Catalpol attenuates osteoporosis in ovariectomized rats through promoting osteoclast apoptosis via the Sirt6-ER-FasL axis

Background: Catalpol, a major active component of the Chinese herb Rehmannia glutinosa, possesses various pharmacological benefits, including anti-inflammatory, antidiabetic, and antitumor properties. Recent studies have reported that catalpol can attenuate bone loss and enhance bone formation. Nevertheless, the molecular mechanisms underlying its effects on osteoporosis pathogenesis remain unclear. Purpose: We investigated whether catalpol had a protective effect against postmenopausal osteoporosis (PMOP) and explored its exact mechanism of action. Methods: Seventy-two rats were randomly divided into six groups: sham, model, low-dose catalpol (5 mg/kg/day), medium-dose catalpol (10 mg/kg/day), high-dose catalpol (20 mg/kg/day), and positive control (alendronate, 2.5 mg/kg). In this experiment, a ovariectomy was performed to establish a female rat model of PMOP. After 12 weeks of gavage, micro-computed tomography (micro-CT) and histochemical staining were performed to evaluate bone mass, bone microstructure and histological parameters. Furthermore, RAW 264.7 cells were induced by RANKL to form mature osteoclasts to investigate the effect of catalpol on osteoclast differentiation and apoptosis in vitro. Additionally, the osteoclast apoptosis-related proteins of Sirt6, ER alpha, FasL, NFATc1, cleaved-caspase 8, cleaved-caspase 3, and Bax were assessed using western blotting. The expressions of NFATc1, Ctsk, Oscar, and Trap were quantified using RT-qPCR. The apoptotic rate of the osteoclasts was determined using flow cytometry. Sirt6 knockdown was performed using siRNA gene silencing in experiments to investigate its role in catalpol-mediated osteoclast apoptosis. The deacetylation of ER alpha in osteoclasts was tested via co-immunoprecipitation. Results: Catalpol (10 and 20 mg/kg) and alendronate (2.5 mg/kg) could significantly improve bone mineral density (BMD) and microstructure and decrease osteoclast density in ovariectomized (OVX) rats. In addition, catalpol (10 and 20 mg/kg) upregulated the expression of Sirt6, ER alpha, FasL, cleaved-caspase 8, cleaved-caspase 3, Bax, and downregulated the expression of NFATc1, Ctsk, Oscar, Trap both in vivo and in vitro. Catalpol also promoted ER alpha deacetylation and stabilized ER alpha protein to enhance the expression of FasL. In addition, Sirt6 knockdown by siRNA prevented ER alpha deacetylation and eliminated catalpol-mediated osteoclast apoptosis. Conclusions: The present study demonstrated that catalpol prevents estrogen deficiency-induced osteoporosis by promoting osteoclast apoptosis via the Sirt6-ER alpha-FasL axis. These findings revealed a novel molecular mechanism underpinning the impact of catalpol in the progression of osteoporosis and provided novel insights into the treatment of osteoporosis.