Progesterone alleviates vascular endothelial senescence through progesterone receptor isoform b–dependent repression of kelch-like family member 38 and stabilisation of sirtuin 1

Background/Introduction Postmenopausal decline in sex hormones contributes to vascular ageing and endothelial dysfunction, yet the role of progesterone (P4) remains underexplored. Sirtuin 1 (SIRT1) is a key anti-senescence deacetylase that preserves endothelial homeostasis by limiting oxidative stress, inflammation and cellular senescence. However, whether P4 exerts vasculoprotective effects through modulation of SIRT1 and the underlying molecular mechanisms during vascular ageing remain unclear. Purpose To determine whether P4 protects against endothelial ageing via SIRT1 and to elucidate the molecular mechanisms by which P4 regulates SIRT1. Methods Ovariectomised (OVX) mice received P4 (4 or 8 mg/kg/day) for 6 months. Human aortic endothelial cells (HAECs) were rendered senescent with D-galactose (D-GAL) and treated with P4 ± the SIRT1 inhibitor EX527. Aortic relaxation to acetylcholine and sodium nitroprusside (SNP) was assessed by wire myography. Nitric oxide (NO) in serum and culture supernatants was quantified by Griess assay; endothelial nitric oxide synthase (eNOS) and SIRT1 by western blotting. Senescence, oxidative stress, and inflammation were assessed by senescence-associated β-galactosidase (SA-β-gal) staining, immunoblotting, immunostaining and ELISA. RNA sequencing (RNA-seq) on flow-sorted mouse aortic endothelial cells identified P4-regulated targets. Protein interaction and proteasome dependence were examined by co-immunoprecipitation, gene overexpression and the proteasome inhibitor MG132. Progesterone receptor (PR) signalling was tested using PR antagonist mifepristone (RU486) and PR-A/PR-B overexpression. Results OVX increased vascular senescence (SA-β-gal, acetyl-p53, p21 and p16), reduced SIRT1 and eNOS, impaired acetylcholine-mediated relaxation (SNP unaffected) and lowered serum NO, with oxidative stress (↓NRF2/PGC1α/SOD2 and ↑NOX2 expression, and ↑8-OHdG immunofluorescence) and inflammatory activation (↑serum TNF-α/IL-6/IL-1β, ↑p-p65 and MCP-1). P4 dose-dependently reversed these changes. In HAECs, P4 attenuated D-GAL–induced senescence and oxidative stress, restored SIRT1/eNOS and increased supernatant NO, whereas EX527 abrogated protection. RNA-seq identified KLHL38 as a prominently P4-downregulated transcript; P4 suppressed KLHL38 mRNA/protein in aorta and HAECs. KLHL38 co-immunoprecipitated with SIRT1; KLHL38 overexpression decreased SIRT1 protein (mRNA unchanged) and MG132 rescued SIRT1, consistent with proteasome-dependent degradation. RU486 prevented P4-mediated KLHL38 repression; under P4, PR-A overexpression increased whereas PR-B suppressed KLHL38. Conclusion(s) P4 alleviates ovx-modelled postmenopausal acceleration of vascular endothelial ageing and dysfunction via PR-B–dependent transcriptional repression of KLHL38, thereby preserving SIRT1 protein stability and endothelial homeostasis. The PR-B–KLHL38–SIRT1 axis links reduced progesterone signalling to vascular ageing in women.