UT受体拮抗剂-urantide抗大鼠心肌缺血再灌注损伤作用及其机制
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摘要
心血管疾病是严重危害人类生命健康的常见病和多发病，具有很高的死亡率。缺血性心脏病是其中最常见的疾病之一，表现为心肌急剧的缺血缺氧。近年来随着动脉搭桥术、溶栓疗法、经皮腔内冠脉血管成形术、体外循环心脏外科手术和心肺脑复苏术等手段的建立和推广应用，能够使缺血的心脏很快恢复血液供应，但同时也发现在心肌缺血基础上恢复血流后，组织损伤加重、甚至发生不可逆性损伤的现象称为心肌缺血再灌注损伤。因此，开发预防和治疗心肌缺血再灌注损伤的有效药物是国内外医药界关注的热点。
Urotensin Ⅱ（U Ⅱ）是一种生长激素样神经环肽，最初从硬骨鱼脊髓尾部分离出来，后来发现在哺乳动物包括人类都普遍存在UⅡ，其广泛分布于脊髓、脑组织和心血管组织。1999年发现U Ⅱ的特异性受体－－UT受体。已证实人类UⅡ（human UⅡ）是迄今已知最强的血管收缩剂，作用强度较ET-1高出1~2个数量级。临床多项研究也显示，在多种心血管疾病中伴有hUⅡ水平的升高，提示hUⅡ及其受体在心血管疾病中可能有着举足轻重的作用。
Urantide是在hUⅡ基础上衍生的肽类UT受体拮抗剂。本课题组前期研究初步表明，urantide对心肌缺血及缺血再灌注损伤具有保护作用，其机制可能与减轻细胞膜脂质过氧化、增加NO生成、减轻钙超载等有关。本研究将分别从整体水平、细胞水平探讨urantide对缺血再灌注大鼠心脏、缺氧再给氧大鼠心肌细胞的保护作用及其机制，重点利用RNA干扰技术下调UT受体表达，在心肌细胞缺氧再给氧模型基础上，探讨urantide对缺氧再给氧心肌细胞保护作用与UT受体之间的关系。
第一部分Urantide对大鼠心肌缺血再灌注损伤的影响及相关机制研究
目的：
观察urantide对缺血再灌注大鼠心肌损伤的影响及其机制，并应用PKC-TK-MAPK信号通路特异性抑制剂白屈菜红碱（CHE）和PI3K-AKT信号通路特异性抑制剂LY294002，重点探讨urantide对缺血再灌注大鼠心肌细胞保护作用与上述两条信号通路的作用机制。
方法：
1.采用大鼠冠状动脉左前降支结扎和松开法（LAD法）制作大鼠缺血再灌注模型，记录心肌缺血/再灌注过程中各组心电图，比较ST段变化情况；在实验结束后，收集各组大鼠血清待测生化指标。
2.实验共分8组，分别是：（1）假手术组；（2）模型组；（3）Urantide3μg·kg~(-1)组；（4）Urantide10μg·kg~(-1)组；（5）Urantide30μg·kg~(-1)组；（6）Verapamil（Ver）1.6mg·kg~(-1)组；（3~6组分别于缺血前10min舌下静脉1min内一次性推注相应药物）；（7）Urantide30μg·kg~(-1)+CHE（Chelerythrine，PKC特异性阻断剂）组：在穿线稳定后，舌下静脉快速推注CHE1mg·kg~(-1)，5min后舌下静脉快速推注urantide30μg·kg~(-1)，稳定10min后再行缺血再灌注操作；（8）Urantide30μg·kg~(-1)+LY294002（PI3K-AKT信号通路阻断剂）组：在穿线稳定后，舌下静脉快速推注LY2940020.3mg·kg~(-1)，5min后舌下静脉快速推注urantide30μg·kg~(-1)，稳定10min后再行缺血再灌注操作；
3.检测血清中NO、MDA、cTnI含量和NOS、SOD、LDH、CK的活性；
4.实验结束后取心脏行伊文思兰和TTC双染色，计算IS/AAR，即梗死面积。
5.实验结束后取心脏常规固定，采用HE染色法观察心肌组织病理结构改变，采用透射电镜观察心肌组织超微结构的改变。
6.采用TUNEL法检测心肌细胞中凋亡指数；
7.采用免疫组织化学方法检测缺血再灌注后心肌组织中Bcl-2和Bax蛋白表达的水平并进行半定量分析；
8.采用免疫印迹方法检测缺血再灌注后心肌组织中Akt和p-Akt蛋白表达的水平并进行半定量分析。
结果：
1.在大鼠冠状动脉左前降支结扎和松开致心肌缺血再灌注损伤的模型上，urantide（10，30μg·kg~(-1)）对心电图中ST段及T波的抬高有明显的抑制作用；urantide（10，30μg·kg~(-1)）能显著降低梗死区与缺血危险区的比值，而CHE与LY294002能取消urantide的这种保护作用。
2. Urantide10，30μg·kg~(-1)两个剂量组可明显降低缺血再灌注大鼠血清中CK、LDH活性，同时可明显降低大鼠血清中升高的cTnI水平。Urantide+CHE与Urantide+LY294002组显示CK、LDH活性及cTnI水平升高，与urantide30μg·kg~(-1)比较有显著性差异。提示urantide对缺血再灌注大鼠心肌细胞的保护作用可被CHE及LY294002取消。
3.HE染色光镜下观察和电镜观察结果表明10，30μg·kg~(-1)urantide与模型组相比可显著减轻缺血再灌注诱导的心肌细胞和亚细胞损伤，而Urantide+CHE与Urantide+LY294002组心肌细胞损伤加重，超微结构破坏明显。
4. Urantide10，30μg·kg~(-1)两个剂量组可明显降低缺血再灌注大鼠血清中MDA水平，提高NO含量，升高SOD、NOS活性。Urantide+CHE与Urantide+LY294002组MDA水平升高，NO含量、SOD及NOS活性下降，与模型组相比无统计学意义。提示这两种抑制剂可取消urantide对缺血再灌注大鼠心肌细胞的抗氧化作用。
5.TUNEL法检测心肌细胞凋亡结果表明，urantide三个剂量组均可以不同程度的降低缺血再灌注大鼠的心肌细胞凋亡指数，而Urantide+CHE组与Urantide+LY294002组心肌细胞凋亡指数与urantide用药组相比明显升高，与模型组相比无统计学意义。
6.免疫组化结果显示，urantide可不同程度增加了心肌组织中bcl-2蛋白的表达水平，降低了心肌组织中bax蛋白的表达水平，Urantide+CHE组与Urantide+LY294002组bcl-2及bax蛋白的表达水平与模型组相比无统计学意义。
7.Western blot结果显示，urantide各剂量组可不同程度的升高p-Akt/Akt蛋白的比值，Urantide+CHE组与Urantide+LY294002组可降低p-Akt/Akt蛋白的比值。
