乙酰水杨酸姜黄素酯通过TLR4/NF-κB通路拮抗LPS诱导的BV2小胶质细胞炎症损伤

Curcumin acetylsalicylate antagonizes LPS-induced inflammatory injury of BV2 microglia through TLR4/NF-κB pathway

目的研究乙酰水杨酸姜黄素酯(curcumin acetylsalicylate,CA)对脂多糖(lipopolysaccharide,LPS)诱导的BV2小胶质细胞炎症的改善作用及机制。方法应用Discovery Studio分子模拟软件将CA与核因子-κB(nuclear factor-κB,NF-κB)、胰岛素诱导基因2(nsulin inducible gene 2,Insig2)、Wnt信号蛋白(Wnt)、尼曼匹克C1型L1(Niemann-Pick C1 like 1,NPC1-L1)、Toll样受体2(Toll-like receptors 2,TLR2)和Toll样受体4(Toll-like receptors 4,TLR4)蛋白进行分子对接,筛选出结合紧密的蛋白。将BV2小胶质细胞分为对照组、模型组、CA干预组。对照组不做处理;模型组加入10 mg/L的LPS作用24 h;CA干预组加入20、40、60μmol/L的CA作用2 h后,加入10 mg/L LPS作用24 h。CCK-8法检测CA对BV2小胶质细胞增殖的影响;ELISA法检测细胞TNF-α、IL-1β;Griess法检测细胞分泌NO的水平;Western blot法检测iNOS、NF-κB p65、p-NF-κB p65、IκB-α、p-IκB-α、TLR4和MYD88蛋白的表达。结果与对照组比较,在0~80μmol/L浓度范围内,CA干预组细胞存活率无显著性差异(P>0.05)。与对照组比较,模型组细胞TNF-α、IL-1β、NO水平明显升高(P<0.05);与模型组比较,CA干预组细胞TNF-α、IL-1β、NO水平明显下降(P<0.05)。与对照组比较,模型组的p-NF-κB水平升高(P<0.05);与模型组比较,CA干预组降低p-NF-κB水平(P<0.05)。与对照组比较,模型组的TLR4、MYD88的蛋白表达水平明显升高(P<0.05);与模型组比较,CA干预组的TLR4、MYD88的蛋白表达水平明显降低(P<0.05)。结论CA能有效抑制LPS诱导的BV2小胶质细胞炎症反应,其作用可能与其抑制NF-κB/TLR4信号通路有关。

Objective To research the effect and mechanism of curcumin acetylsalicylate(CA)on lipopolysaccharide(LPS)-induced BV2 microglial inflammation.Methods Discovery Studio molecular modeling package was used to docking CA with nuclear factor-κB(NF-κB),Toll-like receptor 2(TLR2)and Toll-like receptor 4(TLR4)proteins to screen out the tightly bound proteins.BV2 microglia cells were divided into control group,model group and CA administration group.The control group was not treated;the model group was added with 10 mg/L LPS for 24 h;the CA administration group was treated with 10 mg/L CA for 24 h,and then added 20,40,60μmol/L LPS for 24 h.The effect of CA on the proliferation of BV2 cells was detected by CCK-8.ELISA method was used to detect the cellular TNF-α and IL-1β;the level of NO secreted by cells was detected by Griess method;the expression levels of iNOS,NF-κB p65,p-NF-κB p65,IκB-α,p-IκB-α,TLR4 and MYD88 protein were detected by Western blot.Results Compared with the control group,in the concentration range of 0-80μmol/L,the cell viability of the CA administration group had no significant difference(P>0.05);compared with the control group,the levels of TNF-α,IL-1β and NO in the model group were significantly increased(P<0.05);compared with the model group,the levels of TNF-α,IL-1β and NO in the CA administration group were significantly decreased(P<0.05).Compared with the control group,the level of p-NF-κB in the model group was increased(P<0.05);compared with the model group,the level of p-NF-κB in the CA administration group was reduced(P<0.05).Compared with the control group,the protein expression levels of TLR4 and MYD88 in the model group were significantly increased(P<0.05);compared with the model group,the protein expression levels of TLR4 and MYD88 in the CA administration group were significantly decreased(P<0.05).Conclusion CA can effectively inhibit the inflammatory response of BV2 microglial cells induced by LPS,and its effect may be related to its inhibition of NF-κB/TLR4 signaling pathway.