左祥榮 徐 健 解衛(wèi)平
1.南京醫(yī)科大學(xué)第一附屬醫(yī)院重癥醫(yī)學(xué)科,江蘇南京 210029;2.南京醫(yī)科大學(xué)第一附屬醫(yī)院呼吸內(nèi)科,江蘇南京 210029
4-苯基丁酸抑制內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)改善大鼠肺動脈高壓右心衰竭的實驗研究
左祥榮1徐 健2解衛(wèi)平2
1.南京醫(yī)科大學(xué)第一附屬醫(yī)院重癥醫(yī)學(xué)科,江蘇南京 210029;2.南京醫(yī)科大學(xué)第一附屬醫(yī)院呼吸內(nèi)科,江蘇南京 210029
目的 探討內(nèi)質(zhì)網(wǎng)應(yīng)激(ERS)分子伴侶4-苯基丁酸(4-PBA)對野百合堿(MCT)所致肺動脈高壓(PAH)大鼠右心衰竭的影響。方法32只SD大鼠分為空白對照組(CON組)、空白對照+4-PBA組(CON+PBA組)、MCT模型組(MCT組),MCT+4-PBA治療組(MCT+PBA組),每組各8只,MCT(60 mg/kg)腹腔注射建立PAH大鼠右心衰竭模型,造模后4-PBA(500 mg/kg)灌胃4周,右心導(dǎo)管測量右心室收縮壓(RVSP)和平均肺動脈壓(mPAP),計算右心肥厚指數(shù)(RVHI),HE染色觀察右心形態(tài)學(xué)變化,ELISA法測定氨基末端B型利鈉肽前體(NT-proBNP)水平,Westen blot檢測右心室葡萄糖調(diào)節(jié)蛋白78(GRP78)及C/EBP環(huán)磷酸腺苷反應(yīng)元件結(jié)合轉(zhuǎn)錄因子同源蛋白(CHOP)的表達。結(jié)果與CON組比較,MCT組大鼠mPAP、RVSP、RVHI、NT-proBNP明顯升高,差異有統(tǒng)計學(xué)意義(P<0.05),心肌細胞明顯肥大,GRP78和CHOP蛋白表達明顯增加,差異有統(tǒng)計學(xué)意義(P<0.05);與MCT組比較,MCT+PBA組大鼠mPAP、RVSP、RVHI、NT-proBNP明顯下降,差異有統(tǒng)計學(xué)意義(P<0.05),心肌細胞肥大明顯改善,GRP78和CHOP蛋白表達下調(diào),差異有統(tǒng)計學(xué)意義(P<0.05)。 結(jié)論 ERS在PAH、右心衰竭發(fā)生過程中發(fā)揮重要作用,而4-PBA可以改善右心衰竭,其機制可能與抑制ERS有關(guān)。
肺動脈高壓;右心衰竭;內(nèi)質(zhì)網(wǎng)應(yīng)激;4-苯基丁酸
肺動脈高壓 (pulmonary arterial hypertension,PAH)是一組由多種原因引起的、進展迅速、預(yù)后極差的疾病,其特點是肺血管阻力進行性增加,導(dǎo)致右心室后負荷增加、右心肥厚和衰竭[1]。PAH的治療取得了相當(dāng)大的進展,但仍無法“治愈”[2],右心肥厚、衰竭是PAH患者預(yù)后的主要決定因素[3]。因此,積極探索PAH右心衰竭的發(fā)病機制和治療措施尤為重要。大量研究已證實內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulumstress,ERS)及其介導(dǎo)的凋亡參與代謝性疾病、神經(jīng)退行性疾病、心血管疾病等多種疾病的發(fā)病[4]。此外,ERS在PAH發(fā)病中也起著重要作用,抑制ERS可降低肺血管阻力、減輕肺血管重構(gòu)和右心肥厚[5]。4-苯基丁酸(4-phenylbutyric acid,4-PBA)是一種重要的ERS相關(guān)化學(xué)分子伴侶,具有調(diào)節(jié)ERS的作用。本研究采用野百合堿(monocrotaline,MCT)建立經(jīng)典的大鼠PAH、右心衰竭模型,從ERS的角度觀察4-PBA對右心衰竭大鼠右心室ERS標(biāo)志蛋白葡萄糖調(diào)節(jié)蛋白78(glucose-regulated protein 78,GRP78)及C/EBP環(huán)磷酸腺苷反應(yīng)元件結(jié)合轉(zhuǎn)錄因子同源蛋白(C/EBP homologous protein,CHOP)的影響,以探討4-PBA能否通過抑制ERS對右心衰竭起保護作用。
