牛磺酸抑制瞬时受体电位通道3亚型降低血压及改善血管功能的研究

发布时间：2018-07-06 17:46

本文选题：血压 + 高血压前期　；参考：《第三军医大学》2016年博士论文

【摘要】：背景和目的:高血压(Hypertension)是目前世界范围内影响人类健康的主要公共卫生问题之一,估计有将近20%的人群受高血压影响。目前关于高血压具体的发病原因及机制还尚不清楚。2003年,由美国预防、检测、评价与治疗高血压联合委员会在第7次报告中首次提出了高血压前期(Prehypertension)的概念,其具体是指在未使用降压药的情况下,两次或者两次以上不同时间测得的收缩压(Systolic blood pressure,SBP)波动于120-139mmHg和(或)舒张压(Diastolic blood pressure,DBP)波动于80-89mmHg的阶段。相当一部分高血压前期患者在4年之内将进展为高血压,尤其在中老年人这种现象更普遍。据估计,目前大约有30%至50%人群处于高血压前期的阶段。高血压前期常常伴随其他心血管代谢危险因素,并显著增加冠状动脉心脏疾病、中风和肾功能不全的患病风险。对于高血压前期的患者早期干预,可有效降预防高血压的发病率,并对靶器官的损害起到早期预防的作用。目前,一些关于治疗高血压前期的策略已经开始用于干预高血压前期患者,包括生活方式的改变,如健康的饮食和规律体育运动;以及使用降压药物,如血管紧张素Ⅱ受体阻滞剂。尽管这些干预措施可以改善高血压前期状态,但依从性差和抗高血压药物相关的副作用阻碍了其在高血压前期患者中的应用。因此,目前迫切需要一种可靠而且副作用小的治疗措施以防止高血压前期向高血压的发展。含硫氨基酸(Sulfur amino acid)是哺乳动物体内不可缺少的氨基酸之一,其代谢终产物包括牛磺酸(Taurine),硫化氢(Hydrogen sulfide,H_2S)和二氧化硫(Sulfur dioxide,SO2)。牛磺酸在150年前首次从牛胆汁中分离出来而得名。机体内牛磺酸的来源以摄取为主,可通过膳食动物性食品如蛋、肉及海生动物获得。此外,牛磺酸也可以自身合成,其主要通过半胱氨酸或甲硫氨酸等含硫氨基酸经一系列酶促反应转化而来。H_2S作为一种生物介质,在体内许多生理及病理过程中均发挥潜在的作用。H_2S主要是以半胱氨酸和同型半胱氨酸为底物,由胱硫醚β合酶(Cystathionine-β-synthase,CBS)、胱硫醚γ裂解酶(Cystathionine-γ-lyase,CSE)和3-巯基丙酮酸转硫酶(3-mercaptopyruvatesulfurtransferase,3-mst)促反应生成。之前研究发现,牛磺酸和H_2s在高血压的发生发展中发挥重要的作用。例如,mozaffari等研究发现体内低水平牛磺酸可以加重高盐引起的高血压。此外,vandenberg等关于肾移植后患者的研究发现,尿中低水平H_2s代谢产物与肾移植后患者收缩压升高明显相关。但含硫氨基酸是如何发挥抗高血压作用的机制仍需进一步探讨。瞬时受体电位通道(transientreceptorpotentialchannels,trpcs)是结构同源的一类细胞膜上的非选择性阳离子通道家族。研究发现,trpc3介导的细胞膜上钙信号在高血压发病过程中发挥重要的作用。此外,最近的一项研究发现H_2s具有影响小鼠骨髓间充质干细胞胞膜trpcs并调节其胞内Ca~(2+)平衡的作用。尽管基于目前研究结果并未发现含硫氨基酸、trpc3及钙信号与高血压之间的直接关系,但足以提醒我们四者之间存在密切的关系。因此,基于以上研究结果我们进一步探讨了牛磺酸在高血压前期及改善血管功能中的作用及其具体机制。材料与方法:本研究分为临床试验和动物实验两大部分,第一部分的临床研究方案经第三军医大学大坪医院论理委员会讨论通过,并注册usnationallibraryofmedicine(http://www.clinicaltrials.gov,identifier:nct01816698),所有志愿者签署知情同意书。入选120名高血压前期志愿者(男性51例,女性69例)随机分配至牛磺酸颗粒组(60例)和安慰剂颗粒组(60例),及58名年龄匹配的正常血压志愿者被纳入我们的临床试验研究。第二部分以自发性高血压大鼠(shr)、trpc3-/-小鼠和wt小鼠为模型,分离人和小鼠的肠系膜动脉及小鼠和大鼠的主动脉应用于分子机制的实验研究。1.牛磺酸干预对高血压前期志愿者血压的影响。应用水银血压计测量牛磺酸干预期间志愿者的诊室血压;应用动态血压计监测牛磺酸干预前、后志愿者的24小时动态血压。2.牛磺酸干预对高血压前期志愿者血管功能的影响。应用高分辨率血管超声技术检测牛磺酸干预前、后志愿者内皮依赖性血管舒张功能(flow-mediateddilation,fmd)和非内皮依赖性血管舒张功能(nitroglycerin-mediateddilation,nmd)。3.牛磺酸干预对血浆硫化氢(H_2s)含量的影响。应用反相高效液相色谱法检测牛磺酸干预前后志愿者血浆中牛磺酸和H_2s水平。4.牛磺酸干预对血管组织H_2s合成酶和trpc3的表达的影响。以自发性高血压大鼠(shr,4周龄)为模型,以2%牛磺酸饮用水及对照组干预12周,分离大鼠主动脉应用免疫荧光和免疫印迹(westernblot)技术检测人和动物血管壁的cbs,cse和trpc3的表达。5.牛磺酸干预对野生型小鼠(wt)及trpc3-/-小鼠血管功能的影响。分离小鼠的肠系膜动脉应用微血管张力测定系统检测牛磺酸干预前、后血管的舒缩功能;6.牛磺酸对血管组织及血管平滑肌细胞[Ca~(2+)]i的影响。以应用fura-2am/Ca~(2+)荧光探针技术检测牛磺酸干预前、后血管组织及平滑肌细胞内[Ca~(2+)]i的变化及trpc3的影响。结果:1.与正常血压对照组相比较,高血压前期志愿者诊室血压和24h动态血压显著增高,其脉搏波传导速度(cm/s)及餐后血糖也明显升高。高血压前期志愿者安慰剂组和牛磺酸组的基线特征无显著性差异。2.与基线血压相比较,长期牛磺酸干预显著降低高血压前期志愿者的诊室血压和24h动态血压,且白天平均动态血压下降显著,夜间平均动态血压无明显影响。长期牛磺酸干预对高血压前期志愿者正常高值组的血压下降幅值比正常低值组更为明显。3.长期牛磺酸干预对高血压前期志愿者内皮依赖性血管舒张(fmd)和非内皮依赖性血管舒张(nmd)功能均有显著的改善,而安慰剂干预无作用。4.长期牛磺酸干预对高血压前期志愿者血浆牛磺酸和H_2s水平均有显著升高,且血浆牛磺酸和H_2s水平与收缩压和舒张压下降幅值呈负相关。5.人肠系膜动脉(mas)及野生型小鼠(wt)主动脉有cbs、cse和trpc3共表达,trpc3-/-基因敲除小鼠的主动脉cbs、cse表达比同窝野生型trpc3+/+小鼠表达显著增强。6.长期牛磺酸干预显著上调自发性高血压大鼠(shr)主动脉cbs、cse的表达,而抑制trpc3的表达。离体实验显示,牛磺酸孵育人肠系膜动脉(mas)能显著增加cbs、cse表达,而抑制trpc3的表达,且呈剂量依赖的关系。7.利用毒胡萝卜素(thapsigargin,tg)耗竭细胞内Ca~(2+)后,H_2s的供体硫氢化钠(nahs)显著改善肠系膜动脉kcl诱导的血管收缩反应,而trpc3-/-基因敲除小鼠比同窝野生型trpc3+/+小鼠nahs的作用明显增强。8.nahs能抑制苯肾上腺素(pe)诱导的肠系膜动脉Ca~(2+)内流;trpc3-/-基因敲除或trpc3抑制剂(pyr3)均能显著减少tg诱导的肠系膜动脉Ca~(2+)内流,nahs能显著抑制同窝野生型trpc3+/+小鼠tg诱导的肠系膜动脉Ca~(2+)内流,而trpc3-/-基因敲除小鼠无此作用。结论:1.长期牛磺酸干预可降低高血压前期志愿者的诊室血压和24h动态血压,且白天平均动态血压下降显著,高血压前期志愿者在正常高值时血压下降更明显。2.长期牛磺酸干预能显著改善高血压前期志愿者的内皮依赖性和非内皮依赖性血管舒张功能。3.长期牛磺酸干预能增加高血压前期志愿者血浆牛磺酸和H_2S水平,且血浆牛磺酸和H_2S浓度越高,血压下降越显著。4.CBS、CSE和TRPC3在人、动物的肠系膜动脉和主动脉存在共表达,TRPC3基因敲除或牛磺酸均能显著增加CBS、CSE表达,而抑制TRPC3表达。长期牛磺酸干预降低血压与抑制血管组织TRPC3,减少血管平滑肌[Ca2+]i有关。长期牛磺酸干预可上调CBS、CSE表达,促进H_2S生成,改善高血压前期血压及血管功能。
