| Objective: Biological rhythm is a universal phenomenon in physiological activities of every physiological system of human beings. From molecule biochemistry, physiological activities of tissue organs, up to psychological behaviors of single living organisms, almost all of them have periodic fluctuations in different frequency. Biological rhythms can be divided into three types on the basis of their cycle lengths: 1) circadian (or diurnal) rhythms with a period of approximately 24 hours (20 hours to 28 hours in general); 2) ultradian rhythms, with a period significantly shorter than 24 hours (generally shorter than 20 hours); and 3) infradian rhythms, with a period notable longer than 24 hours (generally longer than 28 hours). Circadian rhythms, which are associated with many physiological and pathological phenomena of cardiovascular systems, are the most common and best studied in human beings. It is discovered in resent years that the blood pressure of human beings is presented typical circadian rhythms feature, and it has important clinical significance for hypertension patients. Therefore, it is very important that to sufficiently understand the chronobiology feature of blood pressure and to further study its regulation mechanism of biological rhythm for the better prevention and treatment of hypertension.The regulation of blood pressure is a complicated process, during which the renin-angiotensin system (RAS), catecholamines, and endothelial vasoactive substances may play important roles. The level or activity changes of vasoactive substances which above mentioned may produce obviously influence for the level of blood pressure. Therefore, it is very important to further study the diurnal fluctuation feature of vasoactive substances for further understanding the regulation of circadian rhythms of blood pressure and its mechanism. It is reasonable to postulate that the changes of circulating or tissue levels of these vasoactive substances would surely result the alternation of blood pressure. Accordingly, the critical perception of the chronobilogical features of vasoactive chemicals would be beneficial to a further understanding of circadian mechanisms of blood pressure. During the past decades, some preliminary results had been reported by scholars abroad in the aspects of diurnal manifestation of RAS, and circadian properties had been suggested, although most these studies were limited to serum level. Before, part of foreign scholar had done initial research for the plasma level and activity of renin, angiotensin-II, it shows that plasma levels or activities of above-mentioned substances presented typical circadian rhythms. But most of the studies were merely involved in the level of blood circulation. It's uncertainty that the circadian rhythms are presented in distribution in tissues and molecular expression of those substances and the relationship between diurnal fluctuation of vasoactive substances and circadian rhythms of blood pressure will be expected to study in further.As we know, biologic rhythm is endogenous and hereditability, and meanwhile, it is conformed and adjusted by environment signals. Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. With the aging process, the cyclical variation of external environment might adjust the phase of the circadian oscillation. In order to accommodating living environment, various kinds process including physiological functions, biochemistry and metabolic process would change continuously in mammals with different sex, and it gradually become an basic vital phenomena and posses hereditability.We research the chronobiological features of RAS in 17-month-old rat and 3-month-old rat of different sex and the effects of age and sex on circadian rhythms of RAS and blood pressure. Therefore in this study, we investigated the levels of renin, angiotensin-converting-enzyme (ACE) and angiotensin II in plasma or serum in rats raised under different age and sex. And meanwhile, we also examined the expression patterns of rennin mRNA, ACE mRNA, and AT1 mRNA in rat myocardial tissue. Also, the chronobiological features of RAS, and its relationship with the circadian rhythm of blood pressure conduced to understand the mechanisms of diurnal rhythm of blood pressure.Methods: Ninety-six SPRD rats were included in this study. They were divided into three groups (with 32 for every group) according to their age and sex: rats that were 17-month-old male rats were designated as the "M group" (weight, 400 to 440 g); rats that were 17-month-old female rats were designated as the "F group" (weight, 380 to 420 g); for the control group, we selected the rats that were 3-month-old male rats were designated as the "D group" (weight, 250 to 275 g). They were kept in a separate environment-controlled