| Objective: In the biological world, any life activities of all life forms---from the primier to senior---have their own specific rythms, which are called as biological rhythm. It is a basic character of life and a kind of mechanism similar to a clock formed inside the body during the long course of biological evolution. It can adjust the physical activities of body when time changes, which will then start, progress and finish at given times. Considering the duration, biological rhythm can be classified into three types, i. e., circadian rhythm or diurnal rhythm, which duration is about 24 hours; ultradian rhythm, with the duration obviously shorter than 24 hours, mostly shorter than 20 hours. It can be as short as several hours or several minutes, even several seconds; infradian rhythm, with the cycle length much longer than 24 hours, and is more than 28 hours in most cases and can be as long as days, months or even longer. Diurnal rhythm is the most common type and people have done the most research work on it. It has been found in recent years that diurnal rhythm variation is an independent risk factor of cardiovascular system diseases. Hypertension is also an independent risk factor of cardiovascular system diseases. Diurnal rhythm of most hypertensive patients tends to go through two types of changes, over dipper or nondipper. These changes are relate to the changes of circadian rhythm of endothelin (ET) and nitric oxide (NO). It has been proved that the unbalance of ET and NO secreted from vascellum endothelial cells plays an important role in the occurring and deterioration of hypertension, which influence and act on each other and go into the vicious cycle. It is a hot topic now on how to control blood pressure and resume healthy blood pressure rhythm, reverse the disequilibrium of endothelium functions, stop the damage on target organs at the same time. At present the results of the researches are paradoxical on the aspects of whether various administrated antihypertensive drugs at different dosing time could affect the diurnal rhythm of blood pressure and endothelium active substances at different manner. At this background, we select bisoprolol to drench rats at different times and then observe its effect on them.Method: One-hundred and twenty-eight male SD rats weighted 250-300 grams (bought from the Laboratory Animal Research Center of Hebei Medical University) were fed in the Laboratory Animal Research Center of Hebei Provincial People's Hospital and let them take food freely, with the room temperature at 22±2℃. These rats are then randomly divided into four groups: one group drenched at 07:00AM (MT), another group drenched at 07:00PM (NT) and one control group for MT (MC) and another for NT (NC) respectively. The infusion groups were given orally 3mg/Kg bisoprolol every day and the control groups were given the same dosage of placebo(water)at 07:00 and 07:00, respectively. Each group has 32 rats and these rats are divided into 4 sub-groups randomly, with 8 rats for each sub-group. After 4 weeks feeding, we measured all the rats'blood pressure with rat-tail manometry at different times of the same day (02:00,08:00,14:00,20:00), euthanatized a sub-group rats in each group (MT, NT, MC, NC) and collected the plasma, blood serum and myocardial tissues. Radioimmunity method, colorimetric method and acid reductase method were adopted to measure the level or activity of ET-1, NO and eNOS in blood and myocardial tissues. Reverse transcription PCR method was utilized to test the eNOS, mRNA expression in myocardial tissues. Cosine fitting method was used to analyze the chronobiologial feature of the level or antivity of these chemicals in the blood and myocardial tissues of the four groups rats. Zero amplitude test was used to evaluate that whether there is diurnal rhythm for these active substances, and analysis of variance was applied to compare the significances. It is considered as statistically significant difference when P<0.05.Results: 1.The analysis of variance showed that the blood serum and tissue eNOS activities of MT, NT, MC and NC have no statistical significance among different groups (P>0.05). Cosine fitting analysis and zero amplitude tests reveal that the blood serum eNOS activities of MT, NT, MC and NC possess the characteristic of circadian rhythm, and their peak times are 09:06, 09:26, 07:51 and 08:40, respectively, while the amplitudes are apparently different from each other. The tissue eNOS activities of the four groups also possess typical characteristic of circadian rhythm and the peak times are at 14:40,14:14,12:37 and 12:26, respectively, but the amplitudes have no difference.2. The analysis of variance shows that the concentrations of NO in the blood serum and tissue of MT, NT, MC and NC groups have no statistical significance(P>0.05). Cosine fitting analysis and zero amplitude tests reveal that the concentrations of blood serum NO of MT, NT, MC and NC possess typical circadian rhythm, and their peak times are 12:47, 12:22, 10:40 and 10:36, respectively. The amplitudes are quite different from each other. The concentrations of tissue NO of the four groups also possess typical circadian rhythm and the peak times are 15:55, 14:59, 14:33 and 15:03, respectively and the amplitudes also are different.3. The analysis of variance shows that the concentrations of ET-1 in the blood serum of MT, NT, MC and NC groups are significantly different from each other, and the ET-1 concentrations of MT and NT groups are much lower than that of MC and NC group, which are statistically significant(P<0.0001). The concentrations of tissue ET-1 of the four groups have no statistical significance (P>0.05). Cosine fitting analysis and zero amplitude tests reveal that the ET-1 concentrations in the blood serum of MT, NT, MC and NC groups possess typical diurnal rhythm, and their peak times are at 22:28, 23:35, 22:28 and 22:22, respectively, and the amplitudes are different from each other. The concentrations of myocardial tissue ET-1 of the four groups also possess typical diurnal rhythm and the peak times are 02:09, 23:14, 01:49 and 02:13, respectively and the amplitudes of the two control groups are much higher than those of both active treating groups.4. The analysis of variance shows that the tissue eNOSmRNA expressions of MT, NT, MC and NC group also have no statistical significance(P>0.05). Cosine fitting analysis and zero amplitude tests reveal that the tissue eNOSmRNA expressions of MT, NT, MC and NC groups possess the characteristic of circadian rhythm, and their peak times are at 11:44, 11:48, 11:41 and 11:55, respectively, while the amplitudes of all the groups are the same.5. The analysis of variance shows that the average blood pressure levels of rats in MT, NT, MC and NC groups have no statistical significance. Cosine fitting analysis and zero amplitude tests reveal that the average blood pressure levels of MT, NT, MC and NC possess the typical characteristic of circadian rhythm, and their peak times are at 21:47, 21:39, 21:10 and 20:40, respectively and the amplitudes of all the groups also are the same.Conclusion: 1. The concentration or activity of ET-1, NO and eNOS in the blood and myocardial tissue of health rats possess typical circadian rhythm. The eNOSmRNA expression in myocardial tissue also shows obvious circadian rhythm. 2. Bisoprolol can decrease the concentration of plasma endothelin-1 without influencing that of tissue ET-1 and the activity or concentration of NO and eNOS in blood and myocardial tissue. 3. Bisoprolol has influence on the chronobiology characteristic of the concentration or the activity of ET-1, NO, eNOS in the blood and myocardial tissue of normal rats. The peak times of the substances above in the active treating groups are different from that of control groups and their amplitudes also differ from each other.4. A middle dosage of bisoprolol might not decrease normal blood pressure significantly.
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