Nrf2信号通路对极寒环境致小鼠心肌损伤影响

丛培芳， 柳云恩， 施 琳， 史秀云， 刘学磊， 刘 颖， 佟 周， 佟昌慈，张玉彪， 金红旭， 侯明晓

沈阳军区总医院 急诊医学部 全军重症(战)创伤救治中心实验室 辽宁省重症创伤和器官保护重点实验室，辽宁 沈阳 110016



·灾难医学·

Nrf2信号通路对极寒环境致小鼠心肌损伤影响

丛培芳， 柳云恩， 施 琳， 史秀云， 刘学磊， 刘 颖， 佟 周， 佟昌慈，张玉彪， 金红旭， 侯明晓

沈阳军区总医院 急诊医学部 全军重症(战)创伤救治中心实验室 辽宁省重症创伤和器官保护重点实验室，辽宁 沈阳 110016

目的 通过建立极寒环境致小鼠心肌损伤模型，探索Nrf2信号通路在极寒环境致小鼠心肌损伤中的作用机制。方法 将20只昆明小鼠分为模型组(10只)与对照组(10只)。模型组构建极寒环境致心肌损伤小鼠模型，对照组常规饲养。HE染色观察心肌病理改变；Masson染色观察心肌组织纤维化；Western blot检测心肌组织过氧化氢酶(CAT)、Nrf2、Keap1蛋白的表达；Real time-PCR检测心肌组织CAT、Nrf2、Keap1基因的表达。结果 与对照组比较，模型组小鼠心肌出现炎症细胞浸润、心肌纤维交错排列及纤维化；模型组小鼠心肌组织CAT、Nrf2蛋白表达显著升高，Keap1蛋白表达显著下降(P<0.05)；模型组小鼠心肌组织CAT、Nrf2mRNA表达显著升高，Keap1mRNA表达显著下降(P<0.05)。结论 极寒环境可导致小鼠心肌炎症及纤维化改变，该病变可能与Nrf2/Keap1信号通路相关。

极寒环境； 心肌损伤； Nrf2； 小鼠

DOI∶10.16048/j.issn.2095-5561.2016.06.08

极寒环境作为特殊天气，对人体的影响极大，可以增加心血管与呼吸系统疾病的发病率及死亡率[1-2]；还会使凝血、神经等系统受到损害，发生冻伤、血液浓缩、体内各种生物因子紊乱等一系列的变化[3-8]。在极寒环境中，人体会更加的不适应，进而产生一系列的反应[9]，循环系统的反应主要为：早期心率加快、心输出量增多、平均动脉压升高，随着体温的持续下降，心肌发生损伤，心率减慢、心肌收缩力减弱、低血压等症状开始逐渐出现，甚至会出现心肌纤维化[10-13]。Nrf2是一种碱性亮氨酸拉链蛋白，在细胞氧化应激反应、炎症以及抗肿瘤中发挥作用[14-16]。Nrf2功能的发挥受Keap1的调控，Keap1是Nrf2在细胞质中的结合蛋白，发生氧化应激时，其半胱氨酸残基被修饰，可使Nrf2在细胞核内积聚，与ARE结合后，促进靶基因的表达[17]。本研究通过建立极寒环境致小鼠心肌损伤模型，探索Nrf2信号通路在极寒环境致小鼠心肌损伤中的作用机制。现报道如下。

1 材料与方法

1.1 材料 成年健康昆明小鼠20只，雌雄各半，体质量(30±1)g，购自沈阳军区总医院动物实验中心。小鼠随机分为模型组(10只)与对照组(10只)，自由饮食饮水(饲料购自江苏省协同医药生物工程有限责任公司)。

1.2 方法 模型组小鼠每天4 h置于-20℃下活动，其余时间回归室温条件饲养，持续1周；对照组小鼠室温条件饲养1周。

2 结果

2.1 HE染色观察心肌损伤病理改变 与对照组比较，模型组小鼠部分心肌出现炎细胞浸润，心肌纤维不再规则、交错排列。见图1。

图1 两组小鼠心肌(HE×200倍;a.对照组;b.模型组)

2.2 Masson染色观察心肌组织纤维化 与对照组比较，模型组小鼠心肌组织被染成青色的纤维略有增多，表明模型组小鼠的心肌组织发生了纤维化。见图2。

图2 两组小鼠心肌(Masson×200倍;a.对照组;b.模型组)

