Xu Guang, Qin Qiaojing, Yang Min, Qiao Zhongdong, Gu Yong, Niu Jianying
Department of Nephrology, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, China.
School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai, China.
Diabetes Res Clin Pract. 2017 Feb;124:30-40. doi: 10.1016/j.diabres.2016.12.016. Epub 2016 Dec 30.
Amounts of macrophages were infiltrated in glomeruli in diabetic nephropathy. Heparanase has been thought to be closely related to proteinuria. Our aims were to determine the effect of heparanase on the inflammation in AGEs-stimulated macrophages and its role on the functions of glomerular endothelial cells (GEnCs).
The expression of inflammation cytokines in macrophages were assayed by q-RT PCR, western, and ELISA. Then western was used to measure the expression of RAGE and key proteins in NF-κB pathway in macrophages. The expression of the adherence molecules and tight junction proteins in GEnCs were assessed by western. The adherence of mononuclear cells to GEnCs were observed by HE staining and transendothelial FITC-BSA were tested for the permeability of GEnCs.
HPA siRNA and heparanase inhibitor sulodexide could attenuate the increasing inflammatory factors (TNF-α and IL-1β) in AGEs-stimulated macrophages. NF-κB inhibitor PDTC could also decrease the augmented inflammation cytokines through inhibiting the activation of the NF-κB pathway induced by AGEs. The phosphorylation of NF-κB signaling pathway could be also attenuated by HPA siRNA and sulodexide, the same to the receptor of AGEs RAGE. When the macrophage-conditioned culture medium were added to the glomerular endothelial cells, we found HPA siRNA and sulodexide groups could decrease the increasing adherence and permeability of GEnCs induced by AGEs.
Heparanase increases the inflammation in AGEs-stimulated macrophages through activating the RAGE-NF-κB pathway. Heparanase driven inflammation from AGEs-stimulated macrophages increases the adherence of GEnCs and augments the permeability of GEnCs.
糖尿病肾病患者肾小球中有大量巨噬细胞浸润。乙酰肝素酶被认为与蛋白尿密切相关。我们的目的是确定乙酰肝素酶对晚期糖基化终末产物（AGEs）刺激的巨噬细胞炎症的影响及其在肾小球内皮细胞（GEnCs）功能中的作用。
通过定量逆转录聚合酶链反应（q-RT PCR）、蛋白质免疫印迹法（western）和酶联免疫吸附测定（ELISA）检测巨噬细胞中炎症细胞因子的表达。然后用蛋白质免疫印迹法检测巨噬细胞中晚期糖基化终末产物受体（RAGE）和核因子κB（NF-κB）信号通路关键蛋白的表达。用蛋白质免疫印迹法评估肾小球内皮细胞中黏附分子和紧密连接蛋白的表达。通过苏木精-伊红（HE）染色观察单核细胞与肾小球内皮细胞的黏附情况，并检测异硫氰酸荧光素标记的牛血清白蛋白（FITC-BSA）跨内皮情况以评估肾小球内皮细胞的通透性。
乙酰肝素酶小干扰RNA（HPA siRNA）和乙酰肝素酶抑制剂舒洛地昔可减轻AGEs刺激的巨噬细胞中炎症因子（肿瘤坏死因子-α和白细胞介素-1β）的增加。NF-κB抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）也可通过抑制AGEs诱导的NF-κB信号通路激活来降低炎症细胞因子的增加。HPA siRNA和舒洛地昔也可减弱NF-κB信号通路的磷酸化，对AGEs受体RAGE的作用相同。当将巨噬细胞条件培养基添加到肾小球内皮细胞中时，我们发现HPA siRNA和舒洛地昔组可降低AGEs诱导的肾小球内皮细胞黏附和通透性的增加。
乙酰肝素酶通过激活RAGE-NF-κB信号通路增加AGEs刺激的巨噬细胞中的炎症反应。乙酰肝素酶驱动的AGEs刺激的巨噬细胞炎症增加了肾小球内皮细胞的黏附并增强了肾小球内皮细胞的通透性。
