Ang Ⅱ誘導(dǎo)大鼠心肌細(xì)胞肥大過(guò)程中分泌型卷曲相關(guān)蛋白5表達(dá)上調(diào)

金 鑫，郭炳彥，李擁軍

(河北醫(yī)科大學(xué)第二醫(yī)院 心內(nèi)科， 河北 石家莊 050000)

研究論文

Ang Ⅱ誘導(dǎo)大鼠心肌細(xì)胞肥大過(guò)程中分泌型卷曲相關(guān)蛋白5表達(dá)上調(diào)

金 鑫，郭炳彥，李擁軍*

(河北醫(yī)科大學(xué)第二醫(yī)院 心內(nèi)科， 河北 石家莊 050000)

目的探討分泌型卷曲相關(guān)蛋白5(sFRP5)在大鼠心肌細(xì)胞肥大中表達(dá)的機(jī)制。方法體外培養(yǎng)乳鼠心肌細(xì)胞，用血管緊張素Ⅱ(AngⅡ 10-6mmol/L,48 h)誘導(dǎo)心肌細(xì)胞肥大。應(yīng)用替米沙坦和Y27632分別阻斷血管緊張素1型受體(AT1R)及Rho/Rho激酶(Rho/ROCK)信號(hào)通路；PD98059、SB203580及SP600125分別阻斷p38 MAPK、ERK1/2及JNK通路； RT-PCR檢測(cè)sFRP5的表達(dá)；Western blot檢測(cè)sFRP5、ROCK1、ROCK2、總MYPT1、p-MYPT1表達(dá)。結(jié)果Ang Ⅱ誘導(dǎo)的心肌細(xì)胞肥大致sFRP5表達(dá)上調(diào)(P<0.05)，Y27632下調(diào)sFRP5的表達(dá)(P<0.05)；替米沙坦下調(diào)ROCK1的表達(dá)(P<0.05)；SP600125明顯降低sFRP5表達(dá)的增加(P<0.05)。結(jié)論在Ang Ⅱ誘導(dǎo)的心肌細(xì)胞肥大過(guò)程中，主要通過(guò)Rho/ROCK1/JNK通路上調(diào)sFRP5的表達(dá)。

分泌型卷曲相關(guān)蛋白 5；血管緊張素Ⅱ；心肌細(xì)胞；肥大；JNK通路

心肌細(xì)胞肥大是心臟重構(gòu)的重要環(huán)節(jié)，心肌細(xì)胞肥大初期有一定的代償意義，后期則嚴(yán)重影響心肌功能，嚴(yán)重可致心力衰竭。 因此，逆轉(zhuǎn)心肌細(xì)胞肥大是心血管研究領(lǐng)域的重要課題之一。分泌型卷曲相關(guān)蛋白5 (secreted frizzled-related protein 5,sFRP5)屬于分泌型卷曲相關(guān)蛋白家族中的一員，是一種新發(fā)現(xiàn)的脂肪細(xì)胞因子和抗炎因子[1]，已證實(shí)它可以在心肌細(xì)胞及心肌細(xì)胞肥大中表達(dá),且主要由AngⅡ 1型受體(angiotensin type 1 receptor,AT1R)介導(dǎo)[2]。

Rho蛋白是一種小分子的G蛋白，Rho激酶又稱為Rho相關(guān)螺旋卷曲蛋白激酶(rho-associated kinase, ROCK)，屬于絲氨酸/蘇氨酸蛋白激酶家族成員，是Rho蛋白下游最主要的效應(yīng)物。目前，大量研究表明Rho/ROCK信號(hào)通路廣泛參與心肌細(xì)胞收縮、遷移、增殖、凋亡、心肌肥厚和心肌梗死后心室重塑等過(guò)程[3- 4]。

絲裂原活化蛋白激酶(mitogen activated protine kinases, MAPKs)信號(hào)通路是細(xì)胞內(nèi)信號(hào)傳導(dǎo)的主要途徑之一，在心肌肥厚的生理及病理過(guò)程中起著重要作用[5]。