第二部分UT受体下调对urantide抗大鼠心肌细胞缺氧再给氧损伤作用的影响
目的：
利用RNA干扰技术，将UTsiRNA转染至原代培养心肌细胞，下调UT受体的表达，在乳鼠原代心肌细胞缺氧复氧模型基础上，观察urantide对心肌细胞缺氧复氧的保护作用与UT受体的关系。
方法：
1.进行乳鼠心肌细胞原代培养，观察不同培养时间心肌细胞的形态及活力，采用免疫组织化学方法进行心肌细胞鉴定；
2.将三种设计合成的UT siRNA用脂质体转染的方法导入原代培养的心肌细胞中，分别用实时定量PCR的方法和Western blot方法检测UT mRNA表达水平和UT蛋白表达水平，筛选出抑制性能最佳的UT siRNA。
3.将UT siRNA转染至原代培养的大鼠乳鼠心肌细胞中，再进行缺氧3h再给氧3h处理，处理前给予urantide10-6mol·L~(-1)，实验结束时收集细胞培养上清，进行相应指标的测定。
4.用台盼蓝染色法和MTT染色法分别检测心肌细胞存活率，PI单染色法流式细胞仪检测心肌细胞凋亡，荧光素染料Hoechst33258测定心肌细胞凋亡，检测心肌细胞培养上清中NO、MDA、cTnI含量和NOS、SOD、LDH、CK的活性。
结果：
1.原代新生大鼠心肌细胞贴壁性能良好，在培养d3细胞搏动明显，搏动频率逐渐加快，至d7天搏动频率开始减少，细胞数量在d3~d4天增长明显；免疫组化方法对心肌细胞进行鉴定，显示心肌细胞纯度可达95%以上。
2.实时荧光定量PCR结果和Western blot结果显示三对UT siRNA对UT mRNA表达水平和UT蛋白表达水平均有抑制作用，其中UT siRNA3组与对照组相比有显著性差异，故后续实验中采用UT siRNA3转染心肌细胞下调UT受体表达。
3.台盼蓝染色法和MTT染色法均显示urantide10-6mol·L~(-1)能够明显增加细胞存活率，但UTsiRNA转染造成UT受体下调对1×10-6mol·L~(-1)urantide的心肌细胞存活率提升有明显的下降作用，MTT染色法显示心肌细胞存活率从其在正常心肌细胞上的81.13±12.46%降至60.89±9.41%。
4.10-6mol·L~(-1)urantide可明显降低细胞培养上清中cTnI含量以及CK、 LDH活性，而在UT受体下调的心肌细胞上，urantide10-6mol·L~(-1)对cTnI含量以及CK、LDH活性的降低作用有明显减弱，cTnI含量以及CK、LDH活性分别从其在正常心肌细胞上的63.73±11.36ng·L~(-1),0.83±0.10U·L~(-1)和109.62±12.66U·L~(-1)分别升高至90.22±12.43ng·L~(-1),1.49±0.08U·L~(-1)和168.90±16.18U·L~(-1)。
5.10-6mol·L~(-1)urantide可降低细胞培养上清中MDA含量，同时提高培养上清中SOD和NOS活性及NO含量，在UTsiRNA介导的UT受体下调的心肌细胞上，10-6mol·L~(-1)urantide对MDA含量降低和SOD活性提高作用均有明显减弱，但对其NOS和NO作用未见明显影响。
6.采用PI单染法流式细胞仪和Hoechst33258荧光染色分别检测了心肌细胞凋亡，而在UTsiRNA3转染使UT受体下调的心肌细胞上，urantide10-6mol·L~(-1)对缺氧再给氧诱导的心肌细胞凋亡抑制作用有明显减弱，凋亡率从其在正常心肌细胞的8.04±0.85%和6.33±1.23%分别升高至13.90±1.11%和9.96±1.41%。
结论：
1.Urantide对大鼠缺血再灌注损伤的心肌组织具有明显的保护作用，该作用可能与抗脂质过氧化、增加NO生成、抑制心肌细胞凋亡等有关；
2.Urantide抗缺血再灌注心肌细胞损伤作用可能与激活PKC-TK-MAPK信号通路及PI3K-AKT信号通路有关；
3.UT受体介入了urantide对大鼠乳鼠心肌细胞缺氧复氧损伤的保护作用；
4.UT受体介导了urantide的保护作用的抗脂质过氧化和抗凋亡机制，但没有涉及其对NO生成和释放作用。
Background：
Cardiovascular diseases are the commonly and frequently encountered diseaseswhich are severely harmful to health. It is well known that cardiovascular diseases havehigh mortality. Ischemic heart disease is one of the high incidence diseases, which ischaracterized by the myocardium lack blood and oxygen supply rapidly. For the pastfew years, Coronary Artery Bypass, thrombolytic therapy, percutaneous transluminalcoronary angioplasty (PTCA), external ciculation of cardiac surgical procedures andcardiopulmonary cerebral resuscitation were established and extensively applied toclinics. All the methods mentioned above can make the ischemic heart regain bloodsupply rapidly. On the contrary, the damage of myocardium was observed to becomeaggratated even proceed irreversibility injury after reperfusion. This phenomemon wasnamed as the ischemical reperfusion injury. Therefore, more and more attention waspaid to develop the effective medicines for prevention or therapy toischemia-reperfusion injury.
