【作者】 赵宇；

【导师】 张晓良；

【作者基本信息】 东南大学 ， 内科学（肾脏病学）， 2019， 博士

【摘要】 目的糖尿病肾病(DN)是一种慢性炎症性疾病,巨噬细胞浸润是DN重要病理特征,也是导致DN进展的关键因素。巨噬细胞主要通过粘附和迁移过程进入肾组织。糖尿病肾组织中的巨噬细胞主要存在两个表型,即M1/M2型巨噬细胞。M1型巨噬细胞介导炎症反应,参与组织损伤;M2型巨噬细胞则具有组织修复作用。研究发现,糖尿病肾病(DN)肾组织存在M1/M2巨噬细胞表型失衡并以M1型浸润为主。因此,探讨巨噬细胞的调节机制,达到减少肾组织M1型巨噬细胞浸润的目的,以及寻找有效可行的调节巨噬细胞的药物是解决问题的关键。活性维生素D3对糖尿病肾病具有显著的肾保护作用,但其机制尚未阐明。本研究通过构建STZ诱导的糖尿病肾病大鼠模型及体外巨噬细胞培养模型两方面,观察活性维生素D3对糖尿病肾病巨噬细胞的调节作用,同时探讨可能机制。方法体内实验:通过建立STZ诱导的糖尿病肾病大鼠模型,将雄性SD大鼠随机分为三组:对照组(NC组)、DN组、VD干预组(DN+VD组,骨化三醇0.1μg·kg-1·d-1灌胃)、VD对照组(骨化三醇0.1μg·kg-1·d-1灌胃),干预后18w末处死,PAS染色观察肾脏病理改变。免疫组化法检测肾组织CD68+巨噬细胞浸润以及;Western Blot检测CD68以及M1巨噬细胞特异性标志物iNOS、TNF-α表达,M2巨噬细胞特异性标志物Arg-1、MR表达以及TREM-1、STAT-1、p-STAT-1表达。免疫共聚焦检测CD68和iNOS,CD68和MR,CD68和TREM-1共表达。体外实验:采用体外培养小鼠永生系RAW264.7巨噬细胞,对巨噬细胞予不同的刺激,观察1,25(OH)2D3、TREM-1siRNA,TREM-1过表达质粒,以及STAT-1siNA,STAT-1激活剂对高糖诱导的巨噬细胞粘附、迁移以及M1/M2表型的影响。粘附实验、transwell迁移实验测巨噬细胞的粘附和迁移能力,免疫荧光和Western Blot检测不同干预条件下TREM-1,STAT-1,p-STAT-1以及M1标志物(iNOS、TNF-α)M2标志物(Arg-1、MR)的表达。结果体内实验:与对照组相比,DN组Scr、BUN、24小时尿蛋白及肾小球系膜基质增生程度显著增加(均P<0.05)。VD干预后能明显改善上述病理现象(均P<0.05)。与对照组相比,DN大鼠组肾组织CD68+巨噬细胞浸润数量明显增加,M1型巨噬细胞标志物iNOS、TNF-α表达,TREM-1表达以及p-STAT-1表达显著增加,VD能显著降低DN肾组织巨噬细胞浸润,降低iNOS、TNF-α、TREM-1以及p-STAT-1的表达。体外实验:为了探究高糖状态下巨噬细胞浸润及表型转化的机制,体外培养RAW264.7巨噬细胞。结果发现,与对照组相比高糖组巨噬细胞粘附迁移数量显著增加(P<0.05);M1型巨噬细胞标志物显著增加(P<0.05);巨噬细胞TREM-1和p-STAT-1表达显著增加(P<0.05)。为进一步确定TREM-1和STAT-1对巨噬细胞粘附迁移以及巨噬细胞M1/M2表型的调节作用,分别用TREM-1siRNA和STAT-1siRNA干预巨噬细胞观察巨噬细胞粘附迁移和M1/M2表型的变化。实验结果显示,高糖环境下,TREM-1siRNA组与TREM-1siRNA阴性对照组相比,巨噬细胞粘附和迁移数量显著下降(P<0.05)。M1型巨噬细胞标志物显著下降(P<0.05),但是巨噬细胞STAT-1表达无显著差异。高糖环境下,STAT-1 siRNA组与STAT-1 siRNA阴性对照组相比,巨噬细胞粘附和迁移数量显著下降(P<0.05);M1型巨噬细胞标志物显著下降(P<0.05)同时,巨噬细胞TREM-1表达显著下降(P<0.05)。上述实验结果证实,TREM-1和STAT-1参与巨噬细胞粘附迁移和M1/M2表型的调节,同时STAT-1是TREM-1上游调控因子。为了观察VD对巨噬细胞的调节作用发现,与高糖组相比,VD能显著降低高糖诱导的巨噬细胞粘附迁移数量(P<0.05);显著降低高糖诱导的巨噬细胞TREM-1和p-STAT-1的表达(P<0.05);显著降低高糖诱导的巨噬细胞M1型巨噬细胞标志物的表达(P<0.05)。为了进一步探究VD调节巨噬细胞的机制发现,在高糖环境下,TREM-1质粒过表达组与质粒空载阴性对照组相比,巨噬细胞粘附和迁移数量显著升高(P<0.05);M1型巨噬细胞标志物显著升高(P<0.05),但是巨噬细胞STAT-1表达并无明显变化。高糖环境下,STAT-1激活剂组与正常对照组相比,巨噬细胞粘附和迁移数量显著升高(P<0.05);M1型巨噬细胞标志物显著升高(P<0.05),同时巨噬细胞TREM-1表达显著升高。结论1.糖尿病肾病大鼠肾组织巨噬细胞浸润增多,并且以M1型浸润为主。2.高浓度葡萄糖环境下,STAT-1/TREM-1信号通路的上调促进巨噬细胞粘附迁移以及向M1型转化。3.活性维生素D3能够通过抑制高糖诱导的STAT-1/TREM-1信号通路,降低巨噬细胞粘附迁移并抑制其向M1型转化,从而缓解糖尿病肾病的进展。更多还原

