新化合物RapA治疗2型糖尿病实验模型GK大鼠糖尿病心脏损伤和糖尿病心肌病的作用及其机制
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摘要
研究背景和目的
大量的研究已经证实,丝氨酸/苏氨酸激酶Akt,也称作蛋白质激酶B (PKB),异常活化的或活化受到抑制的Akt,其参与一系列重要的复杂疾病的病理生理过程,与动脉粥样硬化、2型糖尿病及癌症等密切相关。哺乳动物Akt/PKB家族由三种亚型组成：Akt1, Akt2和Akt3,编码这三个亚型的基因位于三个不同的基因座位,但其在功能结构域上具有相似的保守结构。对Akt基因编码区进行比对之后,相似性达到85%。Akt激酶蛋白大约由480个氨基酸残基组成,从NH2末端到COOH末端依次为PH结构域、催化结构域和调节结构域,均参与Akt调节活化。Akt激酶的PH结构域位于细胞质膜,与磷脂酰肌醇三磷酸(PIP3)结合从而被募集到细胞质膜,继而催化结构域中Thr308和Ser473位点发生磷酸化。Akt在哺乳动物各种组织均有丰富表达,Akt1表达于哺乳动物大多数组织,Akt2主要在胰岛素敏感的器官中表达丰度甚高,例如肝脏,骨骼肌,脂肪组织和心脏等；Akt3主要在脑组织和睾丸中表达丰富。
Akt活化在体内主要依赖两大信号途径来完成。第一种途径依赖PI3-kinase激酶活化的方式,该方式主要是通过PI3-kinase的活化来完成的。PI3-kianse能合成4种3’端-磷酸肌醇3 - A phosphatidylinositol [PI (3) P]、3,4 phosphatidylinositol [PI (3,4) P2]、3,5 - phosphatidylinositol [PI (3,5) P2]和3,4,5-trisphosphate [PI (3,4,5) P3],其中PI(3,4)P2和PI(3,4,5)P3部分激活Akt。通常在静息状态下,Akt激酶主要是停留在细胞胞浆,若受到细胞因子或外界应激等其他因素的刺激,继而转到细胞质膜与PIP3结合,从而活化,接着回到细胞胞浆或转移到细胞核中活化发挥调节作用。抑制剂LY294002和渥曼青霉素均可抑制PI3-kinase活性,起到调节Akt活化的作用。还有一条不依赖上游激酶分子直接活化Akt的途径,这里不再详述。Akt作用底物众多,包括线粒体凋亡相关的Bad、caspase家族,心肌肥厚相关的FOXO家族,组织代谢相关的GSK3α/β,炎症相关的IKK家族,还有与第二信使NO相关的NOS家族,所以Akt不仅仅在调节糖脂和蛋白质等物质代谢方面,而且在对细胞增殖、分化和凋亡坏死等方面均扮演重要的角色。
糖尿病(Diabetes mellitus, DM)是慢性进行性疾病,包括1型糖尿病和2型糖尿病,妊娠期糖尿病及其遗传性糖尿病等多个类型。2型糖尿病患者比例占到整个糖尿病患者90%以上,至今全球的糖尿病流行趋势愈来愈严重。糖尿病并发症严重情况下可致人死亡,例如酮症酸中毒。糖尿病心肌病或糖尿病心脏损害是一类以心脏功能不全但不合并心脏冠脉病变和高血压为特征的疾病。一般认为高糖血症,高脂血症和高胰岛素血症是糖尿病心肌损害和糖尿病心肌病发病机制的重要危险因素,也是诱发一系列不良的反应最后导致心脏的纤维化和胶原的沉积的致病因素。围绕改善这些危险因素,从上世纪五十年代,即有大量的实验性和治疗性化学性药物出现,例如盐酸二甲酸胍(metformin),阿卡波糖(Acrobose),重组人胰岛素(体内埋置微量释放)和AICAR,但这些药物本身定位就是控制某些危险因素,对已经发生的靶器官危害和并发症,治疗效果就不甚乐观。Akt激酶在整个心脏的代谢和生存水平上起到的作用已经日渐明了。化合物RapA是国内首次从菌株中发现和证实,国外未见报道。本研究中我们假设在2型糖尿病发病后的稳定期内,各组织器官尚未出现明显损害和并发症,使用化合物RapA长期口服于2型糖尿病模型动物Goto-kakizaki大鼠,研究其对胰岛素抵抗或长期糖尿病影响下的对心脏产生的损害诸如心脏纤维化、心肌细胞线粒体凋亡的治疗作用和作用的分子机制,同时在体外研究本化合物是直接激活Akt还是由其上游分子激活导致的Akt活化,通过小干扰RNA沉默上游信号分子,以明确化合物RapA是否仍能抑制由游离脂肪酸所导致的心肌氧化应激、线粒体功能障碍和细胞凋亡,进一步明确该化合物的抗击糖尿病心肌损害或糖尿病心肌病的作用靶点和分子信号机制。
一、体内实验化合物RapA口服治疗改善Goto-kakizaki 2型糖尿病大鼠心脏损伤和糖尿病心肌病的作用及其机制
实验目的：探讨化合物RapA长期口服喂养2型糖尿病模型GK大鼠改善糖尿病心肌损害和糖尿病心肌病的作用及其分子信号机制
实验方法：
1.选择2型糖尿病模型Goto-kakizaki大鼠,其对照组实验动物为wistar大鼠；两者遗传背景一致；
2.实验动物GK大鼠从第6周喂养高脂饮食至第40周止,对照wistar大鼠,从第6周喂养普通饮食至第40周止；
3.从第24周开始口服化合物RapA-生理盐水悬液至第40周,周期为16周；其中wistar组为10只正常wistar大鼠,其他30只GK大鼠随即分为3组,每组10只大鼠,分别为：GK大鼠组喂服与喂药等体积的生理盐水,GK大鼠喂服RapA每天药量2.5mg/kg, GK大鼠喂服RapA每天药量5.0mg/kg。实验前留取血液标本以及24小时尿。实验动物按照美国Charles river实验室的GK大鼠资料,每2-4周称取体重。直至实验结束处死前。所有的实验动物操作程序均符合美国NIH Publication NO.85-23(1996)守则,以及华中科技大学同济医学院附属同济医院实验动物中心颁布之规范和规定。
4.实验动物按照经口灌胃之方法给药,给药量分为两个剂量,分别为2.5mg/kg.d-1和5.0mg/kg.d-1. Wistar大鼠对照和GK大鼠对照均口服相同体积的生理盐水。处死前一周检测所有实验动物的心脏功能,采用心脏超声法。留取血液标本和24小时尿；行口服葡萄糖耐量实验,然后采用内置导管和导丝方法检测心率和心脏左室压力；上述实验完成后,处死所有实验动物。取心脏、肝脏、肾脏、脂肪、胰腺、骨骼肌、脑组织和主动脉等重要器官组织放入液氮冷冻数小时候转入超低温冰箱保存,以上所诉组织均留取部分4%甲醛溶液固定,经脱水浸蜡包埋后常温保存待用；
5.采用口服葡萄糖耐量的方法检测化合物RapA长期口服对2型糖尿病GK大鼠葡萄糖耐量的影响；
6.采用酶联免疫技术检测大鼠空腹血浆胰岛素,心脏cyclic AMP、caspase-3和caspase-9的含量；应用化学法检测血浆临床化学指标,心肌NADPH oxidase、SOD和MDA水平,利用相应公式计算出相对活性；
7.腹腔大剂量(10U/kg)注射人重组胰岛素激活实验动物胰岛素敏感器官的胰岛素信号通路；
8.应用免疫印迹法(western blotting)检测wistar大鼠和2型糖尿病模型GK大鼠心脏磷酸化Akt的水平(即Akt活化水平)；检测心肌磷酸化insulin receptor-β、磷酸化insulin receptor substrate-1、磷酸化PI3-kinase-p85α、PI3-kinase-p110α、磷酸化AS160、GLUT4转位、磷酸化GSK3α/β、磷酸化PKCε、gp91phox、p67phox、p47phox、p40phox、磷酸化ERK1/2、磷酸化JNK1、BCL-2、Bad、Bax、剪切caspase-3和剪切caspase-9的水平。
实验结果：
1.各个血浆临床化学指标显示,2型糖尿病模型GK大鼠在空腹胰岛素、空腹甘油三酯、空腹血糖、总胆固醇和游离脂肪酸水平远远高于正常对照wistar大鼠水平,体重则出现明显下降,这表明GK大鼠的2型糖尿病的症状非常明显；通过16周RapA口服喂养GK大鼠后,与口服等体积生理盐水的GK大鼠组的临床化学指标相比,空腹血浆胰岛素和血糖明显下降(p<0.05)；总胆固醇水平明显下降(p<0.05)；给予2.5mg/kg.d-1化合物RapA的GK大鼠血浆游离脂肪酸水平明显下降(p<0.05),但是给予5.0mg/kg.d-1化合物RapA的GK大鼠血浆游离脂肪酸水平则没有明显改善(p>0.05)。口服化合物RapA对糖尿病所致的体重减轻,没有明显作用。
2.经millar导管和导丝检测对照wistar大鼠、对照GK大鼠和口服两种剂量的GK大鼠的心脏左室压力,并无明显的改变(p>0.05)；长期口服化合物RapA没有改变GK大鼠的心率的水平,其快于正常对照wistar大鼠。
3.口服葡萄糖耐量实验结果显示,2型糖尿病模型GK大鼠经过16周口服化合物RapA,其全血葡萄糖水平与正常对照wistar大鼠相比仍然处于较高水平,无明显改善作用(p>0.05); 5.Omg/kg.d-1给药剂量的GK大鼠的全血葡萄糖水平在0、30、60和90分钟时间点与口服等体积生理盐水的GK大鼠相比明显降低(p<0.05),2.5mg/kg.d-1给药剂量的GK大鼠的全血葡萄糖水平仅在60和90分钟时间点相比口服等体积生理盐水的GK大鼠有明显降低(p<0.05)。
4.正常对照wistar大鼠与GK大鼠喂服或不喂服化合物RapA对心脏体重比没有明显作用(p>0.05)；通过HE染色计算各组大鼠心肌细胞面积,确定各组大鼠心肌细胞面积没有明显的差异(p>0.05)。
5.通过MASSON三色染色检测各组大鼠心肌组织横切面的心肌胶原纤维含量,结果为：1)口服两种剂量化合物RapA, GK大鼠心肌胶原纤维水平仍然高于正常wistar大鼠心肌的胶原纤维水平；2)通过16周的口服化合物RapA, 2.5mg/kg.d-1和5.0mg/kg.d-1剂量分别使得GK大鼠的心脏血管周围胶原纤维含量从6.182±0.373(%)减低到5.218±0.409(%)、4.532±0.331(%)水平(p<0.05)；2.5mg/kg.d-1和5.0mg/kg.d-1剂量分别使得GK大鼠的心肌细胞间的胶原纤维含量从9.81±0.68(%)减低到8.77±0.65(%)和4.32±0.68(%)水平(p<0.05),改善作用具有显著性差异。
6.依靠心脏超声检测各分组实验动物的心功能,再依据相应的计算公式计算出实验动物的心功能的各个参数,结果为：1)IVS、LV-Pw和LVs参数无明显差异；2)GK大鼠的部分心功能参数Lvd、EF%和FS%仍然低于正常对照wistar大鼠水平(p<0.05),有显著性差异；3)两个口服剂量2.5mg/kg.d-1和5.0mg/kg.d-1能够明显升高EF%和FS%水平相比口服等体积生理盐水的GK大鼠(p<0.05)。
7.免疫印迹法(western blotting)检测结果显示：
1)口服两个剂量2.5mg/kg.d-1和5.0mg/kg.d-1的大鼠心肌Akt激酶Ser473和Thr308位点在受到外源性大剂量的胰岛素刺激后,相比于口服等体积生理盐水的GK大鼠,其磷酸化水平明显上调(p<0.05)；即使在没有外源性胰岛素刺激下,其磷酸化Akt水平也明显上调(p<0.05)。
2) GK大鼠+2.5mg/kg.d-1组和GK大鼠+5.0mg/kg.d-1组注射外源性胰岛素后Insulin receptorβTyr1152/1153位点磷酸化水平较对照GK大鼠明显上调(p<0.05)；
3) GK大鼠+2.5mg/kg.d-1组和GK大鼠+.5.0mg/kg.d-1组注射外源性胰岛素后insulin receptor substrate-1 Ser307磷酸化水平较对照GK大鼠显著性下调(p>0.05)；
4) Insulin receptor substrate-1 S er612磷酸化水平仅口服较高剂量(5.0mg/kg.d-1)明显较对照GK大鼠下调(p<0.05),但是较低的计量(2.5mg/kg.d-1)同对照GK大鼠相比则没有显著性改变(p>0.05)；
5) Insulin receptor substrate-1 Tyr 895磷酸化水平仅GK大鼠+5.0mg/kg.d-1组明显上调(p<0.05)；
6)心肌组织Thr642 AS160磷酸化水平相比于对照GK大鼠口服两种剂量均上调明显(p<0.05),并且存在剂量依赖性,较高剂量比较低剂量上调更多(p<0.05),而对照GK大鼠心肌Thr642 AS160磷酸化即使在胰岛素刺激后也无法激活,而口服化合物RapA后GK大鼠即使没有外源性胰岛素刺激也可激活AS160；
7) GLUT4转运至心肌细胞质膜水平在长期口服化合物RapA明显上调(p<0.05)；
8)口服RapA对GK大鼠心肌PI3-kinase p110α水平无影响；长期口服RapA同外源性胰岛素激活PI3-kinase p85αTyr 458位点磷酸化程度无明显差异(p<0.05),而相比较于对照GK大鼠上调明显(p<0.05),而对照GK大鼠心肌PI3-kinase p85αTyr 458在外源性胰岛素刺激下也未能明显激活相比较与正常wistar大鼠(p>0.05)；
9)心肌Ser21/9 GSK3α/β和Ser729 PKCε磷酸化水平受到长期服用RapA的影响相比于对照GK大鼠下调明显(p<0.05)；
10)心肌NADPH oxidase各亚基蛋白gp91phox、p67phox、p47phox和p40phox等表达水平受到RapA的作用相比于对照GK大鼠明显下调(p<0.05)；
11)与线粒体相关蛋白Bcl-2、Bax、Bad、剪切caspase-3和剪切caspase-9在RapA作用下相比较于对照GK大鼠,其在心肌的水平明显降低(p<0.05)；
12) GK大鼠+2.5mg/kg.d-1组和GK大鼠+5.0mg/kg.d-1组心肌Thr185/Tyr187 ERK1/2磷酸化水平相比对照GK大鼠组明显升高(p<0.05)；Thr183/Tyr185 JNK磷酸化水平则明显下调(p<0.05)；用药后GK大鼠两者的磷酸化水平,前者仍然低于正常wistar大鼠,后者仍然高于正常wistar大鼠；