Urotensin Ⅱ (UⅡ) is a cyclic peptide initially isolated from the caudalneurosecretory system of teleost fish. The cyclic region of UⅡ, which is responsible forthe biological activity of the peptide, has been fully conserved from fish to human. Thematching of the11amino acid peptide human urotensin-Ⅱ (hU-Ⅱ) with an orphanhuman G-protein coupled receptor with homology to rat GPR14, which was named UTreceptor by Ames in1999. hU-Ⅱ was considered to be the most potent mammalianvasoconstrictor identified so far, which vasoconstrictor potency is higher than ET-1.Many clinical analysis show that the hUⅡ in plasma level is elevated in differentcardiovascular diseases including hypertension, heart failure and so on. No doubt hUⅡ and UT receptors play an important role in cardiovascular diseases.Urantide, is the most potent UT receptor antagonist compound so far reported inthe rat isolated aorta, and is also endowed with high affinity at human UT receptors. Ourprevious studies indicated that urantide had a protective effect against myocardialischemia reperfusion injury in rats and mice. The mechanism may related toantioxidation, reducing calcium overload in cardiomyocytes and augmenting thesynthesis of NO. The present study was going to investigate the effects of urantide usingthe ischemia reperfusion rat model in vivo and hypoxia reoxygenation cellular model invitro respectively. The study was to observe the effects and mechanism of urantide oncardiomyocyte protection. In this study, chelerythrine which is an inhibitor of the PKCsignaling pathway and LY294002which is an inhibitor of the PI3K-Akt signalingpathway were used to assess the relationship between the effects of urantide and relativesignal transduction pathway. On the other hand, by using RNA interference to knockdown the expression of UT receptor it was to be investigated if the UT receptor playsrole in protection of urantide in hypoxia reoxygenation cardiomyocytes.Methods and ResultsPart I Effects of urantide on myocardial ischemia-reperfusion injury in rats andits underlying mechanisms
Methods:
1. The ischemical reperfusion model was set up by ligating and untying left anteriordescending coronary artery in rats. The electrocardiogram was recorded during theexperiment, the serum was collected to measure the content of NO, MDA and cTnI aswell as the activities of CK, LDH and NOS. The heart was harvested to determinemyocardial ischemic size and infarct size by dual staining with Evans blue andtriphenyl-tetrazolium-chloride.
2. The rats were randomized into8groups: All hearts were subjected to theischemia/reperfusion (I/R) protocol consisting of30min of regional ischemia and90 min of reperfusion except for the sham group.
(1) the sham group: the animals’ chest were operated, but the coronary ligatures werenot tied.
(2) the model group: the hearts were subjected to the ischemia/reperfusion (I/R)protocol consisting of30min of regional ischemia and90min of reperfusion
(3) urantide3μg·kg~(-1)group: Urantide (3μg·kg~(-1))was given through intravenous drugperfusion ten minutes before ischemia.
(4) urantide10μg·kg~(-1)group: Urantide(10μg·kg~(-1)) was given through intravenous drugperfusion ten minutes before ischemia.
(5) urantide30μg·kg~(-1)group: Urantide(30μg·kg~(-1)) was given through intravenous drugperfusion ten minutes before ischemia.
(6) verapamil1.6mg·kg~(-1)group: ver (1.6mg·kg~(-1)) was given through intravenous drugperfusion ten minutes before ischemia as the positive control
(7) urantide30μg·kg~(-1)+CHE1mg·kg~(-1)(chelerythrine, the inhibitor of PCK signaling)group: the chelerythrine was given through intravenous drug perfusion afterstabilization, urantide(30μg·kg~(-1)) was given through intravenous drug perfusion fiveminutes after the previous administration, the ischemia reperfusion operation wascarried out ten minutes after the urantide afford.
(8)urantide30μg·kg~(-1)+LY2940020.3mg·kg~(-1)(the inhibitor of PI3K-Akt signaling)group: LY294002was given through intravenous drug perfusion after stabilization,urantide(30μg·kg~(-1)) was given through intravenous drug perfusion five minutesafter the previous administration, the ischemia reperfusion operation was carried outten minutes after the Urantide afford.
3. The hearts were fixed at the end of each experiment. The histopathologic examinationof myocardium in rat was observed by hematoxylin and eosin stain. Theultrastructural morphology in rats cardiomyocyte was measured by transmissionelectron microscope.
4.TUNEL labeling was used for the measurement of apoptosis in myocardium.
5. The expressions of Bcl-2and Bax protein in cardiomyocytes of rats were measuredby immunohistochemistry technology.
6. The expressions of Akt and p-Akt protein in cardiomyocytes of rats were measuredby western blot analysis.
Results:
1. The ST segment of ECG markedly elevated during the process of ischemiareperfusion, while urantide (10,30μg·kg~(-1)) could markedly inhibit the elevation ofST segment of ECG. Urantide (10,30μg·kg~(-1)) could also significantly reduce infarctsize as compared to model hearts, while pharmacological inhibition of PKC andPI3K/Akt signaling completely abrogated urantide-induced inhibition in elevation ofST segment and reduction of infarct size.
