ObjectiveTo study whether 5-ipoxygenase(5-LOX)pathway regulates the polarization of microglia,whether Baicalin(BC),Geniposide(GP)and their 7:3 compatibility(PW)affect the polarization of microglia through 5-LOX pathway,and whether Baicalin,Geniposide and their 7:3 compatibility have protective effects on injured neurons.Method(1)The M1 and M2 typing of BV2 microglia was established in vitro,and the optimum concentrations of Baicalin,Geniposide and their 7:3 compatibility on BV2 microglia were screened.(2)The polarization of M1 microglia was analyzed by the expression of inflammatory factors and M1 and M2 markers.The contents of Transforming growth factor-?1(TGF-?1),Interleukin-10(IL-10),Interleukin-1?(IL-1?)and Tumor necrosis factor(TNF-?)in microglia treated with Baicalin,Geniposide and their 7:3 compatibility were detected by ELISA,and the expressions of M1 markers TNF-?,Inducible nitric oxide synthase(iNOS)and M2 markers IL-10,Mannose receptor(Cluster of Differentiation 206,CD206)in microglia before and after treatment were detected by qPCR method.(3)To explore the changes of 5-LOX pathway before and after administration and to explore whether inhibition of 5-LOX pathway is beneficial to M2 polarization of M1 microglia.qPCR method was used to detect the expression of 5-LOX mRNA in microglia before and after treatment,and the expression of M1 markers TNF-?,iNOS and M2 markers IL-10,CD206 mRNA in microglia after administration of Zileuton.The protein expressions of 5-LOX,Cysteinyl leukotriene receptor 1(CysLT1),Cysteinyl leukotriene receptor 2(CysLT2),Leukotriene B4 receptor 1(BLT1)and Leukotriene B4 receptor 2(BLT2)were detected by WB before and after administration of Zileuton.(4)The purpose of this study was to explore whether the drug exerts its neuroprotective effect by directly acting on the damaged nerve cells or by M2 microglia.Firstly,the conditioned incubator and CCK-8 were used to explore the conditions of oxygen-glucose deprivation-reperfusion,and CM-LPS(LPS acting on microglia supernatant),CM-IL4(IL-4-acting on microglia supernatant),CM-PW(PW acting on microglia supernatant)and PW on SH-SY5 Y,after oxygen-glucose deprivation-reperfusion were used to detect the degree of apoptosis by flow cytometry and WB.Result(1)LPS(100ng/m L),IL-4(20ng/mL)can polarize BV2 microglia into two different types: M1 and M2,and the optimum concentrations of BC,GP and PW on microglia are 125 ?M,62.5 ?M and 31.25 ?M.(2)Medium and low concentration of GP and all concentrations of PW could significantly increase the expression of IL-10(P<0.01),medium and high concentration of BC could significantly increase the expression of TGF-?1(P<0.05),and different concentrations of BC and PW could significantly reduce the expression of TNF-?.QPCR showed that the M1 markers iNOS and TNF-? of BV2 cells pretreated with high and low concentrations of BC,GP and PW were lower than those of M1 group,and the expression of CD206 mRNA was increased in high dose PW group.(3)Western blotting showed that the protein expression of 5-LOX,BLT1 and BLT2 in microglia BC,GP and PW stimulated by LPS was lower than that in M1 group(P<0.05),and the effect of PW group was the best.qPCR showed that after BV2 microglia were cultured with Zileuton and stimulated by BC,GP and PW,the expression of 5-LOX mRNA was inhibited.The high and low concentrations of GP and PW could significantly increase the mRNA expression of M2 markers CD206 and IL-10(P<0.05),and the microglia showed M2 polarization.However,after BV2 cells were pretreated with high and low concentrations of BC,GP and PW,the expression of M1 marker iNOS mRNA was inhibited,and the expression of TNF-? mRNA in low dose BC and PW groups was significantly lower than that in M1 group(P<0.05).(4)Flow cytometry and Western blotting showed that CM-IL4,PW and CM-PW could reduce the proportion of apoptosis of SH-SY5 Y neurons,reduce the protein expression of Bax and Caspase-3,and increase the ratio of Bcl-2/Bax.Conclusion.Baicalin,Geniposide and their 7:3 compatibility can inhibit the expression of 5-LOX inflammatory pathway.Baicalin,Geniposide and their 7:3 can down-regulate the expression of 5-LOX inflammatory pathway to promote M1 microglia to M2 polarization and Baicalin / Geniposide 7:3 can directly protect injured neurons,but also indirectly protect injured neurons through M2 polarization of microglia.