芩丹胶囊及其有效成分槲皮素逆转自发性高血压大鼠心室肥厚的实验研究
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摘要
研究背景
高血压病是严重危害人类健康的常见心血管疾病之一,心室肥厚是高血压病常见的并发症,是心脏猝死和其他心血管事件的一个独立的危险因素,主要病理表现包括心肌细胞肥大、心肌间质细胞增生、细胞外基质的堆积,探讨高血压心室肥厚的发生机制对防治高血压靶器官损害、降低死亡率具有重要意义。
过氧化物酶增殖物激活受体γ (peroxisome proliferater-activated receptor-y, PPAR-γ)是一类配体激活的核转录因子超家族成员,近年来的研究证实,在逆转高血压心室肥厚的过程中起着关键的作用,在配体的激动下,PPAR-γ能抑制相关肥厚基因的表达,对高血压心室肥厚引起的心力衰竭起着预防和治疗的作用。NF-κB的激活在心肌肥厚的发展过程中至关重要,近年来研究表明NF-κB可能是抑制心肌肥厚的重要作用靶点. PPARy可以直接与NF-κB发生蛋白质-蛋白质之间的相互作用,形成转录抑制复合物,同时降低NF-κB与DNA结合活性,抑制NF-κB DNA合成,起到抑制其表达的作用。
芩丹胶囊(Qindan Capsule, QC)是导师根据多年临床经验研制成的治疗高血压病肝热血瘀证的中药复方制剂,前期研究表明其在改善高血压血管重构中起着重要的作用,本研究通过研究芩丹胶囊对自发性高血压大鼠(spontaneously hypertensive rats, SHR)心肌组织的病理学及超微结构的影响,应用免疫组织化学法(immunohistochemistry)检测PPAR-γ和NF-κB的蛋白表达的改变,以及荧光实时定量PCR (real-time PCR)检测对下游肥厚基因ANP、BNP的mRNA表达的影响,探讨芩丹胶囊对改善SHR高血压心室肥厚的作用机制,为芩丹胶囊治疗高血压心室肥厚的临床应用提供有效的实验依据。
目的
1.观察芩丹胶囊对SHR大鼠心肌形态学及超微结构的改变及对大鼠血压的影响,探讨芩丹胶囊抑制SHR大鼠心肌肥厚的疗效。
2.观察芩丹胶囊在抑制SHR大鼠心肌肥厚对PPAR-γ和NF-κB的蛋白表达以及对下游肥厚基因ANP、BNP的mRNA表达的影响,探讨芩丹胶囊抑制SHR大鼠心肌肥厚的信号调控机制。
方法
1.研究对象：8周龄雄性SHR大鼠32只和8周龄Wistar-Kyoto大鼠(WKY)8只。随机分为5组,每组8只：(1)WKY对照组；(2)SHR对照组；(3)SHR+替米沙坦组(SHR+Tel);(4)SHR+芩丹胶囊大剂量组(SHR+QCH);(5)SHR+芩丹胶囊小剂量组(SHR+QCL)。SHR+Tel组给予Tel10mg/(kg·d), SHR+QCH组给予芩丹胶囊750mg/(kg·d), SHR+QCL组给予芩丹胶囊150mg/(kg·d), WKY对照组和SHR对照组分别给予等量蒸馏水。各组大鼠均分别灌胃给药,每日1次,连续给药12周。末次给药后禁食24h,不禁水。3%戊巴比妥钠(30mg/kg)腹腔注射,麻醉后进行后续实验。
2.研究内容：
(1)每两周测量尾动脉收缩压(SBP),每周称取体质量(BW)一次；(2)处死动物后,称取左心室质量(LVM),并计算左室相对质量指数(LVM/BW);(3)HE染色观察各组大鼠心肌组织形态；(4)电镜观察各组大鼠心肌组织超微结构；(5)免疫组织化学测定心肌组织中PPAR-γ和NF-κB蛋白的表达；(6)实时定量RT-PCR法检测心肌组织中肥厚基因ANP、BNP的mRNA的表达。
3.统计学处理计量资料统一采用均数±标准误表示,统计软件SPSS17.0进行数据的相关统计分析。两个组之间的数据比较用独立样本的t检验,多组之间的均数比较用单因素或多因素方差分析(ANOVA)和Bonferroni post-hoc检验。当P<0.05,认为有统计学差异。
结果
1.各组大鼠治疗前后收缩压的动态变化
与8周龄WKY阴性对照组大鼠比较,8周龄SHR阳性对照组大鼠血压已经明显升高(P<0.05)。在实验过程中,阳性对照组SHR大鼠收缩压呈持续升高状态,与20周龄阴性对照组的WKY组相比,20周龄阳性对照SHR组大鼠收缩压显著增加(P<0.05)。从给药第4周起,SHR+QCH组、SHR+QCL组、SHR+Tel组大鼠的血压与SHR对照组相比均有下降,其差异具有统计学意义(P<0.05),但各组下降程度不同；给药7周到12周期间,SHR+QCH组较SHR+QCL组血压下降更为显著,差异具有统计学意义(P<0.05)。20周龄SHR+Tel组大鼠收缩压与同周龄SHR+QCH组相比差异无统计学意义(P>0.05)。
2.左室相对质量(LVM/BW)
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠与对照组WKY大鼠相比LVM/BW明显升高(P<0.05)。与20周龄SHR组相比,SHR+QCH组,SHR+QCL组和SHR+Tel组大鼠LVM/BW显著减小(P<0.05)。两两比较分析,SHR+QCH组与SHR+QCL组差异较显著(P<0.05)。
3.各组大鼠心肌组织切片光镜下HE染色观察
20周龄WKY组大鼠心肌细胞排列整齐,细胞核大小均一,胞浆染色分布均匀。SHR组大鼠可见心肌细胞排列紊乱,细胞核大小不规则,胞浆染色加深,细胞内心肌纤维断裂,单位面积内细胞核数量增多的表现。与SHR组大鼠比较,SHR+Tel组和SHR+QCH组、SHR+QCL组大鼠见心肌细胞排列较为整齐,细胞核偶见大小不规则,细胞内心肌纤维断裂情况明显好转,单位面积细胞核数目略增多。
4.各组大鼠心肌组织超微结构的观察
电镜下,WKY心肌纤维排列清晰、整齐,心肌细胞质膜连续、完整,线粒体结构、闰盘结构正常；粗细肌丝排列整齐,肌小节及明暗各带清晰可见,Z线较清晰,间质纤维无增生,细胞核染色质分布较均匀；SHR组大鼠心肌纤维排列稀疏、紊乱,线粒体肿胀、破坏甚至呈空泡状,Z线增宽模糊,可见细胞核染色质边聚的现象；伴有肌原纤维呈灶性溶解,出现肌节对位不齐,肌丝扭曲、断裂,间质可见大量胶原纤维分布。SHR+Tel组,SHR+QCH组,SHR+QCL组较SHR组好转,大部分肌纤维排列紧密,线粒体结构相对完整,且排列适当,细胞核无明显边聚现象,间质内胶原堆积已不明显。
5.心肌组织免疫组织化学PPARγ、NF-κB蛋白表达水平比较
(1)与20周龄阴性对照组WKY组相比,阳性对照SHR组大鼠PPARγ蛋白的表达较WKY组明显减少(P<0.05)；SHR+Tel组,SHR+QCH组,SHR+QCL组较SHR组PPARy核表达明显增多(P<0.05),有显著性差异；SHR+QCH组与SHR+QCL组差异较显著(P<0.05),但和SHR+Tel组比较,无统计学差异(P>0.05)。
(2)与20周龄正常血压对照组WKY大鼠相比,SHR组NF-κB蛋白与WKY组比较,核表达明显增多(P<0.05),SHR+QCH组、SHR+QCL组和SHR+Tel组表达较SHR组均减少(P<0.05),SHR+QCH组与SHR+QCL组差异具有统计学意义(P<0.05),但和SHR+Tel组比较,差异无统计学意义(P>0.05)。
6.实时定量RT-PCR法检测心肌组织中肥厚基因的ANP, BNP mRNA的表达水平的比较
(1)与20周龄正常血压对照组WKY大鼠相比,SHR组大鼠ANP mRNA与WKY组比较表达明显增多(P<0.05),SHR+QCH组、SHR+QCL组和SHR+Tel组ANP mRNA表达较SHR组均减少(P<0.05)；SHR+QCH组与SHR+QCL组差异较显著(P<0.05),但和SHR+Tel组比较,无统计学差异(P>0.05)。
(2)与20周龄正常血压对照组WKY大鼠相比,SHR组大鼠BNP mRNA与WKY组比较表达明显增多(P＜0.05), SHR+QCH组、SHR+QCL组和SHR+Tel组BNP mRNA表达较SHR组均减少(P＜0.05); SHR+QCH组与SHR+QCL组差异较显著(P<0.05),但和SHR+Tel组比较,无统计学差异(P>0.05)。
结论
(1)芩丹胶囊对SHR大鼠心室肥厚有明显的逆转作用,其具体表现为：改善SHR大鼠心肌形态学指标,降低心肌细胞的体积,改善心肌的超微结构。
(2)芩丹胶囊具有PPARy部分激动剂的作用,改善心肌肥厚可能是通过部分激活PPAR-y抑制NF-κB的表达,进而调节其下游肥厚基因ANP, BNP的mRNA的表达。
研究背景
高血压病是严重危害人类健康的常见心血管疾病之一,其并发症心肌肥厚在世界范围内是导致心力衰竭和心脏猝死的一个独立的危险因素；心肌肥厚反应主要的特征包括心肌细胞的肥大,收缩蛋白含量的增加,以及胚胎基因如心房利钠肽(ANP)和脑钠肽(BNP)的再表达。在各种心脏疾病的发生发展过程中,心肌肥厚早期是对各种心脏疾病血压升高的一种适应性反应,是一种慢性代偿机制；但是持续的细胞外信号刺激会导致心肌重塑而最终导致心力衰竭的发生。
过氧化物酶增殖物激活受体-γ (peroxisome proliferater-activated receptor y, PPAR-γ)是一类配体激活的核转录因子超家族成员,在逆转高血压心室肥厚的过程中起着关键的作用,在相关配体的激动下,PPAR-γ能抑制心肌肥厚基因的表达,对高血压心室肥厚引起的心力衰竭起着预防和治疗的作用。近年来研究发现,AP-1(activator protein-1)的激活在心肌肥厚的发展过程中的作用引起了广泛的关注,AP-1家族成员中以C-Jun及C-Fos为主,且以C-Jun/C-Fos异二聚体形式的AP-1生物学活性最强,可参与调节各种细胞增殖,分化,转化反应的过程。在心血管的疾病中,AP-1是心肌肥厚中重要的转录因子,它调控的心肌肥厚反应基因包括心钠肽(ANP)、脑钠肽(BNP)等心肌早期应答基因。实验研究表明,过量表达c-Jun的显形负突变(dominant negative mutation)可以抑制ET-1和PE诱导的涉及AP-1的活性、蛋白合成、肌细胞生长、ANP、BNP基因的表达等一系列心肌肥厚的变化,这些方面都说明AP-1在调控心肌肥厚中起到了重要的作用。此外,研究发现PPAR-y可通过DNA依赖的方式抑制激活蛋白-1(AP-1)介导的信号通路。
槲皮素(3,3',4',5,7-pentahydroxyflavone, Que),是生物黄酮类化合物,广泛存在于自然界中,在调节心血管系统功能方面有良好的发展前景。槲皮素是芩丹胶囊中桑寄生的有效成分,具有良好的降压作用,同时还具有抗氧化、抗血栓、抗炎、减轻心肌肥厚等多种心血管保护作用。近期研究表明,黄酮类可以通过激活了过氧化物酶体增殖物γ抑制在大鼠巨噬细胞诱导的环氧酶和一氧化氮合酶的活动。本研究通过研究槲皮素对SHR大鼠心肌组织的病理学及超微结构的观察,以及应用荧光实时定量PCR (real-time PCR)和免疫组织化学法(immunohistochemistry)检测PPAR-γ和AP-1(C-Jun, C-Fos)蛋白及mRNA表达的改变,以及对下游肥厚基因ANP、BNP的mRNA表达的影响,探讨槲皮素对改善自发性高血压大鼠(spontaneously hypertensive rats, SHR)心室肥厚的作用机制,为槲皮素治疗高血压心室肥厚的临床应用提供有效的实验依据。
目的
1.观察槲皮素对SHR大鼠心肌形态学及超微结构的改变,及对大鼠血压和心动超声的影响,探讨槲皮素抑制SHR大鼠心肌肥厚的疗效。
2.探讨槲皮素在抑制SHR大鼠心肌肥厚对PPAR-γ和AP-1(C-Jun, C-Fos)的蛋白的表达以及下游基因ANP、BNP的mRNA表达的影响,探讨槲皮素抑制SHR大鼠心肌肥厚的信号调控机制。
方法
1.研究对象：8周龄雄性SHR大鼠24只和8周龄Wistar-Kyoto大鼠(WKY)8只。随机分为4组,每组8只：(1)WKY对照组；(2)SHR对照组；(3)SHR+槲皮素高剂量组(SHR+QH;10mg.kg-1,溶于1ml的二甲基纤维素),(4)SHR+槲皮素低剂量组(SHR+QH;5mg.kg-1,溶于1ml的二甲基纤维素),WKY对照组和SHR对照组分别给予等量蒸馏水。各组大鼠均灌胃给药,每日1次,连续给药12周。末次给药后禁食24h,不禁水。3%戊巴比妥钠(30mg/kg)腹腔注射麻醉后,进行后续相关实验。
2.研究内容：(1)每两周测量尾动脉收缩压(SBP),每周称取体质量(BW)一次；(2)实验开始和结束时行常规超声心动图检查；(3)处死动物后,称取左心室质量(LVM),并计算左室相对质量指数(LVM/BW);(4) HE染色和Masson染色观察大鼠心肌组织形态并测量大鼠心肌纤维直径和进行心肌组织间质胶原定量；(5)电镜观察大鼠心肌组织超微结构；(6)免疫组织化学测定心肌组织中PPAR-γ和AP-1(C-Jun, C-Fos)蛋白的表达及定位；(7)实时定量RT-PCR法检测心肌组织中PPAR-γ和AP-1(C-Jun, C-Fos), ANP、BNP的mRNA的表达；(8)Western-blot检测大鼠心肌组织PPAR-γ和AP-1(C-Jun, C-Fos)的表达。
3.统计学处理计量资料统一采用均数±标准误表示,统计软件SPSS17.0进行数据的相关统计分析。两个组之间的数据比较用独立样本的t检验,多组之间的均数比较用单因素或多因素方差分析(ANOVA)和Bonferroni post-hoc检验。当P<0.05,认为有统计学差异。
结果
1.各组大鼠治疗前后收缩压的动态变化
与8周龄正常血压对照组WKY大鼠比较,8周龄SHR各组大鼠血压已经明显升高(P<0.05)。实验过程中,SHR组大鼠收缩压可呈持续升高状态；与20周龄正常血压的WKY组相比,20周龄SHR组大鼠收缩压显著增加(P<0.05)。给药第4周起,SHR+QH组、SHR+QL组大鼠的血压与模型组SHR组相比均有下降,其差异具有统计学意义(P<0.05)。给药7周到12周期间,槲皮素大剂量组较低剂量组血压下降更为显著,差异具有统计学意义(P<0.05)。
2.左室相对质量(LVM/BW)
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠LVM/BW明显增加(P<0.05)。与20周龄SHR组相比,SHR+QH组和SHR+QL组大鼠LVM/BW显著减小(P<0.05)。两两比较分析,SHR+QH组与SHR+QL组差异较显著(P<0.05)。
3.超声心动图检测