8.ELISA检测cyclic AMP在心肌中的表达水平,利用公式计算出相对活性(%),
结果为：
1)正常wistar大鼠对照：6.35±1.89(%)；
2)对照GK大鼠组：43.07±5.91(%)；
3) GK+2.5mg/kg.d-1组：32.52±5.91(%)；
4) GK+5.0mg/kg.d-1组：26.07±2.63(%)；
两个剂量的RapA作用下,cyclic AMP水平明显下调(p<0.05)；
9.ELISA检测心肌caspase-3和caspase-9的表达水平,并计算相对活性(%),结果为：同对照GK大鼠组比较caspase-3和caspase-9在喂药GK大鼠心肌的相对活性均显著性降低(p<0.05)；
10.采用化学法检测大鼠心肌NADPH oxidase和SOD水平,然后计算出相对活性,结果为：正常对照wistar大鼠NADPH oxidase活性远低于GK大鼠水平,而SOD的相对活性则正好相反；通过16周口服化合物RapA,用药组NADPH oxidase相对活性显著性降低(p<0.05),SOD相对活性则显著性升高(p<0.05)；
11.化学法检测大鼠心肌MDA含量,同未口服RapA GK大鼠相比,喂服RapA的GK大鼠MDA含量显著性降低(p<0.05)
二、体外实验化合物RapA抑制高浓度FFA导致心肌细胞线粒体功能障碍和凋亡
实验目的：体外利用小干扰RNA技术沉默化合物RapA的作用靶点Akt激酶上游信号分子,该化合物能否抑制高浓度游离脂肪酸作用下产生的氧化应激、线粒体功能低下和细胞凋亡的初步信号传导模式.
实验方法：
1.利用wst-1试剂确定化合物RapA对心肌细胞系H9C2(2-1)的IC50；
2.使用PI3-kinase-p85α的小分子干扰RNA抑制心肌细胞系H9C2(2-1) PI3-kinase-p85α基因的表达；
3.高浓度游离脂肪酸干预心肌细胞系H9C2(2-1)检测在化合物RapA与p85a小干扰RNA共同作用下心肌细胞系活性氧产生的水平,线粒体凋亡和细胞凋亡的水平；
4.利用报告基因技术检测在高浓度游离脂肪酸干预心肌细胞系H9C2(2-1)下加入化合物RapA同时转染抗氧化应激反应元件-荧光素酶报告基因质粒和小分子干扰RNA的沉默p85α情况下,检测抗氧化应激反应元件-荧光素酶的相对活性。
实验结果：
1.利用不同浓度RapA干预大鼠心肌细胞系H9C2(2-1),计算出用药浓度与细胞数之间的关联,计算出RapA在大鼠心肌细胞系的IC50为88.31uM；利用western blotting检测RapA体外即可在较小浓度激活Akt,即同单独加入外源性胰岛素孵育细胞,其浓度为5uM时即可明显激活Akt；明显低于该细胞的IC50剂量；
2. PI3-kinase p85α的siRNA在干预大鼠心肌细胞48小时后明显抑制其蛋白水平(p<0.05)；
3.在加入RapA (5uM)作用下FFA(游离脂肪酸)-100uM对心肌细胞产生的活性氧,导致的线粒体和细胞凋亡水平明显下调(p<0.05),即使在沉默PI3-kinase p85a表达情况下；
4.以同样方法,转染心肌细胞系ARE-荧光素酶报告基因质粒,检测其相对活性,结果为RapA (5uM)作用于心肌细胞系不依赖p85a提高其相对活性,显著性抑制FFA所致的ARE相对活性减低(p<0.05)。
统计学分析
所有数据均以均数±标准误表示,应用SPSS13.0软件进行统计学分析,各分组组间差异采用单因素方差分析(ANOVA),以P<0.05为差异具有统计学显著性意义。
结论
1.化合物RapA降低代谢相关的风险因素水平,比如空腹胰岛素和血糖,血总胆固醇和游离脂肪酸。
2. 2型糖尿病所致的糖耐量受损在部分时间点经由化合物RapA口服治疗改善明显。
3.化合物RapA拮抗由长期糖尿病诱导的心脏纤维化导致的心功能不全。
4.经RapA治疗,由心脏胰岛素抵抗导致的心脏纤维化水平降低。
5. RapA的治疗作用抑制由胰岛素抵抗导致的胰岛素信号受阻,提高胰岛素刺激的敏感性,并且激活了PI3K-Akt信号通路,包括直接激活Akt,促进GLUT4向细胞膜转运。
6.经过长期口服RapA治疗2型糖尿病GK大鼠的线粒体功能障碍和由活性氧诱导的心肌细胞凋亡受到明显抑制。
7. RapA诱导Akt激酶的直接性的活化而不再需要通过上游信号分子PI3-kinase的亚基p85α激活抑制高浓度游离脂肪酸所引起的心肌细胞的凋亡。
Numerous studies have shown that serine/threonine kinase kinase Akt, also known as protein kinase B (PKB), is an important upstream signal molecules associated with growth factors, cytokines, and other cellular stimulations, which leads to downstream signaling activation in vivo. Abnormal activation or inhibition of activation of Akt has participated in a number of important pathophysiological processes of complex diseases, including atherosclerosis, type 2 diabetes and cancer. Akt family consists of three mammalian isoforms:Akt1, Akt2, Akt3, the encoding genes of isoforms in the three different gene loci, which have similar conserved domain in functional structure. The coding regions of the Akt family genes were comparised, the similarity to 85%. In principle, Akt, about 480 amino acid residues from the NH2 to the COOH end of the order of the end of PH domain, catalytic domain and regulatory domain are involved in regulating Akt activation. Aktl, recruited to the plasma membrane followed by the PH domain binding to the cell membrane phosphatidylinositol triphosphate (PIP3), has effect on phosphorylation of the catalytic domain Thr308 and Ser473. Akt in mammals are expressed in various tissues; Aktl in most mammalian tissues; Akt2 isforms is mainly high in the insulin-sensitive organs such as liver, skeletal muscle, adipose tissue and heart et al; Akt3 isoform is mainly rich in the male reproductive system and brain.
Diabetes (Diabetes mellitus, DM) is a kind of chronic progressive diseases, including type 1 diabetes and type 2 diabetes, gestational diabetes and other genetic diabetes. Type 2 diabetes which accounts for 90% of the diabetic patients, the incidence numbers are increasing every year. Diabetes as a disease class is the result of its final outcome in all body organs involved, leading to organ dysfunction caused by tissue complications, severe cases can cause death. Cardiac damage or diabetic cardiomyopathy is a kind of damage to cardiac dysfunction, but not including coronary artery disease and hypertension for the combined features of the disease. In general, hyperglycemia, hyperlipidemia and hyperinsulinemia in diabetic myocardial damage are main important rist factors in the pathogenesis of diabetic cardiomyopathy, and they also induced a series of adverse reactions, eventually lead to cardiac fibrosis and collagen deposition. Focus on improving these risk factors, from the fifties of last century, that a large number of experimental and therapeutic drugs appear, such as guanidine hydrochloride acid, acarbose, recombinant human insulin (embedded trace the release of the body) and the AICAR, but Thees drugs themselves are to control the positioning of certain risk factors, target organ damage has occurred and complications, the treatment effect is not very optimistic. Akt kinase in the heart of the level of metabolism and the role of survival has been increasingly clear. However, in specific ways to improve diabetes ultimately lead to myocardial damage or the occurrence of diabetic cardiomyopathy, the incidence of death or the development of heart failure, Akt agonist compounds RapA therapeutic effect and mechanism of the official concerned in this study. This compound is the first found from the strains and confirmed that there are no reports abroad.
Through this study, we assume that at the onset of type 2 diabetes of stable period, tissues and organs have not yet obvious damage and complications. By use of long-term oral compound RapA in the Goto-kakizaki rats, we would study the compound effects on insulin resistance and heart under the influence for the heart, such as cardiac fibrosis, myocardial mitochondrial function and apoptosis. In vitro studies, to determine whether compound RapA could activate Akt kinase directly or through the activation of upstream molecules of Akt kinase by small interfering RNA silencing, we use that free fatty acids could lead to myocardial oxidative stress, loss of mitochondrial function and apoptosis, which was able or unable to protect against damages to cardiac cells.