1.1 實驗動物
SPF級健康雄性SD大鼠32只,體重(250±10)g,上海西普爾-必凱實驗動物有限責(zé)任公司提供,合格證號:2008001642652。實驗過程中對動物的處置符合南京醫(yī)科大學(xué)實驗動物倫理委員會的規(guī)定。
1.2 實驗藥物和試劑
野百合堿(MCT,Sigma,美國),單克隆鼠抗GRP 78抗體(Abcam,美國),單克隆鼠抗CHOP抗體和ECL發(fā)光液(Cell Signaling Technology公司),二抗(Santa Cruz公司),內(nèi)參為單克隆鼠抗 GAPDH(Bioworld Technology,美國),NT-proBNP ELISA檢測試劑盒(武漢華美生物工程有限公司),PVDF膜(Millipore公司,美國)。
1.3 主要儀器
16通道生理信號記錄分析系統(tǒng) MP100型和TSD104A壓力傳感器(BIOPAC Systems,Inc.,美國),高速低溫離心機5810R型(eppendorf,德國),紫外分光光度計756型(上海光學(xué)儀器廠),Bio-Rad VersaDoc凝膠成像系統(tǒng)(Bio-Rad,美國),奧林巴斯光學(xué)顯微鏡BX53型(Olympus,日本)。
1.4 方法
1.4.1 動物分組與模型制備 將32只大鼠隨機分為4組:空白對照組(CON組)、空白對照+4-PBA治療組(CON+ PBA組)、野百合堿模型組(MCT組),野百合堿+4-PBA治療組(MCT+PBA組),每組8只。實驗第1天MCT組和MCT+PBA組,每只大鼠分別給予MCT腹腔注射(60 mg/kg);CON組和PBA組給予等量NS腹腔注射。PBA組和MCT+PBA組每日給予4-PBA 500 mg/(kg·d)灌胃,CON組和MCT組每日等量NS灌胃。
1.4.2 血流動力學(xué)測定 實驗第28天,各組大鼠稱重后,20%烏拉坦腹腔注射麻醉(1.0 g/kg),參考文獻[8]的方法,經(jīng)右頸外靜脈插入微型導(dǎo)管至右心室、肺動脈,導(dǎo)管另一端相連壓力傳感器,用16通道生理信號記錄分析系統(tǒng),記錄右心室收縮壓(RVSP),平均肺動脈壓(mPAP)。然后采用頸椎脫臼法處死大鼠,迅速開胸取出心臟,分離右心室(RV)和左心室+室間隔(LV+ S),吸干水分稱重,計算RV/(LV+S)以反映右心肥厚指數(shù)(RVHI)。取部分右心室組織甲醛固定,另取部分-80℃下保存。
1.4.3 形態(tài)學(xué)觀察 將甲醛固定后的右心室心肌組織制成常規(guī)石蠟制片,行蘇木精-伊紅(HE)染色,光學(xué)顯微鏡下觀察病理形態(tài)學(xué)變化。
1.4.4 氨基末端B型利鈉肽前體(NT-proBNP)測定
開胸取出心臟前,先從大鼠右心室取血3 mL,4℃下離心3000 r/min,15 min,取上層血清按說明書操作步驟測定NT-proBNP水平。
1.4.5 Western blot檢測 從-80℃冰箱取出各組大鼠右心室心肌組織,每組3只,RAPA裂解,提取總蛋白,BCA法測蛋白濃度,各組取30 μg蛋白樣本,以10%SDS-PAGE變性凝膠電泳分離后,電轉(zhuǎn)移到PVDF膜上,用5%脫脂奶粉37℃封閉1 h后,分別加入GRP78抗體(1∶5000稀釋)、CHOP抗體(1∶1000)及GAPDH抗體 (1∶4000)4℃孵育過夜,TBST洗滌后,再加入相應(yīng)HRP標(biāo)記的二抗 (1∶5000稀釋),37℃孵育60 min,TBST洗滌,ECL顯影,以GAPDH為內(nèi)參,采用Quantity One軟件分析結(jié)果。