与8周龄的WKY组比较,8周龄的SHR各组大鼠心室肥厚指标IVSd,LVPWd均明显增加(P<0.05)；与20周龄的SHR组相比较,20周龄SHR+QH组、SHR+QL组心室肥厚指标IVSd, LVPWd等均降低(P<0.05)；20周龄SHR+QH组、SHR+QL组的LVIDd, MFS, E/A等指标均明显升高(P<0.05),提示心脏功能得到改善。
4.各组大鼠心肌组织切片光镜下HE染色观察
20周龄WKY组大鼠心肌细胞排列整齐,胞浆染色均匀,细胞核大小均一。SHR组大鼠则见心肌细胞排列紊乱,胞浆染色加深,且细胞核大小不规则,细胞内可见心肌纤维断裂的现象。与20周龄SHR组大鼠相比,SHR+QH组和SHR+QL组大鼠心肌细胞排列较为整齐,细胞内心肌纤维断裂情况已明显好转,细胞核偶见大小不规则。
5.大鼠心肌纤维直径(MFD)的比较
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠心肌纤维直径明显增加(P<0.05)。与20周龄SHR组相比,SHR+QH组和SHR+QL组大鼠心肌纤维直径显著减小(P<0.05)。两两比较分析,SHR+QH组与SHR+QL组差异较显著(P<0.05)。
6.各组大鼠心肌组织超微结构的观察
电镜下,20周龄WKY组表现为心肌细胞核仁清晰,线粒体、闰盘、Z线清楚完整,心肌纤维排列清晰、整齐,间质纤维无增生,细胞核染色质分布较均匀,无胶原分泌。20周龄SHR组表现为线粒体严重肿胀,嵴消失断裂,肌丝排列紊乱,闰盘及Z线不清,内质网扩大,大量的胶原纤维增生成点片状,部分心肌细胞核可见染色质边集的早期凋亡形态改变.SHR+QL组表现为心肌肌原纤维规则排列,闰盘整齐,细胞间质水肿不明显,肌浆网轻度扩张,肌原纤维整齐排列,有轻度线粒体肿胀,无明显坏死。SHR+QH组表现为偶有点状胶原纤维增生,肌丝排列相对整齐,较SHR组有明显改善,胶原增生受到明显抑制。
7.心肌组织切片光镜下Masson染色观察
光镜下Masson染色显示心肌细胞染色呈红色,间质胶原呈蓝绿色。WKY组大鼠胶原组织分布较稀疏,相邻细胞的胶原纤维网着色淡完好；SHR组心肌内胶原组织明显增多,围绕心肌细胞的胶原纤维网排列紊乱、断裂；与SHR组大鼠相比,SHR+QH组和SHR+QL组胶原纤维明显减少,胶原含量明显下降,排列较规整,且呈一定的剂量依赖性。
8.大鼠心肌胶原面积百分比(VFC)比较
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠心肌胶原面积百分比明显增加(P<0.05)。与SHR组相比,20周龄SHR+QH组和SHR+QL组大鼠心肌胶原面积百分比显著减少(P<0.05),两两比较分析,SHR+QH组与SHR+QL组差异较显著(P<0.05)。
9.心肌组织免疫组织化学PPAR-γ、AP-1(C-Jun, C-Fos)蛋白表达水平比较
1)与20周龄正常WKY大鼠相比,SHR组PPAR-γ的核表达较WKY组明显减少(P<0.05)；SHR+QH组、SHR+QL组较SHR组PPAR-γ核表达明显增多(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
2)与20周龄正常血压对照组WKY大鼠相比,SHR组与WKY组比较,AP-1(C-Jun, C-Fos)核表达明显增多(P<0.05),SHR+QH组、SHR+QL组表达较SHR组均减少(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
10.实时定量RT-PCR法检测心肌组织中肥厚基因的ANP, BNP mRNA的表达水平的比较
1)与20周龄正常血压对照组WKY大鼠相比,SHR组PPAR-γ的mRNA表达量较WKY组明显减少(P<0.05)；与20周龄的SHR大鼠相比较,20周龄SHR+QH组和SHR+QL组较SHR组PPAR-γ mRNA表达明显增多(P<0.05);两两比较分析,SHR+QH组与SHR+QL组比较,差异具有统计学意义(P<0.05)。
2)与20周龄正常WKY大鼠相比,SHR大鼠AP-1(C-Jun, C-Fos), ANP, BNP的mRNA表达明显增多(P<0.05),经槲皮素治疗12周后,SHR+QH组、SHR+QL组AP-1(C-Jun, C-Fos), ANP, BNP的mRNA表达较SHR组均明显减少(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组比较,差异具有统计学意义(P<0.05)。
11.Westernblot检测分析PPAR-γ、AP-1(c-fos, c-jun)蛋白表达水平比较
1)与20周龄正常血压对照组WKY大鼠相比,SHR组PPAR-γ的蛋白表达量较WKY组明显减少(P<0.05)；与20周龄的SHR大鼠相比较,20周龄SHR+QH组和SHR+QL组较SHR组PPAR-γ蛋白表达量明显增多(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
2)与20周龄正常血压对照组WKY大鼠相比,SHR组AP-1(C-Jun, C-Fos)蛋白表达量明显增多(P<0.01),与20周龄的SHR大鼠相比较,20周龄SHR+QH组、SHR+QL组蛋白表达量较SHR组均减少(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
结论
(1)槲皮素对SHR大鼠心室肥厚有明显的逆转作用,其具体表现为：改善SHR大鼠心肌形态学指标,降低心肌组织胶原含量,改善心肌的超微结构和心功能的指标。
(2)槲皮素具有PPAR-γ部分激动剂的作用,在改善心肌肥厚的机制部分可能是激活PPAR-γ抑制肥厚转录因子AP-1(C-Jun, C-Fos)的表达,进而调节其下游ANP、BNP的mRNA的肥厚基因的表达。
研究背景：
心肌肥大是心肌细胞对多种病理刺激如压力过负荷、神经体液因子以及心肌收缩蛋白基因突变等出现的一系列组织细胞形态学的适应性反应,肥大特征表现主要包括心肌细胞体积增大,蛋白质合成增加和胚胎期的表达基因再表达。一方面它是心脏早期的适应性反应,是一种慢性代偿机制,许多神经体液因子和,心肌的旁分泌因子参与到心肌肥厚的适应性反应中。另一方面它又是晚期心脏猝死和其他心血管事件的一个独立的危险因素。因此,逆转高血压心肌肥厚是防治高血压及其靶器官损害的关键所在。
许多研究证实,心脏局部肾素-血管紧张素系统(RAS)在心肌肥厚的发生发展中起着重要作用,而Ang Ⅱ(angiotensin Ⅱ, AngⅡ)是起主要作用的生物活性物质。体内和体外实验都显示AngⅡ是致心肌细胞肥大的重要因子,血管紧张素Ⅱ可以直接促进心脏和血管细胞生长,部分通过各种生长因子和细胞外基质蛋白以及细胞因子的诱导。研究发现包括核转录因子AP-1在内很多信号分子在AngⅡ引发的心肌肥大反应中发挥重要作用,这些转录因子反过来又能调节细胞的生长和细胞外基质的生物合成。大量研究表明,参与构成转录激活因子AP-1家族成员中以C-Jun及C-Fos为主,且以C-Jun/C-Fos异二聚体形式的AP-1生物学活性最强,并且直接抑制AP-1的活性可以直接降低心肌肥厚的发生。因此,当原癌基因如c-fos和c-jun作为初始应答基因时,所编码的产物Fos和Jun作为核内转录调控时因子和第三信使,通过一个亮氨酸拉链形成异源二聚体,与位于5’端上游调控区的AP-1位点(TGACTGA保守序列)结合,进一步促进次级应答基因如心房利尿钠因子(ANF)、脑钠肽(BNP)转录和蛋白质合成,导致心肌细胞蛋白质合成增加,蛋白表型向胚胎型转化等心肌肥厚的特征性变化。因此,对心肌肥大信号转导通路的深入认识,不仅有助于阐明心肌肥厚的细胞分子机制,而且为心肌肥厚的防治提供新的策略。
槲皮素(3,3',4',5,7-pentahydroxyflavone, Que),是自然界中分布最广的生物黄酮类化合物。槲皮素是芩丹胶囊中桑寄生的有效成分,我们前期的实验表明槲皮素能够通过TGF-β1/Smad2信号通路改善TGF-β1诱导的成纤维细胞的增殖。同时,我们的体内动物实验表明槲皮素能有效的抑制SHR大鼠心肌肥厚的发生,但是它对心脏的保护作用的靶点和细胞内的信号转导机制还没有完全的阐述清楚。因此,本研究拟进一步探明槲皮素在抑制心肌肥厚的过程中的相关信号通路。我们通过检测槲皮素对AngⅡ诱导的H9C2心肌细胞肥厚的影响,应用免疫荧光检测PPAR-γ, AP-1(C-Fos, C-Jun)蛋白的表达。同时,采用siRNA阻断PPAR-γ蛋白的表达,应用荧光实时定量RT-PCR检测ANP, BNP mRNA表达情况以及western blot检测C-Fos, C-Jun蛋白表达的改变,探讨槲皮素抑制AngⅡ诱导的H9C2心肌肥厚的生物活性的途径,为其临床防治高血压性心肌肥厚提供科学依据。
目的
1.本研究通过建立血管紧张素Ⅱ诱发的大鼠H9C2心肌细胞肥厚模型,考察槲皮素对心肌细胞表面积、[3H]-亮氨酸的掺入测定的影响。
2.通过siRNA阻断心肌细胞的PPAR-γ途径,考察槲皮素对转录因子C-Fos, C-Jun的蛋白表达及心肌肥厚特异性指标ANP和BNP mRNA表达的影响,探讨槲皮素抑制心肌肥厚的作用机制。
方法
1.常规复苏、培养大鼠心肌细胞H9C2至对数生长期备用,构建血管紧张素Ⅱ诱导的心肌细胞H9C2肥大模型,加入不同浓度的槲皮素,孵育一定时间。
2.采用相差显微镜观察细胞大小,软件分析测量心肌细胞表面积,[3H]-亮氨酸掺入法测定心肌细胞蛋白质合成速率。
3.细胞免疫荧光法测定PPAR-γ、C-Fos及C-Jun的蛋白水平及其细胞内的定位。
4.根据GenebanK中PPAR-γ的序列号,设计并化学合成PPAR-γ siRNA和没有明显同源性的非特异性阴性对照NCsiRNA。
5.常规培养H9C2细胞至对数生长期,种于六孔板中,将实验分为实验组(转染PPAR-γ特异性siRNA),阴性对照组(转染PPAR-γ阴性对照siRNA),空白对照组(不转染任何siRNA,余试剂与其他两组相同),进行转染。
6.采用Western-blot技术分别检测三组细胞转染后PPAR-γ蛋白的表达水平,以检测siRNA对PPAR-γ的阻断效率。
7.细胞转染后,槲皮素与AngⅡ细胞共培养,Western-blot法检测C-Fos, C-Jun的蛋白表达水平,RT-PCR法检测心肌肥厚基因ANP、BNP的mRNA表达水平。
8.统计学处理：计量资料统一采用均数±标准误表示,统计软件SPSS17.0进行数据的相关统计分析。两个组之间的数据比较用独立样本的t检验,多组之间的均数比较用单因素或多因素方差分析(ANOVA)和Bonferroni post-hoc检验。当P<0.05,认为有统计学差异。
结果：
1.各实验组细胞的[3H]-亮氨酸掺入量测定
AngⅡ刺激H9C2细胞建立心肌细胞肥厚模型后,AngⅡ组的[3H]-亮氨酸掺入量与正常对照组相比较明显升高(P<0.01),但是在与槲皮素共培养后,[3H]-亮氨酸掺入量明显降低(P<0.01),且其抑制蛋白合成速率作用呈剂量依赖性。
2.H9C2细胞表面积的测定
AngⅡ刺激H9C2细胞建立心肌细胞肥厚模型后,AngⅡ组的H9C2细胞表面积与正常对照组相比较明显升高(P<0.01),但是与槲皮素共培养后,AngⅡ+Que组H9C2细胞表面积明显降低(P<0.01)。
3.细胞免疫荧光检测
(1)与正常对照组相比,AngⅡ组H9C2心肌细胞免疫荧光法测定PPAR-γ的核表达较正常对照组明显减少(P＜0.01); AngⅡ+Que组H9C2心肌细胞免疫荧光较AngⅡ组PPAR-γ核表达明显增多,有统计学意义(P<0.01)；
(2)与正常对照组相比,AngⅡ组H9C2心肌细胞免疫荧光法测定C-Fos, C-Jun的核表达较正常对照组明显增多(P<0.01)；AngⅡ+Que组H9C2心肌细胞细胞免疫荧光较AngⅡ组的C-Fos, C-Jun核表达明显减少,有统计学意义(P<0.01)。
4. Westernblot检测分析
(1) siRNA转染24h后,与空白对照组相比,实验组PPAR-γ siRNA组的PPAR-γ蛋白表达水平明显下调(P<0.01),与正常细胞组相比,阴性对照组NCPPAR-γ siRNA的表达与空白对照组则无统计学差异(P>0.01)。
(2) siRNA转染48h后,给予刺激AngⅡ和槲皮素的共培养后,与AngⅡ组相比较,PPAR-γ siRNA组的C-Fos, C-Jun无明显下降(P>0.01),阴性对照组NC PPAR-γ siRNA与AngⅡ组比较则明显下降(P<0.01)。
5. RT-PCR检测
(1) siRNA转染24h后,与AngⅡ组相比,实验组PPAR-γ siRNA组的ANPmRNA表达水平无明显差异(P>0.01),阴性对照组NC PPAR-γ siRNA的ANPmRNA表达与AngⅡ组比较则明显下降(P<0.01)。
(2) siRNA转染24h后,与AngⅡ组相比,实验组PPAR-γ siRNA组的BNPmRNA表达水平无明显差异(P>0.01),阴性对照组NC PPAR-γ siRNA的BNPmRNA表达与AngⅡ组比较则明显下降(P<0.01)。
结论
1.槲皮素能够显著抑制AngⅡ诱导的H9C2心肌细胞肥厚。
2.槲皮素可以通过激活PPAR-y,下调AP-1(C-Fos, C-Jun)蛋白及ANP、 BNP mRNA的表达,抑制AngⅡ诱导的H9C2心肌细胞肥厚,从而达到减轻心肌肥厚的作用。
Background
Hypertension is one of the familiar risk factors for the human health. Ventricular hypertrophy is a common complication of hypertension, and it is an independent risk factor for sudden cardiac death and the other cardiovascular events. Ventricular hypertrophy is consist of cardiomyocyte hypertrophy, myocardial interstitial cell proliferation, extracellular matrix accumulation pathologically. Therefore, to explore ventricular hypertrophy mechanisms of hypertension is important for prevention and treatment of hypertensive target organ damage and reduce mortality.