2. The cTnI content and CK、LDH activity elevated in model group. Urantide (10,30μg·kg~(-1)) could markedly inhibit the elevation of cTnI content and CK, LDH activitiesin serum. However, those decreased injury indexes increased evidently in urantide30μg·kg~(-1)+CHE group and urantide30μg·kg~(-1)+LY294002group.
3. Morphological observation by HE staining and transmission electron microscopeshowed that the ischemic reperfusion injury was improved by urantide (10,30μg·kg~(-1))administration. But myocardial injury of the urantide30μg·kg~(-1)+CHEgroup and urantide30μg·kg~(-1)+LY294002group showed to become more seriouscompared to that of urantide30μg·kg~(-1)group.
4. Urantide (10,30μg·kg~(-1)) could markedly inhibit the MDA content, and increase theSOD, NOS activities and NO content in serum. However, pharmacological inhibitionof PKC and PI3K/Akt signaling completely abolished changes of these indexesinduced by urantide administration.
5. Compared with those in the sham group, the number of TUNEL-positive cells in I/R group was significantly increased. Urantide (3,10,30μg·kg~(-1)) markedly decreasedthe number of TUNEL-positive cells. On the contrary, pharmacological inhibition ofPKC and PI3K/Akt signaling markedly inhibited urantide-induced decrease ofTUNEL-positive cells.
6. Expression level of Bax protein was significantly higher in model group than that inurantide administration group, whereas the level of Bcl-2protein and Bcl-2/Bax ratiowere markedly higher in30μg·kg~(-1)urantide group than those in model group,respectively. Two proteins expression in urantide30μg·kg~(-1)+CHE group and urantide30μg·kg~(-1)+LY294002group were no statistical difference in comparison with modelgroup.
7. Levels of Akt (total-Akt) and its phosphorylation at Ser473(p-Akt) were determinedby western blotting and phosphorylation of Akt was expressed as the ratio betweenp-Akt and total-Akt. The results showed that Urantide could rise the ratio at threedifferent dose. Furthermore, both CHE and LY294002significantly reduced Aktphosphorylation and decrease the ratio between p-Akt and total-Akt.Part Ⅱ Effects of knockdown of UT receptor on protection of urantide oncultured neonatal rat cardiomyocytes subjected to hypoxia reoxygenation injury
Methods:
1.Cardiomyocytes of neonatal rat were isolated and cultured in vitro. Morphocytologychanges and cell vitality were observed at different period during cell culture. Cellpurity was evaluated through troponin I expression by immunohistochemistrytechnology.
2.Cardiomyocytes were transfected with three different UT siRNA which were designedby Shanghai Gene Pharma Co. Ltd. The UT mRNA and protein expression weredetected by real-time PCR and Western blotting respectively. Thus choosing the mosteffective UT siRNA to knock down the UT receptor.
3. Cardiomyocytes were transfected with the most effective UT siRNA prior to hypoxiathree hours and reoxygenation three hours. Urantide10-6mol·L~(-1)was incubated beforehypoxia reoxygenation. At the end of experiment, the cell culture medium wascollected and proteins were harvested to detect different index.
4. Cell viability was measured by trypan blue staining and MTT staining respectively.Hoechst33258assay and flow cytometric techniques were used to detect apoptoticcells. The content of MDA,NO and cTnI and activities of SOD, CK, LDH, NOS incell culture medium were measured by commercial kits.
Results:
1. Cardiocytes began to adhere and grow after cell culture. It can be observed thatCardiocytes beat spontaneously and rhythmically on the third day, and the frequencebecome more and more after that. The frequence reduced gradually from the seventhday of cell culture. Cardiocytes proliferate obviously on the third and the fourth dayof cell culture. Immunohistochemistry staining confirmed the cell with purity higherthan95％.
2. The levels of UT mRNA and protein expression showed that three differentUTsiRNA could all inhibit UT mRNA and protein express distinctively. The siRNA3was the most effective one among those siRNA, so siRNA3was adopted in latterexperiments.
3. It was found that10-6mol·L~(-1)urantide increased the viability of cardiocytes subjectedto hypoxia/reoxygenation by using both trypan blue staining and MTT staining assay.However, down-regulation of UT receptor in cardiocytes by using UTsiRNA3led toa decrease to increase of cell viability produced by urantide.
4. On the H/R model of myocardial cells, urantide (10-6mol·L~(-1)) could evidently inhibitthe increase of cTn I content, reduce the rise of CK and LDH activities in the cellculture medium. Whereas down-regulation of UT receptor in cardiocytes could also abolish aforementioned effects of urantide.
5. During H/R, Urantide (10-6mol·L~(-1)) could evidently inhibit the increase of MDA andthe decreases of activities of SOD and NOS and content of NO in the cell culturemedium. UT siRNA3-mediated down-regulation of UT receptor could abolish theinhibitory effect of urantide on increase of MDA and decrease of SOD, but had noeffect on urantide-induced alteration of NO content and NOS activity.
6. By using Hoechst33258assay and flow cytometric techniques, it was observed thatthe apoptosis rate in model group was much higher than that in sham group.Pretreatment of10-6mol·L~(-1)urantide significantly inhibited apoptosis induced by H/R.While knock-down of UT receptor by using UT siRNA3could abolish the inhibitioneffects of urantide on H/R-induced apoptosis.
Conclusions:
1. Urantide has a significant protective effect against myocardial ischemic/reperfusioninjury in rats, which may be related to anti-lipidperoxidation, elevating the NOsynthesis and inhibiting apoptosis via modulating the expression o f Bc l-2and Bax.
