| Objective: Biological rhythms are a universal phenomenon in living organisms. From unicellular algae, advanced propagation to human being, it exists in every levels of biosystem and enable adaptation to the environment. Biological rhythms can be divided into 3 types on the basis of their cycle lengths: 1) circadian (or diurnal) rhythms, with a period of approximately 24 hours; 2) ultradian rhythms, with a period significantly shorter than 24 hours; and 3) infradian rhythms, with a period longer than 24 hours. Circadian rhythms, which are the rhythms associated with many cardiovascular parameters, are the most common and best studied. It might be controlled by biological clocks which is the inherent of biosystem. Recently, it has been found that blood pressure follows a typical circadian rhythm-a diurnal rise and a nocturnal fall. The regulation of blood pressure is a complicated process, which the RAS (renin-angiotension system) may plays important roles. This system plays an integral role in the homeostatic control of arterial pressure, water and electrolyte. The abnormal rhythms of blood pressure not only increase the risks of target organs damage but also increase the morbility and mortality of cardiovascular events, including myocardial infarction, stroke, and so on. Thus, it is very important that maintenance the normal rhythms of blood pressure. Thiazide diuretics have been introduced as a kind of anti-hypertensives for more than half one century, which are more effective on antihypertension, and enable decreasing the morbility and mortality of cardiovascular events, so this type diuretics have more important position in the areas of antihypertension. During the past decades, some preliminary results had been reported by scholars abroad in the aspects of diurnal manifestation of RAS system, and its circadian property had been suggested, although most these studies were limited to plasma level. However, the study has been known less, which the effects of usually used antihypertension drugs on circadian rhythm of blood pressure and RAS system. Therefore, in this study, we investigated the levels of plasma renin activity (PRA), serum angiotensin converting enzyme (ACE), as well as angiotensinⅡ(AngⅡ), in plasma and myocardial tissue in rats which were raised under different time of hydrochlorothiazide-treated schedules. Meanwhile, we also examined the expression patterns of AT-1mRNA in myocardial tissue of rats, to investigate the circadian rhythm of RAS system in the molecular level. So the aim of this study is to investigate the chronobiological properties of rat's RAS system and blood pressure, and the effects of different hydrochlorothiazide dosing time on it.Methods: One hundred and twenty-eight age matched male Sprague-Dawlay rats, which body weight 250-300g, were introduced to this study. After two week's free-running period in natural light-dark cycles, they were housed at 22±2℃and free access to food and water. They were divided randomly into four groups according to their treating scheme: rats that were drenched tap water at 07:00 in the morning were designated as the group MC; rats that were drenched tap water at 19:00 were designated as the group NC; rats that were treated with hydrochlorothiazide at 07:00 in the morning were designated as the group MT; rats that were treated with hydrochlorothiazide at 19:00 were designated as the group NT. Treated groups (group MT and NT) were given hydrochlorothiazide 10mg/kg/day. And the control groups (group MC and NC)were given the same volume of tap water. Each group was maintained in its respective feeding scheme for 4 weeks before the experiment. Animal experiments were conducted in accordances with guidelines of Hebei Geriatric Institute.For the determination of the diurnal pattern of PRA, ACE, AngⅡin plasma and myocardial tissue, as well as AT-1mRNA expressions in rats myocardial tissue, the blood pressure was measured by means of a noninvasive tail-cuff method and rats were sacrificed every 6 hours (at 02:00, 08:00, 14:00, 20:00, respectively) by operating on chest, and the indices above were measured. Cosinor fitting analysis and zero amplitude test were introduced to analyze the chronobiological features of RAS in every group. The compare of circadian values among 4 groups was managed based on Analysis of Variance (ANOVA), and p<0.05 was defined as statistically different borderline.Results: 1 The levels of PRA and AngⅡin plasma were significantly higher in group MT, NT than those in control groups by using ANOVA (p<0.01). The levels of PRA and AngⅡin plasma were not significantly different between group MT and NT. The levels of ACE in serum and the levels of AngⅡin myocardial tissue were not significantly different in 4 groups (p>0.05).2 The levels of AT-1mRNA in myocardial tissue were significantly lowering in group MT, NT than those in control groups by using ANOVA (p<0.01). The levels of AT-1mRNA in myocardial tissue were