運動鍛煉上調(diào)M3R對MI大鼠心臟產(chǎn)生保護效應(yīng)及其機制探討

田振軍，張 可，陳 婷,2

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運動鍛煉上調(diào)M3R對MI大鼠心臟產(chǎn)生保護效應(yīng)及其機制探討

田振軍1，張 可1，陳 婷1,2

目的：探討運動上調(diào)M3受體(M3R)對心肌梗死(MI)大鼠心臟產(chǎn)生保護效應(yīng)及其機制。方法：雄性SD大鼠48只，隨機分為正常對照組(C)，心梗組(MI)，心梗+中強度持續(xù)有氧運動組(ME1)，心梗+高強度間歇運動組(ME2)，每組12只。C組常規(guī)飼養(yǎng)，MI組采用左冠脈前降支結(jié)扎法(LAD)建立MI模型。ME1和ME2組心梗手術(shù)1周后進行跑臺運動， 60 min/次，1次/d，5 d/1周×8周。ME1組以10 m/min×5 min，再以3 m/min速度遞增至16 m/min。ME2組以10 m/min×10 min后，逐漸遞增至25 m/min×7 min；之后以15 m/min×3 min間歇運動，依次交替進行。訓(xùn)練結(jié)束后次日，腹腔麻醉進行頸動脈插管測定LVSP、LVEDP、±dp/dtmax等指標，評定心功能變化。之后迅速開胸摘取心臟，分別進行組織學(xué)制片、Masson染色觀察心肌膠原纖維變化，免疫熒光法觀察分析心肌M3R表達，Western Blot法檢測心肌M3R、MEK1/2、p-ERK1/2、ERK1/2、Bcl-2和Bax蛋白表達。結(jié)果：MI組膠原容積百分比(CVF%)和LVEDP較C組顯著升高(P<0.01)，LVSP和±dp/dtmax較C組均顯著降低(P<0.01)。MI后可見心肌M3R陽性染色，且M3R、MEK1/2、p-ERK1/2/ERK1/2表達較C組均顯著升高(P<0.01)，Bcl-2/Bax比值較C組顯著降低(P<0.01)。ME1和ME2組CVF%和LVEDP較MI組均顯著降低(P<0.01)，ME1組-dP/dt max較MI組顯著升高(P<0.01)，ME2組LVSP較MI組顯著升高(P<0.01)。ME1和ME2組均可見心肌M3R陽性染色，M3R、MEK1/2、p-ERK1/2/ERK1/2表達較MI組顯著增加 (P<0.01)，Bcl-2/Bax比值較MI組顯著升高(P<0.01,P<0.05)，ME1組和ME2組間無顯著差異。結(jié)論：持續(xù)有氧運動和高強度間歇運動均可上調(diào)心梗心肌M3R-MEK1/2- ERK1/2通路，抑制心肌細胞凋亡，改善心肌纖維化程度，保護心梗大鼠心功能。心梗心肌M3R-MEK1/2- ERK1/2-細胞凋亡途徑與持續(xù)和間歇運動方式及運動強度關(guān)系不密切。

M3受體;MEK1/2-ERK1/2通路;心肌細胞凋亡;持續(xù)有氧運動;高強度間歇運動；心肌梗死

1 材料與方法

1.1 實驗動物與分組

3月齡雄性Sprague Dawley大鼠48只，體重190～210 g，購于西安交通大學(xué)醫(yī)學(xué)院實驗動物中心(動物質(zhì)量合格證號：陜醫(yī)動證字08-004號)，隨機分為正常對照組(C)，心梗組(MI)，心梗+中等強度持續(xù)運動組(ME1)，心梗+高強度間歇運動組(ME2)。每組12只。動物室溫為18℃～23℃，濕度為50%～60%，標準嚙齒類動物干燥飼料喂養(yǎng)，自由飲食。C組大鼠常規(guī)籠內(nèi)安靜飼養(yǎng)，MI組采用左冠脈前降支結(jié)扎法(LAD)，建立MI模型。ME1和ME2組進行為期8周的小動物跑臺運動。

1.2 主要儀器和試劑

主要儀器為PowerLab/8s生理信號采集處理系統(tǒng)，ALC-V8動物呼吸機，BM-Ⅱ型病理組織包埋機，生物組織攤烤片機，LEICA-RM 2126切片機，BX51奧林巴斯光學(xué)顯微鏡，Nikon Eclipse 55i熒光顯微鏡，低溫高速離心機，酶標儀，Biorad電泳儀和轉(zhuǎn)移槽，凝膠成像儀等。主要試劑為兔抗大鼠M3R多克隆抗體購于abcam公司，ERK1/2、p-ERK1/2、MEK1/2購于CST公司，Bcl-2和Bax購于博士德生物有限公司。