2. The protective effect of urantide on myocardial ischemic/reperfusion injury in ratsmay be mediated via activation of PKC-TK-MAPK signal transduction pathway andPI3K-Akt signal transduction pathway.
3. UT receptor involves in the protective of urantide on hypoxia reperfusion injury in ratcardiomyocyte.
4. UT receptor mediates the mechanisms of anti-lipidperoxidation and anti-apoptoticeffect of urantide, however UT receptor did not involve urantide-induced synthesisand release of NO.
Urotensin Ⅱ（U Ⅱ）是一种生长激素样神经环肽，最初从硬骨鱼脊髓尾部分离出来，后来发现在哺乳动物包括人类都普遍存在UⅡ，其广泛分布于脊髓、脑组织和心血管组织。1999年发现U Ⅱ的特异性受体－－UT受体。已证实人类UⅡ（human UⅡ）是迄今已知最强的血管收缩剂，作用强度较ET-1高出1~2个数量级。临床多项研究也显示，在多种心血管疾病中伴有hUⅡ水平的升高，提示hUⅡ及其受体在心血管疾病中可能有着举足轻重的作用。
Urantide是在hUⅡ基础上衍生的肽类UT受体拮抗剂。本课题组前期研究初步表明，urantide对心肌缺血及缺血再灌注损伤具有保护作用，其机制可能与减轻细胞膜脂质过氧化、增加NO生成、减轻钙超载等有关。本研究将分别从整体水平、细胞水平探讨urantide对缺血再灌注大鼠心脏、缺氧再给氧大鼠心肌细胞的保护作用及其机制，重点利用RNA干扰技术下调UT受体表达，在心肌细胞缺氧再给氧模型基础上，探讨urantide对缺氧再给氧心肌细胞保护作用与UT受体之间的关系。
第一部分Urantide对大鼠心肌缺血再灌注损伤的影响及相关机制研究
目的：
观察urantide对缺血再灌注大鼠心肌损伤的影响及其机制，并应用PKC-TK-MAPK信号通路特异性抑制剂白屈菜红碱（CHE）和PI3K-AKT信号通路特异性抑制剂LY294002，重点探讨urantide对缺血再灌注大鼠心肌细胞保护作用与上述两条信号通路的作用机制。
方法：
1.采用大鼠冠状动脉左前降支结扎和松开法（LAD法）制作大鼠缺血再灌注模型，记录心肌缺血/再灌注过程中各组心电图，比较ST段变化情况；在实验结束后，收集各组大鼠血清待测生化指标。
2.实验共分8组，分别是：（1）假手术组；（2）模型组；（3）Urantide3μg·kg~(-1)组；（4）Urantide10μg·kg~(-1)组；（5）Urantide30μg·kg~(-1)组；（6）Verapamil（Ver）1.6mg·kg~(-1)组；（3~6组分别于缺血前10min舌下静脉1min内一次性推注相应药物）；（7）Urantide30μg·kg~(-1)+CHE（Chelerythrine，PKC特异性阻断剂）组：在穿线稳定后，舌下静脉快速推注CHE1mg·kg~(-1)，5min后舌下静脉快速推注urantide30μg·kg~(-1)，稳定10min后再行缺血再灌注操作；（8）Urantide30μg·kg~(-1)+LY294002（PI3K-AKT信号通路阻断剂）组：在穿线稳定后，舌下静脉快速推注LY2940020.3mg·kg~(-1)，5min后舌下静脉快速推注urantide30μg·kg~(-1)，稳定10min后再行缺血再灌注操作；
3.检测血清中NO、MDA、cTnI含量和NOS、SOD、LDH、CK的活性；
4.实验结束后取心脏行伊文思兰和TTC双染色，计算IS/AAR，即梗死面积。
5.实验结束后取心脏常规固定，采用HE染色法观察心肌组织病理结构改变，采用透射电镜观察心肌组织超微结构的改变。
6.采用TUNEL法检测心肌细胞中凋亡指数；
7.采用免疫组织化学方法检测缺血再灌注后心肌组织中Bcl-2和Bax蛋白表达的水平并进行半定量分析；
8.采用免疫印迹方法检测缺血再灌注后心肌组织中Akt和p-Akt蛋白表达的水平并进行半定量分析。
结果：
1.在大鼠冠状动脉左前降支结扎和松开致心肌缺血再灌注损伤的模型上，urantide（10，30μg·kg~(-1)）对心电图中ST段及T波的抬高有明显的抑制作用；urantide（10，30μg·kg~(-1)）能显著降低梗死区与缺血危险区的比值，而CHE与LY294002能取消urantide的这种保护作用。
2. Urantide10，30μg·kg~(-1)两个剂量组可明显降低缺血再灌注大鼠血清中CK、LDH活性，同时可明显降低大鼠血清中升高的cTnI水平。Urantide+CHE与Urantide+LY294002组显示CK、LDH活性及cTnI水平升高，与urantide30μg·kg~(-1)比较有显著性差异。提示urantide对缺血再灌注大鼠心肌细胞的保护作用可被CHE及LY294002取消。
3.HE染色光镜下观察和电镜观察结果表明10，30μg·kg~(-1)urantide与模型组相比可显著减轻缺血再灌注诱导的心肌细胞和亚细胞损伤，而Urantide+CHE与Urantide+LY294002组心肌细胞损伤加重，超微结构破坏明显。
4. Urantide10，30μg·kg~(-1)两个剂量组可明显降低缺血再灌注大鼠血清中MDA水平，提高NO含量，升高SOD、NOS活性。Urantide+CHE与Urantide+LY294002组MDA水平升高，NO含量、SOD及NOS活性下降，与模型组相比无统计学意义。提示这两种抑制剂可取消urantide对缺血再灌注大鼠心肌细胞的抗氧化作用。
5.TUNEL法检测心肌细胞凋亡结果表明，urantide三个剂量组均可以不同程度的降低缺血再灌注大鼠的心肌细胞凋亡指数，而Urantide+CHE组与Urantide+LY294002组心肌细胞凋亡指数与urantide用药组相比明显升高，与模型组相比无统计学意义。
6.免疫组化结果显示，urantide可不同程度增加了心肌组织中bcl-2蛋白的表达水平，降低了心肌组织中bax蛋白的表达水平，Urantide+CHE组与Urantide+LY294002组bcl-2及bax蛋白的表达水平与模型组相比无统计学意义。