本研究觀察在Ang Ⅱ誘導(dǎo)心肌細(xì)胞肥大時(shí)，sFRP5表達(dá)變化的機(jī)制。

1 材料與方法

1.1 材料

出生2～3 d的SD大鼠乳鼠[SPF級(jí)，河北醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心，SCXK(冀)2003- 1- 003]。DMEM、雙抗、D-Hanks、Ⅱ型膠原酶干粉、胰蛋白酶干粉及胎牛血清(Sigma公司)；Ang Ⅱ、 sFRP5抗體、替米沙坦(temisartan, Telm)、Y27632、SP600125、 PD98059、SB203580及二抗IRDye700DX(Gibco公司)；MTT、DMSO、Trizol、PCR擴(kuò)增劑和RT-PCR檢測(cè)試劑盒(Promega公司)；MYPT1、p-MYPT1、JNK、p-JNK、 p38 MAPK、p-p38 MAPK、ERK1/2、p-ERK1/2、ROCK1及ROCK2兔抗鼠抗體和5-BrdU(Santa Cruz公司)。

1.2 方法

1.2.1 原代心肌細(xì)胞分離、培養(yǎng)及藥物干預(yù)：分離和培養(yǎng)心肌細(xì)胞，AngⅡ(10-6mmol/L)干預(yù)心肌細(xì)胞48 h，誘導(dǎo)心肌細(xì)胞肥大；替米沙坦(10 μmol/L)，Y27632(10 μmol/L)，SP600125(10 μmol/L), PD98059(10 μmol/L)及SB203580(10 μmol/L)分別于AngⅡ之前30 min預(yù)干預(yù)。

1.2.2 RT-PCR檢測(cè)sFRP5 mRNA的表達(dá)：Trizol裂解細(xì)胞，提取心肌細(xì)胞總RNA，反轉(zhuǎn)錄成cDNA后，取反轉(zhuǎn)錄產(chǎn)物行多聚酶鏈(PCR)反應(yīng)。sFRP5上游引物 5′-ACTTTGAATGCCGTGAA-3′，下游引物5′-AATCCAGTTGGTGAGCC-3′，擴(kuò)增片段長(zhǎng)度為113 bp；GAPDH上游引物5′-GAGGCTCTCTTCCA GCCTTC-3′,下游引物5′-AGGGTGTAAAAGCAGCT CA-3′。擴(kuò)增條件：94 ℃變性5 min，94 ℃變性30 s，55 ℃退火30 s，72 ℃延伸30 s，30個(gè)循環(huán),72 ℃延伸7 min，得到的多聚酶聯(lián)反應(yīng)產(chǎn)物2%的瓊脂糖電泳并用成像系統(tǒng)掃描攝片。

1.2.3 Western blot檢測(cè)sFRP5、MYPT1、p-MYPT1、ROCK1及ROCK2蛋白表達(dá):收集細(xì)胞，提取細(xì)胞總蛋白。取樣品蛋白50 μg，SDS-PAGE分離，轉(zhuǎn)膜，5% BSA封閉2 h后，用含5%脫脂奶粉的TBST(Tris-HCl，pH=7.4；NaCl，Tween- 20)洗膜，依次加入sFRP5(1∶500)、MYPT1(1∶500)、p-MYPT1(1∶500)、ROCK1(1∶500)及ROCK2抗體(1∶500)，4 ℃孵育過(guò)夜，洗膜，二抗(1∶2 000)孵育2 h，ECL發(fā)光液孵育2 min，使用Bio-Rad圖像分析系統(tǒng)進(jìn)行半定量分析，GAPDH作為內(nèi)參照。

1.3 統(tǒng)計(jì)學(xué)分析

2 結(jié)果

2.1 Rho/ROCK通路抑制劑Y27632對(duì)心肌細(xì)胞sFRP5表達(dá)的影響

Ang Ⅱ干預(yù)心肌細(xì)胞48 h后，sFRP5 mRNA及蛋白表達(dá)均增加，Ang Ⅱ + Y27632組心肌細(xì)胞sFRP5 mRNA及蛋白水平明顯回降(P<0.05)(圖1)。

2.2 AngⅡ 1型受體(AT1R)對(duì)Rho/ROCK通路的影響

2.2.1 AngⅡ 1型受體(AT1R)與Rho/ROCK通路的關(guān)系:與對(duì)照組相比，Ang Ⅱ組p-MYPT1/MYPT1比值明顯升高(P<0.05)，與Ang Ⅱ組相比，Ang Ⅱ+替米沙坦(Telm)干預(yù)組p-MYPT1/MYPT1比值明顯降低(P<0.05)(圖2)。

