【作者】 邵光方；

【导师】 童建；

【作者基本信息】 苏州大学 ， 卫生毒理学， 2018， 博士

【摘要】 目的:以往研究显示,血压昼夜节律异常与高血压靶器官损害和心血管事件发生之间显著相关,是独立于血压水平的重要危险因素;褪黑素具有一定的抗炎、抗氧化和降血压作用。本论文采用时间生物学的研究方法,探讨褪黑素对高血压患者和高血压模型大鼠血压昼夜节律的影响,为阐明褪黑素参与调节血压及其昼夜节律的作用机制,提供基础资料和实验依据。方法:(1)选取10名原发性高血压患者,连续7d服用褪黑素,采用动态血压监测,记录患者24小时动态血压,利用余弦分析软件,分析患者服药前后血压水平及其昼夜节律的变化。(2)将无线发射子植入自发性高血压大鼠(SHR)腹主动脉,测量SHR动态血压,利用余弦分析软件,分析SHR大鼠血压昼夜节律。在收缩压节律峰值前1h,对大鼠分别注射5mg/kg/d、10mg/kg/d、20mg/kg/d褪黑素,绘制褪黑素-降压率剂量反应曲线,确定褪黑素干预剂量。(3)在收缩压节律峰值和谷值前1h(峰值组、谷值组),分别对大鼠注射10mg/kg/d褪黑素,比较不同时点注射褪黑素对大鼠血压及其昼夜节律的影响。(4)在收缩压节律峰值、谷值前1h,对峰值组、谷值组大鼠分别注射10mg/kg/d的褪黑素,在注射后0h、1h、2h,取大鼠尾静脉取血,采用ELISA方法,测定不同时点注射褪黑素对大鼠血清中褪黑素、内皮素、血管紧张素Ⅱ、一氧化氮和一氧化氮合成酶的影响。(5)将人脐静脉血管内皮细胞分三组进行培养,分别是正常气压培养组、加压24KPa培养组、加压24KPa+褪黑素(10μM)培养组。收集各组6h、12h 18h、24h培养液,采用ELISA方法测定不同时点内皮素、血管紧张素Ⅱ、一氧化氮和一氧化氮合成酶的水平变化情况。(6)收集6h和18h加压24KPa培养组、加压24KPa+褪黑素(10μM)培养组细胞,提取RNA进行m RNA基因表达谱芯片测定,筛选褪黑素处理后差异表达的基因,通过GO和KEGG分析,初步获得褪黑素调控血压昼夜节律的可能信号通路。结果:(1)原发性高血压患者连续服用7天褪黑素后,收缩压较服用前降低9.39mm Hg,舒张压较服用前降低6.6mm Hg。一例患者服用褪黑素后,恢复了已经消失的血压昼夜节律。(2)SHR大鼠注射褪黑素后,与对照组比较,收缩压、舒张压、平均压中值变化无统计学差异;血压节律峰值前注射褪黑素后,与对照组比较,收缩压振幅下降46.38%,峰值相位提前3.73小时,舒张压振幅下降30.83%,峰值相位提前6.27h小时,平均压振幅下降47.34%,峰值相位提前4.96小时,差异均具有统计学意义;血压节律谷值前注射褪黑素后,与对照组比较,收缩压振幅下降24.74%(P>0.05),峰值相位延迟了0.66小时(P>0.05),舒张压振幅下降26.96%(P<0.05),峰值相位延迟了2.15小时(P<0.05),平均压振幅下降4.9%(P>0.05),峰值相位延迟了1.58小时(P<0.05);(3)注射褪黑素后2h,与对照组比较,节律峰值组SBP、DBP、MBP分别下降11.56%、12.66%、12.02%,差异均具有统计学意义;节律谷值组SBP、DBP、MBP分别下降10.45%、16.63%、12.88%,差异均具有统计学意义。(3)节律峰值前注射褪黑素后,1h和2h与0h比较,血清MT分别上升了7.80%和10.38%,ET分别下降了23.34%和14.65%,Ang II分别下降了10.06%和5.30%,NO分别上升了101.17%和57.87%,e NOS 1h上升了5.53%,2h下降了5.18%,以上均差异均具有统计学意义。节律谷值前注射褪黑素后,1h和2h与0h比较,血清MT分别上升了17.19%和9.13%,Ang II分别下降了17.43%和7.30%,NO分别上升了35.37%和5.43%,ET 1h较0h下降20.90%(P<0.05),2h较0h上升3.59%(P>0.05),e NOS 1h较0h上升5.53%(P<0.05),2h较0h下降5.18%(P<0.05)。(4)加压培养+褪黑素处理组与单纯加压培养组比较,HUVEC细胞的ET、Ang II含量明显下降,NO和e NOS含量明显升高,差异均具有统计学意义。6h褪黑素处理组与24KPa组相比,共检测出335个差异表达基因,其中上调基因121个,下调基因214个;18 h褪黑素处理组与24KPa组相比,共检测出157个差异表达基因,其中上调基因63个,下调基因94个。经GO和KEGG分析,这些差异表达基因主要参与细胞周期、细胞昼夜节律调节、与NO合成有关的c GMP-PKG通路、以及与血压调节有关的肾素/胰岛素分泌的信号通路。结论:(1)一定剂量的褪黑素能够显著降低高血压患者和高血压大鼠的血压,并影响血压昼夜节律的参数,如血压变化的振幅和峰值相位,从而改变血压昼夜波动的类型。(2)褪黑素对血压昼夜节律的影响,可能通过血管活性因子如ET、Ang II、NO和e NOS的介导实现的。(3)褪黑素可直接作用于血管内皮细胞,上调腺嘌呤环化酶G蛋白偶联受体通路,激活与昼夜节律调控相关的通路(circadian entrainment),从而发挥调节血压昼夜节律的功能。更多还原

