GLP-1受体激动剂干预AngⅡ诱导高血压心脏纤维化的实验研究

发布时间：2018-07-08 09:51

本文选题：高血压 + 心脏纤维化　；参考：《天津医科大学》2017年博士论文

【摘要】：研究背景心脏纤维化可发生于各种心脏疾患,如高血压、缺血性心脏病和心脏瓣膜病。当前我国流行病学调查数据显示,近年来15岁以上人群高血压患病率已高达24%,累计高血压患病人数达2.66亿,每年新增高血压病例约1000万。由于人群中高血压的治疗率(24.7%)与控制率(6.1%)较低,高血压已经逐渐成为心脏纤维化的主要病因。高血压导致的心脏纤维化表现为心脏组织中胶原合成与降解紊乱(包括原有胶原纤维网的降解和胶原纤维过度合成)、心肌细胞间隙胶原纤维异常沉积、心室壁顺应性下降和心脏肥厚,早期造成心脏舒张功能不全,逐渐造成收缩功能不全,最终导致心力衰竭。血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)在高血压心脏纤维化的病理生理过程中发挥着重要作用,AngⅡ通过与AngⅡⅠ型受体(TypeⅠangiotensinⅡreceptor,AT_1R)结合使血管平滑肌收缩、激活炎症细胞诱导机体炎症反应,并刺激成纤维细胞过度表达胶原蛋白致组织间隙胶原纤维沉积造成心脏纤维化。目前针对AngⅡ诱发高血压心脏纤维化的防治药物主要包括血管紧张素转换酶抑制剂(Angiotensin converting enzymeinhibitor,ACEI)和AT_1R拮抗剂(AngiotensinⅡreceptor blocker,ARB)两大类药物,目的在于减少AngⅡ的生成和抑制AT_1R激活。胰高血糖素样肽-1(Glucagon-like peptide 1,GLP-1)是小肠内分泌L细胞分泌的肠促胰岛素激素,在维持人体餐后血糖稳定方面发挥重要作用,但内源性GLP-1分泌后迅速被体内的二肽基肽酶-4(Dipeptidyl peptidase-4,DDP-4)降解,在人体内的半衰期仅为1-2分钟。因此为了延长其半衰期,人们发现了目前应用于临床2型糖尿病治疗的GLP-1受体激动剂:艾塞那肽和利拉鲁肽。临床实践发现,GLP-1受体激动剂除了在糖尿病治疗方面发挥控制血糖、改善胰岛素抵抗等作用外,还在改善人体脂代谢、调节血管内皮功能、预防动脉硬化和心脏保护方面发挥重要作用,提示这类药物可为2型糖尿病患者带来血糖控制与心血管保护的双重获益。动物实验表明,GLP-1受体激动剂可缩小心肌梗死面积、减轻心肌缺血再灌注损伤、改善心脏功能,同时还能改善心肌梗死后心脏纤维化程度。在AngⅡ诱导的高血压心脏纤维化模型中,GLP-1受体激动剂也可抑制血压升高、抑制心脏肥厚并能改善心脏纤维化。此外,GLP-1受体激动剂抑制心脏纤维化的作用也在合并心力衰竭、心肌梗死或高血压的2型糖尿病患者中得到证实。利拉鲁肽与艾塞那肽肽链结构不同,目前尚缺乏研究对比该两种GLP-1受体激动剂对高血压心脏纤维化的影响是否存在不同;且GLP-1受体激动剂对参与心脏纤维化进程中不同细胞(心肌细胞、成纤维细胞、巨噬细胞)的作用也罕见报道。另外,对于GLP-1受体激动剂是否可直接作用于心肌成纤维细胞进而参与心脏纤维化进程,目前学界也存在争议。因此本研究将分别利用利拉鲁肽和艾塞那肽干预AngⅡ诱导的高血压心脏纤维化体外培养细胞和小鼠模型以就上述3个问题进行探讨。研究目的1.观察比较利拉鲁肽和艾塞那肽对AngⅡ诱导的高血压心脏纤维化小鼠心功能和心脏纤维化的作用;2.观察利拉鲁肽和艾塞那肽对于体外培养的心肌细胞、巨噬细胞和成纤维细胞在AngⅡ刺激下的作用;3.通过细胞共培养的方法,观察在细胞水平GLP-1受体激动剂对心脏纤维化过程的作用方式。研究方法1.动物模型建立及药物干预40只健康雄性C57BL/6J小鼠(体重21-26g)适应性饲养1周后随机分为4组。根据分组情况于小鼠背部植入胶囊渗透压泵:对照组植入渗透压泵中含有生理盐水,AngⅡ组、AngⅡ+利拉鲁肽组、AngⅡ+艾塞那肽组植入含有AngⅡ的胶囊渗透压泵以1.44mg/kg/d的速度恒速释放AngⅡ28d;AngⅡ+利拉鲁肽组和AngⅡ+艾塞那肽组分别于术前72h给予利拉鲁肽0.4 mg/kg/d和艾塞那肽0.1mg/kg/d腹腔注射直至实验结束。实验结束后对小鼠进行称重、血糖试纸检测空腹血糖,然后安乐死处死小鼠,留取心脏组织标本。2.超声心动图评价小鼠心脏结构与功能实验末对所有小鼠进行超声心动图检查,利用二维超声和M型超声成像技术测定左心室收缩末期内径(Left ventricular internal dimension in systole,LVIDs)、左心室舒张末期内径(Left ventricular internal dimension in diastole,LVIDd)、室间隔收缩末期厚度(Interventricular septal width in systole,IVSs)、室间隔舒张末期厚度(Interventricular septal width in diastole,IVSd)、左室后壁收缩末期厚度(Left ventricular posterior wall width in systole,LVPWs)、左室后壁舒张末期厚度(Left ventricular posterior wall width in diastole,LVPWd),左心室射血分数(Left ventricular ejection fraction,LVEF)和左心室短轴缩短率(Left ventricular fraction shortening,LVFS)评价小鼠心脏结构与功能。3.小鼠血压测定在术前与术后,每周应用小鼠鼠尾血压仪测量各组小鼠收缩压,根据收缩压的平均值和标准差,应用Graphpad软件绘制时间-收缩压折线图。4.组织学染色将各组小鼠取材后的心脏制作石蜡切片,进行HE和Masson染色,观察心肌细胞形态、组织间隙胶原沉积情况,Masson染色半定量分析比较各组心脏标本的胶原含量。5.小鼠心肌成纤维细胞分离培养本研究利用2-3日龄昆明小鼠乳鼠心脏,剪碎后经胶原酶Ⅱ消化,利用差速贴壁法分离出小鼠心肌成纤维细胞。纯化后培养于DMEM培养基中,选择2-3代细胞用于实验。6.细胞培养探讨两种GLP-1受体激动剂对AngⅡ刺激的小鼠心肌成纤维细胞、小鼠巨噬细胞RAW264.7和大鼠心肌细胞H9C2的作用。根据给予刺激不同进行分组:对照组(无干预)、AngⅡ组(AngⅡ10~(-6)mol/L)、AngⅡ+艾塞纳肽组(AngⅡ10~(-6)mol/L+艾塞纳肽100nmol/L)、AngⅡ+利拉鲁肽组(AngⅡ10~(-6)mol/L+利拉鲁肽100nmol/L),培养24h后收集细胞。7.细胞共培养应用Transwell小室将小鼠巨噬细胞与心肌成纤维细胞共培养,按步骤6中的分组进行干预,观察巨噬细胞与成纤维细胞间的相互作用。8.蛋白印迹(Western blot)收集各组细胞,提取蛋白,分别检测心肌细胞和成纤维细胞Ⅰ型胶原、Ⅲ型胶原、MMP-2和MMP-9的表达及巨噬细胞IL-6、IL-1β和TGF-β1的表达情况。