[Abstract]:Background and purpose: hypertension (Hypertension) is one of the major public health problems affecting human health worldwide. It is estimated that nearly 20% of the population is affected by hypertension. The current causes and mechanisms of hypertension are still not clear for.2003, the United States Joint Committee for the prevention, detection, evaluation and treatment of hypertension. For the first time in the seventh report, the concept of prehypertension (Prehypertension) was first proposed. It specifically refers to the fluctuation of the Systolic blood pressure (SBP) in the 120-139mmHg and / or diastolic pressure (Diastolic blood pressure, DBP) fluctuating in the 80-89mmHg order without the use of antihypertensive drugs at different times. A considerable portion of prehypertensive patients will progress into high blood pressure within 4 years, especially in middle aged and elderly people. It is estimated that about 30% to 50% people are at the stage of prehypertension. Prehypertension is often associated with other cardiovascular metabolic risk factors and significantly increases coronary heart disease, stroke, and stroke. The risk of renal insufficiency. Early intervention in patients with prehypertension can effectively reduce the incidence of hypertension and play an early preventive effect on target organ damage. At present, some strategies for the treatment of prehypertension have begun to intervene in prehypertensive patients, including lifestyle changes, such as health. Diet and regular physical exercise; and the use of antihypertensive drugs, such as angiotensin II receptor blockers. Although these interventions can improve prehypertension, side effects associated with poor compliance and antihypertensive drugs have hindered their application in prehypertensive patients. Small side effects to prevent the development of prehypertension to hypertension. Sulfur amino acid is one of the essential amino acids in mammals, its metabolic products include taurine (Taurine), hydrogen sulfide (Hydrogen sulfide, H_2S) and sulfur dioxide (Sulfur dioxide, SO2). Taurine was first 150 years ago. The source of taurine is derived from the body of the body. The source of taurine is derived mainly through dietary animal foods such as eggs, meat and marine animals. In addition, taurine can also be synthesized by its own, which is converted to.H_2S by a series of enzymatic reactions mainly through a series of sulfur-containing amino acids such as cysteine or methionine. Mediators, which play a potential role in many physiological and pathological processes in the body,.H_2S mainly take cysteine and homocysteine as substrates, cystathine beta synthase (Cystathionine- beta -synthase, CBS), cystathion gamma lyase (Cystathionine- gamma -lyase, CSE) and 3- mercapto pyruvate thiotransferase (3-mercaptopyruvatesulfurtransferase, 3-m). St) promote the formation of the reaction. Previous studies have found that taurine and H_2s play an important role in the development of hypertension. For example, mozaffari and other studies have found that low levels of taurine in the body can aggravate high salt induced hypertension. In addition, the study of Vandenberg and other patients after renal transplantation has found that low levels of H_2s metabolites in urine and renal migration The increase in systolic blood pressure in patients with post implantation is obviously related. But the mechanism of how sulfur containing amino acids play an antihypertensive role still needs to be further explored. The transient receptor potential channel (transientreceptorpotentialchannels, TRPCs) is a non selective cation channel family on a kind of cell membrane of structure homologous. The study found