Effects And Underlying Mechanisms Of FDCA On SU5416 Plus Hypoxia-induced Pulmonary Hypertension In Rats

Background:Pulmonary hypertension（PH）,a common progressive pulmonary vascular disease in clinic,is characterized by pulmonary vascular remodeling,progressive pulmonary artery occlusion,and increased pulmonary artery pressure,resulting in right heart failure or even death.The complexity of etiology and pathogenesis of PH makes it an incurable and life-shortening disease.Therefore,it is of great clinical significance to explore potential mechanisms of PH,and develop novel therapeutic approaches for treating PH.Objective:To investigate the effects of a new oral salt,fasudil dichloroacetate（FDCA）,on SU5416 plus hypoxia（Su Hx）-induced rat model of PH in vivo,and explore the underlying mechanisms of hypoxia-induced pulmonary arterial smooth muscle cell（PASMC）in vitro.Methods:SD rats were randomly divided into 6 groups（n=8 per group）:control group,Su Hx group,Su Hx+FDCA（15,45,135 mg/kg）group,as well as Su Hx+bosentan（100 mg/kg）as a positive control group.The PH rat model was established by a single subcutaneous injection of SU5416（20 mg/kg）followed by hypoxia（10%O₂）exposure for 3 weeks.FDCA（15,45,or 135 mg/kg i.g.daily）or bosentan（100 mg/kg i.g.daily）were administered from the first day after SU5416injection.After 3-week hypoxia,right ventricular systolic pressure（RVSP）,right ventricular hypertrophy index（RVHI）,and histological changes of the pulmonary arterial vessels as well as right ventricle（RV）were assessed.Additionally,in vitro,the effects of FDCA（50μM）,compared with equimolar doses of fasudil,DCA,or fasudil+DCA,on the cell viability,proliferation,migration,and contraction of human pulmonary arterial smooth muscle cell（PASMC）under hypoxia（1%O₂）were evaluated by CCK-8 assay,Ed U incorporation assay,Transwell assay,and cell contraction assay,respectively.The effects of FDCA on mitochondrial homeostasis,Ca²⁺/Ca MK and ROCK signaling pathways of PASMCs were assessed by fluorescent probe and Western Blot.Results:FDCA dose-dependently attenuated Su Hx-induced PH,with significant reductions in RVSP and RVHI.The histopathological results showed that Su Hx-induced PH rat revealed typical pathological changes of pulmonary arterioles and RV.FDCA significantly reduced the pulmonary artery wall thickness（PAWT）,pulmonary vessel muscularization,perivascular fibrosis,as well as RV hypertrophy and fibrosis in a dose dependent manner.In vitro,FDCA inhibited hypoxia-induced PASMC proliferation,migration,and contraction to a greater degree than fasudil or DCA alone by restoring mitochondrial function,reducing intracellular Ca²⁺,and inhibiting calcium/calmodulin-dependent kinase(Ca²⁺/Ca MK)activity as well as Rho-kinase activity.Conclusion:FDCA ameliorates hypoxia-induced PASMC dysfunction by inhibiting both Ca²⁺/Ca MK and Rho-kinase signaling pathways,as well as maintaining mitochondrial homeostasis,thus alleviating Su Hx-induced PH.