Nrf2信號(hào)通路對(duì)極寒環(huán)境致小鼠心肌損傷影響

叢培芳， 柳云恩， 施 琳， 史秀云， 劉學(xué)磊， 劉 穎， 佟 周， 佟昌慈，張玉彪， 金紅旭， 侯明曉

沈陽(yáng)軍區(qū)總醫(yī)院 急診醫(yī)學(xué)部 全軍重癥(戰(zhàn))創(chuàng)傷救治中心實(shí)驗(yàn)室 遼寧省重癥創(chuàng)傷和器官保護(hù)重點(diǎn)實(shí)驗(yàn)室，遼寧 沈陽(yáng) 110016

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·災(zāi)難醫(yī)學(xué)·

Nrf2信號(hào)通路對(duì)極寒環(huán)境致小鼠心肌損傷影響

叢培芳， 柳云恩， 施 琳， 史秀云， 劉學(xué)磊， 劉 穎， 佟 周， 佟昌慈，張玉彪， 金紅旭， 侯明曉

沈陽(yáng)軍區(qū)總醫(yī)院 急診醫(yī)學(xué)部 全軍重癥(戰(zhàn))創(chuàng)傷救治中心實(shí)驗(yàn)室 遼寧省重癥創(chuàng)傷和器官保護(hù)重點(diǎn)實(shí)驗(yàn)室，遼寧 沈陽(yáng) 110016

目的 通過(guò)建立極寒環(huán)境致小鼠心肌損傷模型，探索Nrf2信號(hào)通路在極寒環(huán)境致小鼠心肌損傷中的作用機(jī)制。方法 將20只昆明小鼠分為模型組(10只)與對(duì)照組(10只)。模型組構(gòu)建極寒環(huán)境致心肌損傷小鼠模型，對(duì)照組常規(guī)飼養(yǎng)。HE染色觀察心肌病理改變；Masson染色觀察心肌組織纖維化；Western blot檢測(cè)心肌組織過(guò)氧化氫酶(CAT)、Nrf2、Keap1蛋白的表達(dá)；Real time-PCR檢測(cè)心肌組織CAT、Nrf2、Keap1基因的表達(dá)。結(jié)果 與對(duì)照組比較，模型組小鼠心肌出現(xiàn)炎癥細(xì)胞浸潤(rùn)、心肌纖維交錯(cuò)排列及纖維化；模型組小鼠心肌組織CAT、Nrf2蛋白表達(dá)顯著升高，Keap1蛋白表達(dá)顯著下降(P<0.05)；模型組小鼠心肌組織CAT、Nrf2mRNA表達(dá)顯著升高，Keap1mRNA表達(dá)顯著下降(P<0.05)。結(jié)論 極寒環(huán)境可導(dǎo)致小鼠心肌炎癥及纖維化改變，該病變可能與Nrf2/Keap1信號(hào)通路相關(guān)。

極寒環(huán)境； 心肌損傷； Nrf2； 小鼠

DOI∶10.16048/j.issn.2095-5561.2016.06.08

極寒環(huán)境作為特殊天氣，對(duì)人體的影響極大，可以增加心血管與呼吸系統(tǒng)疾病的發(fā)病率及死亡率[1-2]；還會(huì)使凝血、神經(jīng)等系統(tǒng)受到損害，發(fā)生凍傷、血液濃縮、體內(nèi)各種生物因子紊亂等一系列的變化[3-8]。在極寒環(huán)境中，人體會(huì)更加的不適應(yīng)，進(jìn)而產(chǎn)生一系列的反應(yīng)[9]，循環(huán)系統(tǒng)的反應(yīng)主要為：早期心率加快、心輸出量增多、平均動(dòng)脈壓升高，隨著體溫的持續(xù)下降，心肌發(fā)生損傷，心率減慢、心肌收縮力減弱、低血壓等癥狀開始逐漸出現(xiàn)，甚至?xí)霈F(xiàn)心肌纖維化[10-13]。Nrf2是一種堿性亮氨酸拉鏈蛋白，在細(xì)胞氧化應(yīng)激反應(yīng)、炎癥以及抗腫瘤中發(fā)揮作用[14-16]。Nrf2功能的發(fā)揮受Keap1的調(diào)控，Keap1是Nrf2在細(xì)胞質(zhì)中的結(jié)合蛋白，發(fā)生氧化應(yīng)激時(shí)，其半胱氨酸殘基被修飾，可使Nrf2在細(xì)胞核內(nèi)積聚，與ARE結(jié)合后，促進(jìn)靶基因的表達(dá)[17]。本研究通過(guò)建立極寒環(huán)境致小鼠心肌損傷模型，探索Nrf2信號(hào)通路在極寒環(huán)境致小鼠心肌損傷中的作用機(jī)制。現(xiàn)報(bào)道如下。

1 材料與方法

1.1 材料 成年健康昆明小鼠20只，雌雄各半，體質(zhì)量(30±1)g，購(gòu)自沈陽(yáng)軍區(qū)總醫(yī)院動(dòng)物實(shí)驗(yàn)中心。小鼠隨機(jī)分為模型組(10只)與對(duì)照組(10只)，自由飲食飲水(飼料購(gòu)自江蘇省協(xié)同醫(yī)藥生物工程有限責(zé)任公司)。

