Nrf2调控心肌整合应激反应的机制研究

Integrated stress response (ISR) mediated by protein kinase R-like ER kinase (PERK) regulates protein synthesis and apoptosis. Our previous work found that PERK mediated cardiomyocyte apoptosis induced by endoplasmic reticulum (ER) stress. We hypothesize that "nuclear factor erythroid 2-related factor 2 (Nrf2）integratively regulates PERK-mediated cell survival/apoptosis by promoting survival in the early stage of ISR, and by resulting in apoptosis through ATF4/CHOP pathway in the late stage of ISR". In the present project, Nrf2 expression will be detected by using of real time PCR and Western blot analysis, and immunofluorescence will be used to detect sub-cellular localization of Nrf2 during ER stress. The relation of dynamic alteration of expression and sub-cellular localization of Nrf2 and cardiomyocyte survival/damage will be analysis. Effects of intervention of expression and activity of Nrf2 on ER stress-induced ATF4 expression/activation and cardiomyocyte damage will be investigated to demonstrate that Nrf2 is a key molecule for regulating PERK-mediated survival/apoptosis. Effects of intervention of ATF4 expression on Nrf2-mediated CHOP expression and apoptosis will be studied to demonstrate that ATF4 serves as Nrf2's target on PERK-mediated apoptosis. The results of the project will provide a new target for adaptating mechanism underlying stressed cardiomyocytes and treatment of cardiovascular diseases from integrative regulation of PERK-mediated apoptosis.

蛋白激酶R样内质网激酶（PERK）介导的整合应激反应调控蛋白合成与细胞凋亡。本项目在课题组证实PERK介导心肌内质网应激相关凋亡基础上，提出《核因子红系衍生相关因子2（Nrf2）在内质网应激早期有利于细胞存活，晚期通过正反馈调节ATF4/CHOP途径导致凋亡，从而调控PERK介导的整合应激反应》的假说。采用实时定量PCR和免疫印迹法研究内质网应激中Nrf2表达、免疫荧光技术检测Nrf2亚细胞定位的动态变化及其与心肌细胞存活/凋亡的关系，研究干预Nrf2表达和活性对于内质网应激致ATF4表达/活性以及心肌细胞损伤的影响，证实Nrf2是调控PERK介导凋亡途径的关键分子；进一步研究干预ATF4表达对于Nrf2介导CHOP表达和细胞凋亡作用的影响，证实ATF4是Nrf2调控PERK介导细胞凋亡的靶分子。从整合调控PERK介导细胞凋亡的角度，为心肌细胞应激适应的机制研究和心血管疾病防治提供新靶点。

内质网应激是心血管疾病发生发展的重要亚细胞机制，过度内质网应激是造成心血管损伤的关键环节。本课题以核因子红系相关因子2 (Nrf2)为切入点，主要研究内质网应激过程中蛋白激酶R样内质网激酶 (PERK)通过Nrf2核转位介导内质网应激相关细胞凋亡和自噬的机制。研究胡萝卜素 ( thapsigargin, TG ) 或衣霉素 （TM）诱导心肌细胞内质网应激过程中Nrf2表达、定位的动态变化、证实Nrf2表达和定位的动态变化与内质网应激致心肌细胞存活/损伤相关。研究干预细胞Nrf2核转位对于内质网应激PERK途径及其介导内质网应激相关细胞凋亡分子CHOP表达和细胞凋亡的影响，证实Nrf2通过PERK调控内质网应激相关细胞凋亡。进一步研究内质网应激过程中PERK下游分子ATF4表达的变化，探讨激动剂特丁基对苯二酚 (Tertiary butylhydroquinone, tBHQ) 激活Nrf2核转位，或以抑制剂全反式视黄酸下调Nrf2活性对于内质网应激上调ATF4表达的影响及其机制，证实Nrf2调控PERK介导细胞凋亡靶分子。在此基础上进一步研究内质网应激诱导的H9c2细胞内质网自噬、LC-3II和Beclin 1蛋白表达上调情况，证实内质网应激诱导内质网自噬现象；内质网应激抑制剂减轻内质网应激诱导的H9c2细胞LC-3II和Beclin 1蛋白表达上调和内质网自噬。通过Nrf2抑制剂全反式视磺酸 (ATRA) 抑制Nrf2活性，发现ATRA明显减轻内质网应激引起的LC-3II在内质网的高表达，且LC-3II在内质网内的集聚程度明显降低，下调Beclin 1蛋白表达；提示抑制Nrf2途径减轻内质网应激诱导的内质网自噬。上述结果表明证实Nrf2可以整合调控内质网应激相关细胞自噬和PERK介导的内质网应激相关细胞凋亡，上述研究结果从整合调控PERK介导细胞凋亡的角度，为细胞应激适应的机制研究和细胞应激相关疾病防治提供新靶点。