9.统计学方法实验数据的统计学分析应用SPSS16.0软件。连续变量资料以均数±标准差的形式表示。根据变量特点选择Oneway ANOVA或LSD post-hoc来进行多组数据间的变量比较。p0.05代表差异具有统计学显著性意义。研究结果1.体重和血糖:在实验结束时,AngⅡ+利拉鲁肽组(26.18±1.40g)和AngⅡ+艾塞那肽组的小鼠体重(26.51±1.38g)比对照组(27.65±0.97g)稍下降(p0.05);4个小鼠实验组之间的空腹血糖无差异(p0.05)。2.收缩压的影响:在实验结束时,AngⅡ+利拉鲁肽组(139.0±2.9mm Hg)和AngⅡ+艾塞纳肽组小鼠(141.7±5.4mm Hg)收缩压均低于AngⅡ组(148.3±4.1mm Hg,p0.05),GLP-1受体激动剂干预组小鼠之间收缩压无差异(p3.超声心动图指标:AngⅡ+利拉鲁肽组的LVIDd、LVIDs、IVSd、LVPWd、LVEF和FS分别为3.59±0.38mm、2.01±0.26mm、0.59±0.08mm、0.82±0.05mm、76.3±3.06%、44.4±2.17%;AngⅡ+艾塞那肽组的LVIDd、LVIDs、IVSd、LVPWd、LVEF和FS分别为3.43±0.63mm、1.94±0.41mm、0.69±0.04mm、0.79±0.04mm、75.6±5.23%、44.3±2.75%;以上两组的各指标与AngⅡ组0.87±0.04mm、LVEF 64.5±4.62%和FS 35.5±3.24%)的均有差异(p0.05),GLP-1受体激动剂干预组小鼠之间上述指标无差异(p0.05)。4.组织学HE染色和Masson染色:(1)HE染色切片显示,与对照组相比,AngⅡ组心脏室壁增厚、心腔扩大、心肌细胞肥大、排列紊乱、细胞间隙缩小;而利拉鲁肽和艾塞那肽干预组的上述异常表观较AngⅡ组轻。(2)Masson染色显示,AngⅡ组切片染成蓝色的胶原纤维含量明显高于其余3组,半定量分析显示利拉鲁肽和艾塞那肽干预组切片的胶原含量低于AngⅡ组(p0.05),GLP-1受体激动剂干预组小鼠之间胶原含量无差异(p0.05)。5.GLP-1受体激动剂对大鼠心肌细胞的影响:细胞培养Western blot结果显示,与对照组相比,AngⅡ组H9C2细胞MMP-2、MMP-9和Ⅲ型胶原表达量增加(p0.05);利拉鲁肽和艾塞那肽干预后可抑制AngⅡ对心肌细胞MMP-2和MMP-9表达的上调作用(p0.05)。6.GLP-1受体激动剂对小鼠巨噬细胞的影响:Western blot显示,相比对照组,AngⅡ可上调小鼠巨噬细胞RAW264.7、促炎因子IL-6、IL-1β和TGF-β1的表达(p0.05);相比AngⅡ组,给予利拉鲁肽和艾塞那肽干预后,巨噬细胞表达TGF-β1减少(p0.05)。7.GLP-1受体激动剂对小鼠心肌成纤维细胞的影响:Western blot显示,相比对照组,AngⅡ刺激后心肌成纤维细胞表达Ⅰ型胶原、Ⅲ型胶原、MMP-2、MMP-9增多(p0.05);而相比AngⅡ组,上述蛋白在利拉鲁肽和艾塞那肽干预组均无变化(p0.05)。8.GLP-1受体激动剂对与小鼠巨噬细胞共培养的小鼠心肌成纤维细胞的影响:Western blot显示,和AngⅡ组相比,加入AngⅡ刺激的巨噬细胞可促进成纤维细胞表达更多的胶原蛋白和MMP-2、MMP-9,而加入利拉鲁肽和艾塞那肽干预AngⅡ刺激的巨噬细胞后,心肌成纤维细胞中Ⅰ型、Ⅲ型胶原、MMP-2、MMP-9的表达量减少(p0.05)。研究结论1.利拉鲁肽和艾塞那肽能够降低AngⅡ诱导的小鼠动物模型血压升高的幅度,降压幅度二者之间无差异,对小鼠的空腹血糖水平无影响;2.利拉鲁肽和艾塞那肽能改善AngⅡ诱导的小鼠高血压心脏纤维化模型的心脏肥厚、心腔扩大、心功能减低和心脏间质纤维化,并且二者之间无差别;3.利拉鲁肽和艾塞那肽能够抑制AngⅡ刺激下大鼠心肌细胞H9C2过量表达MMP-2、MMP-9以及小鼠巨噬细胞RAW264.7TGF-β1的过量表达;对于AngⅡ诱导的小鼠心肌成纤维细胞过量表达MMP-2、MMP-9、Ⅰ型胶原、Ⅲ型胶原无抑制作用;4.利拉鲁肽与艾塞那肽干预AngⅡ刺激的巨噬细胞与心肌成纤维细胞共培养可抑制心肌成纤维细胞中Ⅰ型、Ⅲ型胶原、MMP-2、MMP-9的表达。
[Abstract]:Background cardiac fibrosis can occur in all kinds of heart diseases, such as hypertension, ischemic heart disease and heart valvular disease. Current epidemiological data in China show that in recent years, the prevalence of hypertension in people over 15 years of age has reached 24%, the cumulative number of hypertensive patients is 266 million, and about 10 million of the new cases of hypertension are added each year. The treatment rate of hypertension (24.7%) and control rate (6.1%) are low. Hypertension has gradually become the main cause of cardiac fibrosis. The cardiac fibrosis caused by hypertension is manifested in the disorder of collagen synthesis and degradation in the cardiac tissue (including the degradation of the original collagen fibrous net and the oversynthesis of collagen fibers), and the abnormal collagen fibrils in the intercellular space of the cardiac myocytes Deposition, decline of ventricular wall compliance and cardiac hypertrophy, early diastolic dysfunction, gradually causing systolic dysfunction, and eventually leading to heart failure. Angiotensin