Effects Of Melatonin On The Circadian Rhythm Of Hypertension With Underlying Mechanism

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.