梓醇对AGEs刺激肾系膜细胞介导巨噬细胞极化的影响

Effect of catalpol on RAW264. 7 macrophage polarization mediated by AGEs-stimulated mouse mesangial cells

目的探讨梓醇对晚期糖基化终末产物(AGEs)刺激肾系膜细胞介导巨噬细胞极化的影响。方法将巨噬细胞(RAW264.7)置于Transwell上层小室,肾系膜细胞(MMCs)于下层孔板进行体外共培养,设置模型组、0-BSA对照组、梓醇组(0.1、1.0、10.0μmol·L^(-1)),另设氨基胍组(1.0μmol·L^(-1))作为阳性对照。加入各药物预孵下层系膜细胞1 h后,用AGEs(100 mg·L^(-1))刺激,继续孵育23 h,采用ELISA法检测系膜细胞上清液中单核细胞趋化蛋白-1(MCP-1)的分泌水平;Western blot法检测巨噬细胞M1型标记蛋白i NOS、CD16/32、TNF-α、COX-2和M2型标记蛋白CD206、Arg-1的表达水平;流式细胞仪检测巨噬细胞i NOS与CD206蛋白表达百分率。结果 AGEs可提高系膜细胞MCP-1的分泌水平(P<0.01),上调巨噬细胞i NOS、TNF-α、CD16/32、COX-2蛋白表达(P<0.05,P<0.01),下调CD206和Arg-1蛋白表达;而梓醇(0.1、1.0、10.0μmol·L^(-1))预孵能降低系膜细胞MCP-1的分泌水平(P<0.01),下调i NOS、TNF-α、CD16/32、COX-2蛋白表达(P<0.05,P<0.01),上调CD206和Arg-1蛋白表达(P<0.05),并呈浓度依赖性。结论梓醇可通过调控AGEs刺激系膜细胞分泌MCP-1的水平,抑制巨噬细胞的M1型极化,并促进其向M2型极化,减轻炎症反应,缓解糖尿病肾损伤。

Aim To investigate the effect that catalpol intervenes macrophage polarization mediated by mouse mesangial cells（ MMCs） stimulated by advanced glycation end products（ AGEs）. Methods RAW264. 7macrophages and MMCs were co-cultured in vitro and divided into model group（ 100 mg·L（-1）AGEs）,control group（ 100 mg · L（-1）BSA）,catalpol（ 0. 1,1. 0,10. 0 μmol · L（-1）） group,and aminoguanidine（ 1. 0μmol·L（-1）） group which was set as positive control.After being incubated with catalpol for 1 h,MMCs were stimulated by AGEs for 23 h. The proliferationinhibition rate of MMCs was measured by MTT assay.MCP-1 in supernatant liquid of MMCs was detected by ELISA method. The expression of i NOS,CD16/32,TNF-α,COX-2,CD206 and Arg-1 was detected by Western blot. Simultaneously,the percentage of i NOS and CD206 was also measured by flow cytometry. Results AGEs could increase the level of MCP-1 secreted by MMCs. The expression of i NOS, TNF-α,CD16/32 and COX-2 protein of macrophage was upregulated after MMCs stimulated by AGEs,while the expression of CD206 and Arg-1 was down-regulated.After being intervened by catalpol,these effects could be reversed. All the changes were concentration-related. Conclusions Catalpol can inhibit macrophages M1-type polarization process and promote M2-type polarization,which may be mediated through MCP-1 secreted by MMCs after AGEs stimulation. Catalpol can ameliorate inflammation and relieve diabetic kidney injury.