摘要
前言
现有的研究提示,糖尿病心血管病并发症的发病机制可能有多种因素通过多种途径参与。糖尿病引起的内皮功能异常、胰岛素抵抗、心肌代谢异常是糖尿病心肌重构和心肌舒张功能障碍致心功能不全的始动环节。AMP依赖蛋白激酶(AMPK),因可能通过多种途径参与保护心血管效应而受到越来越广泛的关注。目前认为AMPK是胰岛素敏感性的一种正调节因子,它不仅参与细胞内脂肪酸氧化代谢,还可通过调节GLUT4和mTOR的表达影响心肌细胞的糖代谢以及蛋白质合成。
近年来,Ghrelin的心脏保护作用逐渐被人们重视,尽管作用机制仍在探讨之中,但其在心血管系统的作用成为目前研究的热点。Ghrelin在体研究表明,下丘脑内注射外源性Ghrelin,可以通过激活AMPK调节机体的能量代谢;细胞水平的研究表明,Ghrelin可以通过激活AMPK改善血管内皮细胞的氧化应激。总之,越来越多的科学研究表明,Ghrelin可能与细胞代谢的关键酶AMPK有着密切的联系。
本研究应用外源性的Rat-Ghrelin注射于大鼠腹腔,观察其对大鼠心脏功能、血糖血脂代谢、心脏病理结构的影响,系统评价Ghrelin对自发性糖尿病大鼠的干预作用;采用分子生物学方法,观察Ghrelin对糖尿病大鼠心肌代谢关键酶AMPK以及信号转导通路下游所调控的GLUT4和mTOR表达的影响,以探讨Ghrelin确切的心血管效应及其可能的分子机制,为临床防治代谢综合征提供新的理论依据和治疗靶点。
材料与方法
一、实验材料
SPF级12周龄雄性GK大鼠(Goto-Kakisaki Wistar rats)24只和SPF级12周龄雄性Wistar大鼠8只,购于上海斯莱科实验动物有限责任公司。分组干预4周后取血,并采集心肌及胸主动脉标本。Ghrelin ELISA试剂盒购于南京生物技术公司;Ghrelin多克隆抗体购于博士德生物公司;Rat-Ghrelin、P-AMPK多克隆抗体、GLUT4单克隆抗体、mTOR单克隆抗体、β-actin单克隆抗体、RT-PCR试剂盒等购于上海Sirocco Pharmaceutic公司.
二、实验方法
大鼠编号随机分为4组:Wistar组:蒸馏水灌胃4周;GK组:同等容量蒸馏水灌胃4周;二甲双胍组:二甲双胍350mg/kg/d灌胃4周;Ghrelin组:Rat-Ghrelin200μg/kg/d腹腔注射4周。检测各组大鼠血糖、血脂、胰岛素、内皮素、一氧化氮、肿瘤坏死因子-α、Ghrelin等生化指标。12导生理记录仪测量左心室收缩压、左心室舒张末压、左心室压力最大上升速率、左心室压力最大下降速率。采用Masson特染技术以及免疫组化技术对各组大鼠心肌、胸主动脉的组织超微结构和病理学检查。Western bolt方法检测各组大鼠心肌、胸主动脉组织mTOR蛋白、AMPK、GLUT4蛋白表达水平。RT-PCR方法检测各组大鼠心肌、胸主动脉组织mTOR蛋白、AMPK,GLUT4基因表达。
三、统计学处理
计量资料以均数±标准差((?)±s)表示,所有的数据均通过SPSS13.0处理。对于正态分布的变量采用方差分析进行多细问比较,方差不齐者用秩和检验,采用t检验进行两组间比较;非正态分布的变量经对数转换后采用t检验。以P<0.05为差异有统计学意义。
实验结果
一、各组大鼠体重及Lee指数比较
实验干预因素处理前,四组大鼠体重无统计学差异(P>0.05);实验4周后,Ghrelin组和二甲双胍组大鼠的体重增长较GK对照组减少(P<0.05);二甲双胍组大鼠体重增长最缓慢,接近于正常对照组。所有自发性糖尿病大鼠的Lee指数在实验干预因素处理前,均高于Wistar组大鼠(P<0.05);实验4周后,二甲双胍组和Ghrelin组大鼠的Lee指数无显著性差异(P>0.05),均明显低于GK对照组(P<0.05)。
二、各组大鼠收缩压变化
实验干预因素处理前,所有自发糖尿病大鼠血压均高于Wistar大鼠(P<0.05);实验4周后,各组大鼠收缩压水平无显著变(P>0.05)化;Ghrelin组和二甲双胍组收缩压仍显著高于Wistar大鼠,较GK组的收缩压水平略低,但差异不显著(P>0.05)。
三、各组大鼠糖脂代谢相关指标的比较
实验干预因素处理4周后,GK组大鼠的HW/BW、FBG、FINS、HOMA-IR、TC、TG、LDL-C、TNF-a明显高于另外三组(P<0.05);Ghrelin组的上述指标均明显低于GK组(P<0.05);二甲双胍组HW/BW、FBG、FINS、HOMA-IR、TC、TG、TNF-a明显低于GK组(P<0.05),但LDL-C与GK组差异不显著(P>0.05)。
四、各组大鼠内皮功能相关指标的比较
GK组大鼠血浆ET-1、心肌ET-1、心肌iNOS、血清iNOS高于Wistar组(P<0.05),心肌NO、血清cNOS、心肌cNOS、血清NO低于Wistar组(P<0.05);Ghrelin组大鼠血浆ET-1、心肌ET-1、心肌iNOS、血清iNOS较GK组大鼠降低(P<0.05),心肌NO、血清cNOS、心肌cNOS、血清NO较GK组大鼠升高(P<0.05)。二甲双胍组血浆ET-1、心肌ET-1、心肌iNOS、心肌NO、血清cNOS、血清iNOS、心肌cNOS较GK组大鼠降低(P<0.05),心肌iNOS与无显著差别(P>0.05)。
五、各组大鼠血流动力学指标的变化
实验4周后,与Wistar组大鼠比较,GK组大鼠的心率、LVEDP升高(P<0.05);而LVESP、±dp/dtmax降低(P<0.05);Ghrelin四周干预后的GK大鼠心率、LVEDP降低,LVESP、±dp/dtmax较Wistar组大鼠显著升高(P<0.05);与Wistar组大鼠比较,二甲双胍组心率、LVEDP降低,±dp/dtmax显著升高(P<0.05),但LVESP无明显差异(P>0.05)。
六、大鼠心脏HE染色及心肌Masson染色
Wistar组大鼠心肌纤维无肥大,细胞核呈圆形或椭圆形,据于细胞中心,心肌间质结构正常。GK组大鼠心肌细胞肥大、深染,心肌细胞边界不清,细胞核大小不均匀,心肌Masson染色提示心肌间质增生;Ghrelin干预后,光镜下心肌细胞形态基本完整,无肿大,Masson染色提示心肌间质增生较GK对照组明显减轻。二甲双胍组心肌细胞形态基本完整,无肿大,但Masson染色提示仍有较明显心肌间质增生。
七、各组大鼠血浆Ghrelin水平比较及各组大鼠主动脉及心肌免疫组化染色
GK组大鼠血浆及主动脉心肌组织免疫组化半定量显示Ghrelin表达水平明显低于Wistar组(P<0.05)。应用Ghrelin干预组大鼠的血浆Ghrelin水平及免疫组化半定量显示主动脉心肌组织Ghrelin表达水平高于其它三组(P<0.05),二甲双胍组亦高于GK组(P<0.05)。
