摘要
目的探讨内质网应激(ERS)介导的晚期氧化蛋白产物(AOPPs)诱导肾小管上皮细胞(HK-2)的自噬抑制及其信号通路。方法 (1)AOPPs刺激HK-2细胞,检测信号通路腺苷酸活化蛋白激酶/乳动物雷帕霉素靶蛋白(AMPK/mTOR)的表达;(2)AMPK抑制剂Compoun C和激活剂AICAR分别预处理细胞,并予AOPPs刺激,检测自噬标志物LC3Ⅱ/LC3Ⅰ、Beclin1、P62及AMPK/mTOR蛋白的表达;(3)ERS激活剂Thapsigargin与抑制剂Salubrinal分别预处理细胞,并予AOPPs刺激,检测ERS标志蛋白葡萄糖调节蛋白78(GRP78)、自噬及AMPK/mTOR蛋白的表达。结果 AOPPs可抑制AMPK的磷酸化和诱导mTOR的磷酸化(P<0.05)。Compoun C使LC3Ⅱ/LC3Ⅰ、Beclin1表达下降,p62表达上升,同时抑制AMPK的磷酸化和诱导mTOR的磷酸化(P<0.05),而AICAR表现出相反的效果。Thapsigargin能抑制AMPK的磷酸化和诱导mTOR的磷酸化,LC3Ⅱ/LC3Ⅰ、Beclin1表达下降,p62表达上升,而Salubrinal表现出相反的效果。结论ERS可能通过激活AMPK/mTOR信号通路介导AOPPs诱导的自噬抑制的发生。
Objective To investigate the effects of endoplasmic reticulum stress(ERS)on advanced oxidation protein products(AOPPs)-inhibited autophagy in human renal tubular epithelial cells(HK-2)and its possible signaling pathway.Methods (1)HK-2 cells were stimulated by AOPPs,and the expression of AMP-activated protein kinase and mammalian target of rapamycin(AMPK/mTOR)was detected by Western blot.(2)HK-2 cells were treated with AMPK inhibitor(Compoun C)or AMPK activator(AICAR)respectively,and then stimulated by AOPPs.Western blot was used to determine the expression of autophagy related proteins,including LC3Ⅱ/LC3Ⅰ,Beclin1,P62 and AMPK/mTOR signaling pathway related proteins.(3)HK-2 cells were treated with ERS activator(Thapsigargin)or ERS inhibitor(Salubrinal),and then stimulated by AOPPs.The levels of ERS related protein,GRP78,and autopahgy related proteins as well as AMPK/mTOR signaling pathway related proteins were detected via Western blot.Results AOPPs inhibited the phosphorylation of AMPK and induced the phosphorylation of mTOR(P<0.05).Compoun C decreased the protein expression levels of LC3Ⅱ/LC3Ⅰand Beclin 1,increased the expression level of p62 protein,inhibited the phosphorylation of AMPK,and induced the phosphorylation of mTOR(P<0.05),while the AICAR showed the opposite effects.Thapsigargin inhibited the phosphorylation of AMPK and induced the phosphorylation of mTOR,decreased the protein expression levels of LC3Ⅱ/LC3Ⅰand Beclin 1,increased the p62 protein level(P<0.05),while the Salubrinal showed the opposite results.Conclusion ERS could mediate AOPPs-induced autophagy inhibition by activation of the AMPK/mTOR signaling pathway in HK-2 cells.
作者
李敏晖
汤珣
姜婷婷
张雯英
谢唯
章俊
LI Minhui;TANG Xun;JIANG Tingting;ZHANG Wenying;XIE Wei;ZHANG Jun(Blood Purification Center,Nanhai Hospital of Southern Medical University, Foshan,Guangdong 528244,China;Department of Nephrology,Zhujiang Hospital of Southern Medical University,Guangzhou,Guangdong 510280,China)
出处
《重庆医学》
CAS
2018年第29期3735-3738,3742,共5页
Chongqing medicine
基金
2017佛山市卫生和计划生育局科研课题立项项目(20170214)
关键词
晚期氧化蛋白产物
内质网应激
丝氨酸苏氨酸蛋白激酶/哺乳动物雷帕霉素靶蛋白通路
自噬
advanced oxidation protein products
endoplasmic reticulum stress
serine/threonine protein kinase/mammalian target of rapamycin signaling pathway
autophagy
