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剪切型X盒结合蛋白1在单侧输尿管结扎小鼠肾间质纤维化中的作用

邵德翠, 缪乃俊, 李佳嘉

皖南医学院细胞电生理实验室，芜湖 241002；复旦大学基础医学院生理学与病理生理学系，上海 200032；皖南医学院第一附属医院病理科，芜湖 241002

摘要

剪切型X盒结合蛋白1 (spliced X-box binding protein 1, XBP1S)是内质网应激关键信号分子。有研究表明XBP1S在肾脏系膜细胞具有抗氧化应激和抗凋亡作用，而氧化应激是肾纤维化发生发展重要因素。缬沙坦(valsartan)具有改善肾纤维化作用。本研究旨在探讨valsartan能否通过XBP1S进而影响肾间质纤维化发展。C57BL/6J小鼠行左侧输尿管结扎(unilateral ureteral obstruction, UUO)制作肾纤维化模型，valsartan组于造模前1 d起每天给予valsartan (20 mg/kg)灌胃，UUO组和对照组给予等容积生理盐水灌胃，于手术后第7天处死动物，取左侧肾。HE、Masson和天狼星红(Sirius red)染色观察肾间质纤维化；免疫组化染色观察XBP1S在肾间质表达；Western blot检测XBP1S、纤维连接蛋白(fibronectin)、α-平滑肌肌动蛋白(α-smooth muscle actin, α-SMA)、BAX和BCL2蛋白水平；实时荧光定量PCR检测NADPH氧化酶亚基p47-phox与p67-phox mRNA水平。结果显示，与对照组相比，UUO组XBP1S蛋白水平明显降低，valsartan明显升高UUO小鼠XBP1S蛋白水平；HE、Masson和Sirius red染色结果显示valsartan明显改善UUO小鼠肾间质纤维化；Western blot结果显示valsartan明显降低UUO小鼠fibronectin、BAX/BCL2、α-SMA蛋白水平。实时荧光定量PCR结果显示：UUO组p47-phox与p67-phox mRNA水平明显较对照组升高，相比较于UUO组，valsartan明显降低UUO小鼠p47-phox与p67-phox mRNA水平。以上结果提示，XBP1S蛋白水平下调与UUO模型的肾间质纤维化有关，其机制可能与XBP1S抗氧化应激相关。

关键词： 内质网应激; 肾纤维化; 剪切型X盒结合蛋白1; 氧化应激

分类号：R334

Involvement of spliced X-box binding protein 1 in renal fibrosis induced by unilateral ureteral obstruction in mice

SHAO De-Cui, MIAO Nai-Jun, LI Jia-Jia

The Cell Electrophysiology Laboratory, Wannan Medical College, Wuhu 241002, China； Department of Physiology and Pathophysiology, Shanghai Medical College, Fudan University, Shanghai 200032, China； Department of Pathophysiology, The First Affiliated Hospital of Wannan Medical College, Wuhu 241002, China

Abstract

Endoplasmic reticulum (ER) stress is involved in the process of kidney fibrosis. Spliced X-box binding protein 1 (XBP1S) is the key mediator of ER stress while its role in fibrosis is still poorly understood. This study was aimed to investigate the role of XBP1S in renal fibrosis and evaluate whether valsartan could alleviate fibrosis through XBP1S. Renal interstitial fibrosis was induced by unilateral ureteral obstruction (UUO) in C57BL/6 mice, and UUO mice were daily administered with valsartan (20 mg/kg) through oral gavage. After 7 days of UUO, at euthanasia, left kidney was collected to examine the histological alteration by using haematoxylin-eosin staining, Masson’s trichrome staining, Sirius red staining and immunohistochemistry. Western blot was used to assess XBP1S, targets of XBP1S, fibronectin, α-SMA, BAX and BCL2 protein levels. Real-time polymerase chain reaction was performed to assess NADPH oxidase subunits p47-phox and p67-phox mRNA levels. The results showed that XBP1S expression was decreased by about 70% in the UUO mice compared with that in sham mice (P < 0.01), which was reversed by valsartan administration (P < 0.05). Meanwhile, UUO-induced renal interstitial fibrosis was attenuated by valsartan treatment. In addition, the protein levels of fibronectin and α-SMA were upregulated by UUO induction (P < 0.01), and valsartan administration inhibited the protein levels of fibronectin and α-SMA in UUO mice (P < 0.05). Western blot analysis showed that the ratio of BAX to BCL2 protein level was increased in UUO model compared with that in sham mice, and the increment also was diminished by valsartan treatment (P < 0.05). Finally, UUO-induced mRNA levels of p47-phox and p67-phox were significantly attenuated by valsartan administration (P < 0.05). These results showed that valsartan at least partly restores renal interstitial fibrosis by enhancing XBP1S activation through inhibiting oxidative stress and apoptosis in the UUO mice. These results suggest that XBP1S could be a potential therapeutic target for kidney fibrosis.

Key words: endoplasmic reticulum stress; fibrosis; spliced X-box binding protein 1; oxidative stress

收稿日期：2015-11-06　　录用日期：2016-02-23

通讯作者：邵德翠　　E-mail: shaodecui@sina.com

引用本文：

邵德翠, 缪乃俊, 李佳嘉. 剪切型X盒结合蛋白1在单侧输尿管结扎小鼠肾间质纤维化中的作用[J]. 生理学报 2016; 68 (2): 157-164.

SHAO De-Cui, MIAO Nai-Jun, LI Jia-Jia. Involvement of spliced X-box binding protein 1 in renal fibrosis induced by unilateral ureteral obstruction in mice. Acta Physiol Sin 2016; 68 (2): 157-164 (in Chinese with English abstract).