7.Western blot结果显示，urantide各剂量组可不同程度的升高p-Akt/Akt蛋白的比值，Urantide+CHE组与Urantide+LY294002组可降低p-Akt/Akt蛋白的比值。
第二部分UT受体下调对urantide抗大鼠心肌细胞缺氧再给氧损伤作用的影响
目的：
利用RNA干扰技术，将UTsiRNA转染至原代培养心肌细胞，下调UT受体的表达，在乳鼠原代心肌细胞缺氧复氧模型基础上，观察urantide对心肌细胞缺氧复氧的保护作用与UT受体的关系。
方法：
1.进行乳鼠心肌细胞原代培养，观察不同培养时间心肌细胞的形态及活力，采用免疫组织化学方法进行心肌细胞鉴定；
2.将三种设计合成的UT siRNA用脂质体转染的方法导入原代培养的心肌细胞中，分别用实时定量PCR的方法和Western blot方法检测UT mRNA表达水平和UT蛋白表达水平，筛选出抑制性能最佳的UT siRNA。
3.将UT siRNA转染至原代培养的大鼠乳鼠心肌细胞中，再进行缺氧3h再给氧3h处理，处理前给予urantide10-6mol·L~(-1)，实验结束时收集细胞培养上清，进行相应指标的测定。
4.用台盼蓝染色法和MTT染色法分别检测心肌细胞存活率，PI单染色法流式细胞仪检测心肌细胞凋亡，荧光素染料Hoechst33258测定心肌细胞凋亡，检测心肌细胞培养上清中NO、MDA、cTnI含量和NOS、SOD、LDH、CK的活性。
结果：
1.原代新生大鼠心肌细胞贴壁性能良好，在培养d3细胞搏动明显，搏动频率逐渐加快，至d7天搏动频率开始减少，细胞数量在d3~d4天增长明显；免疫组化方法对心肌细胞进行鉴定，显示心肌细胞纯度可达95%以上。
2.实时荧光定量PCR结果和Western blot结果显示三对UT siRNA对UT mRNA表达水平和UT蛋白表达水平均有抑制作用，其中UT siRNA3组与对照组相比有显著性差异，故后续实验中采用UT siRNA3转染心肌细胞下调UT受体表达。
3.台盼蓝染色法和MTT染色法均显示urantide10-6mol·L~(-1)能够明显增加细胞存活率，但UTsiRNA转染造成UT受体下调对1×10-6mol·L~(-1)urantide的心肌细胞存活率提升有明显的下降作用，MTT染色法显示心肌细胞存活率从其在正常心肌细胞上的81.13±12.46%降至60.89±9.41%。
4.10-6mol·L~(-1)urantide可明显降低细胞培养上清中cTnI含量以及CK、 LDH活性，而在UT受体下调的心肌细胞上，urantide10-6mol·L~(-1)对cTnI含量以及CK、LDH活性的降低作用有明显减弱，cTnI含量以及CK、LDH活性分别从其在正常心肌细胞上的63.73±11.36ng·L~(-1),0.83±0.10U·L~(-1)和109.62±12.66U·L~(-1)分别升高至90.22±12.43ng·L~(-1),1.49±0.08U·L~(-1)和168.90±16.18U·L~(-1)。
5.10-6mol·L~(-1)urantide可降低细胞培养上清中MDA含量，同时提高培养上清中SOD和NOS活性及NO含量，在UTsiRNA介导的UT受体下调的心肌细胞上，10-6mol·L~(-1)urantide对MDA含量降低和SOD活性提高作用均有明显减弱，但对其NOS和NO作用未见明显影响。
6.采用PI单染法流式细胞仪和Hoechst33258荧光染色分别检测了心肌细胞凋亡，而在UTsiRNA3转染使UT受体下调的心肌细胞上，urantide10-6mol·L~(-1)对缺氧再给氧诱导的心肌细胞凋亡抑制作用有明显减弱，凋亡率从其在正常心肌细胞的8.04±0.85%和6.33±1.23%分别升高至13.90±1.11%和9.96±1.41%。
结论：
1.Urantide对大鼠缺血再灌注损伤的心肌组织具有明显的保护作用，该作用可能与抗脂质过氧化、增加NO生成、抑制心肌细胞凋亡等有关；
2.Urantide抗缺血再灌注心肌细胞损伤作用可能与激活PKC-TK-MAPK信号通路及PI3K-AKT信号通路有关；
3.UT受体介入了urantide对大鼠乳鼠心肌细胞缺氧复氧损伤的保护作用；
4.UT受体介导了urantide的保护作用的抗脂质过氧化和抗凋亡机制，但没有涉及其对NO生成和释放作用。
Background：
Cardiovascular diseases are the commonly and frequently encountered diseaseswhich are severely harmful to health. It is well known that cardiovascular diseases havehigh mortality. Ischemic heart disease is one of the high incidence diseases, which ischaracterized by the myocardium lack blood and oxygen supply rapidly. For the pastfew years, Coronary Artery Bypass, thrombolytic therapy, percutaneous transluminalcoronary angioplasty (PTCA), external ciculation of cardiac surgical procedures andcardiopulmonary cerebral resuscitation were established and extensively applied toclinics. All the methods mentioned above can make the ischemic heart regain bloodsupply rapidly. On the contrary, the damage of myocardium was observed to becomeaggratated even proceed irreversibility injury after reperfusion. This phenomemon wasnamed as the ischemical reperfusion injury. Therefore, more and more attention waspaid to develop the effective medicines for prevention or therapy toischemia-reperfusion injury.