1.3 心梗模型制備與運動方案

1.4 血流動力學(xué)指標測定

運動8周結(jié)束后次日，腹腔麻醉PowerLab/8s記錄心電圖，右頸總動脈插管同步測試左室收縮壓(LVSP)、左室舒張末壓(LVEDP)、左室壓力最大上升速率(+dp/dtmax)和最大下降速率(-dp/dtmax)等反映心功能的指標。數(shù)據(jù)采集完畢后，迅速開胸摘取心臟，進行后續(xù)實驗。

1.5 取材、樣品處理與組織學(xué)實驗

待心功能指標測試完畢后，每組隨機選取6只大鼠，迅速摘取心臟，置10%中性甲醛溶液固定24 h后，經(jīng)流水沖洗、梯度乙醇脫水、二甲苯透明，石蠟包埋，連續(xù)切片(厚5 μm)，常規(guī)制片，常規(guī)Masson染色，顯微鏡觀察、攝片。另速摘取6只大鼠心臟鋁箔紙包裹，液氮冷凍后移至-80℃低溫冰箱保存，用于Western Blot實驗。

免疫熒光實驗的切片經(jīng)脫蠟至水、PBS清洗和微波抗原修復(fù)后，用正常山羊血清在濕盒中37℃封閉30 min。滴加一抗(兔抗大鼠M3R多克隆抗體，1∶300)，濕盒4℃過夜。室溫復(fù)溫45 min，PBS沖洗。滴加TRITC標記的二抗(1∶200)。37℃濕盒孵育1 h，PBS沖洗。滴加DAPI避光孵育2 min，PBS沖洗、封片。每次染色設(shè)置空白對照(PBS取代一抗和二抗)及陰性對照(PBS取代一抗)。采用熒光顯微鏡觀察，低倍鏡選位，400倍鏡下拍照。各組選取6張石蠟切片，每張選10個視野，統(tǒng)計平均光密度(Mean Optical Density，MOD)。

1.6 Western Blot實驗

采用RIPA試劑提取總蛋白，Bradford方法測定蛋白濃度。等量蛋白采用12% SDS聚丙烯酰胺凝膠垂直電泳分離，NC轉(zhuǎn)膜，麗春紅染膜，室溫搖動封閉(3% BSA，TBST稀釋)60 min后，分別加入兔抗M3R(1∶500)、MEK1/2(1∶1 000)、ERK1/2(1∶1 000)、p-ERK1/2(1∶1 000)、Bcl-2(1∶500)、Bax(1∶100)等多克隆抗體，4℃過夜，室溫洗膜，加入HRP羊抗兔IgG抗體(Jackson，1∶10 000)孵育30 min，室溫洗膜，化學(xué)發(fā)光底物ECL(Millipore)發(fā)光顯跡。內(nèi)參為GAPDH，計算目的蛋白與內(nèi)參蛋白條帶的積分光密度(Integral Optical Density，IOD)。

1.7 數(shù)據(jù)采集與統(tǒng)計學(xué)處理

2 實驗結(jié)果

2.1 心肌M3R表征結(jié)果

免疫熒光觀察顯示，M3R陽性染色為紅色熒光顆粒。C組心肌M3R陽性熒光顆粒極少。MI后可見心肌M3R紅色熒光顆粒呈點狀分布，且較C組顯著增加(P<0.01)；ME1和ME2組M3R陽性顆粒較MI組均顯著增加(P<0.01)，兩組間無顯著差異(圖1)。Western Blot結(jié)果顯示，C組、MI組、ME1組和ME2組心肌M3R表征與免疫熒光觀察統(tǒng)計結(jié)果一致(圖1)。結(jié)果表明，心肌細胞上分布著M3R，心梗可引起心肌應(yīng)激性M3R上調(diào)表達，有氧運動和高強度間歇運動均可顯著上調(diào)M3R表達，且無運動強度差異。

