金 鑫,郭炳彦,李拥军
(河北医科大学第二医院 心内科, 河北 石家庄 050000)
研究论文
Ang Ⅱ诱导大鼠心肌细胞肥大过程中分泌型卷曲相关蛋白5表达上调
金 鑫,郭炳彦,李拥军*
(河北医科大学第二医院 心内科, 河北 石家庄 050000)
目的探讨分泌型卷曲相关蛋白5(sFRP5)在大鼠心肌细胞肥大中表达的机制。方法体外培养乳鼠心肌细胞,用血管紧张素Ⅱ(AngⅡ 10-6mmol/L,48 h)诱导心肌细胞肥大。应用替米沙坦和Y27632分别阻断血管紧张素1型受体(AT1R)及Rho/Rho激酶(Rho/ROCK)信号通路;PD98059、SB203580及SP600125分别阻断p38 MAPK、ERK1/2及JNK通路; RT-PCR检测sFRP5的表达;Western blot检测sFRP5、ROCK1、ROCK2、总MYPT1、p-MYPT1表达。结果Ang Ⅱ诱导的心肌细胞肥大致sFRP5表达上调(P<0.05),Y27632下调sFRP5的表达(P<0.05);替米沙坦下调ROCK1的表达(P<0.05);SP600125明显降低sFRP5表达的增加(P<0.05)。结论在Ang Ⅱ诱导的心肌细胞肥大过程中,主要通过Rho/ROCK1/JNK通路上调sFRP5的表达。
分泌型卷曲相关蛋白 5;血管紧张素Ⅱ;心肌细胞;肥大;JNK通路
心肌细胞肥大是心脏重构的重要环节,心肌细胞肥大初期有一定的代偿意义,后期则严重影响心肌功能,严重可致心力衰竭。 因此,逆转心肌细胞肥大是心血管研究领域的重要课题之一。分泌型卷曲相关蛋白5 (secreted frizzled-related protein 5,sFRP5)属于分泌型卷曲相关蛋白家族中的一员,是一种新发现的脂肪细胞因子和抗炎因子[1],已证实它可以在心肌细胞及心肌细胞肥大中表达,且主要由AngⅡ 1型受体(angiotensin type 1 receptor,AT1R)介导[2]。
Rho蛋白是一种小分子的G蛋白,Rho激酶又称为Rho相关螺旋卷曲蛋白激酶(rho-associated kinase, ROCK),属于丝氨酸/苏氨酸蛋白激酶家族成员,是Rho蛋白下游最主要的效应物。目前,大量研究表明Rho/ROCK信号通路广泛参与心肌细胞收缩、迁移、增殖、凋亡、心肌肥厚和心肌梗死后心室重塑等过程[3- 4]。
丝裂原活化蛋白激酶(mitogen activated protine kinases, MAPKs)信号通路是细胞内信号传导的主要途径之一,在心肌肥厚的生理及病理过程中起着重要作用[5]。
本研究观察在Ang Ⅱ诱导心肌细胞肥大时,sFRP5表达变化的机制。
出生2~3 d的SD大鼠乳鼠[SPF级,河北医科大学实验动物中心,SCXK(冀)2003- 1- 003]。DMEM、双抗、D-Hanks、Ⅱ型胶原酶干粉、胰蛋白酶干粉及胎牛血清(Sigma公司);Ang Ⅱ、 sFRP5抗体、替米沙坦(temisartan, Telm)、Y27632、SP600125、 PD98059、SB203580及二抗IRDye700DX(Gibco公司);MTT、DMSO、Trizol、PCR扩增剂和RT-PCR检测试剂盒(Promega公司);MYPT1、p-MYPT1、JNK、p-JNK、 p38 MAPK、p-p38 MAPK、ERK1/2、p-ERK1/2、ROCK1及ROCK2兔抗鼠抗体和5-BrdU(Santa Cruz公司)。
1.2.1 原代心肌细胞分离、培养及药物干预:分离和培养心肌细胞,AngⅡ(10-6mmol/L)干预心肌细胞48 h,诱导心肌细胞肥大;替米沙坦(10 μmol/L),Y27632(10 μmol/L),SP600125(10 μmol/L), PD98059(10 μmol/L)及SB203580(10 μmol/L)分别于AngⅡ之前30 min预干预。
1.2.2 RT-PCR检测sFRP5 mRNA的表达:Trizol裂解细胞,提取心肌细胞总RNA,反转录成cDNA后,取反转录产物行多聚酶链(PCR)反应。sFRP5上游引物 5′-ACTTTGAATGCCGTGAA-3′,下游引物5′-AATCCAGTTGGTGAGCC-3′,扩增片段长度为113 bp;GAPDH上游引物5′-GAGGCTCTCTTCCA GCCTTC-3′,下游引物5′-AGGGTGTAAAAGCAGCT CA-3′。扩增条件:94 ℃变性5 min,94 ℃变性30 s,55 ℃退火30 s,72 ℃延伸30 s,30个循环,72 ℃延伸7 min,得到的多聚酶联反应产物2%的琼脂糖电泳并用成像系统扫描摄片。
1.2.3 Western blot检测sFRP5、MYPT1、p-MYPT1、ROCK1及ROCK2蛋白表达:收集细胞,提取细胞总蛋白。取样品蛋白50 μg,SDS-PAGE分离,转膜,5% BSA封闭2 h后,用含5%脱脂奶粉的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抗体(1∶500),4 ℃孵育过夜,洗膜,二抗(1∶2 000)孵育2 h,ECL发光液孵育2 min,使用Bio-Rad图像分析系统进行半定量分析,GAPDH作为内参照。
Ang Ⅱ干预心肌细胞48 h后,sFRP5 mRNA及蛋白表达均增加,Ang Ⅱ + Y27632组心肌细胞sFRP5 mRNA及蛋白水平明显回降(P<0.05)(图1)。
2.2.1 AngⅡ 1型受体(AT1R)与Rho/ROCK通路的关系:与对照组相比,Ang Ⅱ组p-MYPT1/MYPT1比值明显升高(P<0.05),与Ang Ⅱ组相比,Ang Ⅱ+替米沙坦(Telm)干预组p-MYPT1/MYPT1比值明显降低(P<0.05)(图2)。