II (Angiotensin II, Ang II) plays an important role in the pathophysiological process of hypertensive cardiac fibrosis, and Ang II passes through the Ang II type I receptor (Type I angi). Otensin II receptor, AT_1R) combined with contraction of vascular smooth muscle, activating inflammatory cells to induce inflammatory response, and stimulating fibroblasts to overexpress collagen induced collagen fibrous deposition of collagen to cause cardiac fibrosis. At present, the prevention and treatment drugs for Ang II induced hypertension mainly include angiotensin converting Enzyme inhibitors (Angiotensin converting enzymeinhibitor, ACEI) and AT_1R antagonists (Angiotensin II receptor blocker, ARB) are the two major drugs to reduce the formation of Ang II and inhibit AT_1R activation. Glucagon like peptide 1 is an enterostimulating hormone secreted by intestinal endocrine cells and is maintained. The human body plays an important role in postprandial blood glucose stability, but the endogenous GLP-1 secretion is rapidly degraded by the two peptidyl peptidase -4 (Dipeptidyl peptidase-4, DDP-4) in the body, and the half-life in the human body is only 1-2 minutes. Therefore, in order to prolong the half-life of the body, a GLP-1 receptor agonist, which should be used for the treatment of type 2 diabetes, is found. The clinical practice has found that GLP-1 receptor agonists have played an important role in improving human lipid metabolism, regulating vascular endothelial function, preventing arteriosclerosis and heart protection in addition to controlling blood sugar and improving insulin resistance in the treatment of diabetes, suggesting that these drugs can be used in type 2 diabetes. Animal experiments have shown that GLP-1 receptor agonists can reduce myocardial infarction area, reduce myocardial ischemia reperfusion injury, improve cardiac function, and improve the degree of cardiac fibrosis after myocardial infarction. In the Ang II induced hypertensive cardiac fibrosis model, GLP-1 receptor excitation Also, the role of GLP-1 receptor agonists in inhibiting cardiac fibrosis is also confirmed in patients with type 2 diabetes with heart failure, myocardial infarction, or hypertension. Lalalupin and alsasin peptide chain structure is different, and there is still a lack of research and comparison at present. The effect of two GLP-1 receptor agonists on hypertensive cardiac fibrosis is different, and the role of GLP-1 receptor agonists in different cells (cardiomyocytes, fibroblasts, macrophages) in the process of cardiac fibrosis is also rarely reported. In addition, whether GLP-1 receptor agonists can directly act on myocardial fibroblasts. This study will discuss the above 3 problems with lealulu and alenenin on Ang II induced hypertensive cardiac fibrosis in vitro culture cells and mouse models respectively. Purpose 1. the purpose of this study was to induce Ang II than lealulu and alenenin. The function of cardiac function and cardiac