A.sFRP5 protein expression; B.sFRP5 mRNA expression;*P<0.05 compared with control;#P<0.05 compared with Ang Ⅱ group

圖1RhoA/ROCK通路抑制劑Y27632對(duì)大鼠心肌肥大細(xì)胞中sFRP5表達(dá)的影響

p-MYPT1, MYPT1 protein expression and p-MYPT1/MYPT1 ratio; *P<0.05 compared with control; #P<0.05 compared with Ang Ⅱ group圖2 AngⅡ 1型受體(AT1R)與Rho/ROCK通路的關(guān)系Fig 2 Relationship between Ang Ⅱ type 1 receptor (AT1R) and Rho/ROCK pathway in rat cardio- myocyte hypertrophy(±s, n=3)

2.2.2 替米沙坦對(duì)ROCK亞基的影響:替米沙坦預(yù)作用心肌細(xì)胞30 min，后用Ang Ⅱ刺激48 h。對(duì)照組相比，Ang Ⅱ刺激心肌細(xì)胞后ROCK1及ROCK2表達(dá)均明顯增加(P<0.05)；但與Ang Ⅱ組相比，Ang Ⅱ +替米沙坦組心肌細(xì)胞ROCK1表達(dá)明顯下調(diào)(P<0.05)，而ROCK2表達(dá)無(wú)明顯變化(圖3)。

2.3 MAPK信號(hào)通路對(duì)心肌細(xì)胞肥大中sFRP5表達(dá)的影響

2.3.1 抑制ERK通路后，sFRP5的表達(dá):用PD98059預(yù)作用心肌細(xì)胞30 min，后用Ang Ⅱ刺激48 h。與對(duì)照組相比，PD98059組sFRP5水平無(wú)明顯變化，與Ang Ⅱ組相比，Ang Ⅱ+PD98059組sFRP5水平無(wú)明顯變化(圖4)。

2.3.2 抑制p38 MAPK通路后，sFRP5的表達(dá):用SB203580預(yù)作用心肌細(xì)胞30 min，后用Ang Ⅱ刺激48 h。與對(duì)照組相比，SB203580組sFRP5水平無(wú)明顯變化，與Ang Ⅱ組相比，Ang Ⅱ+SB203580組sFRP5水平無(wú)明顯變化(圖5)。

2.3.3 抑制JNK 通路后，sFRP5的表達(dá):用SP600125預(yù)作用心肌細(xì)胞30 min，后用Ang Ⅱ刺激48 h。與對(duì)照組相比，SP600125組sFRP5水平無(wú)明顯變化，但與Ang Ⅱ組相比，Ang Ⅱ+SP600125組sFRP5水平明顯降低(P<0.05)(圖6)。

3 討論

Ang Ⅱ通常作為心肌細(xì)胞肥大的誘導(dǎo)劑，主要通過(guò)激活A(yù)ng Ⅱ 1型受體發(fā)揮作用。

A.ROCK1 protein expression; B.ROCK2 protein expression;*P<0.05 compared with control;#P<0.05 compared with Ang Ⅱ group

A.sFRP5 protein expression; B.sFRP5 mRNA expression; *P<0.05 compared with control圖4 ERK通路對(duì)sFRP5表達(dá)的影響Fig 4 Effect of ERK pathway on sFRP5 expression in rat cardiomyocyte hypertrophy(±s, n=3)

Rho/ROCK信號(hào)通路位于多種信號(hào)通路的上游，有“分子開(kāi)關(guān)”的作用[6]，研究證實(shí)Rho/ROCK 通路與血管緊張素Ⅱ 1 型受體(AT1R)的信號(hào)傳導(dǎo)有關(guān)[7]。但是Rho/ROCK通路能否在sFRP5表達(dá)增加的過(guò)程中發(fā)揮作用，以及此過(guò)程中AT1 R與Rho/ROCK 通路的上下游關(guān)系，尚未見(jiàn)報(bào)道。

本研究用特異性阻斷劑Y27632阻斷Rho/ROCK通路后， sFRP5水平明顯下降。用AT1R阻斷劑替米沙坦作用于心肌細(xì)胞后，p-MYPT1明顯下降，而總MYPT1無(wú)明顯變化，p-MYPT1/總MYPT1水平明顯降低，提示Rho/ROCK通路可以被替米沙坦特異性阻斷，Ang Ⅱ-AT1R是Rho/ROCK的上游通路。這與之前的研究結(jié)論一致。

