在自噬功能障碍情况下Nrf2调节失代偿性心脏重构及功能障碍的机制研究

发布时间：2018-05-08 02:11

  本文选题：Nrf2 + 自噬　； 参考：《山东大学》2016年博士论文

【摘要】：研究背景：转录因子NE-2相关因子2(Nrf2)是碱性亮氨酸拉链转录因子Cap'n'Coall(CNC)家族的成员,Nrf2通过与一种顺式增强子序列结合,我们称之为抗氧化反应元件(ARE),其核心核苷酸序列为'5-RTGACNNNGC-3',与这种增强子结合可启动200多种归属于不同领域的基因的表达,包括抗氧化基因、Ⅱ相解毒酶、转录因子、转运蛋白、清道夫受体以及分子伴侣蛋白等等。因此,Nrf2具有广泛而复杂的生物学功能,从经典的抗氧化防御,到细胞周期的调控和蛋白质量的控制。Nrf2对心脏的保护作用已经在许多动物模型中证实,包括以主动脉弓缩窄术(TAC)引发心脏压力负荷增加诱导的失代偿性心脏重构和心功能障碍模型。从分子水平上分析,转录因子Nrf2启动抗氧化蛋白以及解毒蛋白的表达,从而对抗氧化应激压力导致的心脏损伤和功能失常。同时,研究发现Nrf2可促进自噬清除毒性的多聚泛素化蛋白聚集体的功能,从而保护心脏不受细胞内毒性蛋白的损伤。但是,与之前的结论相反,最近的研究发现,在一种蛋白聚集诱发心肌病的小鼠动物模型中,这种模型通过向小鼠基因组内导入人来源的alpha B-晶状体蛋白基因突变体(hCryABR120G)并进行小鼠老龄化实现,敲除Nrf2基因可以抵御心脏的还原压力,减少多聚泛素化蛋白在心脏内的聚集,减缓心脏病理性肥大,改善心力衰竭。分析原因,持续性的Nrf2激活可能引发过度的还原压力导致hCryABR120G诱发的心肌病。然而,也有研究发现心肌特异性敲除Nrf2基因的小鼠对抗了TAC术后4周的压力负荷诱发的失代偿性心脏重构和功能障碍,这与Nrf2介导还原压力的理论相违背。另一方面,前期的研究也证明在自噬功能障碍的状态下,Nrf2的激活导致了肝脏的损伤。另外,自噬功能障碍在小鼠CryABR120G (mCryABR120G)基因诱导的心肌病中起了重要作用,mCryABR120G基因和hCryABR120G基因有着几乎相同的核苷酸序列,且衰老本身也伴随着自噬功能不足或损伤,从这些关联上看,衰老的hCryABR120G小鼠心脏中Nrf2介导的负面作用很可能与自噬功能不足有关。综上所述,在不同的病理状态下,Nrf2对心脏的作用可能是保护性的也可能是损伤性的,这种双面作用确切的机制目前还没有研究清楚。在本次研究中,我们发现心脏中Nrf2基因激活的病理生理学作用与自噬功能的完整性密切相关。自噬功能完整时Nrf2的激活对心脏的适应性反应起着重要作用,然而,在自噬功能障碍时,Nrf2的激活又介导了心脏的失代偿性重构和功能障碍。我们在研究中发现,自噬功能损伤可能是通过阻断核内Fyn控制的Nrf2向细胞核外的转运,从而导致了Nrf2在核内积聚,启动血管紧张素原的大量表达,进而恶化心脏失代偿性重构,加重心功能损伤。研究目的：1、研究Nrf2在TAC手术诱发的压力负荷引起的心脏重构和心功能障碍的病理进展过程中所起的作用；2、研究Nrf2激活对心脏的作用与自噬功能状态之间的关系；3、探讨在不同自噬功能状态下,Nrf2影响心脏功能的具体机制。研究方法：第一部分：1、用Nrf2基因敲除小鼠和野生型对照组小鼠进行主动脉弓缩窄手术,建立心衰模型,检测术后存活状况,在模型的早期EBD检测心肌坏死,模型的晚期阶段,超声、qPCR、WGA、Masson、免疫组化检测心脏重构状况和心功能,分析不同病理生理学阶段Nrf2基因敲除对心脏的影响；2、通过对TAC术后的WT小鼠术后不同时间点进行自噬流的检测,分析TAC术后不同阶段自噬功能的状态;3、运用心肌特异性Atg5基因敲除小鼠建立自噬功能障碍心脏的TAC模型,观察在自噬功能障碍时心脏的病理状态以及Nrf2和其下游基因表达水平。第二部分：1、以qPCR和Western Blot的方法检测白噬功能障碍小鼠心脏承受压力负荷时心衰相关基因Agt的表达状况；2、以Western Blot的方法检测自噬功能障碍对小鼠心脏承受压力负荷时各信号通路激活水平的影响,并与Nrf2基因敲除小鼠心脏压力负荷模型相对比,分析两者之间的联系；3、建立Nrf2/Atg5双敲小鼠心脏压力负荷动物模型,以免疫组化、western blot检测更具体的分析Nrf2对压力诱导心衰的病理生理作用与自噬功能的关系。研究结果：第一部分：1、Nrf2基因敲除加重了TAC手术诱发的心脏急性损伤,但使手术引起的远期的心脏重塑和心力衰竭得到了缓解；2、在压力负荷引起的心脏病程中,Nrf2激活所起的病理生理学作用与自噬功能的完整性有关；3、自噬功能障碍的小鼠心脏压力负荷增加时,存在Nrf2的过度表达和激活。第二部分：1、在自噬功能缺失的压力超负荷心脏中,Nrf2启动Agt的表达；2、在压力负荷诱导损伤的心脏,自噬功能障碍阻断了TAC诱导的Jak/Fyn通路的激活,使Nrf2向细胞核外转运降解减少。研究结论：1、自噬功能完整时Nrf2激活对心脏起保护作用,而当自噬功能障碍时,Nrf2的激活加速了心脏病理性重构和心功能失常的进程；2、在自噬功能完整时,Nrf2激活可以降低心肌坏死率保护心脏,但在自噬功能不足时,Nrf2激活了心脏内血管紧张素Ⅱ通路,使病情进一步恶化；3、自噬功能的障碍导致了Nrf2在细胞核内的聚集,进一步启动了血管紧张素原的高表达,Nrf2在核内的聚集与Jak2/Fyn信号通路的阻断有关。
[Abstract]:Background : The transcription factor NE - 2 - associated factor 2 ( Nrf2 ) is a member of the basic leucine zipper transcription factor Cap ' n ' Coall ( CNC ) family . Nrf2 binds to a sequence of cis - enhancer sequences , and we call it anti - oxidation reaction element ( ARE ) whose core nucleotide sequence is ' 5 - RTMM NNlngc - 3 . It is found that Nrf2 plays an important role in preventing cardiac injury and dysfunction induced by stress stress in mice . The purpose of this study was to study the role of Nrf2 in the pathogenesis of cardiac remodeling and cardiac dysfunction induced by TAC operation .
2 . To study the relationship between the activation of Nrf2 and the function of autophagy in the heart ;