room (25±3°C), in which a strict 12-hour light/12-hour dark cycle regimen was enforced and free access to food and water. Each group was maintained for 12 weeks before the experiments. Animal experiments were conducted in accordance with guidelines of Hebei Geriatric Institute. For the determination of the diurnal pattern of renin, ACE, angiotensin-II in plasma or serum, as well as renin mRNA, ACE mRNA, and AT1 mRNA expressions in rat myocardial tissue, animals were sacrificed every 6 hours (at 2:00, 08:00, 14:00, and 20:00, respectively) by decapitation, 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 3 groups was managed based on Analysis of Variance (ANOVA), and p<0.05 was defined as statistically different borderline.Result:1 The levels of renin activity , ACE, angiotensin-II in plasma or serum were significantly higher in D than those in M and F group by using ANOVA (P=0.0001), and there were not significantly different between F group and M group. It showed the gerontological decline of the renin-angiotensin system. In comparison with adult male group, the plasma or serum levels of renin, ACE, angiotensin-II in elderly group were significantly reduced.2 The expression levels of renin mRNA, and ACE mRNA in myocardial tissue were significantly higher in D than those in M and F group by using ANOVA , and there were not significantly different between F group and M group. Conversely, in comparing with M group and F group, the expression levels of AT1 mRNA in D group were significantly reduced.3 Based on cosinor fitting analysis and zero amplitude test, circadian rhythms were approved in all the three groups (i.e., M group, F group, and D group), and the curves and characteristic values of plasma or serum level of renin activity, ACE, and angiotensin II were obtained, respectively. Each of those RAS component shows significant diurnal variation (P<0.05), with the peak times at 23:17, 23:33, and 23:20, respectively in M group. The results also showed that the plasma renin activity, the plasma level of angiotensin II and the serum level of ACE in both F and D groups present circadian variation, with the peaks occurring at 23:27, 0:00, 23:53 and 21:13, 21:48, 20:36, respectively, with somewhat earlier in D group than those in M and F groups. Furthermore, the amplitudes of vibration in both M group and F group were decreased, which suggested that the oscillations of plasma or serum level of renin activity, ACE, and angiotensin II were attenuated in elderly rat group.4 The expression of renin mRNA, ACE mRNA, and AT1 mRNA were found to oscillate following circadian rhythm in this study. The chronobiology feature of renin mRNA and ACE mRNA were similar to those of the RAS components in plasma or serum, with the peak time at night hours in three groups. Similarly, the peak values of these indices were later in M and F groups than those in D group. The amplitudes of the cosinor curves were decreased in both M and F groups. However, the chronobiology feature of AT1 mRNA was different with renin mRNA and ACE mRNA, with the peaks occurring at 20:44, 20:26, 22:09, respectively, in M, F and D group, with somewhat earlier in M and F group than those in D group. Furthermore, the amplitudes of vibration in both M and F group were increased.5 The circadian characters of renin activity, ACE, and angiotensin II in plasma or serum were almost the same as Renin mRNA, ACE mRNA in myocardial tissue, which suggested that the circulating RAS and the genetic express of components of RAS might possess the same chronobiological mechanism. The peak times of renin mRNA and ACE mRNA were similar to the other components of RAS in plasma or serum, with a little bit later peak time when compared with the renin activity, ACE in plasma or serum.6 Based on cosinor fitting analysis and zero amplitude test, circadian rhythms were approved in all the three groups (i.e., M, F, and D group), and the curves and characteristic values of were obtained respectively. The blood pressure present significant diurnal variation(P<0.05), with the peak times at 0:42, 3:41, and 23:19 in M, F and D group, respectively, with somewhat earlier or later in M group than in F and D groups. Furthermore, the amplitudes of vibration were alike in three groups.In conclusion, the results suggested that the circadian variations of renin, ACE, and angiotensin II in plasma or serum, and of renin mRNA, ACE mRNA, and AT1 mRNA in myocardial tissue were with similar chronobiological characters. Their peak times were almost the same as those of blood pressure. The diurnal nature might be endogenic, which might affect the circadian rhythm of blood pressure. The diurnal oscillation of RAS might begin at the molecular level.
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