Peroxisome proliferator-activated receptor y (peroxisome proliferater-activated receptor-y, PPAR-y) is a ligand-activated nuclear transcription factor superfamily members. Recent studies have confirmed that PPARy plays a critical role in inhibition of cardiac hypertrophy both in vitro and in vivo. NF-κB activation was essential to the development of cardiac hypertrophy in recent years and inhibition of NF-κB activation may be important targets for myocardial hypertrophy. Furthermore, we also have shown previously that PPAR-y regulates gene expression in a DNA-independent fashion by interfering with other signaling pathways, such as the NF-κB pathway
Qindan Capsule(QC), a prepared compound of traditional Chinese medicine, has been used as an anti-hypertensive agent for years in clinic. It has shown that QC plays an important role in attenuating vascular remodeling in our previous study.In the present study,the changes of the pathology of myocardial hypertrophy were observed in SHR, and the variation of ANP and BNP mRNA expression was detected by RT-PCR, and the variation of PPARy and NF-κB was detected by immunohistochemistry. In order to discover novel agents on inhibiting myocardial hypertrophy and improving cardiac function, the possible mechanism of QC on improveing ventricular hypertrophy in the spontaneously hypertensive rats was investigated. It could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.
Aim
1. To investigate the anti-hypertrophy effects of QC through observing the changes of the morphology and the ultrastructure in myocardial tissues.
2. To investigate the possible mechanism of QC in reversing myocardial hypertrophy in hypertension through observing the changes of the PPARy and NF-κB and the variation of ANP and BNP mRNA expression.
Methods
40male (8weeks old) SHRs and8male (8weeks old) Wistar Kyoto (WKY) rats were randomly divided into5groups(n=8):the WKY control group, the SHR control group, the Telmisartan (SHR+Tel)group, the high dosage QC group (SHR+QCL), and the low dosage QC group (SHR+QCL).The SHR+Tel group was treated with Telmisartan for10mg/kg per day, the SHR+QCH group was treated with high dosage (750mg/kg per day) of QC, and the SHR+QCL group was treated with low dosage(150mg/kg per day)of QC. The WKY group and the SHR group were treated with equal volume of distilled water instead. All the treatments were administered by gastrogavage once a day for12successive weeks.
After the last administration, all rats were fasted with free access to water for24h, and anesthetized with intraperitoneal injecfion of3%pentobarbital (30mg/kg)which was followed by other experiments.The detection contents listed below:(1) Measurement of the systolic blood pressure (SBP);(2) The left ventricular weight (LVW) and body weight (BW) ratio was determined;(3) Observation of cardiomyocyte morphological by means of HE;(4) The ultrastrueture of myocardial cell was observed with transmission electron microscope;(5) Determination of the levels of PPARy,NF-κB in myocardial by immunohistochemistry;(6)Analysis of ANP and BNP mRNA expression by real-time PCR.
Statistical analysis
The data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared. Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of SBP
The SBP of rats in the SHR group with8weeks old was much higher than those in the WKY group(P<0.05). The rats in the SHR group continuously developed further hypertension to a high pressure level. After12weeks, the SBP of rats in the SHR group with20weeks old was significantly higher than those in the WKY group (P＜0.05). From the second week of treatment, the SBP was lower in the SHR+Tel group, the SHR+QCH group, and the SHR+QCL group than that in the SHR group(all P＜0.05). After six weeks of treatment,the SBP in the SHR+QCH group was decreased significantly compared with that in the SHR+QCL group(P＜0.05). After twelve weeks of treatment, the SBP in each group was decreased in different degree compared with the SHR group(all P＜0.05). The SBP in the SHR+QCH group and the SHR+Tel group was lower than that in the SHR+QCL group(P＜0.05), but no significant difference was shown in comparison among these two groups (P＜0.05).
2. Left ventricular relative mass(LVM/BW)
At the end of the experiment, the left ventricular weight index (LVW/BW) in SHRs was significantly greater than in WKY rats. This parameter was dose-dependently and significantly decreased in QC or Tel-treated SHRs compared with distilled water-treated SHRs (P＜0.05).
3. Observation of seetions of myocytes stained with HE
Pathological changes were significantly attenuated by QC or Tel. Hematoxylin and eosin staining was performed for all groups. Compared with the WKY group, cardiac muscle fibers were enlarged and disorganized in the SHR group. Hematoxylin and eosin-stained myocardial tissue in the QC or Tel-treated SHR groups showed reductions in the size of the cardiomyocytes and reduced fibrosis. In addition, cardiac muscle fibers were also better-arranged.
4. Ultrastructural changes of myocytes observed by transmission electron microscopy.
In the WKY group, arrays of myofibrils were closely arranged in an orderly manner within the sarcomere, and mitochondria were of normal size and present in normal numbers. However, in the SHR group, the mitochondrial structure was damaged severely and cellular or tissue swelling was apparent in myocardium. Most myofibrils had either disappeared or were poorly arranged. Mitochondria were noticeably swollen and loosely arranged. In addition, mitochondrial membranes were vague or partly ruptured and cristae were clearly loose or dissolved with many vacuoles. The significant change in ultrastructural organization of mitochondria and myofibrils was attenuated in the SHR+QCH group, the SHR+QCL group and SHR+Tel group with more obvious improvements noted in the high-dose group.
5. Immunohistochemistry detection
(1) Comparison of expression of PPAR-y protein:The expression of PPAR-y protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the three treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
(2) Comparison of expression of NF-κB protein:The expression of NF-κB protein was higher in the SHR group than that in the WKY group (P＜0.05).The expression of NF-κB protein was decreased in the three treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
6. Comparison of expression of ANP and BNP mRNA
(1) Comparison of expression of ANP mRNA:The expression of ANP mRNA was higher in the SHR group than that in the WKY group (P＜0.05).The expression of ANP mRNA was decreased in the QC or Tel treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
(2) Comparison of expression of BNP mRNA:The expression of BNP mRNA was higher in the SHR group than that in the WKY group (P＜0.05).The expression of BNP mRNA was decreased in the QC or Tel treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
Conclusions
(1) A reversing effect of Qindan capsule on the hypertensive ventricular hypertrophy in SHR was observed in our present study. Qindan capsule could improve the morphological index of myocardial tissue and reduce myocardial tissue collagen content. Furthermore, it improves cardiac function and myocardial ultrastructure.
(2) Qindan capsule may inhibit cardiac hypertrophy by enhancing PPAR-y expression partly and by suppressing the NF-κB signaling pathway, resulting inhibition the transeription of its downstream gene expression of ANP and BNP.
Background
Hypertension is one of the familiar risk factors for the human health. Cardiac hypertrophy results in congestive heart failure and is a major world-wide cause of sudden death. The hypertrophic response in cardiomyocytes is characterized by an enlargement of myocytes, an increase in the content of contractile proteins, and expression of embryonic genes such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The hypertrophic response is compensatory at an early stage of various cardiac diseases, but sustained extracellular stimuli may lead to excessive cardiac hypertrophy and finally to heart failure.
Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor gene family. In recent study also demonstrated the PPAR-y-dependent pathway is critical in the inhibition of cardiac hypertrophy both in vivo and vitro. One major transcription factor is the activator protein1(AP-1) complex, which is composed of the Jun and Fos family of DNA binding protooncoproteins. The c-Jun and c-Fos proteins form dimmers that bind DNA through specific response elements to transactivate the transcription of genes downstream from these enhancer elements.The activation of AP-1has been shown to be involved in the regulation of a variety of cellular processes such as proliferation, differentiation, and transformation. Moreover, recent studies have demonstrated that myocardial AP-1DNA binding activities are significantly increased in experimental cardiac hypertrophy in cardiovascular disease. It can regulate mang hypertrophy gene including such as ANP and BNP. Recently, it has been reported that a dominant negative mutant lacking the transactivating domain of c-Jun (DNJun) inhibits endothelin1(ET)-1and phenylephrine (PE)-induced cardiomyocyte hypertrophy. Furthermore, we also have shown that PPAR-y regulates gene expression in a DNA-independent fashion by interfering with other signaling pathways previously, such as the activator protein-1(AP-1) pathway.
Quercetin (3,3',4',5,7-pentahydroxyflavone) is a member of a group of naturally occurring compounds and is one of the most widely distributed bioflavonoids. The very extensive biological effects of flavonoids, including their anti-inflammatory, anti-oxidant, anti-atherosclerotic, and anti-hypertensive properties facilitate an important role for quercetin in the prevention of cardiovascular diseases. Furthermore, it has also been revealed to show agonistic effects on peroxisome proliferator activated receptors and attenuates Monocyte Chemoattractant Protein-1gene expression in glucose-primed aortic endothelial cells via AP-1.
In the present study, the changes of the pathologies of myocardial hypertrophy were observed in SHR rats, and the variation of PPAR-y and AP-1(C-Jun, C-Fos) was detected by immunohistochemistry or western blot, and the variation of its downstream target genes including ANP and BNP mRNA expression was detected by real-time RT-PCR. In order to discover novel agents on inhibiting myocardial hypertrophy and improving cardiac function, the possible mechanism of quercetin on improveing ventricular hypertrophy in the spontaneously hypertensive rats was investigated, which could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.
Aim
1. To investigate the anti-hypertrophy effects of quercetin through observing the changes of the morphology and the ultrastructure in myocardial tissues.
2. To investigate the possible mechanism of quercetin in reversing myocardial hypertrophy in hypertension through observing the changes of the PPAR-y and AP-1(c-fos, c-jun) and the variation of ANP and BNP mRNA expression.
Methods
Twenty four8-week-old male SHRs and eight8-week-old male Wistar-Kyoto (WKY) rats obtained from Slaccas (Shanghai, China) were used in this study. Animals were housed in a temperature-controlled (22±0.5℃) room, and rats had free access to standard rodent chow and water. A12-h/12-h light/dark cycle was maintained. Spontaneously hypertensive rats were randomly divided into three groups:one group (SHR) was treated with vehicle (1ml of1%methylcellulose) and the other two groups were treated with either a low dose of quercetin (SHR+QL;5mg.kg-1, mixed in1ml of1%methylcellulose) or a high dose of quercetin (SHR+QH;10mg.kg-1, mixed in1ml of1%methylcellulose). Age-matched WKY rats were treated with vehicle (1ml of1%methylcellulosa) and used as a control group. Quercetin or vehicle was delivered by gavage at the same time once daily for12weeks.