1.5 統(tǒng)計學(xué)方法
采用SPSS l5.0統(tǒng)計軟件進行數(shù)據(jù)統(tǒng)計分析,正態(tài)分布計量資料以均數(shù)±標(biāo)準(zhǔn)差()表示,多組間比較采用方差分析,兩兩比較采用LSD-t檢驗。以P<0.05為差異有統(tǒng)計學(xué)意義。
2.1 4-PBA對MCT引起的大鼠mPAP、RVSP、RVHI和NT-proBNP的影響
與CON組比較,MCT注射4周后MCT組大鼠的mPAP、RVSP、RVHI和NT-proBNP顯著升高,差異有統(tǒng)計學(xué)意義(P<0.05)。灌服4-PBA 4周,CON+PBA組與CON組比較,上述指標(biāo)差異無統(tǒng)計學(xué)意義(P>0.05);MCT+PBA組與MCT組比較,上述指標(biāo)明顯降低,差異有統(tǒng)計學(xué)意義(P<0.05)。見表1。
表1 各組大鼠的mPAP、RVSP、RVHI和NT-proBNP的比較(n=8,)
表1 各組大鼠的mPAP、RVSP、RVHI和NT-proBNP的比較(n=8,)
注:與CON組比較,*P<0.05;與MCT組比較,#P<0.05;CON:空白對照;CON+PBA組:空白對照+4-苯基丁酸治療組;MCT組:野百合堿模型組;MCT+PBA組:百合堿+4-苯基丁酸治療組;mPAP:平均肺動脈壓;RVSP右心室收縮壓;RV/(LV+S):右心室/左心室+室間隔;NT-proBNP:氨基末端B型利鈉肽前體;1 mmHg=0.133 kPa
組別 mPAP(mmHg) (mmHg) RV/(LV+S) NT-proBNP(ng/L)RVSP CON組CON+PBA組MCT組MCT+PBA組13.63±1.92 13.00±1.69 37.75±4.68*18.86±2.95#31.13±3.48 30.38±4.66 78.38±9.68*49.0±8.16#0.25±0.02 0.26±0.02 0.55±0.06*0.36±0.04#179.16±38.26 205.75±102.13 797.45±293.57*351.18±125.45#
2.2 4-PBA對MCT引起的右心室形態(tài)學(xué)變化的影響
光鏡下CON組大鼠右心室心肌細胞排列整齊,分布均勻,大小較為均一,而MCT組心肌細胞出現(xiàn)排列紊亂、肌間隙增寬,心肌細胞明顯肥大、纖維增生;MCT+PBA組心肌細胞接近CON組,排列無紊亂、細胞肥大及核大不明顯、纖維增生不明顯。見圖1。
2.3 4-PBA對MCT誘導(dǎo)的大鼠右心室心肌細胞GRP78和CHOP蛋白表達的影響
MCT注射4周后,與CON組比較,MCT組GRP78和CHOP蛋白表達明顯增加,差異有統(tǒng)計學(xué)意義(P<0.05);而給予4-PBA 4周,與CON組比較,CON+PBA組GRP78和CHOP蛋白表達無明顯變化,差異無統(tǒng)計學(xué)意義(P>0.05);與MCT組比較,MCT+PBA組GRP78和CHOP蛋白表達明顯下降,差異有統(tǒng)計學(xué)意義(P<0.05)。見圖2、表2。
圖1 4-苯基丁酸對各組大鼠右心室心肌組織形態(tài)學(xué)變化的影響(HE染色,400×)
圖2 4-苯基丁酸對各組大鼠右心室心肌GRP78和CHOP蛋白表達的影響
表2 各組GRP78和CHOP蛋白相對表達量(n=3,)
表2 各組GRP78和CHOP蛋白相對表達量(n=3,)