Urotensin Ⅱ (UⅡ) is a cyclic peptide initially isolated from the caudalneurosecretory system of teleost fish. The cyclic region of UⅡ, which is responsible forthe biological activity of the peptide, has been fully conserved from fish to human. Thematching of the11amino acid peptide human urotensin-Ⅱ (hU-Ⅱ) with an orphanhuman G-protein coupled receptor with homology to rat GPR14, which was named UTreceptor by Ames in1999. hU-Ⅱ was considered to be the most potent mammalianvasoconstrictor identified so far, which vasoconstrictor potency is higher than ET-1.Many clinical analysis show that the hUⅡ in plasma level is elevated in differentcardiovascular diseases including hypertension, heart failure and so on. No doubt hUⅡ and UT receptors play an important role in cardiovascular diseases.Urantide, is the most potent UT receptor antagonist compound so far reported inthe rat isolated aorta, and is also endowed with high affinity at human UT receptors. Ourprevious studies indicated that urantide had a protective effect against myocardialischemia reperfusion injury in rats and mice. The mechanism may related toantioxidation, reducing calcium overload in cardiomyocytes and augmenting thesynthesis of NO. The present study was going to investigate the effects of urantide usingthe ischemia reperfusion rat model in vivo and hypoxia reoxygenation cellular model invitro respectively. The study was to observe the effects and mechanism of urantide oncardiomyocyte protection. In this study, chelerythrine which is an inhibitor of the PKCsignaling pathway and LY294002which is an inhibitor of the PI3K-Akt signalingpathway were used to assess the relationship between the effects of urantide and relativesignal transduction pathway. On the other hand, by using RNA interference to knockdown the expression of UT receptor it was to be investigated if the UT receptor playsrole in protection of urantide in hypoxia reoxygenation cardiomyocytes.Methods and ResultsPart I Effects of urantide on myocardial ischemia-reperfusion injury in rats andits underlying mechanisms
Methods:
1. The ischemical reperfusion model was set up by ligating and untying left anteriordescending coronary artery in rats. The electrocardiogram was recorded during theexperiment, the serum was collected to measure the content of NO, MDA and cTnI aswell as the activities of CK, LDH and NOS. The heart was harvested to determinemyocardial ischemic size and infarct size by dual staining with Evans blue andtriphenyl-tetrazolium-chloride.
2. The rats were randomized into8groups: All hearts were subjected to theischemia/reperfusion (I/R) protocol consisting of30min of regional ischemia and90 min of reperfusion except for the sham group.
(1) the sham group: the animals’ chest were operated, but the coronary ligatures werenot tied.
(2) the model group: the hearts were subjected to the ischemia/reperfusion (I/R)protocol consisting of30min of regional ischemia and90min of reperfusion
(3) urantide3μg·kg~(-1)group: Urantide (3μg·kg~(-1))was given through intravenous drugperfusion ten minutes before ischemia.
(4) urantide10μg·kg~(-1)group: Urantide(10μg·kg~(-1)) was given through intravenous drugperfusion ten minutes before ischemia.
(5) urantide30μg·kg~(-1)group: Urantide(30μg·kg~(-1)) was given through intravenous drugperfusion ten minutes before ischemia.
(6) verapamil1.6mg·kg~(-1)group: ver (1.6mg·kg~(-1)) was given through intravenous drugperfusion ten minutes before ischemia as the positive control
(7) urantide30μg·kg~(-1)+CHE1mg·kg~(-1)(chelerythrine, the inhibitor of PCK signaling)group: the chelerythrine was given through intravenous drug perfusion afterstabilization, urantide(30μg·kg~(-1)) was given through intravenous drug perfusion fiveminutes after the previous administration, the ischemia reperfusion operation wascarried out ten minutes after the urantide afford.
(8)urantide30μg·kg~(-1)+LY2940020.3mg·kg~(-1)(the inhibitor of PI3K-Akt signaling)group: LY294002was given through intravenous drug perfusion after stabilization,urantide(30μg·kg~(-1)) was given through intravenous drug perfusion five minutesafter the previous administration, the ischemia reperfusion operation was carried outten minutes after the Urantide afford.
3. The hearts were fixed at the end of each experiment. The histopathologic examinationof myocardium in rat was observed by hematoxylin and eosin stain. Theultrastructural morphology in rats cardiomyocyte was measured by transmissionelectron microscope.
4.TUNEL labeling was used for the measurement of apoptosis in myocardium.
5. The expressions of Bcl-2and Bax protein in cardiomyocytes of rats were measuredby immunohistochemistry technology.
6. The expressions of Akt and p-Akt protein in cardiomyocytes of rats were measuredby western blot analysis.
Results:
1. The ST segment of ECG markedly elevated during the process of ischemiareperfusion, while urantide (10,30μg·kg~(-1)) could markedly inhibit the elevation ofST segment of ECG. Urantide (10,30μg·kg~(-1)) could also significantly reduce infarctsize as compared to model hearts, while pharmacological inhibition of PKC andPI3K/Akt signaling completely abrogated urantide-induced inhibition in elevation ofST segment and reduction of infarct size.
2. The cTnI content and CK、LDH activity elevated in model group. Urantide (10,30μg·kg~(-1)) could markedly inhibit the elevation of cTnI content and CK, LDH activitiesin serum. However, those decreased injury indexes increased evidently in urantide30μg·kg~(-1)+CHE group and urantide30μg·kg~(-1)+LY294002group.
