硫化氢改善高血压动脉内皮功能紊乱的机制研究

发布时间：2018-04-27 06:43

本文选题：高血压 + H_2S　；参考：《河北医科大学》2016年博士论文

【摘要】：第一部分硫化氢激活PPARδ/eNOS通路改善高血压患者肾动脉内皮依赖的舒张功能目的:硫化氢(H2S)被证实为第三种气体信号分子,其作为气体调质在心血管系统发挥重要作用,具有改善血管内皮功能紊乱的作用,但是其保护内皮作用的机制目前还远未阐明清楚。过氧化物酶体增殖物激活受体δ(peroxisome proliferators-activated receptorδ,PPARδ)作为核受体家族的一员,在代谢方面发挥的作用已经为大家所熟知。最近的研究发现,PPARδ能够通过激活eNOS/NO通路增强内皮依赖的舒张功能。因此,我们提出假设认为H2S通过激活PPARδ/eNOS通路发挥改善内皮的作用。本研究选取高血压患者和血压正常患者的肾动脉,旨在探讨H2S对PPARδ/eNOS通路的影响,以及是否通过该通路改善高血压患者的内皮舒张功能紊乱。方法:本实验选取罹患高血压或血压正常并行肾切除患者的肾动脉。高血压患者的入选标准为收缩压≥140 mmHg或舒张压≥90 mmHg,不合并糖尿病。入选患者均已签署知情同意书。高血压组和血压正常组的年龄无差异(64.00±2.892岁vs 59.92±2.058岁)。高血压组和正常血压组的肾动脉在加入或不加入硫氢化钠(NaHS,50μmol/L)的情况下,置于完全DMEM培养基中37℃孵育12 h。孵育后,取肾动脉在器官浴槽中检测乙酰胆碱(acetylcholine,ACh)诱导的内皮依赖性血管舒张功能。利用Western blot检测血管紧张素受体1(AngiotensinⅡreceptor 1,AT1)、胱硫醚-γ-裂解酶(cystathioneγlyase,CSE)、PPARδ、磷酸化内皮型一氧化氮和酶(phosphorylated endothelial nitric oxide synthase,p-eNOS)、p-Akt和p-AMPK等相关蛋白的表达。人脐静脉内皮细胞经过AngII处理后,观察H2S以及各种工具药对一氧化氮(nitric oxide,NO)生成量的影响。应用激光共聚焦显微镜,检测NO探针的荧光强度来反映NO含量。结果:1高血压组肾动脉对ach诱导的内皮依赖性的舒张反应性较血压正常组肾动脉明显下降。nahs慢性孵育高血压患者肾动脉可改善内皮依赖性舒张功能。2高血压组患者的肾动脉at1蛋白表达水平显著升高,cse蛋白表达水平下降。nahs慢性孵育可下调高血压患者肾动脉at1受体表达,增加cse蛋白水平。3高血压组患者肾动脉pparδ蛋白表达下调,p-enos表达水平降低。nahs慢性孵育可上调高血压患者肾动脉pparδ受体表达,提高p-enos表达水平。4nahs慢性孵育可增加高血压患者肾动脉p-akt和p-ampk蛋白表达水平。5在培养的人脐静脉内皮细胞中,nahs可增加血管紧张素ii(angiotensinii,angii)处理的人脐静脉内皮细胞no生成量。nahs促进no生成的作用可被gsk0660(1μmol/l,pparδ受体阻断剂)、ly294002(5μmol/l,pi3k抑制剂)、wortmanin(0.1μmol/l,pi3k抑制剂)和compoundc(10μmol/l,ampkα抑制剂)部分阻断。而应用at1受体的阻断剂可发挥与nahs相似的作用。结论:h2s可改善高血压患者内皮依赖性的血管舒张功能,其改善内皮功能的作用可能与改善血管内源性cse和at1的失衡,以及激活pparδ/enos通路有关。pi3k/akt和ampk信号通路可能参与了h2s激活pparδ/enos通路的作用。第二部分硫化氢激活pparδ改善肾血管性高血压大鼠肾动脉内皮依赖的舒张功能目的:大鼠肾血管性高血压模型是基础研究中常用的高血压模型之一。肾血管性高血压时肾素-血管紧张素系统(renin-angiotensinsystem,ras)的过度激活,最终导致内皮功能受损。本部分研究利用大鼠肾血管性高血压模型,进一步证实h2s通过激活pparδ通路,改善高血压时动脉内皮以来的舒张功能,并探讨其激活pparδ的信号转导通路。方法:本实验选用健康雄性spraguedawley(sd)大鼠,7周龄,190-200克,随机分为四组:sham组、sham+nahs组、2k1c组和2k1c+nahs组。2k1c和2k1c+nahs组大鼠行两肾一夹手术制备肾血管性高血压模型。sham+nahs和2k1c+nahs组大鼠于两肾一夹手术或假手术后第三周开始腹腔注射nahs(56μmol/kg),其余两组于相同时间点腹腔注射生理盐水。每4周通过鼠尾动脉测量动物血压。第20周末利用小动物超声检测肾动脉血流量;各组动物取肾动脉行微血管张力测定,观察内皮和非内皮依赖性血管舒张功能,并且应用pparδ受体阻断剂gsk0660(1μmol/l)、激动剂gw0742(0.1μmol/l)、ampkα磷酸化抑制剂(compoundc10μmol/l)、pi3k的抑制剂ly294002(5μmol/l)和wortmanin(0.1μmol/l)以及no合酶的抑制剂l-name(100μmol/l),通过慢性孵育2k1c组和2k1c+nahs组大鼠肾动脉观察上述阻断剂对nahs作用的影响。利用高压液相质谱法测定血浆中h2s含量;利用硝酸盐还原酶法测定血浆中no含量;he染色观察肾脏形态学变化;利用westernblot检测pparδ、cse、p-enos、p-akt和p-ampk等蛋白的表达水平。结果:1四组大鼠一般情况监测结果显示:2k1c组和2k1c+nahs组大鼠体重在术后12周开始出现增长缓慢,并一直持续至实验结束。四组动物的进食量和饮水量无差异,2k1c组的尿量较其他三组动物显著升高。2慢性外源性给予nahs可显著降低2k1c大鼠的平均动脉压和血浆中angii水平以及肾动脉at1受体蛋白表达水平。3慢性外源性给予nahs可显著降低2k1c大鼠左右肾脏重量比、改善肾功能、减轻肾实质形态学损伤。4慢性外源性给予nahs可显著增加2k1c大鼠未钳夹侧肾血流量。5慢性外源性给予nahs可显著改善2k1c大鼠内皮依赖性的血管舒张功能,而对非内皮依赖性的舒张功能无显著影响6慢性外源性给予nahs可显著升高血浆h2s和no含量,上调肾动脉cse蛋白表达水平。7慢性外源性给予nahs可显著上调2k1c大鼠肾动脉pparδ和p-enos的蛋白表达水平。8慢性外源性给予nahs可显著上调2k1c大鼠肾动脉p-akt和p-ampk的蛋白表达水平。9应用pparδ受体阻断剂、激动剂,ampkα磷酸化抑制剂,pi3k的抑制剂,no合酶的抑制剂,通过慢性孵育2k1c组和2k1c+nahs组大鼠肾动脉,发现上述阻断剂可部分或全部阻断nahs的作用。结论:h2s可改善高血压患者内皮依赖性的血管舒张功能,其改善内皮功能的作用可能与改善血管内源性cse和at1的失衡,以及激活pparδ/enos通路有关。pi3k/akt和ampk信号通路可能参与了h2s激活pparδ/enos通路,并进而改善高血压动脉内皮依赖性血管舒张功能的作用。第三部分硫化氢通过下调bmp4/cox2的表达改善肾血管性高血压大鼠肾动脉内皮依赖的收缩功能目的:高血压时,内皮功能紊乱一方面表现为内皮依赖的血管舒张功能降低,另一方面表现为内皮依赖的血管收缩功能增强。骨形成蛋白4(bonemorphogenicprotein4,bmp4)属于转化生长因子β(transforminggrowthfactor-β,tgf-β)超家族,主要调节骨骼的胚胎发育和再生修复。最近研究发现,bmp4在心血管系统中发挥重要作用。bmp4通过增加nadph氧化酶的表达和活性,促进活性氧自由基(reactiveoxygenspecies,ros)过度生成,进而增加环氧酶2(cyclooxygenase2,cox2)活性,导致前列腺素类物质生成和释放增多,从而损伤血管内皮,增强血管内皮依赖的收缩功能,最终促进高血压形成。