After the last administration, all rats were fasted with free access to water for24h, and anesthetized with intraperitoneal injection of3%pentobarbital(30mg/kg)which was followed by other experiments. The detection contents listed below:(1)Measurement of the systolic blood pressure (SBP);(2)The common echocardiographic indexes were detected before at the first and at the end of the experiment;(3) the left ventricular weight (LVW) and body weight (BW) ratio was determined;(4)Measurement of the morphological index of cardiomyocyte diameter of myocytes and volume fraction of collagen;(5) The ultrastrueture of myocardial cell was observed with transmission electron microscope.(6) Analysis of PPAR-γ、 AP-1(c-fos, c-jun) protein expression and location by immunohistochemical detection.(7)Analysis of PPAR-γ AP-1(C-Jun, C-Fos) protein expression by western blot.(8)Analysis of PPAR-γ,AP-1(C-Jun, C-Fos),ANP and BNP mRNA expression by real-time PCR;
Statistical analysis
The data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared.Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of SBP
In the week prior to treatment with quercetin, systolic blood pressure in the SHRs was significantly increased compared with the WKY (P＜0.05). Twenty weeks of quercetin administration induced a significant reduction in SBP in the SHRs, and this effect reached statistical significance after the first week of treatment (P＜0.05). From the second week of treatment, SBP was significantly decreased in the quercetin-treated groups compared with the SHR group (P＜0.05), and the decline in the SHR+QH group was distinct from that of the SHR+QL group (P＜0.05) and was significantly distinct from the7th to the12th week (P＜0.05).
2. Left ventricular weight index (LVW/BW)
Neither SHRs nor WKY rats showed a change in body weight after treatment with quercetin or vehicle. The left ventricular weight index (LVW/BW) in SHRs was significantly greater than in WKY rats. This parameter was dose-dependently and significantly decreased in quercetin-treated SHRs compared with vehicle-treated SHRs (P＜0.05).
3. Echocardiographic detection
Compared with the WKY group, LVPWd and IVSd were both significantly increased at week1or12of the experiment but LVIDd decreased by the12th week in the SHR group (P＜0.05). After12weeks of quercetin treatment, LVIDd was significantly increased but IVSd and LVPWd were apparently decreased compared with the SHRs (P＜0.05). Furthermore, the mid-wall fractional shortening and E/A decreased significantly in SHRs and were significantly improved at week12in SHRs treated with quercetin (P＜0.05).
4. Observation of seetions of myocytes stained with HE
Pathological changes were significantly attenuated by quercetin. Hematoxylin and eosin staining was performed for all groups. Compared with the WKY group, cardiac muscle fibers were enlarged and disorganized in the SHR group. Hematoxylin and eosin-stained myocardial tissue in the quercetin-treated SHR groups showed reductions in the size of the cardiomyocytes and reduced fibrosis. In addition, cardiac muscle fibers were also better-arranged.
5.Comparison of Mean cardiomyocyte diameter of myocytes
Mean cardiomyocyte diameter was increased significantly in the SHR group compared with the WKY group(P＜0.05). Mean cardiomyocyte diamete was decreased significantly in the SHR+QH and the SHR+QL groups compared with that in the SHR group(P＜0.05).Quercetin administration dramatically and dose-dependently attenuated the enlargement in myocyte size compared with SHRs (P＜0.05).
6.Ultrastructural changes of myocytes observed by transmission electron microscopy.
In the WKY group, arrays of myofibrils were closely arranged in an orderly manner within the sarcomere, and mitochondria were of normal size and present in normal numbers. However, in the SHR group, the mitochondrial structure was damaged severely and cellular or tissue swelling was apparent. Most myofibrils had either disappeared or were poorly arranged. Mitochondria were noticeably swollen and loosely arranged. In addition, mitochondrial membranes were vague or partly ruptured and cristae were clearly loose or dissolved with many vacuoles. The significant change in ultrastructural organization of mitochondria and myofibrils was attenuated in the quercetin-treated SHR groups with more obvious improvements noted in the high-dose group.
7. Observation of sections of myocytes stained with Masson
Masson's trichrome staining was performed to assess the effect of quercetin on cardiac fibrosis. The WKY myocardium showed a normal array of myocardial fibers and very little interstitial collagen. The SHR group showed many newly formed collagen deposits between myocardial interstices and myocardial disarrangement and cellular swelling as compared with the WKY group. Myocardial collagen deposition was greater in the SHR+QH group and the SHR+QL group than that in the WKY group, but was less than that in the SHR group.
8. Comparison of the volume fraction of collagen
The VFC of myocytes in the SHR group was higher than that in the WKY group(P＜0.05). The VFC in the SHR+QH group and the SHR+QL group were lower than that in the SHR group (P＜0.05).Quercetin administration dramatically and dose-dependently attenuated the increase in collagen volume fraction compared with SHRs (P＜0.05).
9.Immunohistochemical detection
(1) Comparison of expression of PPAR-y protein:The nucleus expression of PPAR-y protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The nucleus expression of AP-1(C-Jun, C-Fos) protein was higher in the SHR group than that in the WKY group(P＜0.05).The expression of AP-1(C-Jun, C-Fos) protein was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner.(P＜0.05).
10. The mRNA expression of related to hypertrophy gene were detected by RT-PCR
(1) Comparison of expression of PPAR-y mRNA:The expression of PPAR-γ mRNA was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y mRNA was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
(2) Comparison of expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA:The expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA was higher in the SHR group than that in the WKY group(P＜0.05).The expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
11. The Protein levels of PPAR-y and AP-1(C-Jun, C-Fos) were detected by western blot
(1) Comparison of expression of PPAR-y protein:The expression of PPAR-γ protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
(2) Comparison of expression of AP-1(C-Jun, C-Fos) protein:The expression of AP-1(C-Jun, C-Fos) protein was higher in the SHR group than that in the WKY group (P＜0.05).The expression of AP-1(C-Jun, C-Fos) protein was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
Conclusions:
(1) A reversing effect of quercetin on the hypertensive ventricular hypertrophy in SHR was observed in our present study. Quercetin could improve the morphological index of myocardial tissue and reduce myocardial tissue collagen content. Furthermore improves cardiac function and myocardial ultrastructure.
(2) Quercetin may inhibit cardiac hypertrophy by enhancing PPAR-γ expression partly and by suppressing the AP-1signaling pathway, resulting inhibition the transcription of its downstream gene expression of ANP and BNP.
Background
The changes of cardiac hypertrophy are the adaptive reaction to the disorders of hemodynamics, neurohumor and local endocrinic factors in hypertension.The hypertrophic response in cardiomyocytes is characterized by an enlargement of myocytes, an increase in the content of contractile proteins, and expression of embryonic genes such as atrial natriuretic peptide(ANP) and brain natriuretic peptide (BNP). The hypertrophic response is compensatory at an early stage of various cardiac diseases, but sustained extracellular stimuli may lead to excessive cardiac hypertrophy and finally to heart failure. Therefore, it is important to reverse the cardiac hypertrophy for prevention and treatment of hypertension and target organ damage.
The renin-angiotensin system(RAS) plays a major role in the regulation of blood pressure. AngiotensinⅡ is one of the most important factors in RAS. It is shown one of the important factors to myocyte hypertrophy both in vivo and in vitro experiments. Ang Ⅱ has direct growth-promoting effects on the cardiac and vascular cells, in part via induction of the expression of various growth factors, extracellular matrix proteins and cytokines. In vitro, Ang II could activates a number of inducible transcription factors such as activator protein-1(AP-1), which in turn make impact on the expression of genes that regulate cell growth and extracellular matrix protein biosynthesis. Activator protein-1could be made up of c-fos and c-jun, which together make a heterodimer complex that plays a significant role in cardiomyocyte hypertrophy, and direct inhibition of AP-1activity significantly declined cardiac hypertrophy in myocardial tissue. Therefore, initiation of myocyte hypertrophic growth is accompanied by a rapid and transient expression of immediate-early genes such as c-fos, c-jun at the genetic level, followed by an activation of the fetal gene regulatory program with reexpression of genes for atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP),increase contractile proteins and transformation to embryonal Protein Phenotype.These observations have contributed to speculation that inhibition of these hypertrophic signals may be effective, patent strategies in the treatment of pathological hypertrophy and heart failure.
Quercetin (3,3',4',5,7-pentahydroxyflavone) is a member of a group of naturally occurring compounds and is one of the most widely distributed bioflavonoids. The very extensive biological effects of flavonoids, including their anti-inflammatory, anti-oxidant, anti-atherosclerotic, and anti-hypertensive properties facilitate an important role for quercetin in the prevention of cardiovascular diseases. Furthermore, it has also been revealed to show agonistic effects on peroxisome proliferator activated receptors and attenuates Monocyte Chemoattractant Protein-1gene expression in glucose-primed aortic endothelial cells via AP-1.The above results suggest the quercetin may inhibits cardiac hypertrophy by blocking AP-1(c-fos, c-jun) and activating PPAR-y signaling pathways in vivo, but the possible mechanism of quercetin direct inhibition of cardiac hypertrophy are still unknown in vitro. In the present study, the changes of the protein synthesis and surface area of cells were observed in H9C2cells, and the variation of PPAR-γ and AP-1(c-fos,c-jun) was detected by immunofluorescence. The variation of PPAR-y and AP-1(c-fos,c-jun) was detected by western blotting and real time-PCR, and the variation of ANP and BNP mRNA expression was detected by real-time RT-PCR after transfection with PPAR-γ siRNA in H9C2cells. In order to discover novel agents on inhibiting H9C2hypertrophy, the possible mechanism of quercetin on improveing cardiac hypertrophy in the H9C2cells cell hypertrophy was investigated, which could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.
Aims
(1) This study used the H9C2cells induced by angiotensin Ⅱ as cardiac hypertrophy model to observe the influence of H9C2cell surface area, the [3H] leucine incorporation at different concentration of quercetin stimulus.
(2) To observed the quercetin impact on transcription factor C-Fos, C-Jun protein expression, detection of specific indicators of cardiac hypertrophy such as ANP and BNP mRNA expression by mean of siRNA blocking PPAR-y pathway.
Methods
1. Culture H9C2cell line routinely. The hypertrophy of H9C2cell was induced with Angiotensin Ⅱ, and intervened with quercetin at different concentration.
2. As the index of cardiomyocyte hypertrophy, the cell size was determined by phase contrast microscope and protein synthesis rate was measured by [3H]-Leucine incorporation.
3. Immunofluorescence was used to detect the protein expression and location of PPAR-y and AP-1(c-fos, c-jun).
4. Based on the Genebank cDNA accession number, design and synthesize PPAR-y siRNA oligonueleotides lines. Design and synthesize one negative siRNA oligonueleotides, which are not specific for any known gene.
5. Culture H9C2cells line routinely.Transfect the siRNA to H9C2cells, three groups was designed, negtive, test and control group.
6. After transfection of siRNA24h, western blot was used to detect the expression of PPAR-y transfection efficiency.
7. After H9C2cells were transfected with each siRNA for48h, cells were incubated with quercetin and Angll alone or in combination for24h. Cells were incubated for another24h prior to RNA isolation for quantitative real-time PCR analysis and48h prior to isolating protein samples for western blotting analysis.
8. Statistical analysis
The data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared.Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of [3H]leucine incorporation
[3H]leucine incorporation was increased significantly in the Ang Ⅱ-induced group compared with the control group(P＜0.01). However, in quercetin pretreated cells, Ang Ⅱ-mediated incorporation was markedly reduced in a concentration-dependent manner (P＜0.01). At100μg/ml, compared with Ang Ⅱ-induced group protein synthesis was reduced to almost basal levels (64%reduction of Ang Ⅱ stimulated [3H]leucine incorporation)(P＜0.01).
2. Comparison of the surface area of H9C2cells
The surface area of H9C2cells was increased significantly in the Ang Ⅱ-induced group compared with the control group(P＜0.01). However, Quercetin (100μg/mL) pretreatment significantly attenuated this increase in H9C2cells compared with the Ang Ⅱ group (P＜0.01).
3. Immunofluorescence detection
(1) Comparison of expression of PPAR-γ protein:The expression of PPAR-γ protein was declined in the Ang Ⅱ group than that in the control group(P＜0.01) However, the fluorescence of PPAR-γ protein was significantly increased in the quercetin and AngⅡ combination treatment group compared with the Ang Ⅱ group (P＜0.01;).
(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The expression of AP-1(c-fos, c-jun) protein was increased in the Ang Ⅱ group than that in the control group(P＜0.01) However, the fluorescence of AP-1(c-fos, c-jun) was significantly decreased in the quercetin and AngⅡ combination treatment group compared with the Ang Ⅱ group (P＜0.01).
4. Western blotting detection
(1) Comparison of expression of PPAR-γ protein:The expression of PPAR-γ protein was lower in the PPAR-γ NCsiRNA group than that in the control group(P＜0.01) However, the fluorescence of PPAR-γ protein was significantly decreased in the PPAR-γ siRNA group compared with the Ang Ⅱ group (P＜0.01).
(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The expression of AP-1(c-fos, c-jun) protein was higher in the AngⅡ group than that in the control group(P＜0.01). However, the fluorescence of AP-1(c-fos, c-jun) was significantly decreased in the PPAR-y siRNA reatment group compared with the Ang Ⅱ group (P＜0.01).
5. Comparison of expression of ANP and BNP mRNA in H9C2cells
(1) Comparison of expression of ANP mRNA:Transfection siRNA after24hours, the expression of ANP mRNA of PPAR-γ siRNA group was obviously increased compared with the Ang Ⅱ group (P＜0.01). No significant difference was shown in comparison between the Ang Ⅱ group and the PPAR-y NCsiRNA group (P＞0.01).
(2) Comparison of expression of BNP mRNA:Transfection siRNA after24hours, the expression of BNP mRNA of PPAR-y siRNA group was obviously increased compared with the Ang Ⅱ group (P＜0.01). No significant difference was shown in comparison between the Ang Ⅱ group and the PPAR-y NCsiRNA group (P＞0.01).
Conclusions:
(1) A reversing effect of quercetin on Ang Ⅱ induced H9C2cells hypertrophy was observed in our present study.
(2) Quercetin may inhibit Ang Ⅱ induced H9C2cells hypertrophy by enhancing PPAR-y expression partly and by suppressing the AP-1signaling pathway, resulting inhibiting the transcription of its downstream gene expression of ANP and BNP.