八、各组大鼠心肌AMPK,GLUT4,mTOR相对蛋白水平及mRNA表达量比较
GK组大鼠心肌p-AMPK、GLUT4相对蛋白表达水平较Wistar组低(P<0.05),mTOR相对蛋白表达水平较Wistar组高(P<0.05);Ghrelin及二甲双胍干预后大鼠的心肌p-AMPK、GLUT4相对蛋白表达水平较Wistar组有所增加(P<0.05),mTOR相对蛋白水平较Wistar组有所降低(P<0.05),尤以Ghrelin组更显著(P<0.05)。GK组大鼠心肌AMPKα2、GLUT4 mRNA表达水平较Wistar组低(p<0.05),mTORmRNA表达水平较Wistar组高(P<0.05);Ghrelin及二甲双胍干预后大鼠的心肌AMPKα2、GLUT4 mRNA表达水平较Wistar组有所增加(P<0.05),mTOR mRNA水平较Wistar组有所降低(P<0.05),尤以Ghrelin组更显著(P<0.05)。
结论
自发性糖尿病大鼠存在严重的胰岛素抵抗和心肌重构以及主动脉内皮结构功能改变,生长激素释放肽Ghrelin在自发性糖尿病大鼠血浆及心肌主动脉组织中表达减少。Ghrelin腹腔注射可减轻自发性糖尿病大鼠胰岛素抵抗,改善血管内皮结构和功能。Ghrelin腹腔注射可提高自发性糖尿病大鼠心脏左室舒张和收缩功能,抑制心肌细胞间质增生,其机制可能与较长期Ghrelin在体干预上调心肌细胞AMPK表达而促进葡萄糖转运及抑制蛋白质合成有关。
Background
Ghrelin,a growth hormone releasing peptide,is the endogenous ligand for the growth hormone secretagogue receptor and has been detected in cardiovascular tissues. Recently,some studies indicated that exogenous ghrelin had some protective effects on cardiovascular system.
However,the cardioprotective effect and the mechanism of ghrelin have not been elucidated.Diabetes mellitus frequently produces cardiocyte hypertrophy and collagen deposition,changes referred to as LV remodeling.Recently experimental studies have suggested that the AMP-activated protein kinase(AMPK) participate in the regulation of cardioprotective effect.Some studies have also shown that ghrelin is associated with AMPK.In addition,recent studies show that the AMPK pathway was involved in preventing or suppressing LV remodeling through its effects on glucose transporter type 4 and mammalian TOR(mTOR).
In the present experiment,we studied the effect on cardiovascular system and expression of AMPK in the heart of the Goto-Kakizaki(GK) rats injected with ghrelin. The effects of ghrelin on the GLUT4 and mTOR associated with AMPK and LV remodeling were also investigated.
Methods
Goto-Kakizaki(GK) rats(8-week-old males) were randomly divided into three groups of 8 rats each:Ghrelin(received ghrelin 200 g/kg sc twice daily);Metformin at 350mg/kg sc;saline controls.Normal male Wistar kyoto rats(n=8) served as normal controls.Four weeks later,the effects of ghrelin on cardiac remodeling were evaluated by echocardiographic,hemodynamic,histopathological,and gene expression analysis (AMPK,GLUT4.......,mTOR protein and mRNA expression).The serum level of ghrelin was examined by ELISA.
Results
1、Goto-Kakizaki(GK) rats were combined with cardiovascular system diseases.
2、Ghrelin has the certain protective effects on cardiovascular system.
3、Ghrelin prevented ventricular remodeling,increased AMPK and GLUT4 expression in the myocardium,suppressed mTOR protein and mRNA expression.
Conclusions
Ghrelin has the certain protective effects on cardiovascular system.Ghrelin inhibits ventricular remodeling induced by diabetes mellitus,and the preventive effects of ghrelin may be mediated by the AMPK-depended pathway.
引文
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