| AimsHypertension is a common cardiovascular disease, its complications such as stroke and heart failure have a serious impact on human health. Study on hypertension is still a focus point in cardiovascular field. It has been widely demonstrated that abnormality in rennin-angiotensin system is closely associated with occurrence, development and prognosis of hypertension. The treatments with the target point to RAS are a major therapeutic strategy for hypertension. Enhancement in AngiotensinⅡand impairment in Ang 1-7 in the central nervous system have been reported to be associated with cardiovascular dysfunction in hypertension. Normalization of balance between AngⅡand Ang 1-7 plays an important role in hypertension therapy.It has been suggest that exercise training is capable of reducing the cardiovascular risk factors via e.g. weigh loss, but also it is directly beneficial to cardiovascular dysfunction (e.g. lowering blood pressure) in hypertension. Therefore, the influence of ExT on hypertension has attracted lots of researchers. However, the precise mechanism(s) responsible to ExT-induced cardiovascular protection against hypertension has not been clear yet. Neural plasticity including structure and function in the central nervous system has been suggested to a major mechanism responsible to improvement of ExT on hypertension. Therefore, the present work was designed to determine the role of central angiotensin system in mediating the processing of the effect of ExT on hypertension, and further determine the mechanism of cardiovascular effect of central Ang 1-7.Herein, we first studied the effect of ExT on central angiotensin system in spontaneously hypertensive rat (SHR). The effect of ExT on cardiovascular response to activation and antagonism of AngⅡor Ang 1-7 was tested by intracerebroventricular (ICV) injection of their agonists and antagonists. Using Western blot analysis and immunofluorescence initially examined the target region in the brain responsible for ExT on central angiotensin system. On the other hand, we detected the interaction between the effect of Ang 1-7 and nitric oxide at the level of nucleus tractus solitarii (NTS), and examined the effect of central Ang 1-7 on protein expression of NOS in the NTS. MethodsMale SHR and WKY were performed in Part one of this work, and this part was also divided into two parts. The first part was used for the acute animal experiment. In this part, rats are classed for 3 groups:WKY, SHR, SHR+ExT. Twelve weeks after ExT and non-ExT, the cardiovascular effects were observed following ICV injections of AngⅡ(200 pmol), the AngⅡreceptor antagonist Sarthran (15 nmol), Ang 1-7 (400 pmol) and the Ang 1-7 receptor antagonist A779 (500 pmol). The second part is for the molecular biochemistry experiment. In this part, rats are classed for 4 groups: WKY, WKY+ExT, SHR, and SHR+ExT. Twelve weeks after ExT and non-ExT, the protein expression of AT1 ACE, Mas receptor, and ACE2 in the NTS were detected by immunofluorescence and Western blot analysis.SD rats were used for Part two of this study. The effect of pretreatment with the neural NOS inhibitor 7-Nitroindazole (7-NI,10 and 100 pmol) on the Ang 1-7 (25 pmol) induced cardiovascular response in the NTS was determined. We also observed the cardiovascular response to 25 pmol of Ang 1-7 before and after treatment with the NO precursor L-arginine (L-Arg,2 nmol) in the NTS. Using Western blot analysis, the protein expression of NOS in the NTS was detected after chronic ICV infusion of Ang 1-7 (100 pmol/day,3 days).Results1. Effect of ExT on cardiovascular response to central Angiotensin system in hypertension.1.1 Effect of ExT on basal cardiovascular activity in WKY and SHR.Low-intensity training was subjected to a motor-driven treadmill continuously for a period of 12 weeks. Blood pressure (BP) and heart rate (HR) were monitored 1 time/4 weeks by tail-cut off BP measurement system. It was found that BP elevation coupled with increase in ages was significantly lower in SHR-T than SHR-S (P<0.05). However, there is no difference between SHR-S and SHR-T. ExT also has little effect on BP and HR in WKY-S and WKY-T groups. In freely moving conscious state, the baroreflex sensitivity was significantly increased in SHR-T than in SHR-S (P<0.05).1.2 