已有研究显示,bmp4/cox2表达增加与自发性高血压大鼠内皮依赖性的血管收缩功能增强有关。本研究利用2k1c制备的高血压大鼠模型,旨在观察h2s改善高血压大鼠内皮依赖的血管收缩功能,并观察h2s对bmp4/cox2表达的影响。方法:健康雄性sd大鼠,7周龄,190-200克,随机分为四组:sham组、sham+nahs组、2k1c组和2k1c+nahs组。2k1c和2k1c+nahs组大鼠行两肾一夹手术制备肾血管性高血压模型。sham+nahs和2k1c+nahs组大鼠于两肾一夹手术或假手术后第三周开始腹腔注射nahs(56μmol/kg),其余两组于相同时间点腹腔注射生理盐水。开始应用nahs后每4周通过鼠尾动脉测量动物血压;第20周末各组动物取肾动脉行微血管张力的测定,观察血管内皮依赖性的收缩功能;利用高压液相质谱法测定血浆中h2s的含量;利用westernblot检测cse、at1、bmp4、cox2以及氧化应激相关蛋白的表达水平;利用试剂盒检测sod活性和mda含量。结果1 2K1C高血压大鼠慢性给予NaHS处理,可显著降低大鼠动脉血压,上调CSE蛋白表达,增加血浆中H2S含量。2 2K1C大鼠肾动脉内皮依赖性收缩功能显著增强。外源性给予NaHS可改善高血压大鼠内皮依赖性血管收缩功能。3外源性给予NaHS可显著下调肾血管性高血压大鼠肾动脉中AT1受体和BMP 4的蛋白表达。4外源性给予NaHS可显著降低肾血管性高血压大鼠体内氧化应激水平,氧化应激相关蛋白NOX2、NOX4、P67、Nitrotysine表达增加。5外源性给予NaHS可显著升高SOD活性,降低MDA含量。6外源性给予NaHS可显著改变p38 MAPK和COX-2的蛋白表达水平。结论:H2S可显著改善高血压大鼠肾动脉血管内皮依赖的收缩功能,其改善内皮功能的作用可能与抑制BMP4/ROS/COX2通路有关。
[Abstract]:The first part of hydrogen sulfide activates the PPAR Delta /eNOS pathway to improve the endothelium-dependent diastolic function of renal artery in hypertensive patients: hydrogen sulfide (H2S) is proved to be a third gas signal molecule, which plays an important role in the cardiovascular system as a gas conditioner and has the effect of improving vascular endothelial dysfunction, but it protects the endothelial function. The system is far from clear. As a member of the nuclear receptor family, the peroxisome proliferators-activated receptor delta (PPAR delta), a member of the nuclear receptor family, is known to play a role in metabolism. Recent studies have found that PPAR delta can enhance endothelium dependent relaxation by activating the eNOS/NO pathway. Therefore, we hypothesized that H2S plays the role of improving the endothelium by activating the PPAR Delta /eNOS pathway. In this study, the renal arteries of patients with hypertension and normal blood pressure were selected to explore the effect of H2S on the PPAR Delta /eNOS pathway and to improve the endothelium diastolic dysfunction in hypertensive patients. Methods: this experiment The renal arteries of patients with hypertension or normal blood pressure and normal nephrectomy were selected. The standard of hypertension patients was systolic pressure more than 140 mmHg or diastolic pressure more than 90 mmHg, without diabetes. The patients who were selected had signed informed consent. There was no difference between the hypertension group and the normal blood pressure group (64 + 2.892 years vs 59.92 + 2.058 years old). The renal arteries in the group and the normal blood pressure group were incubated at 37 centigrade for 12 h. under the condition of adding or not adding sodium hydrogen sulfide (NaHS, 50 u mol/L). The endothelium dependent vasodilatation function induced by acetylcholine (acetylcholine, ACh) in the organ bath was detected by the renal artery in the complete DMEM medium. The angiotensin was detected by Western blot. Receptor 1 (Angiotensin II receptor 1, AT1), cystthioether - gamma lyase (cystathione gamma lyase, CSE), PPAR Delta, phosphorylated endothelial nitric oxide and enzyme (phosphorylated endothelial nitric oxide synthase, phosphorylated) and other related proteins. Effect of drug on the production of nitric oxide (nitric oxide, NO). The use of laser confocal microscope to detect the fluorescence intensity of NO probe to reflect the NO content. Results: 1 the renal artery to ach induced endothelium dependent diastolic responsiveness in the