注:與CON組比較,*P<0.05;與MCT組比較,#P<0.05;CON組:空白對照組;CON+PBA組:空白對照+4-苯基丁酸治療組;MCT組:野百合堿模型組;野MCT+PBA組:百合堿+4-苯基丁酸治療組;GRP78:葡萄糖調(diào)節(jié)蛋白78;CHOP:C/EBP環(huán)磷酸腺苷反應(yīng)元件結(jié)合轉(zhuǎn)錄因子同源蛋白
組別 GRP78 CHOP CON組CON+PBA組MCT組MCT+PBA組0.477±0.106 0.420±0.106 0.793±0.170*0.487±0.121#0.337±0.074 0.323±0.067 0.613±0.113*0.413±0.042#
由于左右心室在胚胎起源、結(jié)構(gòu)和功能等方面均存在明顯差異[7],常用于治療左心衰竭的方法在右心衰竭中往往效果不佳,提示兩者發(fā)病機制存在明顯差異,右心肥厚、代償和衰竭可能有著獨特機制[7]。大量研究表明ERS在缺血/再灌注、動脈粥樣硬化及心力衰竭等左心疾病的發(fā)病中具有重要作用[4],但ERS是否參與右心肥厚與衰竭的發(fā)生發(fā)展尚不明確。
GRP78主要分布在內(nèi)質(zhì)網(wǎng)腔,能促進未折疊蛋白正確折疊、修飾、保護內(nèi)質(zhì)網(wǎng)功能[8],它能夠敏感地感受到ERS的激活,是ERS標(biāo)志性分子之一[9]。在心力衰竭動物模型和患者的心肌標(biāo)本中均發(fā)現(xiàn)GRP78蛋白表達明顯增加[10-11]。GRP78可以作為降低ERS程度的一個治療目標(biāo)[12]。GRP78表達增加是機體的一種自我保護和修復(fù)機制,但隨著應(yīng)激強度增加或時間延長,導(dǎo)致ERS過度,無法通過GRP78表達增加來代償,則誘導(dǎo)ERS凋亡相關(guān)分子CHOP、caspase-12、JNK等表達增加[13],細胞凋亡。而ERS介導(dǎo)的細胞凋亡可能是心肌細胞代償性肥厚發(fā)展至衰竭的一個重要的惡化因素[13]。研究已證實,CHOP介導(dǎo)的細胞凋亡,促進壓力負荷誘導(dǎo)的心肌肥厚和衰竭[14],而細胞凋亡也參與右心衰竭的發(fā)生發(fā)展[8]。本實驗結(jié)果顯示,CON+PBA組GRP78、CHOP表達不明顯(P>0.05),而MCT注射4周后大鼠右心室GRP78、CHOP表達明顯增加(P<0.05),這就提示ERS參與了右心衰竭的發(fā)生發(fā)展,并且ERS過度誘導(dǎo)CHOP介導(dǎo)的細胞凋亡,促進了右心衰竭。
研究表明,4-PBA能通過減輕ERS,對左心肥厚和衰竭起保護作用[15-17]。在PAH動物模型,4-PBA可以降低肺血管阻力、肺血管重構(gòu)和右心肥大[18-19]。本研究發(fā)現(xiàn)4-PBA明顯降低MCT誘導(dǎo)的PAH大鼠mPAP、RVSP、RVHI和NT-proBNP的升高,并且抑制GRP78,CHOP蛋白表達上調(diào)。其機制可能是4-PBA作為ERS分子伴侶促進內(nèi)質(zhì)網(wǎng)蛋白質(zhì)的正確折疊,減輕ERS的嚴(yán)重程度,進而促使GRP78和CHOP轉(zhuǎn)錄和翻譯減少,抑制細胞凋亡,發(fā)揮心臟保護作用。因此,抑制ERS相關(guān)的細胞凋亡可能是治療心力衰竭的一個新的靶點[20]。
綜上所述,ERS參與PAH、右心衰竭的發(fā)生發(fā)展,4-PBA顯著減輕GRP78的表達,并抑制CHOP介導(dǎo)的細胞凋亡,改善PAH大鼠的右心功能,具有防治PAH右心肥厚與衰竭的潛能,但有關(guān)分子機制尚需進一步深入研究。
[1]Montani D,Chaumais MC,Guignabert C,et al.Targeted therapiesinpulmonary arterialhypertension[J].Pharmacol Ther,2014,141(2):172-191.