2.2 心肌MEK1/2-ERK1/2通路和凋亡信號Bcl-2/Bax蛋白表征

Western Blot結(jié)果顯示，MI組心肌MEK1/2、p-ERK1/2和ERK1/2蛋白表達較C組顯著增加(P<0.01)，ME1和ME2組較MI組顯著增加(P<0.01)，ME1和ME2組間無顯著差異(圖2)。MI組心肌Bcl-2/Bax比值較C組顯著降低(P<0.01)，ME1和ME2組較MI組顯著增加(P<0.05)，ME1和ME2組間無顯著差異(圖2)。結(jié)果表明，心梗心肌MEK1/2-ERK1/2通路發(fā)生應(yīng)激性激活，而有氧運動和高強度間歇運動均可顯著激活MEK1/2-ERK1/2通路，且無運動強度差異。

2.3 心肌纖維化觀察分析和心功能測試結(jié)果

Masson染色結(jié)果顯示，C組心肌細胞排列整齊，藍色膠原纖維所占比例較少。與C組比較，MI組心肌組織結(jié)構(gòu)紊亂，膠原纖維過度增生，膠原容積百分比(CVF%) 顯著增加(P<0.01)；與MI組比較，ME1和ME2組膠原減少，CVF%顯著降低 (P<0.01)，且兩組間無顯著差異(圖3)。

心臟血流動力學(xué)參數(shù)測定結(jié)果顯示，與C組比較，MI組心臟LVSP和±dp/dtmax顯著降低(P<0.01)，LVEDP顯著升高(P<0.01)。與MI組比較，ME1和ME2組心臟LVSP和±dp/dtmax均顯著升高(P<0.01)，LVEDP顯著降低(P<0.01)。ME1和ME2組間無顯著差異，表明心梗后心功能顯著降低，中等強度有氧運動和高強度間歇運動均可顯著提升心功能，且無運動強度差異(圖4)。

圖1 本研究心肌M3R表征結(jié)果(×400)柱狀圖Figure 1. Characterization Results of Cardiac M3R(×400)

圖2 本研究心肌MEK1/2、p-ERK1/2/ERK1/2和Bcl-2/Bax表達的Western Blot結(jié)果柱狀圖Figure 2. Western Blot Results of Cardiac MEK1/2、p-ERK1/2/ERK1/2 and Bcl-2/Bax

圖3 本研究心肌組織Masson染色與分析(×400)切片圖Figure 3. Masson Dyeing Results in Myocardium(×400)

圖4 本研究血流動力學(xué)參數(shù)變化柱狀圖Figure 4. The Index of Hemodynamic

3 分析與討論

3.1 心梗后進行運動干預(yù)均可上調(diào)心肌M3R表達，激活其下游MEK1/2-ERK1/2通路

迷走神經(jīng)通過刺激MR調(diào)節(jié)心臟起搏、房室傳導(dǎo)和心肌收縮等功能[9,40]。過去認為M2R是心臟唯一的MR亞型[2]，近年發(fā)現(xiàn)心肌細胞上存在著M3R。小鼠心臟竇房結(jié)、心房肌、心室肌均有M3R mRNA表達，且水平依次降低。M3R在竇房結(jié)表達密度是心室肌的2～3倍[17,20]。作為迷走神經(jīng)遞質(zhì)作用的靶點之一，M3R的心肌保護作用日益受到重視[23]，激活M3R可成為治療心臟疾病新的靶點[36]。研究發(fā)現(xiàn)，激活竇房結(jié)M3R可導(dǎo)致竇性節(jié)律降低以及舒張期去極化減慢，增加動作電位上升速度[15]。激活心房和心室肌M3R可顯著縮短動作電位持續(xù)時間，減慢心率，保護心功能。上調(diào)M3R可通過增加熱休克蛋白-70和環(huán)加氧酶-2，調(diào)節(jié)蛋白激酶C-ε和β-鏈蛋白的活化與表達[24]，同時抑制miRNA-1及miRNA-376b-5p的表達[29]，發(fā)揮抗心律失常和改善缺血性心肌損傷的作用[24]。研究表明，心梗后的梗死區(qū)及梗死周邊區(qū)均發(fā)生膽堿能失支配現(xiàn)象，且膽堿能神經(jīng)纖維密度從6 h、12 h到48 h逐漸下降。而適宜運動可上調(diào)心血管疾病引起的心臟副交感神經(jīng)活性降低，改善副交感神經(jīng)重構(gòu)[31,37]，并增加心肌M2R的表達，從而保護心功能。但目前關(guān)于運動訓(xùn)練與心肌M3R表征研究報道少見。本研究發(fā)現(xiàn)，MI組、ME1組和ME2組均可見心肌M3R陽性表達。且與MI組比較，ME1和ME2組心肌M3R表達顯著增加。表明，中強度持續(xù)有氧運動和高強度間歇運動兩種干預(yù)方式，均可上調(diào)心梗心臟的M3R表達，并與運動對心功能的改善關(guān)系密切。