2.3 Western blot检测心肌组织中CAT、Nrf2、Keap1蛋白的表达情况 模型组小鼠心肌组织CAT、Nrf2蛋白表达显著升高，Keap1蛋白表达显著下降，差异有统计学意义(P<0.05)。见图3。

图3 两组小鼠心肌组织中CAT、Nrf2、Keap1蛋白的表达情况

2.4 Real time-PCR检测心肌组织中CAT、Nrf2、Keap1基因的表达情况 模型组小鼠心肌组织CAT、Nrf2 mRNA表达显著升高，Keap1 mRNA表达显著下降，差异有统计学意义(P<0.05)。见图4。

图4 两组小鼠心肌组织中CAT、Nrf2、Keap1基因的表达情况

3 讨论

寒冷所致的氧化应激反应可以导致心肌损伤[10]，本实验中的模型组小鼠心肌出现炎症细胞浸润、心肌纤维交错排列及纤维化。氧化应激反应产生的活性氧可以直接或间接地损伤细胞内蛋白质、脂质以及核酸等大分子物质的生理功能，这是多种疾病发生的病理生理基础。本实验中，氧化应激反应发生的代表产物 CAT在模型组表达升高，说明极寒环境所致的心肌损伤与其造成的氧化应激反应相关。Nrf2是CNC转录因子家族成员，含有6个高度保守的结构域Neh，此区域包含C端亮氨酸拉链结构，与细胞核内小Maf蛋白结合，使Nrf2 能够识别、结合ARE，从而启动目标基因转录[18-19]。有研究表明，Nrf2可作为靶点预防或治疗消化系统疾病、肺纤维化、神经退行性病变、癌症等[20-23]。本实验中，与对照组比较，模型组Nrf2的基因与蛋白表达均升高，而Keap1的表达呈降低趋势，差异具有统计学意义(P<0.05)，实验结果说明Nrf2信号通路在极寒环境造成的氧化应激反应中发挥了作用。

总之，极寒环境造成的氧化应激反应可以导致小鼠心肌炎症甚至纤维化的发生，此损伤的发生与Nrf2信号通路有关。由此推测，通过干预Nrf2信号通路来预防或治疗极寒环境所致的心肌损伤也许会取得良好的效果，为相关药物的研发提供了理论基础。

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Effect of Nrf2 signaling pathway on cold stress induced myocardial injury in mice

CONG Pei-fang,LIU Yun-en,SHI Lin,SHI Xiu-yun,LIU Xue-lei,LIU Ying,TONG Zhou,TONG Chang-ci,ZHANG Yu-biao,JIN Hong-xu,HOU Ming-xiao

(Department of Emergency Medicine,Laboratory of PLA Wound and Trauma Center,The General Hospital of Shenyang Military Command,Shenyang 110016,China)

Objective To investigate the mechanism of Nrf2 signaling pathway on cardiac injury of myocardium tissue which induced by oxidative stress of extremely cold weather by establishing the mice model.Methods A total of 20 mice were divided into the control group(n=10) and the model group(n=10).The mice model of cardiac injury induced by extremely cold weather was established in the model group and mice in the control group were conventionally breeded.HE staining was used to observe the myocardial pathological changes;Masson staining was used to observe the myocardial tissue fibrosis;Western blot detection was used to detect myocardial tissue catalase(CAT),Nrf2 and Keap1 protein expression;Real Time PCR was used to detect myocardial tissue CAT,Nrf2 and Keap1 gene expression.Results Compared with the control group,mice in the model group myocardial inflammatory cells infiltration,myocardial fibers were staggered and fibrosis;myocardial CAT tissue of mice in the model group was with a significant rise in Nrf2 protein expression,while Keap1 protein expression was significantly reduced(P<0.05);the myocardial tissue CAT of mice in the model group,Nrf2 had a significant rise in mRNA expression,while Keap1 mRNA expression was significantly reduced(P<0.05).Conclusion Extreme cold environment change can lead to inflammation and fibrosis in mice,and the disease may be associated with Nrf2/Keap1 signal pathway.

Cold stress; Cardiac injury; Nrf2; Mice

全军十二五面上项目(CWS11J295)

丛培芳(1987-)，辽宁沈阳人，药师，硕士

侯明晓,E-mail:houmingxiao188@163.com; 金红旭，E-mail:hongxuj@126.com

2095-5561(2016)06-0351-04

R541

A

2016-10-21