枸杞多糖通过抑制TLR4/NF-κB信号通路促进LPS诱导的BV2小胶质细胞M2型极化

Lycium barbarum polysaccharide promotes M2 polarization of BV2 microglia induced by LPS via inhibiting the TLR4/NF-κB signaling pathway

目的探讨枸杞多糖(LBP)对脂多糖(LPS)诱导的BV2小胶质细胞由M1型向M2型极化的作用及其机制。方法实验分为对照组、LPS组、(0.6、0.9、1.2)g/L LBP处理组。噻唑蓝(MTT)法检测LBP对BV2细胞活性的影响,Griess法检测细胞一氧化氮(NO)释放量,ELISA检测细胞肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)的分泌,免疫荧光细胞化学染色法检测Toll样受体4(TLR4)、核因子κB(NF-κB)、一氧化氮合酶(iNOS)和精氨酸酶1(Arg1)的表达,Western blot法检测钙离子结合接头蛋白分子1(Iba-1)、TLR4、NF-κB、iNOS和Arg1的蛋白水平。结果不同剂量LBP处理后,细胞存活率无明显变化。与正常组相比,LPS组小胶质细胞活化明显,呈阿米巴样,NO释放量增加;Iba-1、TLR4、NF-κB、iNOS、TNF-α、IL-1β、IL-6含量增加,Arg1和IL-10含量降低。LBP组Iba-1、TLR4、NF-κB、iNOS、TNF-α、IL-1β、IL-6含量降低,与剂量呈负相关,Arg1和IL-10含量增加,与剂量呈正相关。结论LBP可能通过阻断TLR4/NF-κB信号通路抑制LPS诱导的BV2细胞活化并促进激活的BV2细胞由M1型向M2型极化。

Objective To investigate the effect of Lycium barbarum polysaccharide(LBP)on the polarization of BV2 microglia from M1 to M2 induced by lipopolysaccharide(LPS)and its mechanism.Methods The BV2 microglia were divided into control group,LPS group,and LBP treatment group(0.6,0.9,1.2)g/L.MTT assay was used to observe the cell viability of BV2 cells,and Griess assay was used to detect the release of NO.The levels of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by ELISA.The expressions of Toll-like receptor 4(TLR4),nuclear factor kappa B(NF-κB),inducible nitric oxide synthase(iNOS),and arginase-1(Arg1)were detected by immunofluorescence cytochemistry.Western blot was used to evaluate the protein levels of ionized calcium-binding adaptor molecule-1(Iba-1),TLR4,NF-κB,iNOS,and Arg1.Results There was no significant difference of the cell survival rate after treatment with different doses of LBP.Compared to those in the control group,in LPS group the BV2 microglia were activated with amoeba-like shape and increased release of NO,the expressions of Iba-1,TLR4,NF-κB,iNOS,TNF-α,IL-1β,and IL-6 were significantly increased,while the expressions of Arg1 and IL-10 was significantly decreased.In LBP group,Iba-1,TLR4,NF-κB,iNOS,TNF-α,IL-1β,and IL-6 were dramatically decreased and negatively correlated with the dose,while Arg1 and IL-10 were increased and positively correlated with the dose.Conclusion LBP inhibits activation of BV2 microglia induced by LPS and promots the M2 polarization,which may be realized through inhibiting TLR4/NF-κB signaling pathway.