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Notch1对糖尿病肾小管间质细胞外基质沉积的影响

刘兴梅^1,2, 沈燕^2,3, 何玉^1,2, 班晓霞^1,2, 金红君^1,2, 何晓兰⁴, 田禾^5,*

¹贵州省人民医院检验科，贵阳 550002；²贵州省人民医院NHC肺脏免疫疾病重点实验室，贵阳 550002；³贵州省人民医院肾内科，贵阳 550002；⁴贵州省人民医院病理科，贵阳 550002；⁵贵州省临床检验中心，贵阳 550002

摘要

本文旨在明确糖尿病肾小管间质中Notch1和自噬水平的变化对细胞外基质沉积的影响，并探讨其机制。取db/m小鼠(正常对照组)和db/db小鼠(糖尿病组)各8只，饲养12周后测定相应的生化指标；同时，体外在正常糖(normal glucose, NG)和高糖(high glucose, HG)条件下培养大鼠肾小管导管上皮细胞(NRK52E细胞)，用Western blotting检测各组细胞中Notch1和LC3蛋白表达变化；在NG和HG条件下过表达和敲低Notch1，用共聚焦显微镜观察自噬流变化，用细胞免疫荧光化学法观察肾小管上皮细胞中Notch1、I型和III型胶原蛋白(Collagen-I和III)变化。结果显示，与db/m小鼠比较，db/db小鼠肾组织中Notch1和Collagen-III表达增加(P < 0.01)，而LC3表达降低(P < 0.05)；体外实验结果显示，HG组与NG组比较，NRK52E细胞Notch1蛋白表达增加(P < 0.01)，LC3表达明显降低(P < 0.01)，高渗组与NG组比较，以上蛋白表达无明显变化；高糖条件下敲低Notch1，激光共聚焦显示自噬增强，而过表达Notch1使自噬向相反方向转变；高糖条件下敲低Notch1，细胞免疫荧光结果显示细胞外基质沉积减少，相反，过表达Notch1使细胞外基质沉积增加。以上结果提示，糖尿病肾组织及高糖培养的肾小管上皮细胞中Notch1表达增加，自噬被抑制；给予外源Notch1敲低干预，自噬恢复，肾小管间质细胞外基质沉积减轻。

关键词： 肾小管间质; Notch1; 自噬; 细胞外基质沉积

Effect of Notch1 on extracellular matrix deposition in the renal tubulointerstitium of diabetes 

LIU Xing-Mei^1,2, SHEN Yan^2,3, HE Yu^1,2, BAN Xiao-Xia^1,2, JIN Hong-Jun^1,2, HE Xiao-Lan⁴, TIAN He^5,*

¹Department of Clinical Laboratory, Guizhou Provincial People’s Hospital, Guiyang 550002, China；²NHC Key Laboratory of Pulmonary Immunological Diseases, Guizhou Provincial People’s Hospital, Guiyang 550002, China；³Department of Nephrology, Guizhou Provincial People’s Hospital, Guiyang 550002, China；⁴Department of Pathology, Guizhou Provincial People’s Hospital, Guiyang 550002, China；⁵Clinical Laboratory Center of Guizhou Province, Guiyang 550002, China

Abstract

The aim of the present study was to observe the effects of Notch1 and autophagy on extracellular matrix deposition in renal tubulointerstitium of diabetes and to explore the mechanism. The mice were randomly divided into normal control group (db/m mice) and diabetes group (db/db mice). After 12 weeks of feeding, the mice were sacrificed and the corresponding biochemical indexes were measured. Rat renal tubular epithelial cells NRK52E were cultured under normal glucose (NG) and high glucose (HG) respectively, and the expression of Notch1 and LC3 proteins were detected by Western blotting. Autophagosomes in NRK52E cells with overexpressed and knockdown Notch1 under NG and HG conditions were observed by confocal microscope, and the expression changes of Notch1, Collagen-I and III protein were detected by immunofluorescence. The results showed that the Notch1 and Collagen-III expressions were increased (P < 0.01) and the LC3 expression was decreased (P < 0.05) in db/db mice compared with db/m mice. In vitro, the Notch1 was increased (P < 0.01) and the LC3 expression was decreased significantly (P < 0.01) in NRK52E cells of HG group compared with NG group. There was no significant change of Notch1 and LC3 expression between the mannitol (MA) group and the NG group. Autophagy was decreased and extracellular matrix deposition was aggravated when Notch1 was overexpressed. In contrast, autophagy was increased and extracellular matrix deposition was relieved by knockdown of Notch1 under HG conditions. In conclusion, Notch1 protein expression was increased and autophagy was reduced in renal tissue of diabetes and renal tubular epithelial cells under HG. The extracellular matrix deposition in the renal tubulointerstitium was relieved by regulating autophagy after the knockdown of Notch1.

Key words: renal tubulointerstitium; Notch1; autophagy; extracellular matrix deposition

收稿日期：　　录用日期：

通讯作者：田禾　　E-mail: 598802660@qq.com

DOI: 10.13294/j.aps.2022.0041

引用本文：

刘兴梅, 沈燕, 何玉, 班晓霞, 金红君, 何晓兰, 田禾. Notch1对糖尿病肾小管间质细胞外基质沉积的影响[J]. 生理学报 2022; 74 (3): 392-400.

LIU Xing-Mei, SHEN Yan, HE Yu, BAN Xiao-Xia, JIN Hong-Jun, HE Xiao-Lan, TIAN He. Effect of Notch1 on extracellular matrix deposition in the renal tubulointerstitium of diabetes . Acta Physiol Sin 2022; 74 (3): 392-400 (in Chinese with English abstract).