高血压病是严重危害人类健康的常见心血管疾病之一,心室肥厚是高血压病常见的并发症,是心脏猝死和其他心血管事件的一个独立的危险因素,主要病理表现包括心肌细胞肥大、心肌间质细胞增生、细胞外基质的堆积,探讨高血压心室肥厚的发生机制对防治高血压靶器官损害、降低死亡率具有重要意义。
过氧化物酶增殖物激活受体γ (peroxisome proliferater-activated receptor-y, PPAR-γ)是一类配体激活的核转录因子超家族成员,近年来的研究证实,在逆转高血压心室肥厚的过程中起着关键的作用,在配体的激动下,PPAR-γ能抑制相关肥厚基因的表达,对高血压心室肥厚引起的心力衰竭起着预防和治疗的作用。NF-κB的激活在心肌肥厚的发展过程中至关重要,近年来研究表明NF-κB可能是抑制心肌肥厚的重要作用靶点. PPARy可以直接与NF-κB发生蛋白质-蛋白质之间的相互作用,形成转录抑制复合物,同时降低NF-κB与DNA结合活性,抑制NF-κB DNA合成,起到抑制其表达的作用。
芩丹胶囊(Qindan Capsule, QC)是导师根据多年临床经验研制成的治疗高血压病肝热血瘀证的中药复方制剂,前期研究表明其在改善高血压血管重构中起着重要的作用,本研究通过研究芩丹胶囊对自发性高血压大鼠(spontaneously hypertensive rats, SHR)心肌组织的病理学及超微结构的影响,应用免疫组织化学法(immunohistochemistry)检测PPAR-γ和NF-κB的蛋白表达的改变,以及荧光实时定量PCR (real-time PCR)检测对下游肥厚基因ANP、BNP的mRNA表达的影响,探讨芩丹胶囊对改善SHR高血压心室肥厚的作用机制,为芩丹胶囊治疗高血压心室肥厚的临床应用提供有效的实验依据。
目的
1.观察芩丹胶囊对SHR大鼠心肌形态学及超微结构的改变及对大鼠血压的影响,探讨芩丹胶囊抑制SHR大鼠心肌肥厚的疗效。
2.观察芩丹胶囊在抑制SHR大鼠心肌肥厚对PPAR-γ和NF-κB的蛋白表达以及对下游肥厚基因ANP、BNP的mRNA表达的影响,探讨芩丹胶囊抑制SHR大鼠心肌肥厚的信号调控机制。
方法
1.研究对象：8周龄雄性SHR大鼠32只和8周龄Wistar-Kyoto大鼠(WKY)8只。随机分为5组,每组8只：(1)WKY对照组；(2)SHR对照组；(3)SHR+替米沙坦组(SHR+Tel);(4)SHR+芩丹胶囊大剂量组(SHR+QCH);(5)SHR+芩丹胶囊小剂量组(SHR+QCL)。SHR+Tel组给予Tel10mg/(kg·d), SHR+QCH组给予芩丹胶囊750mg/(kg·d), SHR+QCL组给予芩丹胶囊150mg/(kg·d), WKY对照组和SHR对照组分别给予等量蒸馏水。各组大鼠均分别灌胃给药,每日1次,连续给药12周。末次给药后禁食24h,不禁水。3%戊巴比妥钠(30mg/kg)腹腔注射,麻醉后进行后续实验。
2.研究内容：
(1)每两周测量尾动脉收缩压(SBP),每周称取体质量(BW)一次；(2)处死动物后,称取左心室质量(LVM),并计算左室相对质量指数(LVM/BW);(3)HE染色观察各组大鼠心肌组织形态；(4)电镜观察各组大鼠心肌组织超微结构；(5)免疫组织化学测定心肌组织中PPAR-γ和NF-κB蛋白的表达；(6)实时定量RT-PCR法检测心肌组织中肥厚基因ANP、BNP的mRNA的表达。
3.统计学处理计量资料统一采用均数±标准误表示,统计软件SPSS17.0进行数据的相关统计分析。两个组之间的数据比较用独立样本的t检验,多组之间的均数比较用单因素或多因素方差分析(ANOVA)和Bonferroni post-hoc检验。当P<0.05,认为有统计学差异。
结果
1.各组大鼠治疗前后收缩压的动态变化
与8周龄WKY阴性对照组大鼠比较,8周龄SHR阳性对照组大鼠血压已经明显升高(P<0.05)。在实验过程中,阳性对照组SHR大鼠收缩压呈持续升高状态,与20周龄阴性对照组的WKY组相比,20周龄阳性对照SHR组大鼠收缩压显著增加(P<0.05)。从给药第4周起,SHR+QCH组、SHR+QCL组、SHR+Tel组大鼠的血压与SHR对照组相比均有下降,其差异具有统计学意义(P<0.05),但各组下降程度不同；给药7周到12周期间,SHR+QCH组较SHR+QCL组血压下降更为显著,差异具有统计学意义(P<0.05)。20周龄SHR+Tel组大鼠收缩压与同周龄SHR+QCH组相比差异无统计学意义(P>0.05)。
2.左室相对质量(LVM/BW)
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠与对照组WKY大鼠相比LVM/BW明显升高(P<0.05)。与20周龄SHR组相比,SHR+QCH组,SHR+QCL组和SHR+Tel组大鼠LVM/BW显著减小(P<0.05)。两两比较分析,SHR+QCH组与SHR+QCL组差异较显著(P<0.05)。
3.各组大鼠心肌组织切片光镜下HE染色观察
20周龄WKY组大鼠心肌细胞排列整齐,细胞核大小均一,胞浆染色分布均匀。SHR组大鼠可见心肌细胞排列紊乱,细胞核大小不规则,胞浆染色加深,细胞内心肌纤维断裂,单位面积内细胞核数量增多的表现。与SHR组大鼠比较,SHR+Tel组和SHR+QCH组、SHR+QCL组大鼠见心肌细胞排列较为整齐,细胞核偶见大小不规则,细胞内心肌纤维断裂情况明显好转,单位面积细胞核数目略增多。
4.各组大鼠心肌组织超微结构的观察
电镜下,WKY心肌纤维排列清晰、整齐,心肌细胞质膜连续、完整,线粒体结构、闰盘结构正常；粗细肌丝排列整齐,肌小节及明暗各带清晰可见,Z线较清晰,间质纤维无增生,细胞核染色质分布较均匀；SHR组大鼠心肌纤维排列稀疏、紊乱,线粒体肿胀、破坏甚至呈空泡状,Z线增宽模糊,可见细胞核染色质边聚的现象；伴有肌原纤维呈灶性溶解,出现肌节对位不齐,肌丝扭曲、断裂,间质可见大量胶原纤维分布。SHR+Tel组,SHR+QCH组,SHR+QCL组较SHR组好转,大部分肌纤维排列紧密,线粒体结构相对完整,且排列适当,细胞核无明显边聚现象,间质内胶原堆积已不明显。
5.心肌组织免疫组织化学PPARγ、NF-κB蛋白表达水平比较
(1)与20周龄阴性对照组WKY组相比,阳性对照SHR组大鼠PPARγ蛋白的表达较WKY组明显减少(P<0.05)；SHR+Tel组,SHR+QCH组,SHR+QCL组较SHR组PPARy核表达明显增多(P<0.05),有显著性差异；SHR+QCH组与SHR+QCL组差异较显著(P<0.05),但和SHR+Tel组比较,无统计学差异(P>0.05)。
(2)与20周龄正常血压对照组WKY大鼠相比,SHR组NF-κB蛋白与WKY组比较,核表达明显增多(P<0.05),SHR+QCH组、SHR+QCL组和SHR+Tel组表达较SHR组均减少(P<0.05),SHR+QCH组与SHR+QCL组差异具有统计学意义(P<0.05),但和SHR+Tel组比较,差异无统计学意义(P>0.05)。
6.实时定量RT-PCR法检测心肌组织中肥厚基因的ANP, BNP mRNA的表达水平的比较
(1)与20周龄正常血压对照组WKY大鼠相比,SHR组大鼠ANP mRNA与WKY组比较表达明显增多(P<0.05),SHR+QCH组、SHR+QCL组和SHR+Tel组ANP mRNA表达较SHR组均减少(P<0.05)；SHR+QCH组与SHR+QCL组差异较显著(P<0.05),但和SHR+Tel组比较,无统计学差异(P>0.05)。
(2)与20周龄正常血压对照组WKY大鼠相比,SHR组大鼠BNP mRNA与WKY组比较表达明显增多(P＜0.05), SHR+QCH组、SHR+QCL组和SHR+Tel组BNP mRNA表达较SHR组均减少(P＜0.05); SHR+QCH组与SHR+QCL组差异较显著(P<0.05),但和SHR+Tel组比较,无统计学差异(P>0.05)。
结论
(1)芩丹胶囊对SHR大鼠心室肥厚有明显的逆转作用,其具体表现为：改善SHR大鼠心肌形态学指标,降低心肌细胞的体积,改善心肌的超微结构。
(2)芩丹胶囊具有PPARy部分激动剂的作用,改善心肌肥厚可能是通过部分激活PPAR-y抑制NF-κB的表达,进而调节其下游肥厚基因ANP, BNP的mRNA的表达。
研究背景
高血压病是严重危害人类健康的常见心血管疾病之一,其并发症心肌肥厚在世界范围内是导致心力衰竭和心脏猝死的一个独立的危险因素；心肌肥厚反应主要的特征包括心肌细胞的肥大,收缩蛋白含量的增加,以及胚胎基因如心房利钠肽(ANP)和脑钠肽(BNP)的再表达。在各种心脏疾病的发生发展过程中,心肌肥厚早期是对各种心脏疾病血压升高的一种适应性反应,是一种慢性代偿机制；但是持续的细胞外信号刺激会导致心肌重塑而最终导致心力衰竭的发生。
过氧化物酶增殖物激活受体-γ (peroxisome proliferater-activated receptor y, PPAR-γ)是一类配体激活的核转录因子超家族成员,在逆转高血压心室肥厚的过程中起着关键的作用,在相关配体的激动下,PPAR-γ能抑制心肌肥厚基因的表达,对高血压心室肥厚引起的心力衰竭起着预防和治疗的作用。近年来研究发现,AP-1(activator protein-1)的激活在心肌肥厚的发展过程中的作用引起了广泛的关注,AP-1家族成员中以C-Jun及C-Fos为主,且以C-Jun/C-Fos异二聚体形式的AP-1生物学活性最强,可参与调节各种细胞增殖,分化,转化反应的过程。在心血管的疾病中,AP-1是心肌肥厚中重要的转录因子,它调控的心肌肥厚反应基因包括心钠肽(ANP)、脑钠肽(BNP)等心肌早期应答基因。实验研究表明,过量表达c-Jun的显形负突变(dominant negative mutation)可以抑制ET-1和PE诱导的涉及AP-1的活性、蛋白合成、肌细胞生长、ANP、BNP基因的表达等一系列心肌肥厚的变化,这些方面都说明AP-1在调控心肌肥厚中起到了重要的作用。此外,研究发现PPAR-y可通过DNA依赖的方式抑制激活蛋白-1(AP-1)介导的信号通路。
槲皮素(3,3',4',5,7-pentahydroxyflavone, Que),是生物黄酮类化合物,广泛存在于自然界中,在调节心血管系统功能方面有良好的发展前景。槲皮素是芩丹胶囊中桑寄生的有效成分,具有良好的降压作用,同时还具有抗氧化、抗血栓、抗炎、减轻心肌肥厚等多种心血管保护作用。近期研究表明,黄酮类可以通过激活了过氧化物酶体增殖物γ抑制在大鼠巨噬细胞诱导的环氧酶和一氧化氮合酶的活动。本研究通过研究槲皮素对SHR大鼠心肌组织的病理学及超微结构的观察,以及应用荧光实时定量PCR (real-time PCR)和免疫组织化学法(immunohistochemistry)检测PPAR-γ和AP-1(C-Jun, C-Fos)蛋白及mRNA表达的改变,以及对下游肥厚基因ANP、BNP的mRNA表达的影响,探讨槲皮素对改善自发性高血压大鼠(spontaneously hypertensive rats, SHR)心室肥厚的作用机制,为槲皮素治疗高血压心室肥厚的临床应用提供有效的实验依据。
目的
1.观察槲皮素对SHR大鼠心肌形态学及超微结构的改变,及对大鼠血压和心动超声的影响,探讨槲皮素抑制SHR大鼠心肌肥厚的疗效。
2.探讨槲皮素在抑制SHR大鼠心肌肥厚对PPAR-γ和AP-1(C-Jun, C-Fos)的蛋白的表达以及下游基因ANP、BNP的mRNA表达的影响,探讨槲皮素抑制SHR大鼠心肌肥厚的信号调控机制。
方法
1.研究对象：8周龄雄性SHR大鼠24只和8周龄Wistar-Kyoto大鼠(WKY)8只。随机分为4组,每组8只：(1)WKY对照组；(2)SHR对照组；(3)SHR+槲皮素高剂量组(SHR+QH;10mg.kg-1,溶于1ml的二甲基纤维素),(4)SHR+槲皮素低剂量组(SHR+QH;5mg.kg-1,溶于1ml的二甲基纤维素),WKY对照组和SHR对照组分别给予等量蒸馏水。各组大鼠均灌胃给药,每日1次,连续给药12周。末次给药后禁食24h,不禁水。3%戊巴比妥钠(30mg/kg)腹腔注射麻醉后,进行后续相关实验。
2.研究内容：(1)每两周测量尾动脉收缩压(SBP),每周称取体质量(BW)一次；(2)实验开始和结束时行常规超声心动图检查；(3)处死动物后,称取左心室质量(LVM),并计算左室相对质量指数(LVM/BW);(4) HE染色和Masson染色观察大鼠心肌组织形态并测量大鼠心肌纤维直径和进行心肌组织间质胶原定量；(5)电镜观察大鼠心肌组织超微结构；(6)免疫组织化学测定心肌组织中PPAR-γ和AP-1(C-Jun, C-Fos)蛋白的表达及定位；(7)实时定量RT-PCR法检测心肌组织中PPAR-γ和AP-1(C-Jun, C-Fos), ANP、BNP的mRNA的表达；(8)Western-blot检测大鼠心肌组织PPAR-γ和AP-1(C-Jun, C-Fos)的表达。
3.统计学处理计量资料统一采用均数±标准误表示,统计软件SPSS17.0进行数据的相关统计分析。两个组之间的数据比较用独立样本的t检验,多组之间的均数比较用单因素或多因素方差分析(ANOVA)和Bonferroni post-hoc检验。当P<0.05,认为有统计学差异。
结果
1.各组大鼠治疗前后收缩压的动态变化
与8周龄正常血压对照组WKY大鼠比较,8周龄SHR各组大鼠血压已经明显升高(P<0.05)。实验过程中,SHR组大鼠收缩压可呈持续升高状态；与20周龄正常血压的WKY组相比,20周龄SHR组大鼠收缩压显著增加(P<0.05)。给药第4周起,SHR+QH组、SHR+QL组大鼠的血压与模型组SHR组相比均有下降,其差异具有统计学意义(P<0.05)。给药7周到12周期间,槲皮素大剂量组较低剂量组血压下降更为显著,差异具有统计学意义(P<0.05)。
2.左室相对质量(LVM/BW)
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠LVM/BW明显增加(P<0.05)。与20周龄SHR组相比,SHR+QH组和SHR+QL组大鼠LVM/BW显著减小(P<0.05)。两两比较分析,SHR+QH组与SHR+QL组差异较显著(P<0.05)。
3.超声心动图检测
与8周龄的WKY组比较,8周龄的SHR各组大鼠心室肥厚指标IVSd,LVPWd均明显增加(P<0.05)；与20周龄的SHR组相比较,20周龄SHR+QH组、SHR+QL组心室肥厚指标IVSd, LVPWd等均降低(P<0.05)；20周龄SHR+QH组、SHR+QL组的LVIDd, MFS, E/A等指标均明显升高(P<0.05),提示心脏功能得到改善。