Methods
Materials
RapA was obtained from Doctor Zhang (North China pharmaceutical Group Corporation, China). A siRNA pool kit for p85alpha of PI3Ks'subunit was purchased from Thermo fisher scientific (Shanghai, China).Human recombinant Insulin was received from Wanbang Pharmaceutical Corporation (Xuezhou, China).Plasma glucose assay kit; total cholesterol quantitation kit, free fatty acids quantitation kit and, kit were obtained from Biovision corporate (Mountain View, California). Luciferase plasmid of p-ARE (antioxidant response element)-luc was provided from Beyotime Institute of Biotechnology (Shanghai, China).
Animals, experimental design and drug treatment
Adult male diabetic Goto-Kakizaki rats (Slack, China)(n=40) and age-matched non-diabetic Wistar rats(Slack, China) were used for the study. Animals'houses were maintained in temperature (22±2℃) and light-controlled (12h light/dark cycle) environment; standard rodent high fat diet foods, chow foods and fresh water were provided to age-matched diabetic Goto-kakizaki and non-diabetic Wistar rats respectively.
At 20-22 weeks of age, body weight and other physiological indicators (fast insulin, plasma glucose, triglyceride and cholesterol) of diabetic Goto-Kakizaki rats were markedly stable, which were compared with earlier indicators, relative to lean Wistar controls as previously reported.
To determine whether RapA agonist would protect against cardiac injuries, appropriate group (n=10) of rats were oral treated for 16 weeks, from age 24 to 40 weeks, with RapA 2.5mg/kg/day and 5.0mg/kg/day in 0.9%NaCl buffer respectively.
The investigation conformed to the Guide for Care and Use of laboratory Animals published by the US National Institutes of Health (NIH Publication NO.85-23 revised 1996).All experimental protocols were approved by Tongji medical college committee for Animals care and use.
Echocardiography
Echocardiographic analysis was performed using a commercially available echocardiograph (VIVID 7, General Electric) equipped with a 15-MHz linear array ultrasound transducer. Parameters needed for the calculation of cardiac function and dimensions were measured from five systole-diastole cycle at least. According to Teichholz method, short axis view of the left ventricle at the level of the papillary muscles was obtained by the linear transducer as previously described above. Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were measured from LV M-mode tracing (with a sweep speed of 50mm/s) at the papillary muscle level; LV fractional shortening (FS) and ejection fraction (EF), measures of LV systolic function, were calculated from LV M-mode by the following equations:
FS%=[(LVEDD-LVESD)/LVEDD]×100
EF%=[(LVEDV)-(LVESV)/LVEDV]×100.
Measurement of cardiovascular variables
For measurement of heart beat ratio and left ventricular pressure, a 2.0-French ultra miniature conductance catheter (MPVS400; Millar Instruments, USA) was introduced through the right jugular vein into the left ventricle as described previously.
Pathology and Histological analysis
The whole heart was fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin. Paraffin-embedded samples were sliced to 6um-thick sections, which were stained with Hematoxylin-eosin staining and Masson's tri-Chrome staining for detecting collagen. To determine whether RapA could take effect on the size of cardiomyoctyes cross-sectional area and the degree of myocardial fibrosis,10 fields were randomly chosen respectively, and the size of cardiomyocytes was measured by use of Image J 3.0 software, and the cardiac collagen fraction was calculated as the ratio of Masson's trichrome fibrosis area to total myocardium area with the software of Image Pro-plus 4.0.
Biochemical measurements and OGTT
Methods and measurements for plasma glucose, total cholesterol, triglyceride and free fatty acids were performed and calculated as appropriate kits'manuals and instructions respectively. An oral glucose test (OGTT,5g/kg body weight) was performed after all rats fasted 12 hours at least. Blood was drawn from the tail vein at 0,30,60,90 and 120 min after the oral glucose treatment. Whole blood glucose concentrations were measured by the Johnsons'Lifescan Ultra 2 meter instrument (Shanghai, China).
Fast insulin assay
Plasma insulin levels were determined by Rat/mouse Insulin ELISA kit (St.Charles, Millipore).
Insulin signaling in vivo
Male diabetic Goto-kakizakti rats and age-matched non-diabetic Wistar rats were fed with the above mentioned HFD and chow foods for 36 weeks by the oral treatment of RapA (12wk) and saline, respectively. Body weights of all rats were measured before the day for injection of human recombinant insulin. Rats were fasted 12 hours at least and intraper-i toneally injected with insulin (10U/kg body weight) or equal volume of 0.9% NaC1. After 30 min, rat tissues were immediately excised, frozen in liquid nitrogen for 1-2 hours at least, and kept at-80℃until homogenization and other analysis.
Western blots
Western blotting was performed as previously described.
Detection of NADPH oxidase activity, MDA assay, Superoxide Dismutase (SOD) Activity
NADPH oxidase activity, MDA and superoxide dismutase (SOD) activity were measured by the colorimeric assay method with commercial kit (Biovision Inc., USA) according to the manufacturer's instructions.
Caspase activity assay
Caspase 3 and caspase 9 activity assay were performed as above mentioned.
Evalution of cAMP in vivo and invitro
Cyclic AMP in vivo and invitro was evaluated using cAMP assay kit (Cellsignaling, MA, USA) according to the assay protocol.
Primary cardiac cell culture
A method of isolation of rat neonatal cardiomyocytes was as previously reported. The isolated cardiomyocytes were grown and maintained as recommend-ed at 37℃in 5% CO2 in DMEM modified to contain 4Mm L-glutamine,4.5g/L glucose, 1mM sodium pyruvate, 2.0g/L sodium bicarbonate,3.0g/L HEPES, added 10% fetal bovine serum (FBS, Gibco, USA) and appropriate antibiotics.
Determination of IC50 and AKT kinasc activity in primary cardiac cells
To determine the cytotoxic activity and apoptosis dose of RapA, cell proliferation reagent WST-1 (Roche, USA) was used for the measurement of IC50% with the treatment of com-pound RapA in primary cardiac cells. AKT kinase activity in vitro was measured by AKT activity kit (Cellsignaling, MA, USA) as the instruction of the manufacture. Calculation of IC50 in vitro was by use of Excel 2007 (Microsoftware, USA).
Measurement of ROS in vitro and apoptosis assay
Palmitic(C 16:0) was provided from Sigma Aldrich (Shanghai, China).BSA (fatty acids-free) was purchased from Roche (Shanghai, China). A method of preparation of free fatty acids was performed as previously reported. Primary cardiomyocytes (2.0-4.0x105 cells/ml) were incubated with 10-20uM DCF-DA (Invitrogen, USA) for 30-40 min at 37℃. The DCF fluorescence was measured by FACS (Beckman) with ex/em wavelength of 485/525nm. The percentage of neonatal cardiac cells apoptosis was confirmed using Mitochondrial Membrane potential/Annvexin V apoptosis kit (Invitrogen, USA) and the annvexin V-FITC apoptosis Detection kit (Sigma, China). Stained cells were analyzed as above mentioned.
Luciferase assay
Report gene assay was performed as previously described.
Statistical analysis
Data are means±standard deviation (SD). Differences between groups were assessed using Student's t test or ANOVA followed by Least-significant Difference, Neuman-Keuls and Bonferroni post hoc testing, where appropriate. P<0.05 indicated significance. All experiments were carried out at least 3 times.
Results
Metabolic and physiological characteristics in control and diabetic Goto-kakizaki rats After the 12-week oral treatment of compound RapA, there was no significantly difference on body weight between the untreated and the compound-treated RapA Goto-kakizaki rats, however, the body weight of the control non-diabetic wistar rats was significantly higher than that of other groups, at age of 36 weeks, fast glucose, insulin, triglyceride and total cholseterols levels were significantly higher in diabetic than in control rats. Furthermore, compared with the untreated Goto-kakizaki rats group, the compound treatment (2.5mg/kg.d-1,5.0mg/kg.d-1) tended to decrease the levels of fast plasma insulin, fast plasma glucose and total cholesterol. The RapA-treatment did not take effect on fasting triglyceride levels between the untreated and the compound-treated groups. The untreated and compound treated GK rats exhibited significantly higher heart rate compared with the wistar control, but left ventricular pressure did not have difference between the untreated and the RapA administration (2.5mg/kg.d-1,5.0mg/kg.d-1) groups(p>0.05).
The oral administration of compound RapA enhances oral glucose tolerance in the diabetic Goto-kakizaki rats partly
Enhancement of glucose tolerance is one of the most critical standards for determining the effectiveness of drugs for diabetic cardioprotecting; we performed OGTT to evaluate whether the compound RapA could inhibit damage of glucose tolerance. At the appropriate time point the levels of whole blood glucose in the diabetic Goto-kakizaki groups were still greatly higher compared with those of the non-diabetic control group. For 16-week treatment (5.0mg/kg.d-1) of compound RapA to Goto-kakizaki rats, the levels of glucose obtained 0,30,60 and 90 min in the diabetic GK rats after glucose intake were significantly lower than that in the untreated GK rats (5.12±0.94 vs.7.62±1.10 p<0.05; 11.36±1.68 vs.14.60±2.47 p<0.05; 15.86±1.97 vs.20.82±1.74 p<0.05 and 18.04±1.48 vs. 22.22±1.83 p<0.05, respectively). Furthermore, the lower dose treatment (2.5mg/kg.d-1) of compound RapA to the diabetic GK rats in the levels of whole blood glucose was significantly lower than those of the untreated diabetic and the higher dose administration rats at 60 and 90 min (15.58±2.37 vs.20.82±1.74 p<0.05 and 17.38±3.32 vs.22.22±1.83 p<0.05; 15.58±2.37 vs.15.86±1.97 p<0.05 and 17.38±3.32 vs.18.04±1.48 p<0.05, respectively). This data indicate that the oral administration of compound RapA attenuated damage of glucose tolerance and has anti-hyperglycemic effect on Goto-kakizaki rats.
Reducement of diabetic cardiac fibrosis by use of compound RapA
No differences in ratios of heart weight to body weight (Hw/Bw) and the cardiomyocyte size in a cross-sectional area were noted between the wistar control group and the diabetic GK groups.Furthermore, heart cross-sections were stained with Masson Trichrome for the determination of the extent of cardiac fibrosis at age of 40 weeks with and without the oral administration of compound RapA. Morphologically, collagen deposition tended to increase in response to long term diabetes but was inhibited by compound RapA. On quantitative analysis of perivasculum and interstitial fibrosis in the heart by fibrosis area fraction, compound RapA (2.5mg/kg per day,5.0mg/kg per day) significantly decreased perivasculum and interstitium fibrosis compared with the untreated GK rats (5.218±0.409 vs.6.182±0.373 and 4.532±0.331 vs.6.182±0.373 p<0.05; 8.77±0.65 vs.9.81±0.68 and 4.32±0.68 vs.9.81±1.35 p<0.05, respectively).
Compound RapA ameliorates dysfunction of diabetic heart
Evaluation of echocardiography exhibited that the EF %(ejection fraction) and FS %(fractional shortening percentage) diameters in the oral treatment (2.5mg/kg per day, 5.0mg/kg per day) of diabetic GK groups at age of 40 weeks were markedly larger (70.20±1.17 vs.66.43±3.20 p<0.05,77.71±2.37 vs.66.43±3.20 p<0.05; 34.80±0.75 vs. 32.29±2.25 p<0.05,36.29±2.12 vs.32.29±2.25 p<0.05, respectively), however, there were no markedly differences in the indicators of IVS, LVPW, LVd and LVs.
