转化生长因子-β激活激酶1抑制剂对AGEs诱导小鼠骨髓来源巨噬细胞活化作用及机制

Effect of TGF-β activated kinase-1 inhibitor on bone marrow-derived macrophages activation and its mechanism

目的应用转化生长因子-β激活激酶1(TGF-βactivated kinase-1,TAK1)抑制剂(5Z-7-oxozeaenol,OZ)作用于晚期糖基化终末产物(adavnaced glyeation end porudets,AGEs)诱导的小鼠骨髓来源巨噬细胞(bone marrow-derived macrophages,BMMs),探讨TAK1信号通路在AGEs诱导的BMMs活化中作用及机制。方法获取C57小鼠的BMMs,运用流式细胞术鉴定BMMs纯度。检测TAK1抑制剂在不同浓度下对AGEs培养巨噬细胞活力的影响,激光共聚焦显微镜和流式细胞术检测巨噬细胞M1亚型;RT-PCR检测各组细胞中MCP-1与TNF-αmRNA的表达;Western blot法检测TAK1、MAPK及NF-κB通路蛋白的表达。结果 AGEs刺激能增加M1型巨噬细胞百分比,TAK1抑制剂可抑制AGEs诱导下巨噬细胞向M1表型活化;与正常对照组比较,AGEs刺激不仅上调BMMs中MCP-1、TNF-αmRNA的表达(P<0.01),而且p-TAK1、TAB1、p-JNK、p-p38MAPK、NF-κBp65蛋白表达也明显增加(P<0.05);通过TAK1抑制剂下调pTAK1表达的同时AGEs培养BMMs的TAB1、p-JNK、pp38MAPK、NF-κBp65及TNF-α、MCP-1表达均明显降低(P<0.05)。结论 AGEs能诱导BMMs向M1表型活化,TAK1抑制剂可能通过TAK1/MAPKs、MAPKs/NF-κB途径抑制AGEs对巨噬细胞的激活和炎症因子的表达。

Aim We used bone marrow-derived macrophages( BMMs),to explore the mechanism of macrophage activation and the effect of TGF-β activated kinase-1(TAK1) inhibitor 5Z-7-oxozeaenol on it under AGEs conditions.Methods The BMMs were obtained from C57 mice,and purity of BMMs was detected by flow cytometry.Cell viability was tested after treatment with different concentrations of TAK1 inhibitors.Laser confocal microscopy was used to detect macrophage M1 subtype.Flow cytometry was used to analyse the macrophage activated by AGEs.TNF-α and MCP-1 mRNA levels were evaluated by qRT-PCR.Western blot was used to detect the expression levels of TAK1 signal pathway protein.Results AGEs stimulation could increse the activity of M1 macrophages,and 5Z-7-oxozeaenol could inhibit the differentiation of BMMs.Compared with control group,AGEs increased the expression of MCP-1 and TNF-α mRNA(P<0.01).p-TAK1,TAB1,p-JNK,p-p38 MAPK and NF-κBp65 proteins expression also increased significantly(P<0.05).After treatment with inhibitor,transcription levels of MCP-1and TNF-α decreased significantly(P<0.05,P<0.01).5Z-7-oxozeaenol treatment downregulated the expression of p-TAK1,TAB1,p-JNK,p-p38 MAPK and NF-κBp65 proteins(P<0.05).Conclusions AGEs can induce BMMs to M1 phenotypic polarization.5Z-7-oxozeaenol reduces the expression of inflammatory cytokine via inhibiting TAK1/MAPKs,MAPKs/NF-κB pathways.