hypertensive group was better than that of the normal blood pressure group and the renal artery decreased significantly in the renal artery, and the renal artery could be improved in the.Nahs chronic incubated hypertensive patients. The expression of AT1 protein in renal artery was significantly increased in the patients with endothelium dependent diastolic function in.2 hypertension group, and the decrease of CSE protein expression level in.Nahs chronic incubation could reduce the expression of AT1 receptor in renal artery in hypertensive patients, increase the level of PPAR delta protein in renal artery of.3 hypertension group, and decrease the p-enos expression level to reduce the chronic incubation of.Nahs. Raising the expression of PPAR delta receptor in renal artery in hypertensive patients and increasing p-enos expression level.4nahs chronic incubation can increase the expression level of p-Akt and p-ampk protein in renal artery of hypertensive patients in cultured human umbilical vein endothelial cells. NaHS can increase the no production of umbilical vein endothelial cells treated by angiotensin II (AngiotensinII, AngII). .nahs promotes no formation by gsk0660 (1 mu mol/l, PPAR delta receptor blocker), LY294002 (5 mol/l, PI3K inhibitor), wortmanin (0.1 mu mol/l, PI3K inhibitor) and compoundc (10 micron, alpha inhibitor). The vasodilatation function, which improves endothelial function, may improve the imbalance of endogenous CSE and AT1, and the activation of the.Pi3k/akt and AMPK signaling pathways of the PPAR Delta /enos pathway may be involved in the H2S activation of the PPAR Delta /enos pathway. The second part of hydrogen sulfide activates PPAR delta to improve the renal artery in renovascular hypertensive rats The objective of skin dependent diastolic function: the model of renovascular hypertension in rats is one of the commonly used hypertension models in basic research. The excessive activation of the renin angiotensin system (renin-angiotensinsystem, RAS) in renal vascular hypertension eventually leads to the damage of the endothelial function. One step confirmed that H2S could improve the diastolic function of arterial endothelium in hypertension by activating the PPAR delta pathway and explore the signal transduction pathway to activate PPAR Delta. Methods: this experiment selected healthy male spraguedawley (SD) rats, 7 weeks old and 190-200 grams, randomly divided into four groups: sham group, sham+nahs group, 2K1C group and 2k1c+nahs group.2k1c and 2k1c+nahs group Rats were treated with two kidney one clip operation to prepare renal vascular hypertension model.Sham+nahs and 2k1c+nahs rats. NaHS (56 mol/kg) was injected into the abdominal cavity at third weeks after two kidney one clip operation or sham operation. The rest two groups were injected with normal saline at the same time point. The blood pressure was measured by the rat tail moving pulse every 4 weeks. The twentieth weekend was used by small animals. The renal artery blood flow was measured by sound. The microvascular tension of the renal arteries was measured in each group. The endothelium and non endothelium dependent vasodilatation were observed. The PPAR delta receptor blocker gsk0660 (1 mu mol/l), the agonist GW0742 (0.1 mu mol/l), the AMPK alpha phosphorylation inhibitor (compoundc10 mu mol/l), the PI3K inhibitor LY294002 (5 mu mol/l) and wortmanin