Activation of Akt kinase and insulin resistance are enhanced and attenuated by oral administration of compound RapA
Our results showed that the compound RapA promoted myocardial Akt Ser473 and Akt Thr308 phosphorylation in the treated diabetic GK rats but not in the control GK rats following intravenous insulin markedly, expectedly, Ser473 and Thr308 phosphorylation of Akt were significantly increased by the use of compound RapA without the stimulus of insulin (Figure 4). Ser307 and Ser612 phosphorylation of insulin receptor substrate-1 were significantly decreased in the treated GK rats'hearts compared to the untreated GK rats. Furthermore, similar to the effect of oral administration of compound RapA on Akt Ser473 and Thr308 phosphoylation, Tyr1152/1153 Insulin Receptor beta and Tyr895 Insulin Receptor substrate-1 phosphorylation were significantly increased in hearts of the treated GK rats relative to the untreated GK control. These data demonstrated that the cardio-protecting effect of compound RapA attenuated insulin resistance in Goto-kakizaki rats.
AS160 phosphorylation and translocation of cytoplasmic GLUT4 to membrane are promoted by the stimulus of compound RapA
Following in vivo intravenous injection of insulin, we observed markedly effects on phosphorylation of Akt's downstream substrate AS 160 after 16 weeks oral treatment of compound RapA to the diabetic Goto-kakizaki rats. Insulin resistance-mediated glucose uptake to inhibit phorsphorylation of Akt and its downstream target AS 160 led us to believe that GLUT4 translocation was significantly impaired in the diabetic Goto-kakizaki rats compared with the treated GK rats, and the levels of translocation of GLUT4 to myocardium membrane were no difference between the lower dose (2.5mg/kg per day) treatment group and the higher dose (5.0mg/kg per day) treatment group.
Effect on Akt's upstream signaling molecules-p85 and p110alpha subunits of PI3K in vivo
To examine whether Akt's upstream signaling molecules can be activated by compound RapA, we assessed p110alpha and p85 subunits of PI3K activation in heart lysates. At the stimulus status of insulin, p-p85 subunit of PI3K was evaluated in hearts from the lower dose (2.5mg/kg per day) and the higher dose (5.0mg/kg per day) treated GK rats compared with the GK control rats; furthermore, similar to the effect on Akt kinase, Tyr458 phosphorylation of p85 subunit of PI3K was significantly increased by use of compound RapA (2.5mg/kg.d-1, 5.0mg/kg.d-1) without the administration of insulin (Figure 6A). Unexpectedly, expression of pllOalpha subunit of PI3K in the GK rats with treatment of compound RapA was lower than those of the untreated GK control rats in vivo. These results showed that compound RapA can affect the activation of p85 subunit of PI3K-Akt signaling pathway for protecting heart damage not via pllOalpha subunit of PI3K in the diabetes status.
Compound RapA activates Akt kinase's downstream signaling molecules relative to mitochondrial dysfunction in diabetic cardiac myocytes.
To test whether compound RapA protects the heart from long term diabetes mediated by its activation of GSK3α/β, the total expression and phosphorylation of GSK3α/βwere determined from the hearts of the wistar control, the untreated GK control and the treated GK rats. The results showed that compound RapA significantly suppressed Ser21/9 phosphorylation of GSK3α/βinduced by diabetes in the treated GK control rats compared with the untreated diabetic rats; furthermore, we found that compound RapA induced a significant decrease of GSK3a/p phorsphorylation between the non-diabetic wistar group and the treated GK group (Figure 7A), it was an effect that was unexpected. It was observed that phorsphorylation of PKCεchanged by compound RapA was similar to changes of GSK3α/βin vivo. To detect whether cyclic AMP activity was reduced by compound RapA, we examined the concentrations of myocardial cyclic AMP as above method. These results demonstrated that relative cyclic AMP activity in GK control rats was significantly higher (43.07±5.91 vs.6.35±1.89) compared with the non-diabetic wistar rats (Figure 7C); expectedly, the lower dose (2.5mg/kg.d-1) and the higher dose (5.0mg/kg.d-1) treatment were markedly lower (32.52±5.91 vs.43.07±5.91,26.07±2.63 vs. 43.07±5.91, respectively) compared with the untreated GK rats.
The results obtained in the previous experiment promoted us to evaluate whether compound RapA could indirectly or directly affect expression of NADPH oxidase and NADPH oxdiase activity induced by mitochondrial dysfunction in long term diabetes. We assessed expressions of subunits of NADPH oxdiase in heart lysates, including gp91 phox, p67phox, p47phox and p40phox. Expressions of gp91 phox and p47phox in the treated higher dose group were significantly decreased compared with the untreated GK group; it was no difference between the lower dose group and the GK control. Oppositely, expressional levels of p67phox and p40phox were lower in lower dose group than those of GK control and the higher dose group respectively; furthermore, it did not differ between the GK control and the higher dose group in levels of expression of p67phox and p40phox. Expressions of NADPH oxdiase in the treated GK rats approached but did not return to the normal state of the wistar rats. We exhibited that relative NADPH oxidase activity in the oral treatment (2.5mg/kg per day, 5.0mg/kg per day) of diabetic GK groups at age of 40 week were significantly downregulated (2.96±0.08 vs.3.44±0.25,2.53±0.24 vs.3.42±0.25, respectively). As the purpose of evaluating the effect of antioxidant by compound RapA, we investigated relative SOD activity and level of MDA in heart tissue. Figure 7E, indicating myocardial relative SOD activity, showed that it was downregulated by 4-fold in GK control group and enhanced by 2-fold and 3-fold in the lower dose (2.5mg/kg per day) and the higher dose (5.0mg/kg per day),respectively. Myocardial MDA level was increased by 1.7-fold in the GK control and inhibited by 14% and 24% in two dose treatment group, respectively.
To explore whether compound RapA can inhibit apoptosis of mitochondria induced by ROS, we examined myocardial Bax, Bad, Bcl-2, cleaved caspase-3 and cleaved caspase-9 expression by western blotting. Western blotting showed that decreases in Bad, cleaved caspase-3 and cleaved caspase-9 expression in the treated GK rats including the lower and the higher dose groups compared with those of untreated GK rats; furthermore, compound RapA did not reduce the expression of Bax in treated GK rats. Similar to the effect on expression of cleaved caspase-3 and caspase-9, relative activities of caspase-3 and caspase-9 were markedly lower in treated GK rats than those in GK control rats and showed dose dependent way.
Thus, we tended to believe that compound RapA had anti-ROS-formation effect and inhibit the progression of apoptosis of cardiac mitochondria in long term diabetes.
The compound RapA activates p44/42MAPK (ERK1/2) and inhibits JNK
ERK (p44/42MAPK) is an important molecule for protecting diabetic heart in the pathological development of myocyte apoptosis and cardiac fibrosis; oppositely, activation of JNK could inhibit restoration of diabetic cardiac function and increase cardiac apoptosis and fibrosis. So, we examined the activation levels of ERK and JNK in hearts of GK rats with or without treatment of compound RapA and the non-diabetic wistar control rats. The results demonstrated that Thrl85/Tyrl87 phosphorylation of ERK1/2 were significantly upregulated compared with the GK control group without the oral administration of compound RapA for 16 week (Figure 7H); furthermore, by the effect of compound RapA, the activation levels of ERK1/2 in hearts of two oral treatment GK group approached the normal status of phosphorylation of ERK1/2 in the non-diabetic wistar rats. Phosphorylation levels of cardiac JNK1 was significantly lower in the diabetic GK rats treated with 5.0mg/kg per day of compound RapA for 16 week compared with the GK control rats, however,2.5mg/kg per day for 16 week of compound RapA did not affect the phosphorylation of cardiac JNK1 in appropriate diabetic group.
Compound RapA inhibits ROS generation and apoptosis mediated by FFA in primary cardiac cells not via PI3K's subunit-p85a
FFA (free fatty acids) is closely involved in the development of type 2 diabetes, which generates reactive oxygen species in heart and finally leads to myocardium apoptosis. According to the results above, to test whether compound RapA could take effect on activation of AKT for protecting diabetic heart directly, we assessed the expression of PI3K's p85αsubunit with p85αgene siRNA pool in primary cardiac cell. The results showed that p85αprotein expression was unaltered in cells exposed to a negative control siRNA sequence; however, siRNA pool against PI3K's p85αsubunit reduced protein expression markedly compared with cardiac cells transfected with negative control siRNA after 48 hours. Relative intracellular ROS levels generated by FFA (100UM) for 48h were markedly increased (305.67±12.73 vs.5.77±0.36, p<0.05) compared with the negative control in primary cardiac cells; Furthermore, compound RapA (5UM) for 48h added to primary cells reduced relative ROS levels markedly (204.42±6.07 vs.305.67±12.73), compared with the FFA (100UM) group. In accordance with compound RapA treatment, siRNA pool-mediated silence of PI3K's p85a in cardiac cells displayed no significantly difference on relative intracellular ROS levels (204.42±6.07 vs.198.02±11.76, p<0.05) compared with the FFA(100UM)+RapA(5UM) group.
The apoptotic effect of ROS was partly mediated by transcriptional factors responded to ROS. To study whether compound RapA could inhibit transcriptional factor's activity induced by ROS, a luciferase reporter gene construct containing antioxidant response element was transfected into primary cardiac cells with or without compound RapA or p85α-siRNA. Similar to the results above, relative luciferase activities of ARE plasmid of the FFA (100UM)+RapA group and the FFA (100UM)+RapA+p85α-siRNA group were significantly higher (1.42±0.08 vs.0.61±0.12,1.24±0.07 vs.0.61±0.12) than those of the FFA (100UM) group. These data suggested that compound RapA could reduce generation of ROS-mediated by free fatty acids and activities of transcriptional factors induced by ROS by activation of AKT kinase directly not via its upstream signal molecule-PI3K's subunit p85a in vitro.
Previously reports have indicated that ROS mediated by FFA could upregulate apoptosis level of cardiac cells in vitro. Here, we determined whether FFA-induced apoptosis of primary cardiac cells is prevented by compound RapA with or without the treatment of p85α-siRNA in vitro. Two days after the co-treatments of FFA and compound RapA and transfection of p85a siRNA, levels of mitochondrial and cellular apoptosis were assessed by FACS; the methods of FACS were performed as described above. Stimulation of cardiac cells with FFA (100UM) significantly induced the cellular and mitochondrial apoptosis by 5.9-and 4.2-fold, respectively, furthermore, compound RapA largely blocked the cellular and mitochondrial apoptotic effect of free fatty acids, expectedly, and addition of p85a-siRNA did not prevent the anti-apoptotic effect of compound RapA. These results suggested that the anti-apoptotic effect of compound RapA was mediated in a large part through activation of AKT kinase directly not via its upstream signal molecule-PI3K's subunit p85a.
Discussion
This study was undertaken to examine the therapeutic effect of compound RapA on the cardiac damage in the long term diabetes. The important findings in the study were:(i) compound RapA reduces metabolic levels of risk factors, including fast plasma insulin, fast plasma glucose, total cholesterol and free fatty acids (FFA); (ii) the damaged glucose tolerance induced by type 2 diabetes may well be ameliorated by the oral treatment of compound RapA in part; (iv) compound RapA protects against the cardiac dysfunction induced by cardiac fibrosis; (iv) cardiac fibrosis induced by insulin resistance could be weakened by the administration of compound RapA; (Ⅴ) compound RapA inhibits insulin resistance and enhances the senstivity of the stimulation of insulin, and activates the PI3K-Akt signaling pathway, including the phosphorylation of Akt, membrane translocation of GLUT4; (Ⅵ) mitochondrial dysfunction and apoptosis induced by reactive oxygen species in myocardium may well be suppressed by compound RapA; (Ⅶ) the apoptotic effect of free fatty acid in vivo may de repressed by compound RapA through the activation of Akt kinase diretly not via the upstream signaling molecule-PI3-kinase's subunit p85α.