3. Morphological observation by HE staining and transmission electron microscopeshowed that the ischemic reperfusion injury was improved by urantide (10,30μg·kg~(-1))administration. But myocardial injury of the urantide30μg·kg~(-1)+CHEgroup and urantide30μg·kg~(-1)+LY294002group showed to become more seriouscompared to that of urantide30μg·kg~(-1)group.
4. Urantide (10,30μg·kg~(-1)) could markedly inhibit the MDA content, and increase theSOD, NOS activities and NO content in serum. However, pharmacological inhibitionof PKC and PI3K/Akt signaling completely abolished changes of these indexesinduced by urantide administration.
5. Compared with those in the sham group, the number of TUNEL-positive cells in I/R group was significantly increased. Urantide (3,10,30μg·kg~(-1)) markedly decreasedthe number of TUNEL-positive cells. On the contrary, pharmacological inhibition ofPKC and PI3K/Akt signaling markedly inhibited urantide-induced decrease ofTUNEL-positive cells.
6. Expression level of Bax protein was significantly higher in model group than that inurantide administration group, whereas the level of Bcl-2protein and Bcl-2/Bax ratiowere markedly higher in30μg·kg~(-1)urantide group than those in model group,respectively. Two proteins expression in urantide30μg·kg~(-1)+CHE group and urantide30μg·kg~(-1)+LY294002group were no statistical difference in comparison with modelgroup.
7. Levels of Akt (total-Akt) and its phosphorylation at Ser473(p-Akt) were determinedby western blotting and phosphorylation of Akt was expressed as the ratio betweenp-Akt and total-Akt. The results showed that Urantide could rise the ratio at threedifferent dose. Furthermore, both CHE and LY294002significantly reduced Aktphosphorylation and decrease the ratio between p-Akt and total-Akt.Part Ⅱ Effects of knockdown of UT receptor on protection of urantide oncultured neonatal rat cardiomyocytes subjected to hypoxia reoxygenation injury
Methods:
1.Cardiomyocytes of neonatal rat were isolated and cultured in vitro. Morphocytologychanges and cell vitality were observed at different period during cell culture. Cellpurity was evaluated through troponin I expression by immunohistochemistrytechnology.
2.Cardiomyocytes were transfected with three different UT siRNA which were designedby Shanghai Gene Pharma Co. Ltd. The UT mRNA and protein expression weredetected by real-time PCR and Western blotting respectively. Thus choosing the mosteffective UT siRNA to knock down the UT receptor.
3. Cardiomyocytes were transfected with the most effective UT siRNA prior to hypoxiathree hours and reoxygenation three hours. Urantide10-6mol·L~(-1)was incubated beforehypoxia reoxygenation. At the end of experiment, the cell culture medium wascollected and proteins were harvested to detect different index.
4. Cell viability was measured by trypan blue staining and MTT staining respectively.Hoechst33258assay and flow cytometric techniques were used to detect apoptoticcells. The content of MDA,NO and cTnI and activities of SOD, CK, LDH, NOS incell culture medium were measured by commercial kits.
Results:
1. Cardiocytes began to adhere and grow after cell culture. It can be observed thatCardiocytes beat spontaneously and rhythmically on the third day, and the frequencebecome more and more after that. The frequence reduced gradually from the seventhday of cell culture. Cardiocytes proliferate obviously on the third and the fourth dayof cell culture. Immunohistochemistry staining confirmed the cell with purity higherthan95％.
2. The levels of UT mRNA and protein expression showed that three differentUTsiRNA could all inhibit UT mRNA and protein express distinctively. The siRNA3was the most effective one among those siRNA, so siRNA3was adopted in latterexperiments.
3. It was found that10-6mol·L~(-1)urantide increased the viability of cardiocytes subjectedto hypoxia/reoxygenation by using both trypan blue staining and MTT staining assay.However, down-regulation of UT receptor in cardiocytes by using UTsiRNA3led toa decrease to increase of cell viability produced by urantide.
4. On the H/R model of myocardial cells, urantide (10-6mol·L~(-1)) could evidently inhibitthe increase of cTn I content, reduce the rise of CK and LDH activities in the cellculture medium. Whereas down-regulation of UT receptor in cardiocytes could also abolish aforementioned effects of urantide.
5. During H/R, Urantide (10-6mol·L~(-1)) could evidently inhibit the increase of MDA andthe decreases of activities of SOD and NOS and content of NO in the cell culturemedium. UT siRNA3-mediated down-regulation of UT receptor could abolish theinhibitory effect of urantide on increase of MDA and decrease of SOD, but had noeffect on urantide-induced alteration of NO content and NOS activity.
6. By using Hoechst33258assay and flow cytometric techniques, it was observed thatthe apoptosis rate in model group was much higher than that in sham group.Pretreatment of10-6mol·L~(-1)urantide significantly inhibited apoptosis induced by H/R.While knock-down of UT receptor by using UT siRNA3could abolish the inhibitioneffects of urantide on H/R-induced apoptosis.
Conclusions:
1. Urantide has a significant protective effect against myocardial ischemic/reperfusioninjury in rats, which may be related to anti-lipidperoxidation, elevating the NOsynthesis and inhibiting apoptosis via modulating the expression o f Bc l-2and Bax.
2. The protective effect of urantide on myocardial ischemic/reperfusion injury in ratsmay be mediated via activation of PKC-TK-MAPK signal transduction pathway andPI3K-Akt signal transduction pathway.
3. UT receptor involves in the protective of urantide on hypoxia reperfusion injury in ratcardiomyocyte.
4. UT receptor mediates the mechanisms of anti-lipidperoxidation and anti-apoptoticeffect of urantide, however UT receptor did not involve urantide-induced synthesisand release of NO.
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