fibrosis in mice with hypertensive cardiac fibrosis; 2. the effects of lealulu and alenenin on the stimulation of cardiac myocytes, macrophages and fibroblasts in vitro were observed by Ang II. 3. by cell co culture, the cardiac fibrosis process was observed at the level of GLP-1 receptor agonists at the cell level. Method 1. animal model establishment and drug intervention of 40 healthy male C57BL/6J mice (body weight 21-26g) were randomly divided into 4 groups after 1 weeks of adaptive feeding. According to the group situation, the capsule osmotic pressure pump was implanted in the back of the mice: the control group was implanted osmotic pump containing physiological salt water, Ang II group, Ang II + L alalu group, Ang II + Arbor The peptide group implants the capsule osmotic pump containing Ang II to release Ang II 28d at a constant speed of 1.44mg/kg/d; Ang II + learalu group and Ang II + aleninin group were given the lialalu 0.4 mg/kg/d and the 0.1mg/kg/d intraperitoneal injection until the end of the experiment before the operation. After the experimental knot, the mice were weighed and the blood glucose test paper was detected. Fasting blood glucose, then euthanasia was executed in mice, and cardiac tissue specimens were left for.2. echocardiography to evaluate the echocardiographic examination of all mice at the end of the heart structure and function test, and the left ventricular end systolic diameter (Left ventricular internal dimension in systole, LVIDs) was measured by two-dimensional ultrasound and M ultrasound imaging technique. The end diastolic diameter of the left ventricle (Left ventricular internal dimension in diastole, LVIDd), the end systolic thickness of the ventricular septum (Interventricular septal width in systole), the end systolic thickness of the ventricular septum, and the end systolic thickness of the left ventricular wall DTH in systole, LVPWs), the end diastolic thickness of left ventricular posterior wall (Left ventricular posterior wall width in diastole, LVPWd), left ventricular ejection fraction and left ventricular short axis shortening rate in mice The mice tail blood pressure instrument was used to measure the systolic pressure of mice every week before and after the operation. According to the mean and standard deviation of the systolic pressure, the Graphpad software was used to draw the time contraction compression line graph.4. tissue staining to make paraffin sections of the hearts after the mice were obtained by HE and Masson staining, and to observe the morphology of the cardiac myocytes and the interstitial space. Collagen deposition, Masson staining and semi quantitative analysis were used to compare the collagen content of cardiac specimens in each group.5. mouse myocardial fibroblasts isolated and cultured. The heart of 2-3 day old Kunming mice was used to digest the rat heart. After the digestion of collagenase II, the murine myocardial fibroblasts were isolated by the differential adherence method, and then cultured in the DMEM medium. 