大量的研究已经证实,丝氨酸/苏氨酸激酶Akt,也称作蛋白质激酶B (PKB),异常活化的或活化受到抑制的Akt,其参与一系列重要的复杂疾病的病理生理过程,与动脉粥样硬化、2型糖尿病及癌症等密切相关。哺乳动物Akt/PKB家族由三种亚型组成：Akt1, Akt2和Akt3,编码这三个亚型的基因位于三个不同的基因座位,但其在功能结构域上具有相似的保守结构。对Akt基因编码区进行比对之后,相似性达到85%。Akt激酶蛋白大约由480个氨基酸残基组成,从NH2末端到COOH末端依次为PH结构域、催化结构域和调节结构域,均参与Akt调节活化。Akt激酶的PH结构域位于细胞质膜,与磷脂酰肌醇三磷酸(PIP3)结合从而被募集到细胞质膜,继而催化结构域中Thr308和Ser473位点发生磷酸化。Akt在哺乳动物各种组织均有丰富表达,Akt1表达于哺乳动物大多数组织,Akt2主要在胰岛素敏感的器官中表达丰度甚高,例如肝脏,骨骼肌,脂肪组织和心脏等；Akt3主要在脑组织和睾丸中表达丰富。
Akt活化在体内主要依赖两大信号途径来完成。第一种途径依赖PI3-kinase激酶活化的方式,该方式主要是通过PI3-kinase的活化来完成的。PI3-kianse能合成4种3’端-磷酸肌醇3 - A phosphatidylinositol [PI (3) P]、3,4 phosphatidylinositol [PI (3,4) P2]、3,5 - phosphatidylinositol [PI (3,5) P2]和3,4,5-trisphosphate [PI (3,4,5) P3],其中PI(3,4)P2和PI(3,4,5)P3部分激活Akt。通常在静息状态下,Akt激酶主要是停留在细胞胞浆,若受到细胞因子或外界应激等其他因素的刺激,继而转到细胞质膜与PIP3结合,从而活化,接着回到细胞胞浆或转移到细胞核中活化发挥调节作用。抑制剂LY294002和渥曼青霉素均可抑制PI3-kinase活性,起到调节Akt活化的作用。还有一条不依赖上游激酶分子直接活化Akt的途径,这里不再详述。Akt作用底物众多,包括线粒体凋亡相关的Bad、caspase家族,心肌肥厚相关的FOXO家族,组织代谢相关的GSK3α/β,炎症相关的IKK家族,还有与第二信使NO相关的NOS家族,所以Akt不仅仅在调节糖脂和蛋白质等物质代谢方面,而且在对细胞增殖、分化和凋亡坏死等方面均扮演重要的角色。
糖尿病(Diabetes mellitus, DM)是慢性进行性疾病,包括1型糖尿病和2型糖尿病,妊娠期糖尿病及其遗传性糖尿病等多个类型。2型糖尿病患者比例占到整个糖尿病患者90%以上,至今全球的糖尿病流行趋势愈来愈严重。糖尿病并发症严重情况下可致人死亡,例如酮症酸中毒。糖尿病心肌病或糖尿病心脏损害是一类以心脏功能不全但不合并心脏冠脉病变和高血压为特征的疾病。一般认为高糖血症,高脂血症和高胰岛素血症是糖尿病心肌损害和糖尿病心肌病发病机制的重要危险因素,也是诱发一系列不良的反应最后导致心脏的纤维化和胶原的沉积的致病因素。围绕改善这些危险因素,从上世纪五十年代,即有大量的实验性和治疗性化学性药物出现,例如盐酸二甲酸胍(metformin),阿卡波糖(Acrobose),重组人胰岛素(体内埋置微量释放)和AICAR,但这些药物本身定位就是控制某些危险因素,对已经发生的靶器官危害和并发症,治疗效果就不甚乐观。Akt激酶在整个心脏的代谢和生存水平上起到的作用已经日渐明了。化合物RapA是国内首次从菌株中发现和证实,国外未见报道。本研究中我们假设在2型糖尿病发病后的稳定期内,各组织器官尚未出现明显损害和并发症,使用化合物RapA长期口服于2型糖尿病模型动物Goto-kakizaki大鼠,研究其对胰岛素抵抗或长期糖尿病影响下的对心脏产生的损害诸如心脏纤维化、心肌细胞线粒体凋亡的治疗作用和作用的分子机制,同时在体外研究本化合物是直接激活Akt还是由其上游分子激活导致的Akt活化,通过小干扰RNA沉默上游信号分子,以明确化合物RapA是否仍能抑制由游离脂肪酸所导致的心肌氧化应激、线粒体功能障碍和细胞凋亡,进一步明确该化合物的抗击糖尿病心肌损害或糖尿病心肌病的作用靶点和分子信号机制。
一、体内实验化合物RapA口服治疗改善Goto-kakizaki 2型糖尿病大鼠心脏损伤和糖尿病心肌病的作用及其机制
实验目的：探讨化合物RapA长期口服喂养2型糖尿病模型GK大鼠改善糖尿病心肌损害和糖尿病心肌病的作用及其分子信号机制
实验方法：
1.选择2型糖尿病模型Goto-kakizaki大鼠,其对照组实验动物为wistar大鼠；两者遗传背景一致；
2.实验动物GK大鼠从第6周喂养高脂饮食至第40周止,对照wistar大鼠,从第6周喂养普通饮食至第40周止；
3.从第24周开始口服化合物RapA-生理盐水悬液至第40周,周期为16周；其中wistar组为10只正常wistar大鼠,其他30只GK大鼠随即分为3组,每组10只大鼠,分别为：GK大鼠组喂服与喂药等体积的生理盐水,GK大鼠喂服RapA每天药量2.5mg/kg, GK大鼠喂服RapA每天药量5.0mg/kg。实验前留取血液标本以及24小时尿。实验动物按照美国Charles river实验室的GK大鼠资料,每2-4周称取体重。直至实验结束处死前。所有的实验动物操作程序均符合美国NIH Publication NO.85-23(1996)守则,以及华中科技大学同济医学院附属同济医院实验动物中心颁布之规范和规定。
4.实验动物按照经口灌胃之方法给药,给药量分为两个剂量,分别为2.5mg/kg.d-1和5.0mg/kg.d-1. Wistar大鼠对照和GK大鼠对照均口服相同体积的生理盐水。处死前一周检测所有实验动物的心脏功能,采用心脏超声法。留取血液标本和24小时尿；行口服葡萄糖耐量实验,然后采用内置导管和导丝方法检测心率和心脏左室压力；上述实验完成后,处死所有实验动物。取心脏、肝脏、肾脏、脂肪、胰腺、骨骼肌、脑组织和主动脉等重要器官组织放入液氮冷冻数小时候转入超低温冰箱保存,以上所诉组织均留取部分4%甲醛溶液固定,经脱水浸蜡包埋后常温保存待用；
5.采用口服葡萄糖耐量的方法检测化合物RapA长期口服对2型糖尿病GK大鼠葡萄糖耐量的影响；
6.采用酶联免疫技术检测大鼠空腹血浆胰岛素,心脏cyclic AMP、caspase-3和caspase-9的含量；应用化学法检测血浆临床化学指标,心肌NADPH oxidase、SOD和MDA水平,利用相应公式计算出相对活性；
7.腹腔大剂量(10U/kg)注射人重组胰岛素激活实验动物胰岛素敏感器官的胰岛素信号通路；
8.应用免疫印迹法(western blotting)检测wistar大鼠和2型糖尿病模型GK大鼠心脏磷酸化Akt的水平(即Akt活化水平)；检测心肌磷酸化insulin receptor-β、磷酸化insulin receptor substrate-1、磷酸化PI3-kinase-p85α、PI3-kinase-p110α、磷酸化AS160、GLUT4转位、磷酸化GSK3α/β、磷酸化PKCε、gp91phox、p67phox、p47phox、p40phox、磷酸化ERK1/2、磷酸化JNK1、BCL-2、Bad、Bax、剪切caspase-3和剪切caspase-9的水平。
实验结果：
1.各个血浆临床化学指标显示,2型糖尿病模型GK大鼠在空腹胰岛素、空腹甘油三酯、空腹血糖、总胆固醇和游离脂肪酸水平远远高于正常对照wistar大鼠水平,体重则出现明显下降,这表明GK大鼠的2型糖尿病的症状非常明显；通过16周RapA口服喂养GK大鼠后,与口服等体积生理盐水的GK大鼠组的临床化学指标相比,空腹血浆胰岛素和血糖明显下降(p<0.05)；总胆固醇水平明显下降(p<0.05)；给予2.5mg/kg.d-1化合物RapA的GK大鼠血浆游离脂肪酸水平明显下降(p<0.05),但是给予5.0mg/kg.d-1化合物RapA的GK大鼠血浆游离脂肪酸水平则没有明显改善(p>0.05)。口服化合物RapA对糖尿病所致的体重减轻,没有明显作用。
2.经millar导管和导丝检测对照wistar大鼠、对照GK大鼠和口服两种剂量的GK大鼠的心脏左室压力,并无明显的改变(p>0.05)；长期口服化合物RapA没有改变GK大鼠的心率的水平,其快于正常对照wistar大鼠。
3.口服葡萄糖耐量实验结果显示,2型糖尿病模型GK大鼠经过16周口服化合物RapA,其全血葡萄糖水平与正常对照wistar大鼠相比仍然处于较高水平,无明显改善作用(p>0.05); 5.Omg/kg.d-1给药剂量的GK大鼠的全血葡萄糖水平在0、30、60和90分钟时间点与口服等体积生理盐水的GK大鼠相比明显降低(p<0.05),2.5mg/kg.d-1给药剂量的GK大鼠的全血葡萄糖水平仅在60和90分钟时间点相比口服等体积生理盐水的GK大鼠有明显降低(p<0.05)。
4.正常对照wistar大鼠与GK大鼠喂服或不喂服化合物RapA对心脏体重比没有明显作用(p>0.05)；通过HE染色计算各组大鼠心肌细胞面积,确定各组大鼠心肌细胞面积没有明显的差异(p>0.05)。
5.通过MASSON三色染色检测各组大鼠心肌组织横切面的心肌胶原纤维含量,结果为：1)口服两种剂量化合物RapA, GK大鼠心肌胶原纤维水平仍然高于正常wistar大鼠心肌的胶原纤维水平；2)通过16周的口服化合物RapA, 2.5mg/kg.d-1和5.0mg/kg.d-1剂量分别使得GK大鼠的心脏血管周围胶原纤维含量从6.182±0.373(%)减低到5.218±0.409(%)、4.532±0.331(%)水平(p<0.05)；2.5mg/kg.d-1和5.0mg/kg.d-1剂量分别使得GK大鼠的心肌细胞间的胶原纤维含量从9.81±0.68(%)减低到8.77±0.65(%)和4.32±0.68(%)水平(p<0.05),改善作用具有显著性差异。
6.依靠心脏超声检测各分组实验动物的心功能,再依据相应的计算公式计算出实验动物的心功能的各个参数,结果为：1)IVS、LV-Pw和LVs参数无明显差异；2)GK大鼠的部分心功能参数Lvd、EF%和FS%仍然低于正常对照wistar大鼠水平(p<0.05),有显著性差异；3)两个口服剂量2.5mg/kg.d-1和5.0mg/kg.d-1能够明显升高EF%和FS%水平相比口服等体积生理盐水的GK大鼠(p<0.05)。
7.免疫印迹法(western blotting)检测结果显示：
1)口服两个剂量2.5mg/kg.d-1和5.0mg/kg.d-1的大鼠心肌Akt激酶Ser473和Thr308位点在受到外源性大剂量的胰岛素刺激后,相比于口服等体积生理盐水的GK大鼠,其磷酸化水平明显上调(p<0.05)；即使在没有外源性胰岛素刺激下,其磷酸化Akt水平也明显上调(p<0.05)。
2) GK大鼠+2.5mg/kg.d-1组和GK大鼠+5.0mg/kg.d-1组注射外源性胰岛素后Insulin receptorβTyr1152/1153位点磷酸化水平较对照GK大鼠明显上调(p<0.05)；
3) GK大鼠+2.5mg/kg.d-1组和GK大鼠+.5.0mg/kg.d-1组注射外源性胰岛素后insulin receptor substrate-1 Ser307磷酸化水平较对照GK大鼠显著性下调(p>0.05)；
4) Insulin receptor substrate-1 S er612磷酸化水平仅口服较高剂量(5.0mg/kg.d-1)明显较对照GK大鼠下调(p<0.05),但是较低的计量(2.5mg/kg.d-1)同对照GK大鼠相比则没有显著性改变(p>0.05)；
5) Insulin receptor substrate-1 Tyr 895磷酸化水平仅GK大鼠+5.0mg/kg.d-1组明显上调(p<0.05)；
6)心肌组织Thr642 AS160磷酸化水平相比于对照GK大鼠口服两种剂量均上调明显(p<0.05),并且存在剂量依赖性,较高剂量比较低剂量上调更多(p<0.05),而对照GK大鼠心肌Thr642 AS160磷酸化即使在胰岛素刺激后也无法激活,而口服化合物RapA后GK大鼠即使没有外源性胰岛素刺激也可激活AS160；
7) GLUT4转运至心肌细胞质膜水平在长期口服化合物RapA明显上调(p<0.05)；
8)口服RapA对GK大鼠心肌PI3-kinase p110α水平无影响；长期口服RapA同外源性胰岛素激活PI3-kinase p85αTyr 458位点磷酸化程度无明显差异(p<0.05),而相比较于对照GK大鼠上调明显(p<0.05),而对照GK大鼠心肌PI3-kinase p85αTyr 458在外源性胰岛素刺激下也未能明显激活相比较与正常wistar大鼠(p>0.05)；
9)心肌Ser21/9 GSK3α/β和Ser729 PKCε磷酸化水平受到长期服用RapA的影响相比于对照GK大鼠下调明显(p<0.05)；
10)心肌NADPH oxidase各亚基蛋白gp91phox、p67phox、p47phox和p40phox等表达水平受到RapA的作用相比于对照GK大鼠明显下调(p<0.05)；
11)与线粒体相关蛋白Bcl-2、Bax、Bad、剪切caspase-3和剪切caspase-9在RapA作用下相比较于对照GK大鼠,其在心肌的水平明显降低(p<0.05)；
12) GK大鼠+2.5mg/kg.d-1组和GK大鼠+5.0mg/kg.d-1组心肌Thr185/Tyr187 ERK1/2磷酸化水平相比对照GK大鼠组明显升高(p<0.05)；Thr183/Tyr185 JNK磷酸化水平则明显下调(p<0.05)；用药后GK大鼠两者的磷酸化水平,前者仍然低于正常wistar大鼠,后者仍然高于正常wistar大鼠；
8.ELISA检测cyclic AMP在心肌中的表达水平,利用公式计算出相对活性(%),
结果为：