關(guān)于激活M3R保護心臟的機制研究日益增多[24]。上調(diào)心臟M3R抑制心肌缺血和肥厚及心律失常等有益作用，由抗細胞凋亡(增加抗凋亡因子Bcl-2和ERKs表達，降低凋亡因子Fas和P38表達)、緩解細胞內(nèi)鈣超載(調(diào)節(jié)胞內(nèi)Ca2+通道，抑制胞內(nèi)Ca2+超載)、降低氧化應(yīng)激水平(增加SOD表達，減少MDA含量)、糾正離子通道失衡等多條信號通路實現(xiàn)[25,36]。而本研究重點探討M3R及其下游MEK1/2-ERK1/2通路與心臟的保護作用。研究發(fā)現(xiàn)，在唾液腺細胞HSY和人類K652細胞中，活化MR可激活ERK1/2通路[4,22]。在原代新生大鼠心肌細胞中，膽堿不能活化ERK1/2，但在氧化應(yīng)激狀態(tài)下則可明顯活化[40]。通過心肌M3R激活MEK1/2-ERK1/2，可促進細胞生存，提高細胞活力[12]。且有研究表明，M3R激活所發(fā)揮的抑制細胞凋亡效應(yīng)與上調(diào)MEK1/2-ERK1/2通路關(guān)系密切[26,41]。本研究發(fā)現(xiàn)，心梗后運動可上調(diào)心臟M3R并激活其下游信號分子MEK1/2表達。表明有氧運動發(fā)揮心梗心臟保護效應(yīng)與心肌M3R-MEK1/2-ERK1/2通路激活關(guān)系密切。

3.2 心梗后的不同強度運動干預(yù)均可抑制心肌細胞凋亡、減輕心肌纖維化程度、改善心功能

目前，臨床上把運動作為治療及預(yù)防心梗病人的重要干預(yù)措施[11]。心梗后進行中強度持續(xù)有氧運動或高強度間歇運動對提高患者心功能和再重構(gòu)心室均具有積極意義[5,10,27]。運動作為心血管疾病干預(yù)手段，其有益作用備受學(xué)者關(guān)注，但相關(guān)機制研究仍顯不足。運動可通過抑制心肌細胞凋亡[30]，促進心肌細胞增殖和血管再生[44]，改善神經(jīng)體液調(diào)節(jié)[33]，促進心臟發(fā)生生理學(xué)重塑[8]，從而對心臟產(chǎn)生保護作用。目前研究認為，M3R無論在正常生理狀態(tài)或心肌缺血及心律失常以及心衰等病理狀態(tài)下，均可通過參與心率和收縮變力調(diào)節(jié)，心肌復(fù)極化過程、心房顫動的產(chǎn)生和恢復(fù)等[34]，對缺血心臟發(fā)揮重要保護作用[14]。 激活M3R可上調(diào)心肌環(huán)氧化酶-2表達，抑制缺血誘導(dǎo)的Cx43的脫磷酸化，縮小心肌梗死面積，保護心功能[43]。本研究發(fā)現(xiàn)，M3R表征與持續(xù)和間歇運動方式、運動強度無關(guān)。MEK1/2-ERK1/2是細胞內(nèi)重要的信號通路，對促進細胞生長、生存，抑制細胞凋亡等均發(fā)揮關(guān)鍵作用[23,26]。在病理生理條件導(dǎo)致的細胞凋亡中，G-蛋白偶聯(lián)受體可激活心肌細胞MEK1/2-ERK1/2通路發(fā)揮抗凋亡作用[14,23,42]。本研究結(jié)果發(fā)現(xiàn)，兩種運動方式干預(yù)均可增加心梗心肌Bcl-2/Bax比值，抑制膠原過度增生，改善心梗大鼠心功能；同時發(fā)現(xiàn)，運動可激活心梗心臟的M3R-MEK1/2-ERK1/2通路。上述結(jié)果與持續(xù)和間歇運動方式及運動強度無顯著差異。中等強度有氧運動較高強度間歇運動對心梗心臟更安全。提示，采取中等強度有氧運動方式保護心梗心臟具有重要意義。