2-3 generation cells were selected for experimental.6. cell culture to explore the effect of two GLP-1 receptor agonists on Ang II stimulated murine myocardial fibroblasts, mouse macrophages RAW264.7 and rat cardiac myocyte H9C2. The control group was divided into groups: the control group (no intervention), the Ang II Group (Ang II 10~ (-6) mol/L), Ang II + Senna peptide group (Ang) II 10~ (-6) mol/L+ Senna peptide 100nmol/L), Ang II + L alalu peptide group (Ang II 10~ (-6) mol/L+ alalu peptide 100nmol/L). After culture 24h, the cell.7. cells were co cultured and used to co culture mouse macrophages and myocardial fibroblasts, and they were intervened in step 6 and observed between macrophages and fibroblasts. Interaction.8. Western blot (Western blot) was used to collect all the cells and extract protein. The expression of type I collagen, collagen type III, MMP-2 and MMP-9 in cardiomyocytes and fibroblasts and the expression of IL-6, IL-1 beta and TGF- beta 1 in macrophage, IL-6, IL-1 beta and TGF- beta 1 were used for statistical analysis of the statistical analysis of SPSS16.0 software. Oneway ANOVA or LSD post-hoc was selected according to the variable characteristics to compare the variables between the multiple groups of data. The.P0.05 representation difference was statistically significant. The results of the study were 1. weight and blood sugar: at the end of the experiment, the mice of Ang II + learalu group (26.18 + 1.40g) and Ang II + alisin group The body weight (26.51 + 1.38g) was slightly lower than that of the control group (27.65 0.97g) (P0.05), and the effect of the fasting blood glucose (P0.05).2. systolic pressure between the 4 experimental groups: at the end of the experiment, the systolic pressure of the Ang II + alaru group (139 + 2.9mm Hg) and the Ang II + alSenna peptide group (141.7 + 5.4mm Hg) was lower than that of the Ang II Group (148.3 +. There was no difference in systolic pressure between mice in the LP-1 receptor agonist group (p3. echocardiography: LVIDd, LVIDs, IVSd, LVPWd, LVEF and FS were 3.59 + 0.38mm, 2.01 + 0.26mm, 0.59 + 0.08mm, 0.82 +, 76.3 + 3.06%, 44.4 + 2.17%, 3.43 + 0, respectively. .63mm, 1.94 + 0.41mm, 0.69 + 0.04mm, 0.79 + 0.04mm, 75.6 + 5.23%, 44.3 + 2.75%, and the indexes of the above two groups were different from Ang II Group 0.87 + 0.04mm, LVEF 64.5 + 4.62% and FS 35.5 + 3.24%. There was no difference between the GLP-1 receptor agonist intervention group and the mice. Compared with the control group, the cardiac ventricular wall in the Ang II group was thickened, the heart cavity enlarged, the cardiac myocytes hypertrophy, disorder and the gap narrowed, while the above abnormality was lighter than the Ang II group. (2) Masson staining showed that the content of the blue collagen fiber in the Ang II group was significantly higher than the other 3 groups, half quantified. The collagen content of the sliced group of lialalu