1)正常wistar大鼠对照：6.35±1.89(%)；
2)对照GK大鼠组：43.07±5.91(%)；
3) GK+2.5mg/kg.d-1组：32.52±5.91(%)；
4) GK+5.0mg/kg.d-1组：26.07±2.63(%)；
两个剂量的RapA作用下,cyclic AMP水平明显下调(p<0.05)；
9.ELISA检测心肌caspase-3和caspase-9的表达水平,并计算相对活性(%),结果为：同对照GK大鼠组比较caspase-3和caspase-9在喂药GK大鼠心肌的相对活性均显著性降低(p<0.05)；
10.采用化学法检测大鼠心肌NADPH oxidase和SOD水平,然后计算出相对活性,结果为：正常对照wistar大鼠NADPH oxidase活性远低于GK大鼠水平,而SOD的相对活性则正好相反；通过16周口服化合物RapA,用药组NADPH oxidase相对活性显著性降低(p<0.05),SOD相对活性则显著性升高(p<0.05)；
11.化学法检测大鼠心肌MDA含量,同未口服RapA GK大鼠相比,喂服RapA的GK大鼠MDA含量显著性降低(p<0.05)
二、体外实验化合物RapA抑制高浓度FFA导致心肌细胞线粒体功能障碍和凋亡
实验目的：体外利用小干扰RNA技术沉默化合物RapA的作用靶点Akt激酶上游信号分子,该化合物能否抑制高浓度游离脂肪酸作用下产生的氧化应激、线粒体功能低下和细胞凋亡的初步信号传导模式.
实验方法：
1.利用wst-1试剂确定化合物RapA对心肌细胞系H9C2(2-1)的IC50；
2.使用PI3-kinase-p85α的小分子干扰RNA抑制心肌细胞系H9C2(2-1) PI3-kinase-p85α基因的表达；
3.高浓度游离脂肪酸干预心肌细胞系H9C2(2-1)检测在化合物RapA与p85a小干扰RNA共同作用下心肌细胞系活性氧产生的水平,线粒体凋亡和细胞凋亡的水平；
4.利用报告基因技术检测在高浓度游离脂肪酸干预心肌细胞系H9C2(2-1)下加入化合物RapA同时转染抗氧化应激反应元件-荧光素酶报告基因质粒和小分子干扰RNA的沉默p85α情况下,检测抗氧化应激反应元件-荧光素酶的相对活性。
实验结果：
1.利用不同浓度RapA干预大鼠心肌细胞系H9C2(2-1),计算出用药浓度与细胞数之间的关联,计算出RapA在大鼠心肌细胞系的IC50为88.31uM；利用western blotting检测RapA体外即可在较小浓度激活Akt,即同单独加入外源性胰岛素孵育细胞,其浓度为5uM时即可明显激活Akt；明显低于该细胞的IC50剂量；
2. PI3-kinase p85α的siRNA在干预大鼠心肌细胞48小时后明显抑制其蛋白水平(p<0.05)；
3.在加入RapA (5uM)作用下FFA(游离脂肪酸)-100uM对心肌细胞产生的活性氧,导致的线粒体和细胞凋亡水平明显下调(p<0.05),即使在沉默PI3-kinase p85a表达情况下；
4.以同样方法,转染心肌细胞系ARE-荧光素酶报告基因质粒,检测其相对活性,结果为RapA (5uM)作用于心肌细胞系不依赖p85a提高其相对活性,显著性抑制FFA所致的ARE相对活性减低(p<0.05)。
统计学分析
所有数据均以均数±标准误表示,应用SPSS13.0软件进行统计学分析,各分组组间差异采用单因素方差分析(ANOVA),以P<0.05为差异具有统计学显著性意义。
结论
1.化合物RapA降低代谢相关的风险因素水平,比如空腹胰岛素和血糖,血总胆固醇和游离脂肪酸。
2. 2型糖尿病所致的糖耐量受损在部分时间点经由化合物RapA口服治疗改善明显。
3.化合物RapA拮抗由长期糖尿病诱导的心脏纤维化导致的心功能不全。
4.经RapA治疗,由心脏胰岛素抵抗导致的心脏纤维化水平降低。
5. RapA的治疗作用抑制由胰岛素抵抗导致的胰岛素信号受阻,提高胰岛素刺激的敏感性,并且激活了PI3K-Akt信号通路,包括直接激活Akt,促进GLUT4向细胞膜转运。
6.经过长期口服RapA治疗2型糖尿病GK大鼠的线粒体功能障碍和由活性氧诱导的心肌细胞凋亡受到明显抑制。
7. RapA诱导Akt激酶的直接性的活化而不再需要通过上游信号分子PI3-kinase的亚基p85α激活抑制高浓度游离脂肪酸所引起的心肌细胞的凋亡。
Numerous studies have shown that serine/threonine kinase kinase Akt, also known as protein kinase B (PKB), is an important upstream signal molecules associated with growth factors, cytokines, and other cellular stimulations, which leads to downstream signaling activation in vivo. Abnormal activation or inhibition of activation of Akt has participated in a number of important pathophysiological processes of complex diseases, including atherosclerosis, type 2 diabetes and cancer. Akt family consists of three mammalian isoforms:Akt1, Akt2, Akt3, the encoding genes of isoforms in the three different gene loci, which have similar conserved domain in functional structure. The coding regions of the Akt family genes were comparised, the similarity to 85%. In principle, Akt, about 480 amino acid residues from the NH2 to the COOH end of the order of the end of PH domain, catalytic domain and regulatory domain are involved in regulating Akt activation. Aktl, recruited to the plasma membrane followed by the PH domain binding to the cell membrane phosphatidylinositol triphosphate (PIP3), has effect on phosphorylation of the catalytic domain Thr308 and Ser473. Akt in mammals are expressed in various tissues; Aktl in most mammalian tissues; Akt2 isforms is mainly high in the insulin-sensitive organs such as liver, skeletal muscle, adipose tissue and heart et al; Akt3 isoform is mainly rich in the male reproductive system and brain.
Diabetes (Diabetes mellitus, DM) is a kind of chronic progressive diseases, including type 1 diabetes and type 2 diabetes, gestational diabetes and other genetic diabetes. Type 2 diabetes which accounts for 90% of the diabetic patients, the incidence numbers are increasing every year. Diabetes as a disease class is the result of its final outcome in all body organs involved, leading to organ dysfunction caused by tissue complications, severe cases can cause death. Cardiac damage or diabetic cardiomyopathy is a kind of damage to cardiac dysfunction, but not including coronary artery disease and hypertension for the combined features of the disease. In general, hyperglycemia, hyperlipidemia and hyperinsulinemia in diabetic myocardial damage are main important rist factors in the pathogenesis of diabetic cardiomyopathy, and they also induced a series of adverse reactions, eventually lead to cardiac fibrosis and collagen deposition. Focus on improving these risk factors, from the fifties of last century, that a large number of experimental and therapeutic drugs appear, such as guanidine hydrochloride acid, acarbose, recombinant human insulin (embedded trace the release of the body) and the AICAR, but Thees drugs themselves are to control the positioning of certain risk factors, target organ damage has occurred and complications, the treatment effect is not very optimistic. Akt kinase in the heart of the level of metabolism and the role of survival has been increasingly clear. However, in specific ways to improve diabetes ultimately lead to myocardial damage or the occurrence of diabetic cardiomyopathy, the incidence of death or the development of heart failure, Akt agonist compounds RapA therapeutic effect and mechanism of the official concerned in this study. This compound is the first found from the strains and confirmed that there are no reports abroad.
Through this study, we assume that at the onset of type 2 diabetes of stable period, tissues and organs have not yet obvious damage and complications. By use of long-term oral compound RapA in the Goto-kakizaki rats, we would study the compound effects on insulin resistance and heart under the influence for the heart, such as cardiac fibrosis, myocardial mitochondrial function and apoptosis. In vitro studies, to determine whether compound RapA could activate Akt kinase directly or through the activation of upstream molecules of Akt kinase by small interfering RNA silencing, we use that free fatty acids could lead to myocardial oxidative stress, loss of mitochondrial function and apoptosis, which was able or unable to protect against damages to cardiac cells.
