A-485 alleviates postmenopausal osteoporosis by activating GLUD1 deacetylation through the SENP1-Sirt3 signal pathway.

OBJECTIVE

Postmenopausal osteoporosis (OP) is a bone disease caused by estrogen deficiency. A-485 is a selective inhibitor of p300/CBP histone acetyltransferase (HAT) with potential regulatory effects on bone remodeling. This study aims to investigate the effects of A-485 on postmenopausal OP and its underlying mechanisms.

METHODS

For animal experiments, 61 female Wistar rats were used to establish an OP model through ovariectomy (OVX). The rats were administered with A-485 (100 mg/kg/day) via intraperitoneal injection for six weeks. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DXA). Histopathological changes were observed using HE and Masson's trichrome staining. ELISA was used to measure bone resorption markers (CTX-1, DPD) and the bone formation marker (P1NP) in rats. Osteoblast differentiation markers (Runx2, OCN), SENP1, Sirt3 expression levels, and GLUD1 acetylation were assessed via Western blot (WB) and RT-qPCR. In vitro, MC3T3-E1 osteogenic progenitor cells were cultured in osteogenic differentiation medium supplemented with ascorbic acid, β-glycerophosphate, dexamethasone, and fulvestrant. CCK-8 was performed to evaluate cell proliferation. Flow cytometry was selected to measure apoptosis and mitochondrial membrane potential. WB and RT-qPCR were employed to analyze ERα, ERβ, Runx2, Sirt3, and GLUD1 acetylation. Additionally, Alizarin red staining was applied to monitor osteoblast mineralization. ATP levels were detected using a commercial kit, and ROS levels were measured by MitoSOX Red.

RESULTS

In vivo, ovariectomized rats exhibited lower BMD, impaired bone trabeculae, increased CTX-1 and DPD, and altered expression of Runx2 and OCN, all of which were reversed by A-485 treatment. In vitro, A-485 activated GLUD1 deacetylation, enhanced osteogenic differentiation, and improved mitochondrial function. Regarding the mechanism, A-485 activated the SENP1-Sirt3 signal pathway, with SENP1 knockdown negating the effects of A-485. In vivo, A-485 reduced GLUD1 acetylation and promoted improvement of OP, which were reversed by SENP1 knockdown.

CONCLUSION

A-485 ameliorates postmenopausal OP by activating GLUD1 deacetylation via the SENP1-Sirt3 signal pathway, thus improving mitochondrial function, and promoting osteogenic differentiation and mineralization.