4.各组大鼠心肌组织切片光镜下HE染色观察
20周龄WKY组大鼠心肌细胞排列整齐,胞浆染色均匀,细胞核大小均一。SHR组大鼠则见心肌细胞排列紊乱,胞浆染色加深,且细胞核大小不规则,细胞内可见心肌纤维断裂的现象。与20周龄SHR组大鼠相比,SHR+QH组和SHR+QL组大鼠心肌细胞排列较为整齐,细胞内心肌纤维断裂情况已明显好转,细胞核偶见大小不规则。
5.大鼠心肌纤维直径(MFD)的比较
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠心肌纤维直径明显增加(P<0.05)。与20周龄SHR组相比,SHR+QH组和SHR+QL组大鼠心肌纤维直径显著减小(P<0.05)。两两比较分析,SHR+QH组与SHR+QL组差异较显著(P<0.05)。
6.各组大鼠心肌组织超微结构的观察
电镜下,20周龄WKY组表现为心肌细胞核仁清晰,线粒体、闰盘、Z线清楚完整,心肌纤维排列清晰、整齐,间质纤维无增生,细胞核染色质分布较均匀,无胶原分泌。20周龄SHR组表现为线粒体严重肿胀,嵴消失断裂,肌丝排列紊乱,闰盘及Z线不清,内质网扩大,大量的胶原纤维增生成点片状,部分心肌细胞核可见染色质边集的早期凋亡形态改变.SHR+QL组表现为心肌肌原纤维规则排列,闰盘整齐,细胞间质水肿不明显,肌浆网轻度扩张,肌原纤维整齐排列,有轻度线粒体肿胀,无明显坏死。SHR+QH组表现为偶有点状胶原纤维增生,肌丝排列相对整齐,较SHR组有明显改善,胶原增生受到明显抑制。
7.心肌组织切片光镜下Masson染色观察
光镜下Masson染色显示心肌细胞染色呈红色,间质胶原呈蓝绿色。WKY组大鼠胶原组织分布较稀疏,相邻细胞的胶原纤维网着色淡完好；SHR组心肌内胶原组织明显增多,围绕心肌细胞的胶原纤维网排列紊乱、断裂；与SHR组大鼠相比,SHR+QH组和SHR+QL组胶原纤维明显减少,胶原含量明显下降,排列较规整,且呈一定的剂量依赖性。
8.大鼠心肌胶原面积百分比(VFC)比较
与20周龄正常血压对照组WKY大鼠相比,20周龄SHR组大鼠心肌胶原面积百分比明显增加(P<0.05)。与SHR组相比,20周龄SHR+QH组和SHR+QL组大鼠心肌胶原面积百分比显著减少(P<0.05),两两比较分析,SHR+QH组与SHR+QL组差异较显著(P<0.05)。
9.心肌组织免疫组织化学PPAR-γ、AP-1(C-Jun, C-Fos)蛋白表达水平比较
1)与20周龄正常WKY大鼠相比,SHR组PPAR-γ的核表达较WKY组明显减少(P<0.05)；SHR+QH组、SHR+QL组较SHR组PPAR-γ核表达明显增多(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
2)与20周龄正常血压对照组WKY大鼠相比,SHR组与WKY组比较,AP-1(C-Jun, C-Fos)核表达明显增多(P<0.05),SHR+QH组、SHR+QL组表达较SHR组均减少(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
10.实时定量RT-PCR法检测心肌组织中肥厚基因的ANP, BNP mRNA的表达水平的比较
1)与20周龄正常血压对照组WKY大鼠相比,SHR组PPAR-γ的mRNA表达量较WKY组明显减少(P<0.05)；与20周龄的SHR大鼠相比较,20周龄SHR+QH组和SHR+QL组较SHR组PPAR-γ mRNA表达明显增多(P<0.05);两两比较分析,SHR+QH组与SHR+QL组比较,差异具有统计学意义(P<0.05)。
2)与20周龄正常WKY大鼠相比,SHR大鼠AP-1(C-Jun, C-Fos), ANP, BNP的mRNA表达明显增多(P<0.05),经槲皮素治疗12周后,SHR+QH组、SHR+QL组AP-1(C-Jun, C-Fos), ANP, BNP的mRNA表达较SHR组均明显减少(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组比较,差异具有统计学意义(P<0.05)。
11.Westernblot检测分析PPAR-γ、AP-1(c-fos, c-jun)蛋白表达水平比较
1)与20周龄正常血压对照组WKY大鼠相比,SHR组PPAR-γ的蛋白表达量较WKY组明显减少(P<0.05)；与20周龄的SHR大鼠相比较,20周龄SHR+QH组和SHR+QL组较SHR组PPAR-γ蛋白表达量明显增多(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
2)与20周龄正常血压对照组WKY大鼠相比,SHR组AP-1(C-Jun, C-Fos)蛋白表达量明显增多(P<0.01),与20周龄的SHR大鼠相比较,20周龄SHR+QH组、SHR+QL组蛋白表达量较SHR组均减少(P<0.05)；两两比较分析,SHR+QH组与SHR+QL组差异具有统计学意义(P<0.05)。
结论
(1)槲皮素对SHR大鼠心室肥厚有明显的逆转作用,其具体表现为：改善SHR大鼠心肌形态学指标,降低心肌组织胶原含量,改善心肌的超微结构和心功能的指标。
(2)槲皮素具有PPAR-γ部分激动剂的作用,在改善心肌肥厚的机制部分可能是激活PPAR-γ抑制肥厚转录因子AP-1(C-Jun, C-Fos)的表达,进而调节其下游ANP、BNP的mRNA的肥厚基因的表达。
研究背景：
心肌肥大是心肌细胞对多种病理刺激如压力过负荷、神经体液因子以及心肌收缩蛋白基因突变等出现的一系列组织细胞形态学的适应性反应,肥大特征表现主要包括心肌细胞体积增大,蛋白质合成增加和胚胎期的表达基因再表达。一方面它是心脏早期的适应性反应,是一种慢性代偿机制,许多神经体液因子和,心肌的旁分泌因子参与到心肌肥厚的适应性反应中。另一方面它又是晚期心脏猝死和其他心血管事件的一个独立的危险因素。因此,逆转高血压心肌肥厚是防治高血压及其靶器官损害的关键所在。
许多研究证实,心脏局部肾素-血管紧张素系统(RAS)在心肌肥厚的发生发展中起着重要作用,而Ang Ⅱ(angiotensin Ⅱ, AngⅡ)是起主要作用的生物活性物质。体内和体外实验都显示AngⅡ是致心肌细胞肥大的重要因子,血管紧张素Ⅱ可以直接促进心脏和血管细胞生长,部分通过各种生长因子和细胞外基质蛋白以及细胞因子的诱导。研究发现包括核转录因子AP-1在内很多信号分子在AngⅡ引发的心肌肥大反应中发挥重要作用,这些转录因子反过来又能调节细胞的生长和细胞外基质的生物合成。大量研究表明,参与构成转录激活因子AP-1家族成员中以C-Jun及C-Fos为主,且以C-Jun/C-Fos异二聚体形式的AP-1生物学活性最强,并且直接抑制AP-1的活性可以直接降低心肌肥厚的发生。因此,当原癌基因如c-fos和c-jun作为初始应答基因时,所编码的产物Fos和Jun作为核内转录调控时因子和第三信使,通过一个亮氨酸拉链形成异源二聚体,与位于5’端上游调控区的AP-1位点(TGACTGA保守序列)结合,进一步促进次级应答基因如心房利尿钠因子(ANF)、脑钠肽(BNP)转录和蛋白质合成,导致心肌细胞蛋白质合成增加,蛋白表型向胚胎型转化等心肌肥厚的特征性变化。因此,对心肌肥大信号转导通路的深入认识,不仅有助于阐明心肌肥厚的细胞分子机制,而且为心肌肥厚的防治提供新的策略。
槲皮素(3,3',4',5,7-pentahydroxyflavone, Que),是自然界中分布最广的生物黄酮类化合物。槲皮素是芩丹胶囊中桑寄生的有效成分,我们前期的实验表明槲皮素能够通过TGF-β1/Smad2信号通路改善TGF-β1诱导的成纤维细胞的增殖。同时,我们的体内动物实验表明槲皮素能有效的抑制SHR大鼠心肌肥厚的发生,但是它对心脏的保护作用的靶点和细胞内的信号转导机制还没有完全的阐述清楚。因此,本研究拟进一步探明槲皮素在抑制心肌肥厚的过程中的相关信号通路。我们通过检测槲皮素对AngⅡ诱导的H9C2心肌细胞肥厚的影响,应用免疫荧光检测PPAR-γ, AP-1(C-Fos, C-Jun)蛋白的表达。同时,采用siRNA阻断PPAR-γ蛋白的表达,应用荧光实时定量RT-PCR检测ANP, BNP mRNA表达情况以及western blot检测C-Fos, C-Jun蛋白表达的改变,探讨槲皮素抑制AngⅡ诱导的H9C2心肌肥厚的生物活性的途径,为其临床防治高血压性心肌肥厚提供科学依据。
目的
1.本研究通过建立血管紧张素Ⅱ诱发的大鼠H9C2心肌细胞肥厚模型,考察槲皮素对心肌细胞表面积、[3H]-亮氨酸的掺入测定的影响。
2.通过siRNA阻断心肌细胞的PPAR-γ途径,考察槲皮素对转录因子C-Fos, C-Jun的蛋白表达及心肌肥厚特异性指标ANP和BNP mRNA表达的影响,探讨槲皮素抑制心肌肥厚的作用机制。
方法
1.常规复苏、培养大鼠心肌细胞H9C2至对数生长期备用,构建血管紧张素Ⅱ诱导的心肌细胞H9C2肥大模型,加入不同浓度的槲皮素,孵育一定时间。
2.采用相差显微镜观察细胞大小,软件分析测量心肌细胞表面积,[3H]-亮氨酸掺入法测定心肌细胞蛋白质合成速率。
3.细胞免疫荧光法测定PPAR-γ、C-Fos及C-Jun的蛋白水平及其细胞内的定位。
4.根据GenebanK中PPAR-γ的序列号,设计并化学合成PPAR-γ siRNA和没有明显同源性的非特异性阴性对照NCsiRNA。
5.常规培养H9C2细胞至对数生长期,种于六孔板中,将实验分为实验组(转染PPAR-γ特异性siRNA),阴性对照组(转染PPAR-γ阴性对照siRNA),空白对照组(不转染任何siRNA,余试剂与其他两组相同),进行转染。
6.采用Western-blot技术分别检测三组细胞转染后PPAR-γ蛋白的表达水平,以检测siRNA对PPAR-γ的阻断效率。
7.细胞转染后,槲皮素与AngⅡ细胞共培养,Western-blot法检测C-Fos, C-Jun的蛋白表达水平,RT-PCR法检测心肌肥厚基因ANP、BNP的mRNA表达水平。
8.统计学处理：计量资料统一采用均数±标准误表示,统计软件SPSS17.0进行数据的相关统计分析。两个组之间的数据比较用独立样本的t检验,多组之间的均数比较用单因素或多因素方差分析(ANOVA)和Bonferroni post-hoc检验。当P<0.05,认为有统计学差异。
结果：
1.各实验组细胞的[3H]-亮氨酸掺入量测定
AngⅡ刺激H9C2细胞建立心肌细胞肥厚模型后,AngⅡ组的[3H]-亮氨酸掺入量与正常对照组相比较明显升高(P<0.01),但是在与槲皮素共培养后,[3H]-亮氨酸掺入量明显降低(P<0.01),且其抑制蛋白合成速率作用呈剂量依赖性。
2.H9C2细胞表面积的测定
AngⅡ刺激H9C2细胞建立心肌细胞肥厚模型后,AngⅡ组的H9C2细胞表面积与正常对照组相比较明显升高(P<0.01),但是与槲皮素共培养后,AngⅡ+Que组H9C2细胞表面积明显降低(P<0.01)。
3.细胞免疫荧光检测
(1)与正常对照组相比,AngⅡ组H9C2心肌细胞免疫荧光法测定PPAR-γ的核表达较正常对照组明显减少(P＜0.01); AngⅡ+Que组H9C2心肌细胞免疫荧光较AngⅡ组PPAR-γ核表达明显增多,有统计学意义(P<0.01)；
(2)与正常对照组相比,AngⅡ组H9C2心肌细胞免疫荧光法测定C-Fos, C-Jun的核表达较正常对照组明显增多(P<0.01)；AngⅡ+Que组H9C2心肌细胞细胞免疫荧光较AngⅡ组的C-Fos, C-Jun核表达明显减少,有统计学意义(P<0.01)。
4. Westernblot检测分析
(1) siRNA转染24h后,与空白对照组相比,实验组PPAR-γ siRNA组的PPAR-γ蛋白表达水平明显下调(P<0.01),与正常细胞组相比,阴性对照组NCPPAR-γ siRNA的表达与空白对照组则无统计学差异(P>0.01)。
(2) siRNA转染48h后,给予刺激AngⅡ和槲皮素的共培养后,与AngⅡ组相比较,PPAR-γ siRNA组的C-Fos, C-Jun无明显下降(P>0.01),阴性对照组NC PPAR-γ siRNA与AngⅡ组比较则明显下降(P<0.01)。
5. RT-PCR检测
(1) siRNA转染24h后,与AngⅡ组相比,实验组PPAR-γ siRNA组的ANPmRNA表达水平无明显差异(P>0.01),阴性对照组NC PPAR-γ siRNA的ANPmRNA表达与AngⅡ组比较则明显下降(P<0.01)。
(2) siRNA转染24h后,与AngⅡ组相比,实验组PPAR-γ siRNA组的BNPmRNA表达水平无明显差异(P>0.01),阴性对照组NC PPAR-γ siRNA的BNPmRNA表达与AngⅡ组比较则明显下降(P<0.01)。
结论
1.槲皮素能够显著抑制AngⅡ诱导的H9C2心肌细胞肥厚。
2.槲皮素可以通过激活PPAR-y,下调AP-1(C-Fos, C-Jun)蛋白及ANP、 BNP mRNA的表达,抑制AngⅡ诱导的H9C2心肌细胞肥厚,从而达到减轻心肌肥厚的作用。
Background
Hypertension is one of the familiar risk factors for the human health. Ventricular hypertrophy is a common complication of hypertension, and it is an independent risk factor for sudden cardiac death and the other cardiovascular events. Ventricular hypertrophy is consist of cardiomyocyte hypertrophy, myocardial interstitial cell proliferation, extracellular matrix accumulation pathologically. Therefore, to explore ventricular hypertrophy mechanisms of hypertension is important for prevention and treatment of hypertensive target organ damage and reduce mortality.
