| Background: Chronic heart failure(CHF) is the syndrome caused by impaired ventricular filling and(or) emptying, which results from myocardial injury and/or structural or functional disorders of the heart. At the beginning of myocardial impairment, the excitability of sympathetic nervous system and rennin- angiotensin-aldosterone(RAAS) increases, leading to the activation of various endogenous neuroendocrinal factors and cytokines. The long-standing, chronic activation triggers myocardial remodeling, aggravates myocardial impairment, and leads to the deterioration of cardiac function, further activating the deterioration of cardiac function and resulting in a vicious cycle. Despite some progress in the mechanism of HF, the mortality of the patients is on the rise, which is a serious public health and social problem. Therefore, the research into HF is in urgent need of new breakthroughs, providing new ideas and theoretical basis for the prevention and treatment of HF.The natriuretic peptide(NP) family plays a pivotal regulatory role in the physiology and pathology of heart and vessels. The discovery of NPs opens a new field of cardiac endocrinology. With the progress of research, researchers have found that NPs possess not only natriuretic diuretic, vasodilatory effects but also exert lots of physiological and pharmacological effects, such as anti-proliferation, anti-apoptosis and immune regulation. The regular variation of NPs in pathophysiological conditions has become a new marker in the diagnosis and prognosis of chronic heart failure(CHF). Today, new types of NPs have been used in the treatment of CHF.In previous experiments, the five amino acid residues in the C-terminal of h-ANP did play an important role in promoting urine excretion. Therefore, the C-terminal of ANP was retained, and it was combined differently with the N-terminal and the ring structure of natural NPs, including ANP, BNP and CNP. We designed all the seven possible structure combinations, which were compared with the vasodilation of natural NPs. And we selected four chimeric NPs of relatively higher activity: ANCAC, BNAAC, BNBAC, CNAAC. In this study, we compare the vasodilatory activity of the four chimeric NPs and selected the chimera, which has the highest activity. Then, the chimera was used in ischemia heart failure animal model in vivo. Main research content: ANP, CNP and VNP was served as comparison: 1. Vascular ring perfusion was employed to determine the vasodilatory effect and the underlying mechanism of CNAAC; 2. MAP, urine volume and urine c GMP levels after intravenous injection of CNAAC were determined; 3. The cardiac structure and function, related factors in body fluid and protein expression were detected after intraperitoneal injection of CNAAC for 4 weeks. The aim is to throw light upon the protective role of the new type of natriuretic peptide chimera against HF, and provide theoretical and experimental basis for the prevention and treatment of HF.Aims:To investigate the activity of the four chimeric NPs, and to screen out one chimera that exhibits the highest activity. The vasodilatory mechanism of the chimeric NP that has the highest activity was determined in vitro. The diuretic, hypotensive effect and the role ofchimeric NP in the treatment of HF was determined in vivo.Methods: 1. Abdominal aorta of rats was isolated and vascular ring perfusion was employed to screen out one chimeric NP that has the highest activity from seven innovatively-designed NPs. 2. To study whether the relaxation of chimeric NP is via NPR/GC/c GMP pathway and the underlying mechanism, abdominal aorta rings were pretreated with ab14356(anti- NPR-A antibody, 1:1000), MB(an inhibitor of unspecific guanylyl cyclase, 10μM), ODQ(a potent inhibitor of soluble guanylyl cyclase, s GC, 10 μM), KT5823(an inhibitor of c GMP-dependent protein kinase, PKG, 10 μM), Gly(an inhibitor of ATP-sensitive potassium channel, KATP, 1 μM), TEA(an inhibitor of calcium-activated potassium channel, Kca, 1 m M), or L-NAME(an inhibitor of nitric oxide synthase, NOS, 100 μM) to determine the vasodilatory activity of NPs. 3. To determine the temporal profile of NPs-induced abdominal aortic c GMP excretion, the c GMP content of rat abdominal aorta sample was measured both under basal condition(incubation in K-H solution) and after the stimulus of natural or chimeric NPs in different time point. The inhibitor of GC was added 15 min prior to NPs incubation to determine the relaxant effect of NPs and the role of GC/c GMP pathway. 4. Hemodynamic parameters were recorded; urine was collected; serum c GMP and urine c GMP level were measured after intravenous injection of ANP, CNP, VNP or CNAAC in rats. 5. Three HF models were used: abdominal aorta stenosis, coronary ligation and abdominal stenosis plus coronary ligation. According to the general conditions, heart/body weight ratio, hemodynamic index, echocardiogram and pathological changes, the best HF model was selected from these three models. 6. On the day of surgery, ANP, CNP, VNP or CNAAC was injected intraperitoneally(one time per two days, 20 nmol/kg/2d). Four weeks later, echocardiogram and hemodynamic assessment through left ventricular intubation were performed to detect the effect of NPson cardiac function in rats with HF. 7. Vascular ring perfusion was employed to determine the vasodilatory effect of CNAAC on abdominal aorta isolated from HF rats. 8. Hematoxylin eosine staining and Masson’s staining were employed to detect the effect of NPs on myocardial infarction size and collagen volume fraction. 9. The level of BNP, Ang II, ALD, ET-1, c GMP, IL-1, IL-6, IL-8, IL-10, and TNF-α in serum were detected by ELISA kit in each group.10. Expression of NPR-A and NPR-C in left ventricle were detected by western blot in each group.Results: 1. Four chimeric NPs were identified of higher activity from seven, they were : ANCAC, BNAAC, BNBAC and CNAAC. The four chimeric NPs relaxed abdominal artery from rats in a dose-dependent manner. The vasodilatory activity from strong to weak was: CNAAC>BNAAC>ANCAC>BNBAC. Then, we found out that CNAAC also exhibited the higher activity than ANP, CNP or VNP. The vasodilatory activity from strong to weak was: CNAAC>VNP≥ANP>CNP. CNAAC was then chosen in the further study. 