Therapeutic Effects And Mechanisms Of Fasudil Derivatives FNO And FDCA On Pulmonary Hypertension

Pulmonary arterial hypertension(PAH)is an unusual disease characterized by intimal hyperplasia,medial hypertrophy and adventitia fibrosis of distal pulmonary artery.It is a severe disease with elevated pulmonary artery pressure,leading to right ventricular failure and death.Vasoconstriction,excessive proliferation and the appearance of anti-apoptotic phenotype of pulmonary artery smooth muscle cells(PASMCs),endothelial dysfunction,inflammation and in situ thrombosis of small pulmonary arteries can promote the development of PAH.Complex etiology involves endothelin signaling pathway,NO signaling pathway and prostacyclin signaling pathway.Targeted drugs related to these pathways such as arizontan,riociguat,and asprostol have been adopted in clinical practice.However,the prognosis of PAH patients is still poor.Therefore,new therapeutic strategies are urgently needed for the treatment of PAH.Drugs targeting Rho/ROCK signaling pathway and potassium channel signaling pathway have been used to treat PAH animal models.In our study,the therapeutic effects of fasudil nitric oxide(FNO)and fasudil dichloroacetate(FDCA)on hypoxia and monocrotaline(MCT)induced PAH rats and their possible mechanisms were investigated.Part I Therapeutic effect and mechanism of fasudil nitric oxide(FNO)on MCT-induced PAHAIM: To investigate the therapeutic effects of FNO on MCT-induced PAH and the potential mechanisms.METHODS: In vitro,the optimal compound of FNO series drugs(FNO2-5)to reduce the expression of inflammatory factors of pulmonary artery smooth muscle cells(PASMCs)and pulmonary artery endothelial cells(PAECs)was assessed by ELISA.In vivo,male SD rats were subcutaneously injected with monocrotaline(MCT 60 mg/kg)and the optimum dosage(0.2mg/kg,1mg/kg,5mg/kg)of FNO for aerosolinhalation was explored.After four weeks,rats were sacrificed for hemodynamic parameters(right ventricular systolic pressure,mean pulmonary artery pressure,systemic circulation systolic pressure)measurement,morphological evaluation by HE staining,Masson trichrome staining(MTS),?-SMA immunohistochemical staining,molecular mechanism investigation by RT-qPCR and Western Blot.RESULTS: In vitro,FNO4 showed the most inhibitory effect on the expression of inflammatory factors(IL-6 and TNF-?)in PASMCs and PAECs induced by hypoxia and PGDF-BB.In vivo,FNO4(5mg/kg)therapeutic aerosol inhalation significantly improved the hemodynamic parameters of MCT-induced PAH rats,including RVSP,and mPAP,but excluding systemic systolic pressure(SAP),increased PASMCs apoptosis,inhibited the activation of RhoA/ROCK signaling pathway,activated NO signaling pathway and apoptotic signaling pathway,inhibited pulmonary vascular muscularization,alleviated pulmonary vascular remodeling and right heart hypertrophy,compared with fasudil(F),nitric oxide(NO)and the combination of two compounds(F+NO).CONCLUSION: FNO4 can effectively inhibit the development and progress of PAH both in vitro and in vivo.It alleviates MCT-induced pulmonary vascular remodeling,decreases right heart hypertrophy,increases the apoptosis of PASMCs in vivo and reduces hypoxia and PGDF-BB induced high expression of inflammatory factors IL-6 and TNF-? in vitro.The potential mechanism is attributed to the inhibition of RhoA/ROCK signaling pathway,activation of NO signaling pathway and apoptotic signaling pathway.Part II Therapeutic effects and mechanisms of fasudil nitric oxide4(FNO4)on hypoxia-induced PAHAIM: To evaluate the therapeutic effects of FNO4 in hypoxia-induced PAH and investigate its potential mechanisms.METHODS: In vivo,male SD rats were continuously placed in a hypoxic chamber(oxygen concentration maintained at 10%)for 28 days.After therapeutic FNO4 aerosol inhalation,hemodynamic parameters were measured,right ventricularhypertrophy index was calculated,and morphological changes were observed by HE staining,Masson trichrome staining(MTS),?-SMA immunohistochemical staining.The expression of related molecules were detected by Western Blot.Results: Compared with fasudil,NO and the combination of two compounds,aerosol inhalation of FNO4(5mg/kg)remarkably improved the hemodynamic parameters,inhibited pulmonary vascular muscularization,alleviated pulmonary vascular remodeling and right heart hypertrophy,decreased ROCK expression of hypoxia-induced PAH ratsCONCLUSION: FNO4 aerosol inhalation relieve the developement of hypoxia induced PAH.It can effectively alleviate pulmonary vascular remodeling and right heart hypertrophy.The potential mechanism is attributed to the inhibition of RhoA/ROCK signaling pathway.PART III Therapeutic effects and mechanisms of fasudil derivative(FDCA)on MCT-induced pulmonary hypertensionAIM: To observe the therapeutic effects of fasudil derivative FDCA on MCT-induced pulmonary hypertension and explore its potential mechanisms.METHODS: Male SD rats were subcutaneously injected with monocrotaline(MCT)60 mg/kg to establish MCT-induced PAH animal model,and then given therapeutic(once a day for 14 days)FDCA intragastrically.After 4 weeks,hemodynamic parameters were measured.Pulmonary remodeling,right ventricular myocardial hypertrophy and fibrosis and apoptosis of pulmonary artery smooth muscle cells(PASMCs)were evaluated by HE staining,Masson trichrome staining(MTS),elastic fiber staining(GAF),immunohistochemical staining(anti-?-SMA).In vivo,expression of inflammatory factors IL-6 and TNF-? in PGDF-BB and hypoxia induced PASMCs and pulmonary artery endothelial cells(PAECs)were detected.RESULTS: FDCA significantly improved the hemodynamic parameters of MCT-induced PAH rats,including right ventricular systolic pressure(RVSP)and mean pulmonary artery pressure(mPAP),decreased right ventricular hypertrophyindex,reduced pulmonary vascular and right ventricle remodeling,increased apoptosis of PASMCs,and alleviated hypoxia and PGDF-BB induced high expression of inflammatory factors IL-6 and TNF-? in PAECs and PASMCs.CONCLUSION: FDCA effectively inhibits the progress of MCT-induced PAH by inhibiting pulmonary vascular inflammation and remodeling,aliviating right ventricle remodeling and fibrosis.