Sacubitril/valsartan Treatment Relieved The Progression Of Established Pulmonary Hypertension In Rat Model And Its Mechanism

Part 1 The role of Sacubitril/valsartan treatment in rats of pulmonary hypertensionObjective: To investigate the effects of sacubitril/valsartan on pulmonary hemodynamics,right ventricle hypertrophy and hypertrophy of pulmonary arteries in both monocrotaline(MCT)-induced and hypoxia-induced pulmonary hypertension(PH)rats.Methods:(1)Male Sprague-Dawley rats(200-250g)were divided into groups including the control(n=6),control + sacubitril/valsartan(control + sac/val,n=6),MCT + vehicle(n=10),MCT + sacubitril/valsartan(MCT + sac/val,n=9)groups and the normoxia(n=6),normoxia + sacubitril/valsartan(normoxia + sac/val,n=6),hypoxia + vehicle(n=6),hypoxia + sacubitril/valsartan(hypoxia + sac/val,n=6)groups.PH models were treated with MCT(60mg/Kg,intraperitoneal injection)or hypoxic environment for 14 days.After that Sacubitril/valsartan at 68 mg/kg(the control + sac/val group,normoxia + sac/val groups and the MCT + sac/val group,normoxia + sac/val groups)or saline at the same volume(the control group,normoxia groups and the MCT + vehicle,hypoxia + vehicle groups)were gavage once daily for 2 weeks.(2)After the expiration of these models,the data of survival were collected.The hemodynamic parameters(pulmonary arterial pressure,systemic pressure,cardiac output,pulmonary vascular resistance)were measured by the acquisition system of hemodynamic data.(3)the ratio of right ventricular/left ventricular + septal weight(RV/LV + S)was obtained to evaluate right heart hypertrophy.Paraffin sections of lung tissues were stained with hematoxylin and eosin(HE)to observe the changes of pulmonary medial thickness and the relative pulmonary vascular muscularization.Results:(1)MCT-induced and hypoxia-induced PH models were successfully established.The survival of MCT-induced rats was improved after the treatment of sacubitril/valsartan(P < 0.05).Compared with the control or normoxic rats,the body weight of rats in MCT/hypoxia + vehicle groups decreased significantly,and the body weight in the groups of intervention were further reduced(P < 0.05).(2)The results of hemodynamic measurement showed that compared with the control and normoxic rats,the pulmonary arterial pressure and cardiac output in MCT/hypoxia + vehicle groups were significantly increased and the cardiac output was decreased(P < 0.05).Systemic pressure had no significant change between the above groups(P > 0.05),and RV/LV + S ratio was also increased(P < 0.05).After the intervention of sacubitril/valsartan,the pulmonary arterial pressure,pulmonary vascular resistance and systemic pressure decreased compared with the PH rats(P < 0.05),and the RV/LV + S ratio also declined(P < 0.05).(3)Histopathologically,the pulmonary medial thickness in the MCT/hypoxia + vehicle groups were significantly increased than that in the control/normoxia groups(P < 0.05),and the relative pulmonary vascular muscularization was raised(increased fully muscularized small arterioles and decreased non-muscularized small arterioles,P < 0.05).sacubitril/valsartan relieved the remodeled small pulmonary artery with medial thickening induced by MCT or hypoxia,and the relative pulmonary vascular muscularization was also declined(decreased fully muscularized small arterioles and raised non-muscularized small arterioles,P < 0.05).Conclusion: Sacubitril/valsartan alleviated PH in MCT-induced and hypoxia-induced rat models.Part 2 The neurohormonal regulation of Sacubitril/valsartan in rats of pulmonary hypertensionObjective: To investigate the effects of sacubitril/valsartan on renin?angiotensin?aldosterone system(RAAS)and natriuretic peptide(NP)system in both monocrotaline(MCT)-induced and hypoxia-induced pulmonary hypertension(PH)rats.Methods:(1)Male Sprague-Dawley rats(200-250g)were divided into groups including the control,MCT + vehicle,MCT + sacubitril/valsartan(MCT + sac/val)groups and the normoxia,hypoxia + vehicle,hypoxia + sacubitril/valsartan(hypoxia + sac/val)groups.PH models were treated with MCT(60mg/Kg,intraperitoneal injection)or hypoxic environment for 14 days.After that Sacubitril/valsartan at 68 mg/kg(the MCT + sac/val group and normoxia + sac/val groups)or saline at the same volume(the control group,normoxia groups and the MCT + vehicle,hypoxia + vehicle groups)were gavage once daily for 2 weeks.(2)After the expiration of these models,the protein expression levels of AT1 receptor and its downstream targets--extra cellular regulated kinase 1/2(ERK1/2)and tyrosine kinase SRC were detected by western blot.(3)The concentrations of NPs(including ANP,BNP and CNP)were estimated using enzyme linked immunosorbent assay(ELISA).The expression levels of NPs in lung tissues were measured by real-time PCR.The protein expression levels of natriuretic peptide receptors(NPRs,including NPR-A,NPR-B and NPR-C)were detected by western blot.(4)The concentrations of c GMP in the supernatant of the lung tissues was detected by ELISA.Results:(1)The results of western blot showed that the protein expression level of AT1 receptor and its downstream targets--ERK1/2 and SRC were raised in PH rats compare with the control/normoxic rats(P < 0.05),and sacubitril/valsartan suppressed the protein expression of them(P < 0.05).(2)The intervention of sacubitril/valsartan increased the upregulated circulating concentrations and the m RNA expression levels in lung tissues of ANP and CNP(P < 0.05)and therefore upregulated the protein expression of NPR-A and NPR-B(P < 0.05),raised the concentrations of c GMP in lung tissues(P < 0.05).(P < 0.05).(3)the protein expression of NPR-C in lung tissues in PH rats were also upregulated after the sacubitril/valsartan treatment.Conclusion: Sacubitril/valsartan alleviated PH in MCT-induced and hypoxia-induced rat models by inhibiting the activated RAAS,promoting ANP/NPR-A/c GMP and CNP/NPR-B/c GMP pathway,restoring the NPR-C signaling.Part 3 The effects of Sacubitril/valsartan on inflammatory factors in rats of pulmonary hypertensionObjective: To investigate the effects of sacubitril/valsartan on serum inflammatory factors in both monocrotaline(MCT)-induced and hypoxia-induced pulmonary hypertension(PH)rats.Methods:(1)Male Sprague-Dawley rats(200-250g)were divided into groups including the control,MCT + vehicle,MCT + sacubitril/valsartan(MCT + sac/val)groups and the normoxia,hypoxia + vehicle,hypoxia + sacubitril/valsartan(hypoxia + sac/val)groups.PH models were treated with MCT(60mg/Kg,intraperitoneal injection)or hypoxic environment for 14 days.After that Sacubitril/valsartan at 68 mg/kg(the MCT + sac/val group and normoxia + sac/val groups)or saline at the same volume(the control group,normoxia groups and the MCT + vehicle,hypoxia + vehicle groups)were gavage once daily for 2 weeks.(2)After the expiration of these models,the concentrations of IL-1?,IL-6,TNF-? and TGF-?1 were estimated using enzyme linked immunosorbent assay(ELISA).Results: sacubitril/valsartan suppressed the elevated serum concentrations of IL-1?,IL-6 and TNF-? in PH rats(P < 0.05),but have no effects on TGF-?1(P > 0.05).Conclusion: Sacubitril/valsartan alleviated PH in MCT-induced and hypoxia-induced rat models by the anti-inflammatory effects.