not significantly different between group MT and NT.3 Based on cosinor fitting analysis and zero amplitude test, circadian rhythm were found in all the 4 groups, and the curves and characteristic values of plasma level of PRA, ACE, AngⅡand AngⅡin myocardial tissue were obtained, respectively. The plasma level of PRA, ACE, AngⅡshows significantly diurnal variation(p<0.05), with the peak times at 13:33, 17:28, and 16:31, respectively in group MC. Group NC also shows diurnal variation of these indices, peak times at 13:36, 17:27and 16:05, respectively. These indices from Group MC and group NC have no difference. The results also showed in both group MT and NT present circadian variation, with the peak times occurring at 04:06, 13:45, 09:52 and 00:30, 10:18, 08:30, respectively, with somewhat ahead of control groups, especially in group MT. Furthermore, the amplitudes of vibration in both group MT and NT were augmented, which suggested that the oscillations of plasma level of PRA and AngⅡwere augmented in groups treated with hydrochlorothiazide. AngⅡin myocardial tissue showed significantly diurnal variation (p<0.05), with the peak times occurring at 15:55, 15:41, 05:16 and 05:53, respectively in group MC, NC, MT and NT. The amplitudes of vibration were not different.4 The expression of AT-1mRNA were found to oscillate following circadian rhythm in this study, with the peak times at 12:18, 12:15, 00:44 and 05:36, respectively in 4 groups. The amplitudes of the cosinor curves of treated groups were diminished and the acrophase were ahead of the control group.5 The levels of average nycterohemeral blood pressure were significantly lower in group MT and NT than those in control groups by using ANOVA (p<0.01). But the levels of average nycterohemeral blood pressure were not significantly different between group MT and NT. At 02:00 and 20:00 time points blood pressure were significantly lower in treated groups than control groups by using ANOVA (p<0.01). at the indices obstained at 08:00 and 14:00 time points there were no different in 4 groups (p>0.05). Based on consinor fitting analysis and zero amplitude test, circadian rhythm of blood pressure were shown in all the 4 groups, with the peak times at 20:40 ,21:10, 19:28 and 20:05,respectively. The amplitudes of the cosinor curves in treated groups were significantly lower than that in control groups. The acrophase were ahead of the control groups.Conclusion: 1 Administrating hydrochlorothiazide at different time schedule, the level of PRA and AngⅡin plasma were significantly higher in group MT, NT than those in control groups. Administrating hydrochlorothiazide at morning or at night had similar effect on PRA and AngⅡ. The levels of ACE in serum and the levels of AngⅡin myocardial tissue were not significantly different in 4 groups. This suggested us that hydrochlorothiazide can active some of components of the RAS system at some extent.2 PRA, ACE and AngⅡin plasma and myocardial tissue showed significantly diurnal variation. Circadian rhythm of PRA, ACE and AngⅡwere altered at different hydrochlorothiazide-treated time schedule (mainly reflected in the shift of acrophase and the change of amplitude). The different hydrochlorothiazide-treated time schedule might affect the diurnal variation of RAS system components.3 The levels of AT-1mRNA in myocardial tissue were significantly lower in group MT, NT than those in control groups. Administrating hydrochlorothiazide at morning or at night had similar effect on the levels of AT-1mRNA. This suggested us that administrating hydrochlorothiazide not only lowering the blood pressure but also protecting myocardial tissue at some extent. AT-1mRNA in myocardial tissue showed significantly diurnal variation. The circadian rhythm of AT-1mRNA was altered at different hydrochlorothiazide-treated time schedule. The amplitudes of the cosinor curces of treated groups were diminished and the acrophase were ahead of the control groups.4 The blood pressure of rats shows the characteristics of circadian rhythm. Administrating hydrochlorothiazide at different time significantly lowed the averaged blood pressure and especially lowed the nocturnal blood pressure. Morever, administrating hydrochlorothiazide at different time enable altered the diurnal variation of blood pressure, which the amplitudes were diminished and the acrophase were ahead.In conclusion, the results suggested that blood pressure and PRA, ACE, AngⅡin plasma and myocardial tissue and AT-1mRNA in myocardial tissue showed significantly circadian rhythm. The diurnal nature might be endogenous, which cannot be diminished by drug, although the chronobiological values could be altered at some extent. Administrating hydrochlorothiazide at different time enable alter the diurnal variation of above indices at some extent, which can shift the acrophase of cosinor curves early, especially after the morning dose.
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