Effect of ExT on cardiovascular response to central Angiotensin system.1.2.1 Acute ICV injection of 200 pmol of AngⅡproduced a significant elevation in BP in four groups (WKY-S:10±3 mmHg; SHR-S:32±4 mmHg; SHR-T:18±3 mmHg). The degree and maintaining time of BP elevation induced by Ang II was greater and longer in SHR-S than WKY-S, but the enhanced sensitivity to AngⅡin SHR-S were significantly blunted following ExT. However, no significant change in HR following ICV injection of AngⅡwas observed in four groups.1.2.2 Acute ICV injection of 15 nmol of the AngⅡreceptor antagonist Sarthran produced a significant fall in BP in four groups (WKY-S:-19±2 mmHg; SHR-S:-40±3 mmHg; SHR-T:-28±3 mmHg). The degree of BP reduction induced by Sarthran was lower in SHR-T than SHR-S. However, no significant change in HR following ICV injection of Sarthran was observed in four groups.1.2.3 Following ICV injection of Ang 1-7 (400 pmol), MAP was slightly increased (5±1 mmHg) in WKY-S group. After ICV injection of Ang 1-7 in SHR-S group, MAP was slowly and continuously lowered (-10±3 mmHg) within 30 min; In SHR-T group, the maximum elevation in MAP (10±3 mmHg) was produced within 10 min, and gradually returned to baseline level after Ang 1-7 injection. No change in HR was found in four groups after injection of Ang 1-7.1.2.4 Following ICV injection of A779 (500 pmol), MAP was significantly increased (22±4 mmHg) in SHR-T group. After ICV injection of A779 in WKY-S and SHR-S groups, MAP was slowly and continuously lowered (WKY-S:-11±4; SHR-S:-15±4 mmHg) within 30 min. No change in HR was found in four groups after injection of Ang 1-7.1.3 Effect of ExT on protein expression of major Angiontensin signaling components in the NTS.The data from Western blot and immunofluoscence suggested that protein expression of Angiotensin converting enzyme (required for AngⅡgeneration) and AT1R within the NTS was significantly (P<0.05) upregulated in SHR than in WKY. In contrast, there was no difference (P>0.05) of ACE2 and Mas receptor expression between SHR and WKY groups. However, no change (P>0.05) in the protein expression of ACE, AT1R, ACE2 and Mas receptor was observed between WKY-S and WKY or between SHR-S and SHR-T.2. Role of NO mechanism in mediating the cardiovascular effect of central Ang 1-7.2.1 Chronic ICV infusion of Ang 1-72.1.1 Compared with aCSF group, chronic ICV injection of Angl-7 (100 pmol/d, 3 days) significantly (P<0.05) decreased MAP (aCSF:122±4 vs.121±3 mmHg; Ang 1-7:125±1 vs.119±1 mmHg) without altering HR.2.1.2 Data from molecular biologyThe concentration of NO in the NTS was significantly (P<0.05) increased after the ICV infusion of Ang 1-7 for 3 days. The data from Western blot analysis showed that protein expression of nNOS was significantly (P<0.005)upregulated following ICV infusion of Ang 1-7 compared with aCSF. However, neither eNOS nor iNOS expression was changed (P>0.05) in the Ang 1-7 infusion group.2.2 Effect of nNOS blockade on the effects of Ang 1-7 in the NTSAfter pretreatment with aCSF, unilateral injection of 25 pmol of Ang 1-7 produced a significant decrease in MAP (103±1 vs.91±1 mmHg, P<0.05) and HR (-14±5 bpm, P<0.05). The decreas in MAP and HR evoked by the NTS Ang1-7 was significant blunted (MAP:-12±1 vs.-4±1 mmHg; HR:-14±5 bpm vs.-8±2 bpm P<0.05) following pretreatment with 100 pmol of the nNOS inhibitor 7-NI.2.3 Effect of Ang 1-7 on the effects of L-Arg in the NTSPretreatment with Ang 1-7 (25 pmol) significantly (P<0.05) attenuated the reduction in MAP (-29±5 vs.-19±3 mmHg), HR(-16±3 vs.-11±2 beats/min), and renal sympathetic nerve activity (-44±5 vs.-26±4%) evoked by microinjection of 2 nmol of L-Arg into the NTS. Chronic infusion of Ang 1-7 significantly (P<0.05) enhanced the decreases in MAP (-33±3 vs.-25±2 mmHg), HR (-21±3 vs.-14±2 beats/min), and renal sympathetic nerve activity (-63±4 vs.-43±4%) evoked by microinjection of 2 nmol of L-Arg into the NTS.ConclusionThe present study has confirmed that ExT improves dysfunction of hypertension via downreguation of AngⅡand upregulation of Ang 1-7 in the central nervous system. Furthermore, we also confirm that nNOS-derived NO plays an important role in mediating the central cardiovascular effect of Ang 1-7 in the NTS.
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