were used. (0.1 mu mol/l) and the inhibitor of NO synthase inhibitor L-NAME (100 mol/l), the effects of these blockers on NaHS were observed by chronic incubation of 2K1C group and rat renal artery in group 2k1c+nahs. The content of H2S in plasma was measured by high pressure liquid chromatography-mass spectrometry, and the content of no in plasma was measured by nitrate reductase; he staining was used to observe the morphological changes of kidney; The expression level of PPAR Delta, CSE, p-enos, p-Akt and p-ampk was detected by Westernblot. Results: 1 the general monitoring results of the four groups of rats showed that the weight of the 2K1C group and the 2k1c+nahs group began to grow slowly at 12 weeks after the operation, and continued until the end of the experiment. There was no difference between the intake of the four groups of animals and the amount of drinking water, and the urine volume of the 2K1C group was more than that of the group of 2K1C. The other three groups of animals significantly increased.2 chronic exogenous NaHS can significantly reduce the average arterial pressure and plasma AngII level in 2K1C rats and the expression level of AT1 receptor protein in renal artery,.3 chronic exogenous administration of NaHS can significantly reduce the kidney weight ratio of 2K1C rats, improve renal function and alleviate the chronic renal parenchyma morphological injury.4 chronic exogenous exogenous disease. Sexual administration of NaHS significantly increased the non clamp side renal blood flow of 2K1C rats.5 chronic exogenous NaHS could significantly improve the endothelium-dependent vasodilatation of 2K1C rats, but there was no significant effect on the non endothelium-dependent diastolic function. 6 chronic exogenous administration of NaHS could significantly increase the content of H2S and no in the plasma and up regulation of the expression of CSE protein in the renal artery. Horizontal.7 chronic exogenous administration of NaHS can significantly increase the protein expression level of PPAR Delta and p-enos in renal artery of 2K1C rats,.8 chronic exogenous administration of NaHS can significantly increase the protein expression level of p-Akt and p-ampk in renal artery of 2K1C rats,.9 application of PPAR delta receptor blockers, agonists, inhibitors, inhibitors, inhibition of synthase Agents, through chronic incubation of 2K1C and 2k1c+nahs rats' renal arteries, found that the above blockers could partially or completely block the role of NaHS. Conclusion: H2S can improve the endothelium dependent vasodilatation function of hypertensive patients. Its effect on improving endothelial function may be associated with the improvement of the imbalance of endovascular CSE and AT1, and the activation of the PPAR Delta /enos pathway. The.Pi3k/akt and AMPK signaling pathways may participate in H2S activation of the PPAR Delta /enos pathway and further improve the role of vascular endothelium dependent vasodilatation in hypertensive arteries. The third part of hydrogen sulfide can improve the systolic function of the renal artery endothelium of renal vascular hypertensive rats by downregulating the expression of bmp4/cox2: endothelial function in hypertension On the one hand, the dysfunction of endothelium dependent vasodilatation is reduced, on the other hand, the endothelial dependent vasoconstrictor function is enhanced. Bone morphogenetic