【Abstract】 Objectives:Previous studies have indicated that abnormal circadian rhythm of blood pressure is significantly associated with target organs damage of hypertension and incidence of cardiovascular events,and is considered as an independent risk factor of hypertension.Melatonin has been shown effects of anti-inflammation,anti-oxidation and antihypertension.The aim of this study is to explore the effect of melatonin on circadian rhythm of hypertension in patients and model rats by using the method of chronobiology,and to clarify the molecular mechanism underlying the regulatory role of melatonin in modulation of circadian rhythm of blood pressure.Methods:(1)10 primary hypertension patients were selected to take melatonin continuouslyfor one week.Ambulatory blood pressure was monitored for 24 h and the Cosine analysis software was used to fit the cosine curve of the circadian rhythm of blood pressure.(2)Wireless transmitter was transplanted into the abdominal aorta of SHR rats to measure the dynamic blood pressure.One hour before the peak time of systolic blood pressure,5mg/kg/d,10mg/kg/d and 20mg/kg/d melatonin was administrated to the SHR rats.A dose-effect curve was drawn to determine the experimental dose of melatonin.(3)One hour before the peak and valley time of the systolic blood pressure,10 mg/kg/d melatonin was injected to the rats to compare the time effect of melatonin.(4)At 0 h,1 h,2 h after melatonin injection,the levels of melatonin,endothelin,angiotensin II,NO and e NOS in rats serum were measured by ELISA assay.(5)Human HEVEC cells were divided into three groups of normal pressure group,24 Kpa pressure group and 24 Kpa pressure plus melatonin(10 μM)group.The cell supernatant collected at 6 h,12 h,18 h and 24 h after melatonin treatment were used to measure the level of endothelin,angiotensin II,NO and e NOS by ELISA assay.(6)The m RNA from cells of two groups,i.e.,6 h and 18 h after melatonin treatment was extracted,and the gene spectrum chip was applied to screen out genes of differential expressions.The GO and KEGG analysis was conducted to reveal the genes and pathways involved in the circadian rhythm regulation by melatonin.Results:(1)After 7 days of continuous melatonin administration,the systolic blood pressure(SBP)of the primary hypertension patients decreased 9.39 mm Hg,and the diastolic blood pressure(DSP)decreased 6.6 mm Hg.The circadian rhythm of blood pressure in one patient was restored by the melatonin treatment.(2)Injection of melatonin 1 h before peak time and trough time in the SHR rats resulted in no significant difference in SBP,DBP and MBP(mean blood pressure)compared to the control.For the circadian parameters,the amplitude of systolic pressure decreased 46.38%,and the peak phase advanced 3.73 h;the amplitude of diastolic pressure decreased 30.83%,and the peak phase advanced 6.27 h;the amplitude of mean pressure decreased 47.34%,and the peak phase advanced 4.96 h,all with significant difference.For the group of injection before trough time,the amplitude of systolic pressure decreased 24.74%(P>0.05),and the peak phase delayed 0.66 h(P>0.05);the amplitude of diastolic pressure decreased 26.96%(P<0.05),and the peak phase delayed 2.15 h(P<0.05);the amplitude of mean pressure decreased 4.9%(P>0.05),and the peak phase delayed 1.58 h(P<0.05);(3)At 2 h after injection,the SBP,DSP and MBP decreased 11.56%,12.66%,12.02%,respectively in the peak treatment group,with significant difference.the SBP,DSP and MBP decreased 10.45%,16.63%,12.88%,respectively in the trough treatment group,with significant difference.(4)In groups with melatonin injection before 1 h and 2 h peak time point,the MT level in serum increased 7.8% and 10.38% compared to that of 0 h;the level of ET decreased 23.34% and 14.65%;the level of Ang II decreased 10.06% and 5.3%;the level of NO increased 101.17% and 57.8%;and the level of e NOS increased 5.53% in 1 h,and decreased 5.18% in 2h,all with significant difference.In groups with melatonin injection before 1 h and 2 h trough time point,the MT level in serum increased 17.19% and 9.13% compared to that of 0 h;the level of Ang II decreased 17.43% and 7.3%;the level of NO increased 35.73% and 5.43%;the level of ET decreased 20.90% in 1 h(P<0.05),and increased 3.59% in 2 h(P>0.05),and the level of e NOS increased 5.53% in 1 h(P<0.05),and decreased 5.18% in 2h(P<0.05).(5)In HUVEC cells,compared to the 24 KPa pressure group,the level of ET and Ang II decreased significantly,and NO and e NOS increased significantly in melatonin treatment group.In 6 h melatonin treatment group,a total of 335 differentially expressed genes were detected,including 121 up-regulated and 214 down-regulated genes.In 18 h melatonin treatment group,a total of 157 differentially expressed genes were detected,including 63 up-regulated and 94 down-regulated genes.The GO and KEGG analysis revealed that most of the differentially expressed genes were involved in pathways functioning in cell cycle,circadian rhythm regulation,c GMP-PKG mediated NO synthesis,and renin/insulin related blood pressure regulation.Conclusions:(1)Melatonin at appropriate dose could significantly decrease the blood pressure of hypertension patients and HSR rats,and modulate the parameters of circadian rhythm of the blood pressure such as the rhythmic amplitude and peak phase,so as to modify the circadian types of blood pressure.(2)The regulatory effect of melatonin on the circadian rhythm of blood pressure might be mediated via the vasoactive factors including ET,Ang II,e NOS and NO.(3)Melatonin could target the human umbilical endothelial cells(HUVECs)to up-regulate the adenylate cyclase-modulating G-protein coupled receptor signaling pathway and to activate the circadian entrainment pathway,thereby to function in regulation of circadian blood pressure.更多还原