2. CNAAC relaxed abdominal aorta in a dose-dependent manner. Incubation of tissues with ab14356, MB, ODQ, Gly or TEA all decreased the potency of CNAAC. Inhibition of PKG completely abolished the relaxant effect of CNAAC. However, the NOS inhibitor, L-NAME, was unable to reverse the relaxation caused by CNAAC. There was no significant difference between abdominal aortic rings with or without endothelium. 3. A single concentration(10-6 M) of NPs generated a larger increase in c GMP level at different time points. CNAAC, compared with other NPs, produced a larger increase in c GMP level, not only at 5 min or 15 min, but also at 30 min. It was showed that c GMP excretion was increased in a time-dependent manner. After incubation with ODQ, the c GMP excretion induced by CNAAC was decreased. 4. ANP, VNP and CNAAC markedly enhanced the UV in rats. The increase in UV after CNP injection showed no difference compared with the correspondence baseline period inthe present study. The diuretic effect from strong to weak was: CNAAC>ANP≥VNP>CNP. All of four types of NPs remarkably enhanced the level of urine c GMP and plasma c GMP and decreased the MAP in rats. 5. In vivo study, ischemia HF and sham animal model were established. On the day of surgery, the rats were administrated with 0.85% Na Cl or NP(i.p. 20nmol/kg/2d, for 4 weeks). CNAAC relaxed abdominal aorta isolated from HF rats in a dose-dependent manner. There was no significant difference between abdominal aortic rings with or without endothelium. 6. The rats with HF were in lower spirits, decreased mobility, yellowed fur color, increased BW/HW, and PW/HW in the HF group. The injection of ANP, CNP, VNP or CNAAC improved the activity and general sign and decreased HW/BW ratio. The PW/BW ratio was decreased only after VNP or CNAAC injection. Hemodynamics examinations revealed that LVSP, ±dp/dtmax was significantly decreased, and LVEDP was significantly increased in the HF group. After NPs administration, left ventricular function, reflected by these parameters, was all reversed. The impulses of ventricular anterior wall in the HF group weakened significantly and the heart volume enlarged, which were observed by echocardiography. The echocardiogram suggested that the injection of ANP, CNP, VNP or CNAAC(i.p. 4 weeks) ameliorates EF, FS, LVEDV and LVESV. Only injection of CNAAC decreased the LVEDD and LVESD of the heart. The efficacy of CNAAC on LVEDD, LVESD, LVEDV and LVESV was superior to ANP, CNP or VNP. 7. As shown in HE staining, the myocardium was well-arranged with clearly band in control group. Inflammatory cell infiltration and infarcted area with serious fibrosis were observed in the HF and NPs-treated group. The area of infarction was pale and thin in the HF and NPs-treated group with Masson’s trichrome staining. The HF group showed a diffused brick-red staining in cytoplasm, with a large amout of blue collagen. Myocardial infarction size and collagen volume fraction in each treated group were significantly decreased compared with HF group. The effect on infarct size of VNP or CNAAC was superior to ANP or CNP. The effect on CVF of VNP was superior to ANP, and the effect on CVF of CNAAC was superior to other treated-group.8. The serum level of BNP, Ang II, ALD and ET-1 in HF rats increased. The serum level of Ang II and ET-1 decreased after ANP, CNP, VNP or CNAAC administration. The serum level of BNP decreased only after VNP or CNAAC administration, and the serum level of ALD decreased after ANP, VNP and CNAAC administration. The serum level of c GMP in the HF rats decreased dramatically compared with that in the control group. The level of c GMP increased after ANP, CNP, VNP or CNAAC admnistration. 9. The serum level of IL-1α, IL-6, IL-8 and TNF-α in HF rats increased compared with those levels in the control group. The serum level of IL-1α was decreased after ANP, CNP, or CNAAC administrtion. The serum level of IL-6 was decreased after ANP, CNP, VNP or CNAAC administration. The serum level of IL-8 and TNF-α were decreased only after CNAAC administration. The effect of CNAAC was superior to the other NPs in increasing the levels of IL-8 and TNF-α in serum. 10. The expression of NPR-A decreased and NPR-C increased dramatically in HF rats. After administration of ANP, VNP or CNAAC, the expression of NPR-A increased. The expression of NPR-A after ANP administration was more than that after CNP administration. Expression of NPR-A didn’t change after CNP administraion. After administration of ANP, CNP, VNP or CNAAC, the expression of NPR-C decreased.Conclusions: 1. We combined the COOH-terminus of ANP with different ring and NH2-terminus from natural NPs, and obtain seven NPs chimera that had biological activity. Four of the chimera NPs were proved to have potential vasorelaxant effect, they are: ANCAC, BNAAC, BNBAC and CNAAC. The four NPs chimera have potent vasodilatory activity in a dose-dependent manner. CNAAC has the highest activity in relaxation of abdominal aorta in a dose-dependent manner and an endothelium-independent manner. Our results suggest that CNAAC has a potent vasodilating effect, probably by activating K+ channels via NPR-A/s GC/c GMP pathway. 2. CNAAC(i.v.) has potent diuretic, and hypotensive effect, which involves c GMP in its aforementioned process.3. Exogenous administrations of CNAAC(i.p.) ameliorated diastolic and systolic dysfunction, hypertrophic index, pulmonary congestion, myocardial infarction and myocardial fibrosis, decreased the elevated level of cytokines, increased the expression of NPR-A and decreased the expression of NPR-C in HF rats.In general, CNAAC has potential cardioprotection, diuresis and vasodilation effects, suggesting that it may have broad prospects in the prevention and treatment of CHF. |
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