知母皂苷元对脂多糖诱导的BV2小胶质细胞亚型M1/M2的极化及其抗炎活性

Effect of Sarsasapogenin on Lipopolysaccharide-induced BV2 Microglial Subtype M1/M2 Polarization and Sarsasapogenin’s Anti-inflammatory Activity

本研究探讨了知母皂苷元(SAR)对脂多糖(lipopolysaccharide,LPS)诱导的BV2小胶质细胞M1向M2亚型的极化及其抗炎作用机制。采用MTT法评价SAR对小胶质细胞存活率的影响;Griess法检测NO释放水平;ELISA法检测细胞上清中TNFα、IL1β、IL6和IL10的表达;免疫细胞化学法检测原代小胶质细胞M1和M2亚型,检测p-p65(磷酸化核转录因子亚基65)、p-p38(磷酸化促分裂素原活化蛋白激酶38)和PPARγ在小胶质细胞中的表达。SAR 0.01~1μmol/L浓度时对正常状态和LPS 100 ng/mL诱导的炎症状态下BV2小胶质细胞存活率无显著影响,10和25μmol/L浓度时表现出一定的细胞毒活性;与正常组相比,LPS组NO释放量增加了22.64%,TNFα、IL1β和IL6的表达量上调了25.5%、13.28%和48.21%,而IL10的表达量下调了14.97%,Cox2阳性和Ym1/2阴性表达的M1亚型小胶质细胞显著增加,p38和p65的磷酸化水平分别是正常组的2.80倍和1.86倍,PPARγ的表达量降低了73.90%;与LPS模型组比较,SAR在0.1和1μmol/L浓度时能抑制NO的释放和炎症因子TNFα、IL1β、IL6的分泌;0.1和1μmol/L SAR促进小胶质细胞向M2亚型极化,显著降低p38和p65的磷酸化水平并上调PPARγ的表达。知母皂苷元的抗炎作用机制为通过激活PPARγ而抑制p65和p38的磷酸化,促进小胶质细胞由M1向M2亚型极化,减少炎症因子的表达和炎症效应分子NO的释放。

In this study,the effect of sarsasapogenin(SAR)on the polarization of BV2 microglia from M1 to M2 subtypes induced by lipopolysaccharide as well as the mechanism underlying SAR’s anti-inflammatory activity were investigated.The MTT method was used to evaluate the effect of SAR on the cell viability of microglia.The release of NO was measured by the Griess method.The expressions of TNFα,IL1β,IL6,and IL10 in the cell culture supernatants were measured by the ELISA.The immunocytochemistry method was used to detect the primary microglia M1 and M2 phenotypes,and the expression levels of p65,p38 and PPARγin microglia.The results showed that SAR(0.01~1μmol/L)had no effect on the cell viability of BV2 microglia under normal conditions and in LPS(100 ng/mL)-induced inflammatory state,however,high doses of SAR(10 and 25μmol/L)showed certain cytotoxic effects.Compared with the normal group,LPS-treated model group exhibited an increased level of released NO(by 22.64%),upregulated expressions of TNFα,IL1βand IL6(by 25.5%,13.28%,and 48.21%,respectively),downregulated expression of IL10(by 14.97%),and significantly increased levels of Cox2 positive and Ym1/2 negative expressed M1 microglia.Furthermore,the phosphorylation levels of p38 and p65 were 2.80 times and 1.86 times,respectively,that of the normal group,while the expression of PPARγdecreased by 73.90%in the LPS-treated group.Compared with the LPS model group,the treatment with SAR(0.1 and 1μmol/L)inhibited the release of NO and secretion of TNFα,IL1βand IL6,and promoted LPS-induced polarization of microglia to M2 phenotype,decreased significantly the phosphorylation of p38 and p65,and up-regulated the PPARγexpression.The mechanisms underlying SAR’s anti-inflammatory function were attributed to the inhibition of the phosphorylation of p65 and p38 via the activation of PPARγ,promotion of the polarization of microglia from M1 to M2 subtype,and reduction of the expression of inflammatory cytokines and release of NO.