絲裂原活化蛋白激酶(mitogen activated protine kinase, MAPKs)有4種亞型。其中，ERK與p38 MAPK信號(hào)通路已被證實(shí)與心肌肥厚的應(yīng)答相關(guān)，而JNK通路的作用則不確定[8- 9]。利用腺病毒介導(dǎo)的基因轉(zhuǎn)染技術(shù)將SEK- 1 (SAPK kinase- 1,抑制JNK激活) 導(dǎo)入心肌細(xì)胞阻斷了壓力超負(fù)荷引起的JNK激活, 減輕了心肌肥厚, 減少了標(biāo)志基因ANF的表達(dá)[10]。而另有報(bào)道 JNK通路可以在抑制心肌肥厚中發(fā)揮作用[11- 12]。JNK究竟在心肌細(xì)胞肥厚及凋亡過(guò)程中發(fā)揮怎樣的作用，尚需進(jìn)一步研究。

A.sFRP5 protein expression; B.sFRP5 mRNA expression; *P<0.05 compared with control圖5 p38 MAPK通路對(duì)sFRP5表達(dá)的影響Fig 5 Effect of p38 MAPK pathway on sFRP5 expression in rat cardiomyocyte hypertrophy(±s, n=3)

A.sFRP5 protein expression; B.sFRP5 mRNA expression;*P<0.05 compared with control;#P<0.05 compared with Ang Ⅱ group

本研究應(yīng)用SB203580, PD98059及SP600125分別阻斷p38 MAPK、ERK1/2及JNK通路后，SP600125明顯阻斷了Ang Ⅱ誘導(dǎo)的心肌細(xì)胞肥大過(guò)程中sFRP5表達(dá)的增加，SB203580和PD98059對(duì)sFRP5表達(dá)增加無(wú)明顯作用，提示JNK在心肌細(xì)胞肥大sFRP5表達(dá)增加過(guò)程中起主要作用。

綜上所述,本研究表明，在Ang Ⅱ誘導(dǎo)的心肌細(xì)胞肥大過(guò)程中，分泌型卷曲相關(guān)蛋白5表達(dá)的增加可能由AT1R/Rho/ROCK1/JNK信號(hào)通路介導(dǎo)。

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Ang Ⅱ induces up-expression of secreted frizzled-related protein 5 in cardiomyocyte hypertrophy of rats

JIN Xin, GUO Bing-yan, LI Yong-jun*

(Dept. of Cardiology, the Second Hospital of Hebei Medical University，Shijiazhuang 050000, China)

ObjectiveTo investigate the mechanism of secreted frizzled-related Protein 5 (sFRP5) expression in cardiomyocyte hypertrophy.MethodsInvivoexperiment, neonatal rat ventricular myocytes were exposed to Ang Ⅱ(10-6mmol/L, 48 h). Telmisartan, Y27632, PD98059，SB203580 and SP600125 were used to block angiotensin type 1 receptor(AT1R), Rho/ROCK, p38 MAPK, ERK1/2 and JNK pathway, respectively. Western blot was applied to determine the expressions of sFRP5, ROCK1, ROCK2, total MYPT1, p-MYPT1. RT-PCR was used to determine sFRP5 expression.ResultsThere was significant inhibition of sFRP5 expression when treated with Y37632 and SP699125, but less with SB203580 and PD98059 in Ang Ⅱ-induced cardiomyocytes. Moreover, telmisartan down-regulated the expression of ROCK1, but no effect on the expression of ROCK2.ConclusionsThe expression of sFRP5 is up-regulated mainly by Rho/ROCK1/JNK pathway in cardiomyocyte hypertrophy induced by Ang Ⅱ.

secreted frizzled-related protein 5; angiotensin Ⅱ; cardiomyocyte; hypertrophy; JNK pathway

2016- 11- 17

2017- 03- 24

國(guó)家自然科學(xué)基金(81570345)；國(guó)家自然科學(xué)基金青年科學(xué)基金(81400217)

*通信作者(correspondingauthor)：lyjbs2009@yeah.net

1001-6325(2018)01-0020-06

R542.2

A