Catalpol attenuates postmenopausal osteoporosis by activating SIRT1-mediated P53 deacetylation and inhibiting CASPASE-3-driven osteoblast apoptosis

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BACKGROUND: Postmenopausal osteoporosis (PMOP) is primarily characterized by a disruption in the balance between bone formation and resorption, predominantly due to osteoblast apoptosis induced by estrogen deficiency. Sirtuin 1 (SIRT1), a pivotal deacetylase known for inhibiting P53-mediated apoptosis, is observed to be downregulated in PMOP. Catalpol (CAT), an iridoid glycoside derived from Rehmannia glutinosa, has demonstrated antiapoptotic and antioxidative properties; however, its potential role in modulating the SIRT1-P53 signaling pathway in the context of PMOP remains to be elucidated. METHODS: Utilizing a network pharmacology approach, SIRT1 was identified as a central target of CAT in the context of PMOP, a finding further substantiated through molecular docking and 100-ns molecular dynamics simulations. To validate this proposed mechanism, ovariectomized (OVX) mice were administered CAT with or without the SIRT1 inhibitor EX527. Bone mass, strength, and remodeling were assessed using micro-computed tomography (micro-CT), three-point bending tests, enzyme-linked immunosorbent assays (ELISA) for procollagen type I N-terminal propeptide (P1NP) and C-terminal telopeptide of type I collagen (CTXI), and immunohistochemical analysis. In vitro, hydrogen peroxide (H₂O₂)-induced MC3T3-E1 cell models were employed to evaluate osteoblast differentiation and apoptosis via alkaline phosphatase (ALP) staining, flow cytometry, and Western blot analysis focusing on the SIRT1-P53-CASPASE-3 axis. RESULTS: Molecular docking and 100-ns MD predicted a plausible CAT-SIRT1 interface. In cells, CETSA showed ligand-induced thermal stabilization of SIRT1, supporting intracellular target engagement. In H₂O₂-challenged osteoblasts, CAT reduced P53 (Lys382) acetylation, cleaved CASPASE-3, and Annexin V-positive cells; these effects were attenuated by the SIRT1 inhibitor EX527. In ovariectomized mice, CAT improved trabecular microarchitecture on micro-CT and rebalanced bone turnover-increasing serum P1NP and decreasing CTX-I-with concomitant reductions in AcP53 and cleaved CASPASE-3 in bone. CONCLUSIONS: These findings support a SIRT1-centered mechanism whereby CAT engages SIRT1 in cells to restrain the P53-CASPASE-3 apoptotic program in osteoblasts and mitigate estrogen-deficiency bone loss in OVX mice.