巨噬细胞诱导糖尿病肾病足细胞凋亡及其机制  

Effect of macrophages on podocytes apoptosis in diabetic nephropathy

作　　者：郭银凤[1] 赵宇[1] 姜彧滕 朱小东[1] 刘必成[1] 张晓良[1] 

机构地区：[1]东南大学医学院,东南大学附属巾大医院,江苏省肾脏病临床研究中心,南京210009

出　　处：《中华内分泌代谢杂志》2017年第8期680-686,共7页Chinese Journal of Endocrinology and Metabolism

基　　金：国家自然科学基金（81370826、81570612）;江苏省十二五医学重点人才项目（RC2011124）;江苏省临床医学研究中心项目（BL2014080）;江苏省普通高校研究生科研创新计划项目（KYLX15-0180、KYLX16_0297）

摘　　要：目的探讨巨噬细胞在糖尿病肾病（DN）足细胞凋亡中的作用及其机制。方法体外培养小鼠巨噬细胞系（RAW264.7），将其分为3组：正常葡萄糖组（NC）、甘露糖组（Man）、高葡萄糖组（HG），并收集上述NC组巨噬细胞上清液（NC-CM）和HG组巨噬细胞上清液（HG-CM）。采用Western印迹和免疫荧光染色检测M1型巨噬细胞标志物iNOS和M2型巨噬细胞标志物MR表达，ELISA检测巨噬细胞上清液中TNF-α水平。体外培养小鼠永生系足细胞MPC5，将其分为以下几组：正常葡萄糖对照组（NC）、NC-CM组、HG-CM组、HG-CM＋ROS抑制剂组（HG-CM＋Tempo）、HG-CM＋p38MAPK抑制剂组（HG-CM＋SB203580）、HG-CM＋抗TNF-α中和抗体组（HG-CM＋TNF-α neutralizing antibody）、HG-CM＋中和抗体对照剂IgG组（HG-CM＋IgG）、重组小鼠TNF-α组。采用Annexin V-FITC/PI和Hoechst33342染色检测足细胞凋亡率，DCFH-DA染色检测足细胞ROS水平，Western印迹检测足细胞cleaved casepase-3、p38MAPK和p38MAPK磷酸化水平（p-p38MAPK）表达。结果（1）与对照组相比，HG组巨噬细胞iNOS表达明显增加，MR表达降低，呈促炎性M1型活化。（2）正常对照组与NC-CM组组间足细胞凋亡、cleaved casepase-3、ROS及p-p38MAPK表达差异无统计学意义。与对照组和NC-CM组相比，HG-CM组足细胞凋亡、cleaved casepase-3、ROS及p-p38MAPK明显增加。（3）与HG-CM组相比，HG-CM＋Tempo，HG-CM＋SB203580组足细胞凋亡和cleaved casepase-3表达明显降低。（4）巨噬细胞上清液HG-CM中TNF-α水平较NC-CM明显增加，且予以高糖刺激后，巨噬细胞TNF-α表达明显增多。（5）与HG-CM组相比，加入抗TNF-α中和抗体能显著减少足细胞凋亡，抑制cleaved casepase-3、ROS及p-p38MAPK表达。（6）单独TNF-α刺激明显上调足细胞凋亡，增加ROS和p-p38 MAPK表达。结论高糖状态下活化的M1型巨噬细胞释放TNF-α，激活足细胞ROS-p38MAPK信号通路进而诱�ObjectiveTo investigate the effect of macrophages on podocytes apoptosis in diabetic nephropathy.MethodsDifferentiated mouse macrophages （RAW264.7） were exposed to normal glucose, high glucose, then the conditioned media （CM） was collected and considered as NC-CM or HG-CM, respectively. Western blotting and immunofluorescent staining were used to detect the specific markers for M1 macrophages （iNOS） and M2 macrophages （MR）. ELISA was used to detect the concentration of TNF-α in the CM. Then normal PRMI 1640 media （control）, NC-CM or HG-CM was added to podocytes. In some experiments, ROS inhibitors （Tempo）, p38 MAPK inhibitor （SB203580）, anti-TNF-α neutralizing antibody, and IgG1 isotype control were respectively added to cells with HG-CM. Besides, recombinant mouse TNF-α alone was applied to incubate podocytes. Podocytes apoptosis was accessed by Annexin V-FITC/PI and Hoechst33342 staining. DCFH-DA staining was used to analyse ROS level. Western blotting was used to detect cleaved casepase-3, p38MAPK, and p-p38MAPK protein.ResultsMacrophages were activated when exposed to high glucose, displaying pro-inflammatory M1 polarization with higher iNOS and lower MR expression. HG-CM but not NC-CM trigged podocytes apoptosis, up-regulated ROS, cleaved casepase-3 and p-p38MAPK. However, the podocytes apoptosis trigged by HG-CM was abolished by either a ROS inhibitor （Tempo） or a p38 MAPK inhibitor （SB203580）. Additionally, TNF-α was increased in the HG-CM. TNF-α protein in macrophage was aslo increased when exposed to high glucose. Anti-TNF-α neutralizing antibody blunted the apoptotic response, excess ROS generation and p-p38 MPAK expression in podocytes induced by HG-CM. Moreover, addition of recombinant TNF-α similarly led to podocytes apoptosis, increased ROS and p38 MPAK expression.ConclusionM1 macrophages activated by high glucose released TNF-α to promote podocytes apoptosis via ROS-p38 MAPK pathway.

关 键 词：巨噬细胞 足细胞 细胞凋亡 糖尿病肾病 

分 类 号：R587.2[医药卫生—内分泌]