and aleninin intervention group was lower than that of the Ang II Group (P0.05). There was no difference in the collagen content between the mice of the GLP-1 receptor agonist intervention group (P0.05) and the effect of.5.GLP-1 receptor agonist on the rat cardiac myocytes: the cell culture Western blot results showed that the H9C2 cell MMP-2, MMP-9 of the Ang II group was compared with the control group. The expression of collagen type III and type III increased (P0.05), and the effects of Ang II on the expression of MMP-2 and MMP-9 (P0.05).6.GLP-1 receptor agonist on murine macrophages were inhibited by learalu and alenenin: Western blot showed that Ang II could increase RAW264.7, IL-6, IL-1 beta, and IL-1 beta in mice compared with the control group And the expression of TGF- beta 1 (P0.05); compared with the Ang II group, the effects of the macrophage on the expression of TGF- beta 1 decreased (P0.05).7.GLP-1 receptor agonists on the myocardial fibroblasts in the Ang II Group: Western blot showed that compared with the control group, the myocardial fibroblasts expressed type I collagen, type III collagen, MMP-2, MMP after Ang II stimulation. -9 increased (P0.05), but compared with the Ang II group, the above protein in the lialalu peptide and the alenenin intervention group had no change (P0.05) the effect of.8.GLP-1 receptor agonist on murine myocardial fibroblasts co cultured with mouse macrophages: Western blot showed that the macrophages added to the Ang II stimulated macrophage can promote fibroblast cell surface compared with the Ang II group. More collagen and MMP-2, MMP-9, and the expression of type I, type III collagen, MMP-2, MMP-9 in myocardial fibroblasts decreased (P0.05) after adding lealupeptide and alenenin to Ang II stimulated macrophages (P0.05). Conclusion 1. lealaru and alenenin could reduce the elevation of blood pressure in the mouse model induced by Ang II. There was no difference between the two degrees and the amplitude of blood pressure, and there was no effect on the level of fasting blood glucose in mice. 2. lalalu and aleninin could improve the cardiac hypertrophy, enlargement of heart cavity, decrease of heart function and interstitial fibrosis in the model of Ang II induced hypertensive cardiac fibrosis in mice, and there was no difference between the two and 3. llalupeptis and aleninin. The overexpression of H9C2 overexpression of MMP-2, MMP-9 and RAW264.7TGF- beta 1 of murine macrophages was inhibited by Ang II stimulation, and the excess expression of MMP-2, MMP-9, type I collagen and type III collagen in murine myocardial fibroblasts induced by Ang II was not inhibited; 4. lyalurin and alenenin interfered with the macrophages and macrophages stimulated by Ang II. Co culture of cardiac fibroblasts inhibited the expression of type I, III collagen, MMP-2 and MMP-9 in cardiac fibroblasts.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R541.3

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