4 結(jié)論

持續(xù)有氧運動和高強度間歇運動均可上調(diào)心肌M3R-MEK1/2- ERK1/2通路，抑制心梗后心肌細胞凋亡，改善心肌纖維化程度，保護心梗大鼠心功能。心梗心臟的M3R-MEK1/2- ERK1/2-細胞凋亡途徑與持續(xù)和間歇運動方式及運動強度關(guān)系不密切。

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Cardioprotective Effect and Its Mechanism of Exercise Training Up-Regulating the Expression of M3Receptor on Myocardial Infarction in Rats

TIAN Zhen-jun1,ZHANG Ke1,CHEN Ting1,2

objective:Cardioprotective effect and mechanism of exercise training up-regulating the expression of M3receptor on myocardial infarction.Methods:48 male Sprague-Dawley rats were randomly assigned to four groups (n=12,per group):control group (C),myocardial infarction group (MI),moderate-intensity aerobic exercise with myocardial infarction group (ME1) and high-intensity aerobic interval exercise with myocardial infarction group (ME2),Rats in C group are breed normally.MI was induced by ligation of the left anterior descending (LAD) coronary artery in MI group。Rats in ME1 and ME2 group take treadmill exercise for 8wk after 1wk post-operation.ME1 group running began at the speed of 10m/min for 5min,then accelerate from 3m/min to 16m/min.ME2 group running began at the speed of 10m/min for 10min,then the speed increases to 25 m/min,after 7min,slow down at the speed of 15m/min for 3min.Take the process alternatively.The total exercise time of ME1 and ME2 are both 60min,5d/1wk×8wk.LVSP,LVEDP,±dp/dtmaxand the cardiac function changes are measured after training.Myocardial collagen fibers were observed by histological section and Masson staining.The expression of myocardial M3R was observed and analyzed by immunoflourecence.The myocardial protein content of M3R,MEK1/2,P-ERK1/2,ERK1/2and apoptosis related Bcl-2 and Bax were assayed by Western Blot.Results:MI increased CVF and LVEDP (P<0.01),but decreased LVSP and -dp/dtmax(P<0.01).After MI myocardial M3positive staining,after MI M3protein expression significantly higher (P<0.01),MEK1/2,P-ERK1/2/ERK1/2protein expression were significantly increased (P<0.01,P<0.01),after the MI the Bcl-2/Bax expression significantly reduced (P<0.01).ME1 and ME2 group CVF%,LVEDP significantly reduced (P<0.01),ME1 -dp/dtmaxsignificantly increased (P<0.01),ME2 LVSP increased significantly (P<0.01).ME1 and ME2 groups were identified myocardial M3,ME1 and ME2 M3protein expression significantly increased (P<0.01,P<0.01).ME1 and ME2 group Bcl-2/Bax expression significantly reduced in the MI group (P<0.01,P<0.01).ME1 and ME2 index had no significant difference.Conclusions:Moderate-intensity aerobic exercise and high-intensity aerobic interval exercise can upregulate the M3R-MEK1/2-ERK1/2signaling pathway,thus inhibit the apoptosis of myocardial cells,reduce myocardial interstitial fibrosis and promote cardiac function after MI.

M3receptor(M3R);MEK1/2-ERK1/2signalingpathways;cardiomyocyteapoptosis;aerobicexercise;high-intensityintervalexercise;myocardialinfarction

1000-677X(2015)05-0055-07

10.16469/j.css.201505007

2014-07-08；

2015-03-16

國家自然科學(xué)基金資助項目(31171141)，陜西師范大學(xué)“211工程”重點建設(shè)學(xué)科——運動生物學(xué)學(xué)科建設(shè)項目(2014-12)。

田振軍(1965-)，男，陜西綏德人，教授，博士研究生導(dǎo)師，主要研究方向為運動心血管生物學(xué)，E-mail：tianzj611@hotmail.com；張可(1989-)，女，陜西西安人，在讀碩士研究生，主要研究方向為運動心血管生物學(xué)；陳婷(1984-)，女，陜西西安人，講師，在讀博士研究生，主要研究方向為運動心血管生物學(xué)。

1.陜西師范大學(xué) 體育學(xué)院 暨運動生物學(xué)研究所 運動與心血管研究室，陜西 西安 710062；2.西藏民族學(xué)院 體育學(xué)院，陜西 咸陽 712082 1.Shaanxi Normal University，Xi’an 710062，China；2.Tibet University for Nationalities，Xianyang 712082，China.

G 804.2

A