Peroxisome proliferator-activated receptor y (peroxisome proliferater-activated receptor-y, PPAR-y) is a ligand-activated nuclear transcription factor superfamily members. Recent studies have confirmed that PPARy plays a critical role in inhibition of cardiac hypertrophy both in vitro and in vivo. NF-κB activation was essential to the development of cardiac hypertrophy in recent years and inhibition of NF-κB activation may be important targets for myocardial hypertrophy. Furthermore, we also have shown previously that PPAR-y regulates gene expression in a DNA-independent fashion by interfering with other signaling pathways, such as the NF-κB pathway
Qindan Capsule(QC), a prepared compound of traditional Chinese medicine, has been used as an anti-hypertensive agent for years in clinic. It has shown that QC plays an important role in attenuating vascular remodeling in our previous study.In the present study,the changes of the pathology of myocardial hypertrophy were observed in SHR, and the variation of ANP and BNP mRNA expression was detected by RT-PCR, and the variation of PPARy and NF-κB was detected by immunohistochemistry. In order to discover novel agents on inhibiting myocardial hypertrophy and improving cardiac function, the possible mechanism of QC on improveing ventricular hypertrophy in the spontaneously hypertensive rats was investigated. It could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.
Aim
1. To investigate the anti-hypertrophy effects of QC through observing the changes of the morphology and the ultrastructure in myocardial tissues.
2. To investigate the possible mechanism of QC in reversing myocardial hypertrophy in hypertension through observing the changes of the PPARy and NF-κB and the variation of ANP and BNP mRNA expression.
Methods
40male (8weeks old) SHRs and8male (8weeks old) Wistar Kyoto (WKY) rats were randomly divided into5groups(n=8):the WKY control group, the SHR control group, the Telmisartan (SHR+Tel)group, the high dosage QC group (SHR+QCL), and the low dosage QC group (SHR+QCL).The SHR+Tel group was treated with Telmisartan for10mg/kg per day, the SHR+QCH group was treated with high dosage (750mg/kg per day) of QC, and the SHR+QCL group was treated with low dosage(150mg/kg per day)of QC. The WKY group and the SHR group were treated with equal volume of distilled water instead. All the treatments were administered by gastrogavage once a day for12successive weeks.
After the last administration, all rats were fasted with free access to water for24h, and anesthetized with intraperitoneal injecfion of3%pentobarbital (30mg/kg)which was followed by other experiments.The detection contents listed below:(1) Measurement of the systolic blood pressure (SBP);(2) The left ventricular weight (LVW) and body weight (BW) ratio was determined;(3) Observation of cardiomyocyte morphological by means of HE;(4) The ultrastrueture of myocardial cell was observed with transmission electron microscope;(5) Determination of the levels of PPARy,NF-κB in myocardial by immunohistochemistry;(6)Analysis of ANP and BNP mRNA expression by real-time PCR.
Statistical analysis
The data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared. Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of SBP
The SBP of rats in the SHR group with8weeks old was much higher than those in the WKY group(P<0.05). The rats in the SHR group continuously developed further hypertension to a high pressure level. After12weeks, the SBP of rats in the SHR group with20weeks old was significantly higher than those in the WKY group (P＜0.05). From the second week of treatment, the SBP was lower in the SHR+Tel group, the SHR+QCH group, and the SHR+QCL group than that in the SHR group(all P＜0.05). After six weeks of treatment,the SBP in the SHR+QCH group was decreased significantly compared with that in the SHR+QCL group(P＜0.05). After twelve weeks of treatment, the SBP in each group was decreased in different degree compared with the SHR group(all P＜0.05). The SBP in the SHR+QCH group and the SHR+Tel group was lower than that in the SHR+QCL group(P＜0.05), but no significant difference was shown in comparison among these two groups (P＜0.05).
2. Left ventricular relative mass(LVM/BW)
At the end of the experiment, the left ventricular weight index (LVW/BW) in SHRs was significantly greater than in WKY rats. This parameter was dose-dependently and significantly decreased in QC or Tel-treated SHRs compared with distilled water-treated SHRs (P＜0.05).
3. Observation of seetions of myocytes stained with HE
Pathological changes were significantly attenuated by QC or Tel. Hematoxylin and eosin staining was performed for all groups. Compared with the WKY group, cardiac muscle fibers were enlarged and disorganized in the SHR group. Hematoxylin and eosin-stained myocardial tissue in the QC or Tel-treated SHR groups showed reductions in the size of the cardiomyocytes and reduced fibrosis. In addition, cardiac muscle fibers were also better-arranged.
4. Ultrastructural changes of myocytes observed by transmission electron microscopy.
In the WKY group, arrays of myofibrils were closely arranged in an orderly manner within the sarcomere, and mitochondria were of normal size and present in normal numbers. However, in the SHR group, the mitochondrial structure was damaged severely and cellular or tissue swelling was apparent in myocardium. Most myofibrils had either disappeared or were poorly arranged. Mitochondria were noticeably swollen and loosely arranged. In addition, mitochondrial membranes were vague or partly ruptured and cristae were clearly loose or dissolved with many vacuoles. The significant change in ultrastructural organization of mitochondria and myofibrils was attenuated in the SHR+QCH group, the SHR+QCL group and SHR+Tel group with more obvious improvements noted in the high-dose group.
5. Immunohistochemistry detection
(1) Comparison of expression of PPAR-y protein:The expression of PPAR-y protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the three treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
(2) Comparison of expression of NF-κB protein:The expression of NF-κB protein was higher in the SHR group than that in the WKY group (P＜0.05).The expression of NF-κB protein was decreased in the three treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
6. Comparison of expression of ANP and BNP mRNA
(1) Comparison of expression of ANP mRNA:The expression of ANP mRNA was higher in the SHR group than that in the WKY group (P＜0.05).The expression of ANP mRNA was decreased in the QC or Tel treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
(2) Comparison of expression of BNP mRNA:The expression of BNP mRNA was higher in the SHR group than that in the WKY group (P＜0.05).The expression of BNP mRNA was decreased in the QC or Tel treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).
Conclusions
(1) A reversing effect of Qindan capsule on the hypertensive ventricular hypertrophy in SHR was observed in our present study. Qindan capsule could improve the morphological index of myocardial tissue and reduce myocardial tissue collagen content. Furthermore, it improves cardiac function and myocardial ultrastructure.
(2) Qindan capsule may inhibit cardiac hypertrophy by enhancing PPAR-y expression partly and by suppressing the NF-κB signaling pathway, resulting inhibition the transeription of its downstream gene expression of ANP and BNP.
Background
Hypertension is one of the familiar risk factors for the human health. Cardiac hypertrophy results in congestive heart failure and is a major world-wide cause of sudden death. The hypertrophic response in cardiomyocytes is characterized by an enlargement of myocytes, an increase in the content of contractile proteins, and expression of embryonic genes such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The hypertrophic response is compensatory at an early stage of various cardiac diseases, but sustained extracellular stimuli may lead to excessive cardiac hypertrophy and finally to heart failure.
Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor gene family. In recent study also demonstrated the PPAR-y-dependent pathway is critical in the inhibition of cardiac hypertrophy both in vivo and vitro. One major transcription factor is the activator protein1(AP-1) complex, which is composed of the Jun and Fos family of DNA binding protooncoproteins. The c-Jun and c-Fos proteins form dimmers that bind DNA through specific response elements to transactivate the transcription of genes downstream from these enhancer elements.The activation of AP-1has been shown to be involved in the regulation of a variety of cellular processes such as proliferation, differentiation, and transformation. Moreover, recent studies have demonstrated that myocardial AP-1DNA binding activities are significantly increased in experimental cardiac hypertrophy in cardiovascular disease. It can regulate mang hypertrophy gene including such as ANP and BNP. Recently, it has been reported that a dominant negative mutant lacking the transactivating domain of c-Jun (DNJun) inhibits endothelin1(ET)-1and phenylephrine (PE)-induced cardiomyocyte hypertrophy. Furthermore, we also have shown that PPAR-y regulates gene expression in a DNA-independent fashion by interfering with other signaling pathways previously, such as the activator protein-1(AP-1) pathway.
Quercetin (3,3',4',5,7-pentahydroxyflavone) is a member of a group of naturally occurring compounds and is one of the most widely distributed bioflavonoids. The very extensive biological effects of flavonoids, including their anti-inflammatory, anti-oxidant, anti-atherosclerotic, and anti-hypertensive properties facilitate an important role for quercetin in the prevention of cardiovascular diseases. Furthermore, it has also been revealed to show agonistic effects on peroxisome proliferator activated receptors and attenuates Monocyte Chemoattractant Protein-1gene expression in glucose-primed aortic endothelial cells via AP-1.
In the present study, the changes of the pathologies of myocardial hypertrophy were observed in SHR rats, and the variation of PPAR-y and AP-1(C-Jun, C-Fos) was detected by immunohistochemistry or western blot, and the variation of its downstream target genes including ANP and BNP mRNA expression was detected by real-time RT-PCR. In order to discover novel agents on inhibiting myocardial hypertrophy and improving cardiac function, the possible mechanism of quercetin on improveing ventricular hypertrophy in the spontaneously hypertensive rats was investigated, which could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.
Aim
1. To investigate the anti-hypertrophy effects of quercetin through observing the changes of the morphology and the ultrastructure in myocardial tissues.
2. To investigate the possible mechanism of quercetin in reversing myocardial hypertrophy in hypertension through observing the changes of the PPAR-y and AP-1(c-fos, c-jun) and the variation of ANP and BNP mRNA expression.
Methods
Twenty four8-week-old male SHRs and eight8-week-old male Wistar-Kyoto (WKY) rats obtained from Slaccas (Shanghai, China) were used in this study. Animals were housed in a temperature-controlled (22±0.5℃) room, and rats had free access to standard rodent chow and water. A12-h/12-h light/dark cycle was maintained. Spontaneously hypertensive rats were randomly divided into three groups:one group (SHR) was treated with vehicle (1ml of1%methylcellulose) and the other two groups were treated with either a low dose of quercetin (SHR+QL;5mg.kg-1, mixed in1ml of1%methylcellulose) or a high dose of quercetin (SHR+QH;10mg.kg-1, mixed in1ml of1%methylcellulose). Age-matched WKY rats were treated with vehicle (1ml of1%methylcellulosa) and used as a control group. Quercetin or vehicle was delivered by gavage at the same time once daily for12weeks.