Methods
Materials
RapA was obtained from Doctor Zhang (North China pharmaceutical Group Corporation, China). A siRNA pool kit for p85alpha of PI3Ks'subunit was purchased from Thermo fisher scientific (Shanghai, China).Human recombinant Insulin was received from Wanbang Pharmaceutical Corporation (Xuezhou, China).Plasma glucose assay kit; total cholesterol quantitation kit, free fatty acids quantitation kit and, kit were obtained from Biovision corporate (Mountain View, California). Luciferase plasmid of p-ARE (antioxidant response element)-luc was provided from Beyotime Institute of Biotechnology (Shanghai, China).
Animals, experimental design and drug treatment
Adult male diabetic Goto-Kakizaki rats (Slack, China)(n=40) and age-matched non-diabetic Wistar rats(Slack, China) were used for the study. Animals'houses were maintained in temperature (22±2℃) and light-controlled (12h light/dark cycle) environment; standard rodent high fat diet foods, chow foods and fresh water were provided to age-matched diabetic Goto-kakizaki and non-diabetic Wistar rats respectively.
At 20-22 weeks of age, body weight and other physiological indicators (fast insulin, plasma glucose, triglyceride and cholesterol) of diabetic Goto-Kakizaki rats were markedly stable, which were compared with earlier indicators, relative to lean Wistar controls as previously reported.
To determine whether RapA agonist would protect against cardiac injuries, appropriate group (n=10) of rats were oral treated for 16 weeks, from age 24 to 40 weeks, with RapA 2.5mg/kg/day and 5.0mg/kg/day in 0.9%NaCl buffer respectively.
The investigation conformed to the Guide for Care and Use of laboratory Animals published by the US National Institutes of Health (NIH Publication NO.85-23 revised 1996).All experimental protocols were approved by Tongji medical college committee for Animals care and use.
Echocardiography
Echocardiographic analysis was performed using a commercially available echocardiograph (VIVID 7, General Electric) equipped with a 15-MHz linear array ultrasound transducer. Parameters needed for the calculation of cardiac function and dimensions were measured from five systole-diastole cycle at least. According to Teichholz method, short axis view of the left ventricle at the level of the papillary muscles was obtained by the linear transducer as previously described above. Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were measured from LV M-mode tracing (with a sweep speed of 50mm/s) at the papillary muscle level; LV fractional shortening (FS) and ejection fraction (EF), measures of LV systolic function, were calculated from LV M-mode by the following equations:
FS%=[(LVEDD-LVESD)/LVEDD]×100
EF%=[(LVEDV)-(LVESV)/LVEDV]×100.
Measurement of cardiovascular variables
For measurement of heart beat ratio and left ventricular pressure, a 2.0-French ultra miniature conductance catheter (MPVS400; Millar Instruments, USA) was introduced through the right jugular vein into the left ventricle as described previously.
Pathology and Histological analysis
The whole heart was fixed with 4% paraformaldehyde, dehydrated and embedded in paraffin. Paraffin-embedded samples were sliced to 6um-thick sections, which were stained with Hematoxylin-eosin staining and Masson's tri-Chrome staining for detecting collagen. To determine whether RapA could take effect on the size of cardiomyoctyes cross-sectional area and the degree of myocardial fibrosis,10 fields were randomly chosen respectively, and the size of cardiomyocytes was measured by use of Image J 3.0 software, and the cardiac collagen fraction was calculated as the ratio of Masson's trichrome fibrosis area to total myocardium area with the software of Image Pro-plus 4.0.
Biochemical measurements and OGTT
Methods and measurements for plasma glucose, total cholesterol, triglyceride and free fatty acids were performed and calculated as appropriate kits'manuals and instructions respectively. An oral glucose test (OGTT,5g/kg body weight) was performed after all rats fasted 12 hours at least. Blood was drawn from the tail vein at 0,30,60,90 and 120 min after the oral glucose treatment. Whole blood glucose concentrations were measured by the Johnsons'Lifescan Ultra 2 meter instrument (Shanghai, China).
Fast insulin assay
Plasma insulin levels were determined by Rat/mouse Insulin ELISA kit (St.Charles, Millipore).
Insulin signaling in vivo
Male diabetic Goto-kakizakti rats and age-matched non-diabetic Wistar rats were fed with the above mentioned HFD and chow foods for 36 weeks by the oral treatment of RapA (12wk) and saline, respectively. Body weights of all rats were measured before the day for injection of human recombinant insulin. Rats were fasted 12 hours at least and intraper-i toneally injected with insulin (10U/kg body weight) or equal volume of 0.9% NaC1. After 30 min, rat tissues were immediately excised, frozen in liquid nitrogen for 1-2 hours at least, and kept at-80℃until homogenization and other analysis.
Western blots
Western blotting was performed as previously described.
Detection of NADPH oxidase activity, MDA assay, Superoxide Dismutase (SOD) Activity
NADPH oxidase activity, MDA and superoxide dismutase (SOD) activity were measured by the colorimeric assay method with commercial kit (Biovision Inc., USA) according to the manufacturer's instructions.
Caspase activity assay
Caspase 3 and caspase 9 activity assay were performed as above mentioned.
Evalution of cAMP in vivo and invitro
Cyclic AMP in vivo and invitro was evaluated using cAMP assay kit (Cellsignaling, MA, USA) according to the assay protocol.
Primary cardiac cell culture
A method of isolation of rat neonatal cardiomyocytes was as previously reported. The isolated cardiomyocytes were grown and maintained as recommend-ed at 37℃in 5% CO2 in DMEM modified to contain 4Mm L-glutamine,4.5g/L glucose, 1mM sodium pyruvate, 2.0g/L sodium bicarbonate,3.0g/L HEPES, added 10% fetal bovine serum (FBS, Gibco, USA) and appropriate antibiotics.
Determination of IC50 and AKT kinasc activity in primary cardiac cells
To determine the cytotoxic activity and apoptosis dose of RapA, cell proliferation reagent WST-1 (Roche, USA) was used for the measurement of IC50% with the treatment of com-pound RapA in primary cardiac cells. AKT kinase activity in vitro was measured by AKT activity kit (Cellsignaling, MA, USA) as the instruction of the manufacture. Calculation of IC50 in vitro was by use of Excel 2007 (Microsoftware, USA).
Measurement of ROS in vitro and apoptosis assay
Palmitic(C 16:0) was provided from Sigma Aldrich (Shanghai, China).BSA (fatty acids-free) was purchased from Roche (Shanghai, China). A method of preparation of free fatty acids was performed as previously reported. Primary cardiomyocytes (2.0-4.0x105 cells/ml) were incubated with 10-20uM DCF-DA (Invitrogen, USA) for 30-40 min at 37℃. The DCF fluorescence was measured by FACS (Beckman) with ex/em wavelength of 485/525nm. The percentage of neonatal cardiac cells apoptosis was confirmed using Mitochondrial Membrane potential/Annvexin V apoptosis kit (Invitrogen, USA) and the annvexin V-FITC apoptosis Detection kit (Sigma, China). Stained cells were analyzed as above mentioned.
Luciferase assay
Report gene assay was performed as previously described.
Statistical analysis
Data are means±standard deviation (SD). Differences between groups were assessed using Student's t test or ANOVA followed by Least-significant Difference, Neuman-Keuls and Bonferroni post hoc testing, where appropriate. P<0.05 indicated significance. All experiments were carried out at least 3 times.
Results
Metabolic and physiological characteristics in control and diabetic Goto-kakizaki rats After the 12-week oral treatment of compound RapA, there was no significantly difference on body weight between the untreated and the compound-treated RapA Goto-kakizaki rats, however, the body weight of the control non-diabetic wistar rats was significantly higher than that of other groups, at age of 36 weeks, fast glucose, insulin, triglyceride and total cholseterols levels were significantly higher in diabetic than in control rats. Furthermore, compared with the untreated Goto-kakizaki rats group, the compound treatment (2.5mg/kg.d-1,5.0mg/kg.d-1) tended to decrease the levels of fast plasma insulin, fast plasma glucose and total cholesterol. The RapA-treatment did not take effect on fasting triglyceride levels between the untreated and the compound-treated groups. The untreated and compound treated GK rats exhibited significantly higher heart rate compared with the wistar control, but left ventricular pressure did not have difference between the untreated and the RapA administration (2.5mg/kg.d-1,5.0mg/kg.d-1) groups(p>0.05).
The oral administration of compound RapA enhances oral glucose tolerance in the diabetic Goto-kakizaki rats partly
Enhancement of glucose tolerance is one of the most critical standards for determining the effectiveness of drugs for diabetic cardioprotecting; we performed OGTT to evaluate whether the compound RapA could inhibit damage of glucose tolerance. At the appropriate time point the levels of whole blood glucose in the diabetic Goto-kakizaki groups were still greatly higher compared with those of the non-diabetic control group. For 16-week treatment (5.0mg/kg.d-1) of compound RapA to Goto-kakizaki rats, the levels of glucose obtained 0,30,60 and 90 min in the diabetic GK rats after glucose intake were significantly lower than that in the untreated GK rats (5.12±0.94 vs.7.62±1.10 p<0.05; 11.36±1.68 vs.14.60±2.47 p<0.05; 15.86±1.97 vs.20.82±1.74 p<0.05 and 18.04±1.48 vs. 22.22±1.83 p<0.05, respectively). Furthermore, the lower dose treatment (2.5mg/kg.d-1) of compound RapA to the diabetic GK rats in the levels of whole blood glucose was significantly lower than those of the untreated diabetic and the higher dose administration rats at 60 and 90 min (15.58±2.37 vs.20.82±1.74 p<0.05 and 17.38±3.32 vs.22.22±1.83 p<0.05; 15.58±2.37 vs.15.86±1.97 p<0.05 and 17.38±3.32 vs.18.04±1.48 p<0.05, respectively). This data indicate that the oral administration of compound RapA attenuated damage of glucose tolerance and has anti-hyperglycemic effect on Goto-kakizaki rats.
Reducement of diabetic cardiac fibrosis by use of compound RapA
No differences in ratios of heart weight to body weight (Hw/Bw) and the cardiomyocyte size in a cross-sectional area were noted between the wistar control group and the diabetic GK groups.Furthermore, heart cross-sections were stained with Masson Trichrome for the determination of the extent of cardiac fibrosis at age of 40 weeks with and without the oral administration of compound RapA. Morphologically, collagen deposition tended to increase in response to long term diabetes but was inhibited by compound RapA. On quantitative analysis of perivasculum and interstitial fibrosis in the heart by fibrosis area fraction, compound RapA (2.5mg/kg per day,5.0mg/kg per day) significantly decreased perivasculum and interstitium fibrosis compared with the untreated GK rats (5.218±0.409 vs.6.182±0.373 and 4.532±0.331 vs.6.182±0.373 p<0.05; 8.77±0.65 vs.9.81±0.68 and 4.32±0.68 vs.9.81±1.35 p<0.05, respectively).
Compound RapA ameliorates dysfunction of diabetic heart
Evaluation of echocardiography exhibited that the EF %(ejection fraction) and FS %(fractional shortening percentage) diameters in the oral treatment (2.5mg/kg per day, 5.0mg/kg per day) of diabetic GK groups at age of 40 weeks were markedly larger (70.20±1.17 vs.66.43±3.20 p<0.05,77.71±2.37 vs.66.43±3.20 p<0.05; 34.80±0.75 vs. 32.29±2.25 p<0.05,36.29±2.12 vs.32.29±2.25 p<0.05, respectively), however, there were no markedly differences in the indicators of IVS, LVPW, LVd and LVs.