that TRPC3 mediated cell membrane Calcium signals play an important role in the pathogenesis of hypertension. In addition, a recent study found that H_2s has an effect on the cell membrane TRPCs of mouse bone marrow mesenchymal stem cells and regulates its intracellular Ca~ (2+) balance. Although the current research results have not found the direct correlation between amino acid, TRPC3 and calcium signals and hypertension It is enough to remind us that there is a close relationship between the four. Therefore, based on the above results, we further explore the role and specific mechanisms of taurine in prehypertension and the improvement of vascular function. Materials and methods: This study is divided into two parts of clinical trial and animal testing, and the first part of the clinical research program Through the discussion of the theory Committee of Daping Hospital of Third Military Medical University, and registered usnationallibraryofmedicine (http://www.clinicaltrials.gov, identifier:nct01816698), all volunteers signed the informed consent. 120 pre hypertensive volunteers (51 male and 69 female) were randomly assigned to the Taurine Granules group (60 cases) and consolation. The drug granule group (60 cases) and 58 age matched normal blood pressure volunteers were included in our clinical trial. The second part was a model of spontaneously hypertensive rats (SHR), trpc3-/- mice and WT mice. The experimental study of the molecular mechanisms of the mesenteric artery and the main arteries of mice and rats and the main arteries of mice and rats was applied to the molecular mechanism of.1. taurine. Effect of pre hypertensive blood pressure in pre hypertensive volunteers. Using a mercury sphygmomanometer to measure the blood pressure of the volunteers in the taurine intervention period; the effect of the 24 hour ambulatory blood pressure.2. taurine intervention on the vascular function of prehypertensive volunteers before the intervention of taurine intervention. High resolution vascular ultrasound was applied. The effects of endothelium-dependent vasodilatation (flow-mediateddilation, FMD) and non endothelium-dependent vasodilatation (nitroglycerin-mediateddilation, NMD).3. taurine on plasma hydrogen sulfide (H_2s) content before and after taurine intervention. The effect of taurine and H_2s level.4. taurine on the expression of H_2s synthetase and TRPC3 in vascular tissue was used in the volunteers. The rats of spontaneously hypertensive rats (SHR, 4 weeks old) were used as models, the drinking water of 2% taurine and the control group were intervened for 12 weeks. The isolated rat aorta was separated by immunofluorescence and Western blot (Westernblot) to detect human and animal. The expression of CBS, CSE and TRPC3 of the vascular wall on the effects of.5. taurine on the vascular function of wild type mice (WT) and trpc3-/- mice. The vasomotor function of the posterior vessels before taurine intervention was detected by the microvascular tension measurement system in the mesenteric artery of mice and the effect of 6. taurine on the blood tube tissue and the]i of vascular smooth muscle cells [Ca~ (2+). Fura-2am/Ca~ (2+) fluorescence probe was used to detect the changes of [Ca~ (2+)]i in the posterior vascular tissue and smooth muscle cells before taurine intervention and the effect of TRPC3. Results: 1. compared with the normal blood pressure control group, the blood pressure and 24h dynamic blood pressure in the early hypertensive volunteers were significantly increased, and the pulse wave conduction velocity (cm/s) and postprandial blood glucose were also observed in the pre hypertensive volunteers. There