After the last administration, all rats were fasted with free access to water for24h, and anesthetized with intraperitoneal injection of3%pentobarbital(30mg/kg)which was followed by other experiments. The detection contents listed below:(1)Measurement of the systolic blood pressure (SBP);(2)The common echocardiographic indexes were detected before at the first and at the end of the experiment;(3) the left ventricular weight (LVW) and body weight (BW) ratio was determined;(4)Measurement of the morphological index of cardiomyocyte diameter of myocytes and volume fraction of collagen;(5) The ultrastrueture of myocardial cell was observed with transmission electron microscope.(6) Analysis of PPAR-γ、 AP-1(c-fos, c-jun) protein expression and location by immunohistochemical detection.(7)Analysis of PPAR-γ AP-1(C-Jun, C-Fos) protein expression by western blot.(8)Analysis of PPAR-γ,AP-1(C-Jun, C-Fos),ANP and BNP mRNA expression by real-time PCR;
Statistical analysis
The data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared.Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of SBP
In the week prior to treatment with quercetin, systolic blood pressure in the SHRs was significantly increased compared with the WKY (P＜0.05). Twenty weeks of quercetin administration induced a significant reduction in SBP in the SHRs, and this effect reached statistical significance after the first week of treatment (P＜0.05). From the second week of treatment, SBP was significantly decreased in the quercetin-treated groups compared with the SHR group (P＜0.05), and the decline in the SHR+QH group was distinct from that of the SHR+QL group (P＜0.05) and was significantly distinct from the7th to the12th week (P＜0.05).
2. Left ventricular weight index (LVW/BW)
Neither SHRs nor WKY rats showed a change in body weight after treatment with quercetin or vehicle. The left ventricular weight index (LVW/BW) in SHRs was significantly greater than in WKY rats. This parameter was dose-dependently and significantly decreased in quercetin-treated SHRs compared with vehicle-treated SHRs (P＜0.05).
3. Echocardiographic detection
Compared with the WKY group, LVPWd and IVSd were both significantly increased at week1or12of the experiment but LVIDd decreased by the12th week in the SHR group (P＜0.05). After12weeks of quercetin treatment, LVIDd was significantly increased but IVSd and LVPWd were apparently decreased compared with the SHRs (P＜0.05). Furthermore, the mid-wall fractional shortening and E/A decreased significantly in SHRs and were significantly improved at week12in SHRs treated with quercetin (P＜0.05).
4. Observation of seetions of myocytes stained with HE
Pathological changes were significantly attenuated by quercetin. Hematoxylin and eosin staining was performed for all groups. Compared with the WKY group, cardiac muscle fibers were enlarged and disorganized in the SHR group. Hematoxylin and eosin-stained myocardial tissue in the quercetin-treated SHR groups showed reductions in the size of the cardiomyocytes and reduced fibrosis. In addition, cardiac muscle fibers were also better-arranged.
5.Comparison of Mean cardiomyocyte diameter of myocytes
Mean cardiomyocyte diameter was increased significantly in the SHR group compared with the WKY group(P＜0.05). Mean cardiomyocyte diamete was decreased significantly in the SHR+QH and the SHR+QL groups compared with that in the SHR group(P＜0.05).Quercetin administration dramatically and dose-dependently attenuated the enlargement in myocyte size compared with SHRs (P＜0.05).
6.Ultrastructural changes of myocytes observed by transmission electron microscopy.
In the WKY group, arrays of myofibrils were closely arranged in an orderly manner within the sarcomere, and mitochondria were of normal size and present in normal numbers. However, in the SHR group, the mitochondrial structure was damaged severely and cellular or tissue swelling was apparent. Most myofibrils had either disappeared or were poorly arranged. Mitochondria were noticeably swollen and loosely arranged. In addition, mitochondrial membranes were vague or partly ruptured and cristae were clearly loose or dissolved with many vacuoles. The significant change in ultrastructural organization of mitochondria and myofibrils was attenuated in the quercetin-treated SHR groups with more obvious improvements noted in the high-dose group.
7. Observation of sections of myocytes stained with Masson
Masson's trichrome staining was performed to assess the effect of quercetin on cardiac fibrosis. The WKY myocardium showed a normal array of myocardial fibers and very little interstitial collagen. The SHR group showed many newly formed collagen deposits between myocardial interstices and myocardial disarrangement and cellular swelling as compared with the WKY group. Myocardial collagen deposition was greater in the SHR+QH group and the SHR+QL group than that in the WKY group, but was less than that in the SHR group.
8. Comparison of the volume fraction of collagen
The VFC of myocytes in the SHR group was higher than that in the WKY group(P＜0.05). The VFC in the SHR+QH group and the SHR+QL group were lower than that in the SHR group (P＜0.05).Quercetin administration dramatically and dose-dependently attenuated the increase in collagen volume fraction compared with SHRs (P＜0.05).
9.Immunohistochemical detection
(1) Comparison of expression of PPAR-y protein:The nucleus expression of PPAR-y protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The nucleus expression of AP-1(C-Jun, C-Fos) protein was higher in the SHR group than that in the WKY group(P＜0.05).The expression of AP-1(C-Jun, C-Fos) protein was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner.(P＜0.05).
10. The mRNA expression of related to hypertrophy gene were detected by RT-PCR
(1) Comparison of expression of PPAR-y mRNA:The expression of PPAR-γ mRNA was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y mRNA was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
(2) Comparison of expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA:The expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA was higher in the SHR group than that in the WKY group(P＜0.05).The expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
11. The Protein levels of PPAR-y and AP-1(C-Jun, C-Fos) were detected by western blot
(1) Comparison of expression of PPAR-y protein:The expression of PPAR-γ protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
(2) Comparison of expression of AP-1(C-Jun, C-Fos) protein:The expression of AP-1(C-Jun, C-Fos) protein was higher in the SHR group than that in the WKY group (P＜0.05).The expression of AP-1(C-Jun, C-Fos) protein was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).
Conclusions:
(1) A reversing effect of quercetin on the hypertensive ventricular hypertrophy in SHR was observed in our present study. Quercetin could improve the morphological index of myocardial tissue and reduce myocardial tissue collagen content. Furthermore improves cardiac function and myocardial ultrastructure.
(2) Quercetin may inhibit cardiac hypertrophy by enhancing PPAR-γ expression partly and by suppressing the AP-1signaling pathway, resulting inhibition the transcription of its downstream gene expression of ANP and BNP.
Background
The changes of cardiac hypertrophy are the adaptive reaction to the disorders of hemodynamics, neurohumor and local endocrinic factors in hypertension.The hypertrophic response in cardiomyocytes is characterized by an enlargement of myocytes, an increase in the content of contractile proteins, and expression of embryonic genes such as atrial natriuretic peptide(ANP) and brain natriuretic peptide (BNP). The hypertrophic response is compensatory at an early stage of various cardiac diseases, but sustained extracellular stimuli may lead to excessive cardiac hypertrophy and finally to heart failure. Therefore, it is important to reverse the cardiac hypertrophy for prevention and treatment of hypertension and target organ damage.
The renin-angiotensin system(RAS) plays a major role in the regulation of blood pressure. AngiotensinⅡ is one of the most important factors in RAS. It is shown one of the important factors to myocyte hypertrophy both in vivo and in vitro experiments. Ang Ⅱ has direct growth-promoting effects on the cardiac and vascular cells, in part via induction of the expression of various growth factors, extracellular matrix proteins and cytokines. In vitro, Ang II could activates a number of inducible transcription factors such as activator protein-1(AP-1), which in turn make impact on the expression of genes that regulate cell growth and extracellular matrix protein biosynthesis. Activator protein-1could be made up of c-fos and c-jun, which together make a heterodimer complex that plays a significant role in cardiomyocyte hypertrophy, and direct inhibition of AP-1activity significantly declined cardiac hypertrophy in myocardial tissue. Therefore, initiation of myocyte hypertrophic growth is accompanied by a rapid and transient expression of immediate-early genes such as c-fos, c-jun at the genetic level, followed by an activation of the fetal gene regulatory program with reexpression of genes for atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP),increase contractile proteins and transformation to embryonal Protein Phenotype.These observations have contributed to speculation that inhibition of these hypertrophic signals may be effective, patent strategies in the treatment of pathological hypertrophy and heart failure.
Quercetin (3,3',4',5,7-pentahydroxyflavone) is a member of a group of naturally occurring compounds and is one of the most widely distributed bioflavonoids. The very extensive biological effects of flavonoids, including their anti-inflammatory, anti-oxidant, anti-atherosclerotic, and anti-hypertensive properties facilitate an important role for quercetin in the prevention of cardiovascular diseases. Furthermore, it has also been revealed to show agonistic effects on peroxisome proliferator activated receptors and attenuates Monocyte Chemoattractant Protein-1gene expression in glucose-primed aortic endothelial cells via AP-1.The above results suggest the quercetin may inhibits cardiac hypertrophy by blocking AP-1(c-fos, c-jun) and activating PPAR-y signaling pathways in vivo, but the possible mechanism of quercetin direct inhibition of cardiac hypertrophy are still unknown in vitro. In the present study, the changes of the protein synthesis and surface area of cells were observed in H9C2cells, and the variation of PPAR-γ and AP-1(c-fos,c-jun) was detected by immunofluorescence. The variation of PPAR-y and AP-1(c-fos,c-jun) was detected by western blotting and real time-PCR, and the variation of ANP and BNP mRNA expression was detected by real-time RT-PCR after transfection with PPAR-γ siRNA in H9C2cells. In order to discover novel agents on inhibiting H9C2hypertrophy, the possible mechanism of quercetin on improveing cardiac hypertrophy in the H9C2cells cell hypertrophy was investigated, which could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.
Aims
(1) This study used the H9C2cells induced by angiotensin Ⅱ as cardiac hypertrophy model to observe the influence of H9C2cell surface area, the [3H] leucine incorporation at different concentration of quercetin stimulus.
(2) To observed the quercetin impact on transcription factor C-Fos, C-Jun protein expression, detection of specific indicators of cardiac hypertrophy such as ANP and BNP mRNA expression by mean of siRNA blocking PPAR-y pathway.
Methods
1. Culture H9C2cell line routinely. The hypertrophy of H9C2cell was induced with Angiotensin Ⅱ, and intervened with quercetin at different concentration.
2. As the index of cardiomyocyte hypertrophy, the cell size was determined by phase contrast microscope and protein synthesis rate was measured by [3H]-Leucine incorporation.
3. Immunofluorescence was used to detect the protein expression and location of PPAR-y and AP-1(c-fos, c-jun).
4. Based on the Genebank cDNA accession number, design and synthesize PPAR-y siRNA oligonueleotides lines. Design and synthesize one negative siRNA oligonueleotides, which are not specific for any known gene.
5. Culture H9C2cells line routinely.Transfect the siRNA to H9C2cells, three groups was designed, negtive, test and control group.
6. After transfection of siRNA24h, western blot was used to detect the expression of PPAR-y transfection efficiency.
7. After H9C2cells were transfected with each siRNA for48h, cells were incubated with quercetin and Angll alone or in combination for24h. Cells were incubated for another24h prior to RNA isolation for quantitative real-time PCR analysis and48h prior to isolating protein samples for western blotting analysis.
8. Statistical analysis
The data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared.Differences were considered to be statistically significant when the P values were less than0.05.
Results
1. Comparison of [3H]leucine incorporation
[3H]leucine incorporation was increased significantly in the Ang Ⅱ-induced group compared with the control group(P＜0.01). However, in quercetin pretreated cells, Ang Ⅱ-mediated incorporation was markedly reduced in a concentration-dependent manner (P＜0.01). At100μg/ml, compared with Ang Ⅱ-induced group protein synthesis was reduced to almost basal levels (64%reduction of Ang Ⅱ stimulated [3H]leucine incorporation)(P＜0.01).
2. Comparison of the surface area of H9C2cells
The surface area of H9C2cells was increased significantly in the Ang Ⅱ-induced group compared with the control group(P＜0.01). However, Quercetin (100μg/mL) pretreatment significantly attenuated this increase in H9C2cells compared with the Ang Ⅱ group (P＜0.01).
3. Immunofluorescence detection
(1) Comparison of expression of PPAR-γ protein:The expression of PPAR-γ protein was declined in the Ang Ⅱ group than that in the control group(P＜0.01) However, the fluorescence of PPAR-γ protein was significantly increased in the quercetin and AngⅡ combination treatment group compared with the Ang Ⅱ group (P＜0.01;).
(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The expression of AP-1(c-fos, c-jun) protein was increased in the Ang Ⅱ group than that in the control group(P＜0.01) However, the fluorescence of AP-1(c-fos, c-jun) was significantly decreased in the quercetin and AngⅡ combination treatment group compared with the Ang Ⅱ group (P＜0.01).
4. Western blotting detection
(1) Comparison of expression of PPAR-γ protein:The expression of PPAR-γ protein was lower in the PPAR-γ NCsiRNA group than that in the control group(P＜0.01) However, the fluorescence of PPAR-γ protein was significantly decreased in the PPAR-γ siRNA group compared with the Ang Ⅱ group (P＜0.01).
(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The expression of AP-1(c-fos, c-jun) protein was higher in the AngⅡ group than that in the control group(P＜0.01). However, the fluorescence of AP-1(c-fos, c-jun) was significantly decreased in the PPAR-y siRNA reatment group compared with the Ang Ⅱ group (P＜0.01).
5. Comparison of expression of ANP and BNP mRNA in H9C2cells
(1) Comparison of expression of ANP mRNA:Transfection siRNA after24hours, the expression of ANP mRNA of PPAR-γ siRNA group was obviously increased compared with the Ang Ⅱ group (P＜0.01). No significant difference was shown in comparison between the Ang Ⅱ group and the PPAR-y NCsiRNA group (P＞0.01).
(2) Comparison of expression of BNP mRNA:Transfection siRNA after24hours, the expression of BNP mRNA of PPAR-y siRNA group was obviously increased compared with the Ang Ⅱ group (P＜0.01). No significant difference was shown in comparison between the Ang Ⅱ group and the PPAR-y NCsiRNA group (P＞0.01).
Conclusions:
(1) A reversing effect of quercetin on Ang Ⅱ induced H9C2cells hypertrophy was observed in our present study.
(2) Quercetin may inhibit Ang Ⅱ induced H9C2cells hypertrophy by enhancing PPAR-y expression partly and by suppressing the AP-1signaling pathway, resulting inhibiting the transcription of its downstream gene expression of ANP and BNP.
引文
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