SIRT1-FOXO3a Mediate The Protective Effect Of Estrogen On Hypoxic Pulmonary Hypertension

Objective: To investigate the role of SIRTl-FOXO3 a in the regulation of estrogen(E2)on pulmonary vascular remodeling(PVR)and pulmonary artery smooth muscle cells(PASMCs)proliferation in hypoxia-induced pulmonary hypertension(HPH).Methods:Female Sprague-Dawley rats treated with bilateral ovariectomy were divided randomly into 4 groups(n=8 each): normoxia group,normoxia+E2 group,hypoxia group and hypoxia + E2 group.Normoxia+E2 and hypoxia + E2 group received subcutaneous injection of 17?-Estradiol [20?g/(kg·d)],and the other groups received subcutaneous injection of physiological saline(0.1 ml/d)respectively.The hypoxic groups were housed at hypoxic chamber,and the normoxic groups were kept in the room air.The rats were fed continuously for 8 weeks to establish hypoxic pulmonary hypertension model.The mean pulmonary arterial pressure(mPAP),right ventricle hypertrophy index(RVHI),and pulmonary vascular remodeling indexes were observed.Enzyme-linked immunosorbent assay(ELISA)was adopted to detect the E2 levels in serum.Immunohistochemistry(IHC)was adopted to test the expression of silent information regulator 2 homolog 1(SIRT1),FOXO3 a transcription factor,proliferated cell nuclear antigen(PCNA)in pulmonary artery.The Universal SIRT Activity Assay Kit was adopted to test SIRT1 activity in lung tissue.In vitro,human PASMCs(hPASMCs)were cultured under normoxic or hypoxic condition,and different concentrations of E2 were administered,then MTT assays were employed to observe the effects of E2 on hypoxia-induced hPASMCs proliferation.Further,cells under hypoxic condition were co-treated with nicotinamide(SIRT1 inhibitor)and E2,or treated with resveratrol(SIRT1 activator),then MTT assays were performed to explore the changes of hPASMCs proliferation.Western blotting and Realtime-PCR assays were adopted to test the protein and mRNA levels of SIRT1,FOXO3 a and PCNA changes in hPASMCs.The Universal SIRT Activity Assay Kit was adopted to test SIRT1 activity in hPASMCs.Results: Chronic hypoxia significantly increased mPAP,RVHI,medial width of pulmonary arterioles,accompanied with decreased expression of SIRT1,decreased expression of FOXO3 a and increased expression of PCNA in rats.Whereas,E2 treatment repressed the elevation of mPAP,RVHI,attenuated the pulmonary artery remodeling induced by chronic hypoxia,and normalized the expression of above-mentioned indexes.In vitro,E2 significantly inhibited hypoxia-induced hPASMCs proliferation,which was also associated with improvements in SIRT1 and FOXO3 a expression.In addition,SIRT1 inhibition attenuated the effects of E2 on hPASMCs proliferation and the expression of FOXO3 a.Moreover,resveratrol,a SIRT1 activator,mimiced the effects of E2.Conclusions: E2 effectively attenuated HPH and PVR of rat models.The underlying mechanism may be through inhibiting PASMCs proliferation via regulating SIRT1-FOXO3 a pathway.