protein 4 (bonemorphogenicprotein4, BMP4) belongs to the superfamily of transforming growth factor beta (transforminggrowthfactor- beta, tgf- beta), which mainly regulates the embryonic development and regenerative repair of bone. Recent studies have found that BMP4 plays an important role in the cardiovascular system,.Bmp4 promotes the overproduction of reactive oxygen free radicals (reactiveoxygenspecies, ROS) by increasing the expression and activity of NADPH oxidase, and then increases the activity of epoxy enzyme 2 (cyclooxygenase2, COX2), which leads to the increase of the formation and release of the precursor adenins, thus damaging the vascular endothelium. Enhancing the vasoconstrictor function of vascular endothelium-dependent and ultimately promoting hypertension. Studies have shown that the increase of bmp4/cox2 expression is related to the enhancement of endothelium dependent vasoconstrictor function in spontaneously hypertensive rats. The aim of this study was to observe the endothelium dependent vasoconstriction in hypertensive rats by using the 2K1C model. Function, and observe the effect of H2S on the expression of bmp4/cox2. Methods: healthy male SD rats, 7 weeks old, 190-200 grams, were randomly divided into four groups: sham, sham+nahs, 2K1C and 2k1c+nahs groups.2k1c and 2k1c+nahs rats were treated with two kidney one clip operation to prepare the renal vascular hypertension model.Sham+nahs and 2k1c+nahs group rats in two kidney one clip operation or artificial hand Third weeks after the operation, the intraperitoneal injection of NaHS (56 mol/kg) and the other two groups were intraperitoneally injected with saline at the same time. The blood pressure was measured by the rat tail artery every 4 weeks after the application of NaHS. The renal artery was measured at the end of the twentieth week, and the vasoconstriction of the vascular endothelial dependence was observed, and the high pressure liquid mass spectrometry was used. The content of H2S in plasma was measured and the expression level of CSE, AT1, BMP4, COX2, and oxidative stress related proteins was detected by Westernblot. The activity of SOD and the content of MDA were detected by the kit. Results the arterial blood pressure, the expression of CSE protein and the H2S content in the plasma could be significantly reduced in 1 2K1C hypertensive rats. Endothelium dependent contractile function of renal artery in rats was significantly enhanced. Exogenous administration of NaHS could improve endothelium-dependent vasoconstrictor function of hypertensive rats.3 exogenous NaHS could significantly downregulate the protein expression of AT1 receptor and BMP 4 in renal artery of renovascular hypertensive rats. Exogenous NaHS could significantly reduce renal vascular hypertension. Oxidative stress level, oxidative stress related protein NOX2, NOX4, P67, Nitrotysine expression increased.5 exogenous NaHS can significantly increase SOD activity, decrease MDA content.6 exogenous NaHS can significantly change p38 MAPK and protein expression level. Conclusion: it can significantly improve the renal artery endothelium dependence in hypertensive rats The role of systolic function in improving endothelial function may be related to inhibition of BMP4/ROS/COX2 pathway.

【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1

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