Molecular Mechanism Study Of Homocysteine Regulating Blood Pressure Rhythm

Background:Abnormal circadian rhythm of blood pressure is closely related to the prognosis of patients with hypertension.It is of great significance to study the mechanism of abnormal circadian rhythm of blood pressure in the treatment of hypertension.Previous studies have suggested that folic acid hypohomocysteine(Hcy)therapy is accompanied by a decrease in nocturnal blood pressure,thus restoring normal blood pressure circadian rhythm.In the previous population-based clinical study,our group found that there was a significant correlation between high Hcy levels and abnormal circadian rhythm of blood pressure in patients with hypertension,and in female patients,the worse the vascular endothelial function,the smaller the decrease in nocturnal blood pressure.However,so far,there has been no study on the pathophysiological molecular mechanism of hyperhomocysteine(HHcy)interfering with blood pressure circadian rhythm.The circadian rhythm of blood pressure is affected by many factors.more and more studies have shown that the endogenous biological clock in mammals can regulate the circadian rhythm of blood pressure through the expression of clock genes.clock genes are mainly located in the hypothalamic suprachiasmatic nucleus and peripheral tissue,including the vascular system.Therefore,from the perspective of vascular endothelial function,we explore whether HHcy can interfere with normal vascular endothelial function by affecting the expression rhythm of clock genes in endothelial cells,and finally affect the circadian rhythm of blood pressure.Objective:To clarify the effects of high homocysteine culture on the expression rhythm of core clock genes Bmal1,Clock,Per2 and endothelial nitric oxide synthase(e NOS)in vascular endothelial cells,and to determine whether HHcy can disrupt the expression rhythm of vascular clock genes,interfere with the normal expression of e NOS in endothelial cells,and then change vascular endothelial function,and finally affect the circadian rhythm of blood pressure.Methods:1.Human umbilical vein endothelial cells(HUVECs)were selected as the experimental model,and the 24 h and 48 h cell viability of HUVECs at different Hcy concentrations(0μmol/L、50μmol/L、100μmol/L、200μmol/L、400μmol/L、800μmol/L)were detected by CCK8,and the concentration level of the Hcy treatment group was determined by combining with relevant preliminary experiments.2.HUVECs were incubated for 2h in a medium with high concentration of fetal bovine serum(50% FBS)to establish that the clock gene expression of endothelial cells cultured in vitro showed a cyclical fluctuation rhythm similar to that caused by light stimulation.3.The m RNA and protein expression of clock genes Bmal1,Clock,Per2 and e NOS in HUVECs at different time points(0 h,4 h,8 h,12 h,16 h,20 h,24 h,28 h,32 h,36 h,40 h,44 h,48h)in the blank control group and high concentration Hcy(400μmol/L)treatment group were detected by RT-PCR and Western bloting.4.The nitrate reductase method was used to detect the NO content in HUVECs culture medium at different time points(0h,6h,12 h,18h,24 h,30h,36 h,42h,48h)in the blank control group and high concentration Hcy(400μmol/L)treatment group.Results:1.The results of CCK8 showed that when the concentration of Hcy was 0μmol/L,50 μmol/L,100 μmol/L,200 μmol/L,and 400 μmol/L the cell survival rate of HUVECs was greater than 80% and the cell condition was good;when the concentration of Hcy was 800μmol/L,the survival rate of HUVECs was less than80% and the cell condition was slightly worse.2.The results of RT-PCR and Western bloting showed that after 2h incubation in high-concentration fetal bovine serum(50% FBS)medium,the rhythm of the clock genes expression in HUVECs was successfully induced,which showed that the clock genes Bmal1,Clock,Per2 were at 0h,4h,16 h,20h,24 h,28h,40 h,44h,48 h showed low expression level,and at 8h,12 h,32h,36 h showed high expression level,showing regular periodic rhythm fluctuations in general.Compared with the blank control group,the m RNA expression and protein expression of clock genes Bmal1,Clock,Per2 at 8h,12 h,32h,and 36 h in the HHcy group were all down-regulated,Compared with the blank control group,there were no significant differences in the m RNA expression and protein expression of clock genes Bmal1,Clock,Per2 at 0h,4h,16 h,20h,24 h,28h,44 h,and 48 h in the HHcy group;Compared with the blank control group,there were no significant differences in the m RNA expression and protein expression of clock genes Bmal1,Clock and Per2 m RNA expression in the HHcy group at 40 hours;The difference is that at 40 h,the expression level of Per2 protein in the HHcy treatment group was significantly down-regulated compared with the blank control group.3.The results of RT-PCR and Western bloting showed that after 2 hours of incubation in high-concentration fetal bovine serum(50% FBS)medium,the expression of e NOS in HUVECs showed a regular periodic rhythm consistent with the expression of clock genes.Compared with the blank control group,the e NOS m RNA expression and protein expression at 8h,12 h,32h,and 36 h in the HHcy group were all down-regulated.And there was no significant difference in e NOS m RNA expression and protein expression in the HHcy group at 0h,4h,16 h,20h,24 h,28h,44 h,40h,and 48 h.4.The results of nitrate reductase detection of NO content showed that the NO content in the HUVECs culture medium of the blank control group did not change significantly within 48h;compared with the blank control group,the NO content of the HUVECs culture medium of the HHcy group increased significantly within 48 h.Conclusions:Hyperhomocysteine inhibits the expression rhythm of clock genes Bmal1,Clock,Per2 and e NOS in human umbilical vein endothelial cells and promotes the secretion of NO in human umbilical vein endothelial cells.