[2]Lai YC,Potoka KC,Champion HC,et al.Pulmonary arterial hypertension:the clinical syndrome[J].Circ Res,2014,115(1):115-130.
[3]van Wolferen SA,Marcus JT,Boonstra A,et al.Prognostic value of right ventricular mass,volume,and function in idiopathic pulmonary arterial hypertension[J].Eur Heart J,2007,28(10):1250-1257.
[4]Millott RI,Dudek E,Michalak M.The endoplasmic reticulum in cardiovascular health and disease[J].Can J Physiol Pharmacol,2012,90(9):1209-1217.
[5]徐健,左祥榮,解衛(wèi)平.內(nèi)質(zhì)網(wǎng)應(yīng)激與肺動脈高壓[J].江蘇醫(yī)藥,2015,41(1):74-77.
[6]Zuo XR,Wang Q,Cao Q,et al.Nicorandil prevents right ventricular remodeling by inhibiting apoptosis and lowering pressure overload in rats with pulmonary arterial hypertension[J].PLoS One,2012,7(9):e44485.
[7]Guihaire J,Bogaard HJ,F(xiàn)lécher E,et al.Experimental models of right heart failure:a window for translational research in pulmonary hypertension[J].Semin Respir Crit Care Med,2013,34(5):689-699.
[8]Sozen E,Karademir B,Ozer NK.Basic mechanisms in endoplasmic reticulum stress and relation to cardiovascular diseases[J].Free Radic Biol Med,2015,78:30-41.
[9]Zhao Q,Hu X,Shao L,et al.LipoxinA4 attenuates myocardial ischemia reperfusion injury via a mechanism related to downregulation of GRP-78 and caspase-12 in rats[J].Heart Vessels,2014,29(5):667-678.
[10]ArumugamS,ThandavarayanRA,VeeraveeduPT,etal.Beneficial effects of edaravone,a novel antioxidant,in rats with dilated cardiomyopathy[J].J Cell Mol Med,2012,16(9):2176–2185.
[11]Ortega A,Roselló-Lletí E,Tarazón E,et al.Endoplasmic reticulumstressinducesdifferentmolecularstructuralalterations in human dilated and ischemic cardiomyopathy[J]. PLoS One,2014,9(9):e107635.
[12]Fu HY,Minamino T,Tsukamoto O,et al.Overexpression of endoplasmic reticulum-resident chaperone attenuates cardiomyocyte death induced by proteasome inhibition[J]. Cardiovasc Res,2008,79(4):600–610.
[13]劉秀華.心力衰竭的內(nèi)質(zhì)網(wǎng)應(yīng)激機制[J].中國醫(yī)學(xué)前沿雜志:電子版,2010,2(1):6-12.[14]Fu HY,Okada K,Liao Y,et al.Ablation of C/EBP homologous protein attenuates endoplasmic reticulum-mediated apoptosis and cardiac dysfunction induced by pressure overload[J].Circulation,2010,122(4):361-369.
[15]Ayala P,Montenegro J,Vivar R,et al.Attenuation of endoplasmic reticulum stress using the chemical chaperone 4-phenylbutyric acid prevents cardiac fibrosis induced by isoproteren ol[J].Exp Mol Pathol,2011,92(1):97-104.