Activation of Akt kinase and insulin resistance are enhanced and attenuated by oral administration of compound RapA
Our results showed that the compound RapA promoted myocardial Akt Ser473 and Akt Thr308 phosphorylation in the treated diabetic GK rats but not in the control GK rats following intravenous insulin markedly, expectedly, Ser473 and Thr308 phosphorylation of Akt were significantly increased by the use of compound RapA without the stimulus of insulin (Figure 4). Ser307 and Ser612 phosphorylation of insulin receptor substrate-1 were significantly decreased in the treated GK rats'hearts compared to the untreated GK rats. Furthermore, similar to the effect of oral administration of compound RapA on Akt Ser473 and Thr308 phosphoylation, Tyr1152/1153 Insulin Receptor beta and Tyr895 Insulin Receptor substrate-1 phosphorylation were significantly increased in hearts of the treated GK rats relative to the untreated GK control. These data demonstrated that the cardio-protecting effect of compound RapA attenuated insulin resistance in Goto-kakizaki rats.
AS160 phosphorylation and translocation of cytoplasmic GLUT4 to membrane are promoted by the stimulus of compound RapA
Following in vivo intravenous injection of insulin, we observed markedly effects on phosphorylation of Akt's downstream substrate AS 160 after 16 weeks oral treatment of compound RapA to the diabetic Goto-kakizaki rats. Insulin resistance-mediated glucose uptake to inhibit phorsphorylation of Akt and its downstream target AS 160 led us to believe that GLUT4 translocation was significantly impaired in the diabetic Goto-kakizaki rats compared with the treated GK rats, and the levels of translocation of GLUT4 to myocardium membrane were no difference between the lower dose (2.5mg/kg per day) treatment group and the higher dose (5.0mg/kg per day) treatment group.
Effect on Akt's upstream signaling molecules-p85 and p110alpha subunits of PI3K in vivo
To examine whether Akt's upstream signaling molecules can be activated by compound RapA, we assessed p110alpha and p85 subunits of PI3K activation in heart lysates. At the stimulus status of insulin, p-p85 subunit of PI3K was evaluated in hearts from the lower dose (2.5mg/kg per day) and the higher dose (5.0mg/kg per day) treated GK rats compared with the GK control rats; furthermore, similar to the effect on Akt kinase, Tyr458 phosphorylation of p85 subunit of PI3K was significantly increased by use of compound RapA (2.5mg/kg.d-1, 5.0mg/kg.d-1) without the administration of insulin (Figure 6A). Unexpectedly, expression of pllOalpha subunit of PI3K in the GK rats with treatment of compound RapA was lower than those of the untreated GK control rats in vivo. These results showed that compound RapA can affect the activation of p85 subunit of PI3K-Akt signaling pathway for protecting heart damage not via pllOalpha subunit of PI3K in the diabetes status.
Compound RapA activates Akt kinase's downstream signaling molecules relative to mitochondrial dysfunction in diabetic cardiac myocytes.
To test whether compound RapA protects the heart from long term diabetes mediated by its activation of GSK3α/β, the total expression and phosphorylation of GSK3α/βwere determined from the hearts of the wistar control, the untreated GK control and the treated GK rats. The results showed that compound RapA significantly suppressed Ser21/9 phosphorylation of GSK3α/βinduced by diabetes in the treated GK control rats compared with the untreated diabetic rats; furthermore, we found that compound RapA induced a significant decrease of GSK3a/p phorsphorylation between the non-diabetic wistar group and the treated GK group (Figure 7A), it was an effect that was unexpected. It was observed that phorsphorylation of PKCεchanged by compound RapA was similar to changes of GSK3α/βin vivo. To detect whether cyclic AMP activity was reduced by compound RapA, we examined the concentrations of myocardial cyclic AMP as above method. These results demonstrated that relative cyclic AMP activity in GK control rats was significantly higher (43.07±5.91 vs.6.35±1.89) compared with the non-diabetic wistar rats (Figure 7C); expectedly, the lower dose (2.5mg/kg.d-1) and the higher dose (5.0mg/kg.d-1) treatment were markedly lower (32.52±5.91 vs.43.07±5.91,26.07±2.63 vs. 43.07±5.91, respectively) compared with the untreated GK rats.
The results obtained in the previous experiment promoted us to evaluate whether compound RapA could indirectly or directly affect expression of NADPH oxidase and NADPH oxdiase activity induced by mitochondrial dysfunction in long term diabetes. We assessed expressions of subunits of NADPH oxdiase in heart lysates, including gp91 phox, p67phox, p47phox and p40phox. Expressions of gp91 phox and p47phox in the treated higher dose group were significantly decreased compared with the untreated GK group; it was no difference between the lower dose group and the GK control. Oppositely, expressional levels of p67phox and p40phox were lower in lower dose group than those of GK control and the higher dose group respectively; furthermore, it did not differ between the GK control and the higher dose group in levels of expression of p67phox and p40phox. Expressions of NADPH oxdiase in the treated GK rats approached but did not return to the normal state of the wistar rats. We exhibited that relative NADPH oxidase activity in the oral treatment (2.5mg/kg per day, 5.0mg/kg per day) of diabetic GK groups at age of 40 week were significantly downregulated (2.96±0.08 vs.3.44±0.25,2.53±0.24 vs.3.42±0.25, respectively). As the purpose of evaluating the effect of antioxidant by compound RapA, we investigated relative SOD activity and level of MDA in heart tissue. Figure 7E, indicating myocardial relative SOD activity, showed that it was downregulated by 4-fold in GK control group and enhanced by 2-fold and 3-fold in the lower dose (2.5mg/kg per day) and the higher dose (5.0mg/kg per day),respectively. Myocardial MDA level was increased by 1.7-fold in the GK control and inhibited by 14% and 24% in two dose treatment group, respectively.
To explore whether compound RapA can inhibit apoptosis of mitochondria induced by ROS, we examined myocardial Bax, Bad, Bcl-2, cleaved caspase-3 and cleaved caspase-9 expression by western blotting. Western blotting showed that decreases in Bad, cleaved caspase-3 and cleaved caspase-9 expression in the treated GK rats including the lower and the higher dose groups compared with those of untreated GK rats; furthermore, compound RapA did not reduce the expression of Bax in treated GK rats. Similar to the effect on expression of cleaved caspase-3 and caspase-9, relative activities of caspase-3 and caspase-9 were markedly lower in treated GK rats than those in GK control rats and showed dose dependent way.
Thus, we tended to believe that compound RapA had anti-ROS-formation effect and inhibit the progression of apoptosis of cardiac mitochondria in long term diabetes.
The compound RapA activates p44/42MAPK (ERK1/2) and inhibits JNK
ERK (p44/42MAPK) is an important molecule for protecting diabetic heart in the pathological development of myocyte apoptosis and cardiac fibrosis; oppositely, activation of JNK could inhibit restoration of diabetic cardiac function and increase cardiac apoptosis and fibrosis. So, we examined the activation levels of ERK and JNK in hearts of GK rats with or without treatment of compound RapA and the non-diabetic wistar control rats. The results demonstrated that Thrl85/Tyrl87 phosphorylation of ERK1/2 were significantly upregulated compared with the GK control group without the oral administration of compound RapA for 16 week (Figure 7H); furthermore, by the effect of compound RapA, the activation levels of ERK1/2 in hearts of two oral treatment GK group approached the normal status of phosphorylation of ERK1/2 in the non-diabetic wistar rats. Phosphorylation levels of cardiac JNK1 was significantly lower in the diabetic GK rats treated with 5.0mg/kg per day of compound RapA for 16 week compared with the GK control rats, however,2.5mg/kg per day for 16 week of compound RapA did not affect the phosphorylation of cardiac JNK1 in appropriate diabetic group.
Compound RapA inhibits ROS generation and apoptosis mediated by FFA in primary cardiac cells not via PI3K's subunit-p85a
FFA (free fatty acids) is closely involved in the development of type 2 diabetes, which generates reactive oxygen species in heart and finally leads to myocardium apoptosis. According to the results above, to test whether compound RapA could take effect on activation of AKT for protecting diabetic heart directly, we assessed the expression of PI3K's p85αsubunit with p85αgene siRNA pool in primary cardiac cell. The results showed that p85αprotein expression was unaltered in cells exposed to a negative control siRNA sequence; however, siRNA pool against PI3K's p85αsubunit reduced protein expression markedly compared with cardiac cells transfected with negative control siRNA after 48 hours. Relative intracellular ROS levels generated by FFA (100UM) for 48h were markedly increased (305.67±12.73 vs.5.77±0.36, p<0.05) compared with the negative control in primary cardiac cells; Furthermore, compound RapA (5UM) for 48h added to primary cells reduced relative ROS levels markedly (204.42±6.07 vs.305.67±12.73), compared with the FFA (100UM) group. In accordance with compound RapA treatment, siRNA pool-mediated silence of PI3K's p85a in cardiac cells displayed no significantly difference on relative intracellular ROS levels (204.42±6.07 vs.198.02±11.76, p<0.05) compared with the FFA(100UM)+RapA(5UM) group.
The apoptotic effect of ROS was partly mediated by transcriptional factors responded to ROS. To study whether compound RapA could inhibit transcriptional factor's activity induced by ROS, a luciferase reporter gene construct containing antioxidant response element was transfected into primary cardiac cells with or without compound RapA or p85α-siRNA. Similar to the results above, relative luciferase activities of ARE plasmid of the FFA (100UM)+RapA group and the FFA (100UM)+RapA+p85α-siRNA group were significantly higher (1.42±0.08 vs.0.61±0.12,1.24±0.07 vs.0.61±0.12) than those of the FFA (100UM) group. These data suggested that compound RapA could reduce generation of ROS-mediated by free fatty acids and activities of transcriptional factors induced by ROS by activation of AKT kinase directly not via its upstream signal molecule-PI3K's subunit p85a in vitro.
Previously reports have indicated that ROS mediated by FFA could upregulate apoptosis level of cardiac cells in vitro. Here, we determined whether FFA-induced apoptosis of primary cardiac cells is prevented by compound RapA with or without the treatment of p85α-siRNA in vitro. Two days after the co-treatments of FFA and compound RapA and transfection of p85a siRNA, levels of mitochondrial and cellular apoptosis were assessed by FACS; the methods of FACS were performed as described above. Stimulation of cardiac cells with FFA (100UM) significantly induced the cellular and mitochondrial apoptosis by 5.9-and 4.2-fold, respectively, furthermore, compound RapA largely blocked the cellular and mitochondrial apoptotic effect of free fatty acids, expectedly, and addition of p85a-siRNA did not prevent the anti-apoptotic effect of compound RapA. These results suggested that the anti-apoptotic effect of compound RapA was mediated in a large part through activation of AKT kinase directly not via its upstream signal molecule-PI3K's subunit p85a.
Discussion
This study was undertaken to examine the therapeutic effect of compound RapA on the cardiac damage in the long term diabetes. The important findings in the study were:(i) compound RapA reduces metabolic levels of risk factors, including fast plasma insulin, fast plasma glucose, total cholesterol and free fatty acids (FFA); (ii) the damaged glucose tolerance induced by type 2 diabetes may well be ameliorated by the oral treatment of compound RapA in part; (iv) compound RapA protects against the cardiac dysfunction induced by cardiac fibrosis; (iv) cardiac fibrosis induced by insulin resistance could be weakened by the administration of compound RapA; (Ⅴ) compound RapA inhibits insulin resistance and enhances the senstivity of the stimulation of insulin, and activates the PI3K-Akt signaling pathway, including the phosphorylation of Akt, membrane translocation of GLUT4; (Ⅵ) mitochondrial dysfunction and apoptosis induced by reactive oxygen species in myocardium may well be suppressed by compound RapA; (Ⅶ) the apoptotic effect of free fatty acid in vivo may de repressed by compound RapA through the activation of Akt kinase diretly not via the upstream signaling molecule-PI3-kinase's subunit p85α.
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