线粒体自噬在糖尿病肾病肾组织巨噬细胞表型转化中的作用

Mitophagy and macrophage M1/M2 phenotype transforming in diabetic nephropathy rats

目的:探讨糖尿病肾病(DN)大鼠肾组织线粒体自噬特征与巨噬细胞M1/M2表型的相关性及在其分化中的作用。方法:建立DN大鼠模型分别于8周、12周、18周、24周末处死,病理染色观察肾脏病理改变,电镜观察肾组织线粒体形态数量和线粒体自噬。Western Blot检测肾组织巨噬细胞及线粒体自噬相关蛋白标志物表达。体外培养RAW264.7细胞,Western Blot和免疫荧光共聚焦比较自噬体生成抑制剂3-甲基腺嘌呤(3-MA)和自噬体生成激活剂雷帕霉素干预前后,巨噬细胞表型标志物和线粒体自噬相关指标的变化。结果:体内实验(1)从12周起,随着时间延长DN大鼠肾组织巨噬细胞浸润M1型增多,并且存在线粒体自噬障碍,表现为iNOS升高,LC3蛋白逐渐降低,p62升高;(2)相关性分析显示,iNOS与LC3成负相关(r=-0.617,P<0.05),而与p62成正相关(r=0.894,P<0.05);(3)电镜结果显示DN大鼠肾组织线粒体肿胀、线粒体嵴消失或降低,且存在线粒体自噬障碍。(4)免疫荧光共聚焦结果显示,DN大鼠肾组织线粒体标记蛋白VDAC和LC3较对照组均降低,共定位表达减少。体外实验(1)高糖干预后,Western Blot结果显示RAW264.7细胞肿瘤坏死因子α、iNOS随着时间延长逐渐升高,同时LC3、Beclin-1表达显著降低,p62明显升高,VDAC逐渐降低。(2)3-MA抑制自噬体生成能促进高糖诱导的巨噬细胞进一步向M1型巨噬细胞转化;(3)雷帕霉素激活自噬体生成能降低高糖诱导的M1型巨噬细胞活化。结论:线粒体自噬可能参与DN大鼠肾组织巨噬细胞M1/M2表型转化。

Objective： To investigate the effects of mitophage on macrophage polarization in rats with DN.Methodology： In vivo,DN model rats were established by intraperitoneal injection with streptozocin（ STZ）. Rats were sacrificed respectively at 8 w、12 w、18 w、24 w for histological and molecular analysis. In vitro,RAW264. 7 cells were cultured with 30 m M glucose with or without mitophage inhibitor（ 3-MA） and activator（ rapamycin） intervention. Mitophagy-related proteins expression of LC3,Beclin-1,p62,VDAC,i NOS（ M1 marker） and MR（ M2 marker） were detected by immunofluorescence and Western Blot. Results： Macrophage phenotype and mitophage had no obvious change in renal tissue of DN at 8 th weeks. However,macrophages were exhibited to M1 phenotype at 12 weeks after induction of DN and displayed a lower level of mitophagy. Additionally,i NOS expression was positive correlated with the p62 expression（ r =0. 894,P〈0. 05）,while negative correlated with LC3（ r =-0. 617,P〈 0. 05）. Electronic microscopic analysis showed mitochondria swelling,crista decrease and lysosome reduction in DN rats compared with NC rats. Moreover,RAW264. 7 macrophages switched to M1 phenotype under high glucose conditions. Meanwhile,mitophagy was downregulated in such high glucose induced M1 macrophages. Furthermore,macrophages expressed more i NOS and lower MR when impaired mitophagy by 3-MA. Rapamycin could significantly blocke high-glucose induced i NOS and enhance MR expression.Conclusion： Mitophage may participate in the regulation of M1/M2 macrophage phenotype in diabetic nephropathy.