【Abstract】 BackgroundDiabetic nephropathy(DN)is an inflammatory chronic disease.Macrophage accumulation is a vital feature in DN.Macrophage infiltration process mainly includes adhesion and migration in renal tissue.Imbalance of M1/M2 macrophages phenotype activation is a key point in diabetic nephropathy(DN).M1 macrophages promote tissue inflammation and fibrosis while M2 macrophages mediate tissue repair.Macrophages mainly exhibit M1 phenotype,whcih contribute to the inflammation and fibrosis in DN.Therefore,the key to solve the problem is to explore the mechanism of macrophages regulation and to find effective and feasible intervention drug.Recent investigations demonstrated that active vitamin D3 reduced albuminuria and improve renal function.The present study aimed to provide a mechanistic insight into the potential effects of 1,25-dihydroxyvitamin D3(VD)in vivo and in vitro high glucose induced macrophages regulation.MethodsExperimental rat model were established and random distributed into four groups as follows: NC,VD,DN and DN+VD(DN+ Calcitriol 0.1 μg·kg-1·d-1 by gavages).Animals were sacrificed respectively at 18 w after treatment.Pathological changes in kidney tissue were detected and the expression of CD68,TREM-1,pSTAT-1 were acquired by Immunohistochemistry stain and Western Blot.In vitro,RAW 264.7 cells were treated by high glucose with or without 10-8 mol/L 1,25(OH)2D3.TREM-1 siRNA,STAT-1 siRNA and high expression plasmid of TREM-1 and STAT-1 activtor were explored to elucidate the underlying mechanism.The expression of TREM-1 and STAT-1 and the changes of macrophage phenotype were examined separately by Western Blot,immunofluorescence stain.The ability of macrophage in adhesion and migration were examined by adhesion experiment and migration assay.Results In vivo: In DN group,levels of BUN,Scr,24-hour urinary protein and glomerular mesangial matrix proliferation were significantly higher,and the expressions of nephrin,podocin were significantly decreased compared with NC groups(P<0.05).These above changes were significantly improved in VD group(P<0.05).In DN group,number of macrophage with CD68+ and expression of iNOS,TNF-α,TREM-1,p-STAT-1 protein were significantly increased in comparation with NC group(P<0.05),while above changes were markedly decreased in DN+VD group(P<0.05).In vitro: In order to explore the mechanism of macrophage infitration and phenotype regulation in high glucose condition,the RAW264.7 cells were cultured.The capacity of adhesion and migration in macrophage and the amount of iNOS,TNF-α,TREM-1,p-STAT-1 protein were obviously increased in HG group,compared with NC group(P<0.05).In order to further determine the regulatory effects of TREM-1 and STAT-1 on the ability of adhesion and migration and the M1/M2 phenotype transformation,TREM-1siRNA and STAT-1siRNA were used respectively.Compared with NTC groups,the number of macrophage adhesion and migration and the expression of iNOS,TNF-α were notably reduced after TREM-1 siRNA and STAT-1 siRNA intervention(P<0.05).While,TREM-1 siRNA did not affect STAT-1 expression.The above results demonstrated that TREM-1 and STAT-1 are involved in the macrophage adhesion and migration and M1/M2 phenotype regulation.In order to observe the effect of VD on macrophage in high glucose condition,the 1,25(OH)2D3 was used.Compared with HG groups,VD significantly decreased the amount of macrophage adhesion and migration and expression of iNOS,TNF-α,TREM-1,p-STAT-1 caused by high glucose(P <0.05).In order to explore the mechanism of VD on macrophage regulation in high glucose condition,high expression plasmid of TREM-1 and STAT-1 activtor were used.The effect that VD significantly decreased the amount of macrophage adhesion and migration and expression of iNOS,TNF-α,TREM-1,p-STAT-1 caused by high glucose were abolished when TREM-1 was overexpressed by TREM-1 plasmid and STAT-1 was activated by STAT-1 activator.Conclusion 1.Macrophage infiltration was increased in STZ-induced DN rats,especially M1 macrophage in renal tissue of DN rats.2.The up-regulation of STAT-1/ TREM-1 signaling pathway promoted the capacity of macrophage adhesion and migration and M1 macrophage transformation under high glucose concentration.3.VD can inhibit the capacity of macrophage adhesion and migration and and M1 macrophage transformation through the STAT-1/TREM-1 pathway under high glucose condition,eventually alleviating the progress of diabetic nephropathy.更多还原