1.2 方法 模型組小鼠每天4 h置于-20℃下活動(dòng)，其余時(shí)間回歸室溫條件飼養(yǎng)，持續(xù)1周；對(duì)照組小鼠室溫條件飼養(yǎng)1周。

2 結(jié)果

2.1 HE染色觀察心肌損傷病理改變 與對(duì)照組比較，模型組小鼠部分心肌出現(xiàn)炎細(xì)胞浸潤(rùn)，心肌纖維不再規(guī)則、交錯(cuò)排列。見(jiàn)圖1。

圖1 兩組小鼠心肌(HE×200倍;a.對(duì)照組;b.模型組)

2.2 Masson染色觀察心肌組織纖維化 與對(duì)照組比較，模型組小鼠心肌組織被染成青色的纖維略有增多，表明模型組小鼠的心肌組織發(fā)生了纖維化。見(jiàn)圖2。

圖2 兩組小鼠心肌(Masson×200倍;a.對(duì)照組;b.模型組)

2.3 Western blot檢測(cè)心肌組織中CAT、Nrf2、Keap1蛋白的表達(dá)情況 模型組小鼠心肌組織CAT、Nrf2蛋白表達(dá)顯著升高，Keap1蛋白表達(dá)顯著下降，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖3。

圖3 兩組小鼠心肌組織中CAT、Nrf2、Keap1蛋白的表達(dá)情況

2.4 Real time-PCR檢測(cè)心肌組織中CAT、Nrf2、Keap1基因的表達(dá)情況 模型組小鼠心肌組織CAT、Nrf2 mRNA表達(dá)顯著升高，Keap1 mRNA表達(dá)顯著下降，差異有統(tǒng)計(jì)學(xué)意義(P<0.05)。見(jiàn)圖4。

圖4 兩組小鼠心肌組織中CAT、Nrf2、Keap1基因的表達(dá)情況

3 討論

寒冷所致的氧化應(yīng)激反應(yīng)可以導(dǎo)致心肌損傷[10]，本實(shí)驗(yàn)中的模型組小鼠心肌出現(xiàn)炎癥細(xì)胞浸潤(rùn)、心肌纖維交錯(cuò)排列及纖維化。氧化應(yīng)激反應(yīng)產(chǎn)生的活性氧可以直接或間接地?fù)p傷細(xì)胞內(nèi)蛋白質(zhì)、脂質(zhì)以及核酸等大分子物質(zhì)的生理功能，這是多種疾病發(fā)生的病理生理基礎(chǔ)。本實(shí)驗(yàn)中，氧化應(yīng)激反應(yīng)發(fā)生的代表產(chǎn)物 CAT在模型組表達(dá)升高，說(shuō)明極寒環(huán)境所致的心肌損傷與其造成的氧化應(yīng)激反應(yīng)相關(guān)。Nrf2是CNC轉(zhuǎn)錄因子家族成員，含有6個(gè)高度保守的結(jié)構(gòu)域Neh，此區(qū)域包含C端亮氨酸拉鏈結(jié)構(gòu)，與細(xì)胞核內(nèi)小Maf蛋白結(jié)合，使Nrf2 能夠識(shí)別、結(jié)合ARE，從而啟動(dòng)目標(biāo)基因轉(zhuǎn)錄[18-19]。有研究表明，Nrf2可作為靶點(diǎn)預(yù)防或治療消化系統(tǒng)疾病、肺纖維化、神經(jīng)退行性病變、癌癥等[20-23]。本實(shí)驗(yàn)中，與對(duì)照組比較，模型組Nrf2的基因與蛋白表達(dá)均升高，而Keap1的表達(dá)呈降低趨勢(shì)，差異具有統(tǒng)計(jì)學(xué)意義(P<0.05)，實(shí)驗(yàn)結(jié)果說(shuō)明Nrf2信號(hào)通路在極寒環(huán)境造成的氧化應(yīng)激反應(yīng)中發(fā)揮了作用。

總之，極寒環(huán)境造成的氧化應(yīng)激反應(yīng)可以導(dǎo)致小鼠心肌炎癥甚至纖維化的發(fā)生，此損傷的發(fā)生與Nrf2信號(hào)通路有關(guān)。由此推測(cè)，通過(guò)干預(yù)Nrf2信號(hào)通路來(lái)預(yù)防或治療極寒環(huán)境所致的心肌損傷也許會(huì)取得良好的效果，為相關(guān)藥物的研發(fā)提供了理論基礎(chǔ)。

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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

全軍十二五面上項(xiàng)目(CWS11J295)

叢培芳(1987-)，遼寧沈陽(yáng)人，藥師，碩士

侯明曉,E-mail:houmingxiao188@163.com; 金紅旭，E-mail:hongxuj@126.com

2095-5561(2016)06-0351-04

R541

A

2016-10-21