A.sFRP5 protein expression; B.sFRP5 mRNA expression;*P<0.05 compared with control;#P<0.05 compared with Ang Ⅱ group
图1RhoA/ROCK通路抑制剂Y27632对大鼠心肌肥大细胞中sFRP5表达的影响
p-MYPT1, MYPT1 protein expression and p-MYPT1/MYPT1 ratio; *P<0.05 compared with control; #P<0.05 compared with Ang Ⅱ group图2 AngⅡ 1型受体(AT1R)与Rho/ROCK通路的关系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 替米沙坦对ROCK亚基的影响:替米沙坦预作用心肌细胞30 min,后用Ang Ⅱ刺激48 h。对照组相比,Ang Ⅱ刺激心肌细胞后ROCK1及ROCK2表达均明显增加(P<0.05);但与Ang Ⅱ组相比,Ang Ⅱ +替米沙坦组心肌细胞ROCK1表达明显下调(P<0.05),而ROCK2表达无明显变化(图3)。
2.3.1 抑制ERK通路后,sFRP5的表达:用PD98059预作用心肌细胞30 min,后用Ang Ⅱ刺激48 h。与对照组相比,PD98059组sFRP5水平无明显变化,与Ang Ⅱ组相比,Ang Ⅱ+PD98059组sFRP5水平无明显变化(图4)。
2.3.2 抑制p38 MAPK通路后,sFRP5的表达:用SB203580预作用心肌细胞30 min,后用Ang Ⅱ刺激48 h。与对照组相比,SB203580组sFRP5水平无明显变化,与Ang Ⅱ组相比,Ang Ⅱ+SB203580组sFRP5水平无明显变化(图5)。
2.3.3 抑制JNK 通路后,sFRP5的表达:用SP600125预作用心肌细胞30 min,后用Ang Ⅱ刺激48 h。与对照组相比,SP600125组sFRP5水平无明显变化,但与Ang Ⅱ组相比,Ang Ⅱ+SP600125组sFRP5水平明显降低(P<0.05)(图6)。
Ang Ⅱ通常作为心肌细胞肥大的诱导剂,主要通过激活Ang Ⅱ 1型受体发挥作用。
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图4 ERK通路对sFRP5表达的影响Fig 4 Effect of ERK pathway on sFRP5 expression in rat cardiomyocyte hypertrophy(±s, n=3)
Rho/ROCK信号通路位于多种信号通路的上游,有“分子开关”的作用[6],研究证实Rho/ROCK 通路与血管紧张素Ⅱ 1 型受体(AT1R)的信号传导有关[7]。但是Rho/ROCK通路能否在sFRP5表达增加的过程中发挥作用,以及此过程中AT1 R与Rho/ROCK 通路的上下游关系,尚未见报道。
本研究用特异性阻断剂Y27632阻断Rho/ROCK通路后, sFRP5水平明显下降。用AT1R阻断剂替米沙坦作用于心肌细胞后,p-MYPT1明显下降,而总MYPT1无明显变化,p-MYPT1/总MYPT1水平明显降低,提示Rho/ROCK通路可以被替米沙坦特异性阻断,Ang Ⅱ-AT1R是Rho/ROCK的上游通路。这与之前的研究结论一致。
丝裂原活化蛋白激酶(mitogen activated protine kinase, MAPKs)有4种亚型。其中,ERK与p38 MAPK信号通路已被证实与心肌肥厚的应答相关,而JNK通路的作用则不确定[8- 9]。利用腺病毒介导的基因转染技术将SEK- 1 (SAPK kinase- 1,抑制JNK激活) 导入心肌细胞阻断了压力超负荷引起的JNK激活, 减轻了心肌肥厚, 减少了标志基因ANF的表达[10]。而另有报道 JNK通路可以在抑制心肌肥厚中发挥作用[11- 12]。JNK究竟在心肌细胞肥厚及凋亡过程中发挥怎样的作用,尚需进一步研究。
A.sFRP5 protein expression; B.sFRP5 mRNA expression; *P<0.05 compared with control图5 p38 MAPK通路对sFRP5表达的影响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
本研究应用SB203580, PD98059及SP600125分别阻断p38 MAPK、ERK1/2及JNK通路后,SP600125明显阻断了Ang Ⅱ诱导的心肌细胞肥大过程中sFRP5表达的增加,SB203580和PD98059对sFRP5表达增加无明显作用,提示JNK在心肌细胞肥大sFRP5表达增加过程中起主要作用。
综上所述,本研究表明,在Ang Ⅱ诱导的心肌细胞肥大过程中,分泌型卷曲相关蛋白5表达的增加可能由AT1R/Rho/ROCK1/JNK信号通路介导。
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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
国家自然科学基金(81570345);国家自然科学基金青年科学基金(81400217)
*通信作者(correspondingauthor):lyjbs2009@yeah.net
1001-6325(2018)01-0020-06
R542.2
A