摘要
目的:本研究通过调节巨噬细胞自噬体生成探究其对巨噬细胞黏附及迁移功能的影响,以降低糖尿病肾病(DN)巨噬细胞浸润。方法:体内实验,12w末处死SD正常大鼠和DN组大鼠,病理染色观察肾脏病理改变,检测肾组织巨噬细胞标志物及自噬相关标志物表达。体外实验,检测正常与高糖(HG)组条件下,RAW 264.7巨噬细胞自噬体数量变化,自噬相关标志物LC3和Beclin-1、自噬体清除指标P62的表达,以及巨噬细胞粘附和迁移数量。分别加用自噬体生成激活剂雷帕霉素(RAPA),自噬体生成抑制剂3-甲基腺嘌呤(3-MA),观察其对巨噬细胞自噬标记物表达及其粘附和迁移功能的影响,电镜观察巨噬细胞自噬体数量的变化。结果:体内实验,DN大鼠肾损伤严重,肾组织巨噬细胞标志物CD68表达增加,LC3表达减少,P62表达增加(P<0.05)。体外实验,电镜观察发现HG组自噬体数量减少,Western与免疫荧光显示自噬相关蛋白LC3和Beclin-1表达降低,P62表达升高(P<0.05);3-MA组自噬体数量减少,Western与免疫荧光显示LC3和Beclin-1表达降低(P<0.05);HG组和3-MA组巨噬细胞粘附和迁移数量增多(P<0.05);电镜观察RAPA增加巨噬细胞自噬体数量,Western与免疫荧光显示RAPA提高了被HG抑制的巨噬细胞自噬水平(P<0.05),减少HG诱导的巨噬细胞黏附和迁移数量(P<0.05)。结论:HG抑制自噬水平,促进巨噬细胞黏附迁移;激活自噬体生成能提高自噬水平,减轻巨噬细胞粘附和迁移;减少自噬体生成可降低自噬水平,促进巨噬细胞粘附和迁移。
Objective:This study modulated the formation of macrophage autophagosome to investigate the impact of autophagosome on the adhesion and migration capacity,so as to reduce the macrophage infiltration of diabetic nephropathy(DN).Methods:In vivo,rats were randomly distributed into control(NC)and DN groups.The pathological changes in renal tissue were assessed,and expression of CD68,LC3 and P62 were analyzed.In vitro,RAW 264.7 cells were divided into normal and high glucose(HG)groups.The capacity for macrophage adhesion and migration and the expression of autophagy markers(LC3,Beclin-1 and P62)were observed with and without autophagy modulators(rapamycin and 3-methyladenine for RAPA and 3-MA).The numbers of autophagosomes were observed by electron microscopy.Results:In vivo,renal injury was aggravated in diabetic rats compared with NC group.The expression levels of CD68 and P62 of renal tissues increased in DN group,while expression level of LC3 decreased(P<0.05).In vitro,HG reduced the numbers of autophagosomes with less expression of LC3 and Beclin-1,but increase expression of P62(P<0.05).3-MA reduced the numbers of autophagosomes with less expression of LC3 and Beclin-1(P<0.05).HG or 3-MA promoted the adhesion and migration capacity of macrophages(P<0.05).However,RAPA increased the numbers of macrophage autophagosomes,resulting in an increase expression of LC3 and Beclin-1,whereas a reduction expression of P62,which lead to inhibition of adhesion and migration of macrophages induced by HG(P<0.05).Conclusion:HG inhibits level of autophagy and promotes macrophage adhesion and migration.Activating numbers of autophagosomes can improve the level of autophagy and reduce the adhesion and migration of macrophages,while reducing numbers of autophagosomes can reduce the level of autophagy and promote the adhesion and migration of macrophages.
作者
姜彧滕
赵宇
朱小东
刘玉秋
郭银凤
吴贝贝
张晓良
Jiang Yuteng;Zhao Yu;Zhu Xiaodong;Liu Yuqiu;Guo Yinfeng;Wu Beibei;Zhang Xiaoliang(School of Medicine,Southeast University,Nanjing 210009,China;Department of Nephrology,Zhong Da Hospital,Southeast University,Nanjing 210009,China)
出处
《巴楚医学》
2018年第3期6-12,共7页
Bachu Medical Journal
基金
国家自然科学基金项目(No:81570612)
江苏省临床医学研究中心项目(No:BL2014080)
江苏省普通高校研究生科研创新计划资助项目
中央高校基本科研业务费专项基金(No:SJCX17_0056)
关键词
巨噬细胞
高糖
自噬体
粘附
迁移
macrophages
high glucose
autophagy
adhesion
migration