3 . To investigate the specific mechanism of Nrf2 on cardiac function in different autophagy states . The study was as follows : 1 . Using Nrf2 gene knockout mice and wild - type control group mice to perform aortic arch narrowing operation , establish a heart failure model , detect the survival status of the model , detect myocardial necrosis at the early stage of the model , detect cardiac remodeling in the advanced stage of the model , detect cardiac remodeling and cardiac function by immunohistochemistry , and analyze the effects of Nrf2 gene knock - out on the heart in different pathological stages ;
2 . Through the detection of autophagy flow at different time points after TAC operation in WT mice , a TAC model of autophagy dysfunction was established by using the myocardial specificity Atg5 gene knockout mice . The pathological states of the heart and the expression level of Nrf2 and its downstream genes were observed at the time of autophagy dysfunction .
2 . Western Blot was used to detect the effect of autophagy dysfunction on the activation level of each signal pathway when the heart of mice was subjected to pressure load , and compared with the model of Nrf2 knockout mice ' s heart pressure load model , the relationship between them was analyzed .
3 . To establish an animal model of cardiac stress load of Nrf2 / Atg5 double - tapping mice . The relationship between the pathological physiology and the autophagy function of Nrf2 on stress - induced heart failure was detected by immunohistochemistry and western blot . The results showed that the first part : 1 , Nrf2 knockout aggravated the acute myocardial injury induced by TAC operation , but the long - term cardiac remodeling and heart failure caused by operation were relieved .
2 . In the course of heart disease caused by pressure load , the pathological physiology of Nrf2 activation is related to the integrity of autophagy function ;
3 . Over - expression and activation of Nrf2 were present in the mice with autophagy dysfunction . The second part : 1 . The expression of Agt was activated by Nrf2 in the pressure overload heart deficient in autophagy function .
2 . The activation of the TAC - induced Jak / Fyn pathway was blocked by autophagy dysfunction in the heart and autophagy dysfunction induced by pressure overload . Conclusion : 1 . Activation of Nrf2 plays a role in protecting the heart when the autophagy function is complete , and the activation of Nrf2 accelerates the process of cardiac remodeling and cardiac dysfunction when autophagy dysfunction occurs ;
2 . When the autophagy function is complete , the activation of Nrf2 can reduce the myocardial necrosis rate and protect the heart , but when the autophagy function is insufficient , the Nrf2 activates the angiotensin II pathway in the heart to further deteriorate the condition ;
3 . The dysfunction of autophagy resulted in the accumulation of Nrf2 in the nucleus , further the high expression of angiotension , and the accumulation of Nrf2 in the nucleus was related to the blocking of the Jak2 / Fyn signal pathway .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54
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本文编号：1859488