was no significant difference in baseline characteristics between the placebo group and the taurine group at prehypertension. Compared with the baseline blood pressure,.2. was significantly lower in the consulting room and the ambulatory blood pressure in the pre hypertensive volunteers, and the mean daytime ambulatory blood pressure decreased significantly and the mean nocturnal ambulatory blood pressure had no significant influence. The decrease of the blood pressure in the normal high value group of prehypertensive volunteers was more obvious than that of the normal low value group..3. long-term taurine intervention improved the function of endothelium-dependent vasodilatation (FMD) and non endothelium-dependent vasodilatation (NMD) in pre hypertensive volunteers, while placebo intervention had no effect on.4. long-term cows. The levels of taurine and H_2s in patients with prehypertension were significantly increased by sulfonic acid intervention, and the levels of plasma taurine and H_2s were negatively correlated with.5. human mesenteric artery (MAS) and wild type mouse (WT) aorta CBS, CSE and TRPC3 co expression, trpc3-/- knockout mice's aorta CBS, CSE table. The expression of Dabi fossa trpc3+/+ mice significantly enhanced.6. long-term taurine intervention significantly up-regulated the expression of CBS and CSE in the aorta of spontaneously hypertensive rats (SHR), and inhibited the expression of TRPC3. In vitro experiments showed that taurine incubated the mesenteric artery (MAS) to significantly increase CBS, CSE expression and inhibit the expression of TRPC3 in a dose dependent manner. Relationship.7. using Ca~ (2+) in the depleted cells of thapsigargin (TG), H_2s donor sodium hydrogen sulfide (NaHS) significantly improved the vasoconstrictor response induced by the mesenteric artery KCl, while trpc3-/- knockout mice significantly enhanced the inhibition of phenylephrine induced intestines than the NaHS of the same nest wild type trpc3+/+ mice. Ca~ (2+) internal flow, trpc3-/- gene knockout or TRPC3 inhibitor (pyr3) can significantly reduce the Ca~ (2+) flow in the mesenteric artery induced by TG, and NaHS can significantly inhibit the TG induced mesenteric artery Ca~ (trpc3-/-) flow in the same nest wild type trpc3+/+ mice, but there is no effect in the knockout mice. Conclusion: 1. long-term taurine intervention can reduce the high blood pressure. The blood pressure and 24h dynamic blood pressure in the consulting room in the pre pressure volunteers and the daytime mean ambulatory blood pressure decreased significantly. The decrease of blood pressure in the pre hypertensive volunteers was more obvious at the normal high value..2. long-term taurine intervention could significantly improve the endothelium-dependent and non endothelium dependent vasodilatation function.3. long-term taurine intervention in prehypertensive volunteers. The plasma taurine and H_2S levels were increased in pre hypertensive volunteers, and the higher the concentration of taurine and H_2S in plasma, the more significant the blood pressure decreased.4.CBS, CSE and TRPC3 were co expressed in human mesenteric arteries and aorta. TRPC3 gene knockout or taurine could significantly increase the expression of CBS, CSE, and inhibit the expression of TRPC3. Predescending hypotension is related to inhibiting vascular tissue TRPC3 and reducing vascular smooth muscle [Ca2+]i. Long-term taurine intervention can increase CBS, CSE expression, promote H_2S production, improve blood pressure and vascular function in prehypertensive period.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1

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