[16]Park CS1,Cha H,Kwon EJ,et al.The chemical chaperone 4-phenylbutyric acid attenuates pressure-overload cardiac hypertrophy by alleviating endoplasmic reticulum stress[J].Biochem Biophys Res Commun,2012,421(3):578-584.
[17]Takada A,Miki T,Kuno A,et al.Role of ER stress in ventricular contractile dysfunction in type 2 diabetes[J]. PLoS One,2012,7(6):e39893.
[18]Dromparis P,Paulin R,Stenson TH,et al.Attenuating endoplasmic reticulum stress as a novel therapeutic strategy in pulmonary hypertension[J].Circulation,2013,127(1):115-125.
[19]Koyama M,F(xiàn)uruhashi M,Ishimura S,et al.Reduction of endoplasmic reticulum stress by 4-phenylbutyric acid prevents the development of hypoxia-induced pulmonary arterial hypertension[J].Am J Physiol Heart Circ Physiol,2014,306(9):H1314-1323.
[20]Wang J,Hu X,Jiang H.ER stress-induced apoptosis:anovel therapeutic target in heart failure[J].Int J Cardiol,2014,177(2):564-565.
Study of 4-phenylbutyric acid ameliorating right ventricular failure in pulmonary artery hypertension rats by reducing endoplasmic reticulum stress
ZUO Xiangrong1XU Jian2XIE Weiping2
1.Department of Critical Care Medicine,the First Affiliated Hospital of Nanjing Medical University,Jiangsu Province, Nanjing 210029,China;2.Department of Respiratory Medicine,the First Affiliated Hospital of Nanjing Medical University,Jiangsu Province,Nanjing 210029,China
Objective To investigate the effects of endoplasmic reticulum stress(ERS)molecular chaperone 4-phenyl butyric acid (4-PBA)on right ventricular failure in pulmonary artery hypertension rats induced by monocrotaline(MCT). Methods 32 SD rats were divided into four groups:black control group (CON group),black control+4-PBA group (CON+PBA group),MCT model group(MCT group),MCT+4-PBA treatment group(MCT+PBA group),with eight rats in each group.Right ventricular failure was induced in rats by intraperitoneal injection of MCT(60 mg/kg),after molding, 4-PBA (500 mg/kg)gavage were given for 4 weeks,mean pulmonary arterial pressure (mPAP)and right ventricular systolic pressure (RVSP)were measured by right cardiac catheterization,right ventricular hypertrophy index(RVHI) was calculated as the weight ratio of right ventricle to left ventricle plus septum,the right ventricular histopathological changes were observed by HE,the level of serum N-terminal B type natriuretic peptide (NT-proBNP)was determined by ELISA,and the level of ERS related proteins glucose-regulated protein 78(GRP78)and C/EBP homologous protein (CHOP)protein expressions in right ventricle were determined by Westen blot.Results Compared with CON group,the levels of mPAP,RVSP,RVHI and NT-proBNP were obviously increased in MCT group,the differences were statistically significant(P<0.05),the degree of cardiomyocyte hypertrophy was significantly,and the levelof GRP78 and CHOP protein expression were upregulated significantly,the differences were statistically significant(P<0.05).Compared with MCT group,the level of mPAP,RVSP,RVHI,and NT proBNP in MCT+PBA group obviously decreased,the differences were statistically significant(P<0.05),the degree of cardiomyocyte hypertrophy showed improved,and the GRP78 and CHOP protein expression were lower,the differences were statistically significant (P<0.05).Conclusion ERS plays an important role in the process of right heart failure induced by PAH,and 4-PBA can improve right heart failure,its mechanism may be related to inhibiting ERS.
Right ventricular failure;Pulmonary artery hypertension;Endopiasmic reticuium stress;4-phenylbutyric acid
R332
A
1673-7210(2015)11(c)-0004-05
2015-08-25本文編輯:蘇 暢)
國家自然科學(xué)基金資助項目(81200159);江蘇省“六大人才高峰”項目(2012-WS-028)。
左祥榮(1978-),男,副主任醫(yī)師,副教授,碩士研究生導(dǎo)師;研究方向:重癥醫(yī)學(xué)以及肺動脈高壓。