Based on NF-κB and Notch1/Hes1 Signaling Pathways, the Mechanism of Artesunate on Inflammation in Osteoporosis in Ovariectomized Rats was Investigated.

ABSTRACT

BACKGROUND: Artesunate (ART) has the potential to modulate the nuclear factor kappa B (NF-κB) and Notch1/Hes1 signaling pathways, which play crucial roles in the pathogenesis of osteoporosis. This study aims to explore whether ART participates in the progression of osteoporosis by regulating these signaling pathways. METHODS: In the in vitro experiments, we treated bone marrow mesenchymal stem cells (BMSCs) with different concentrations of ART (0, 3, 6, 12 µM) and evaluated osteogenic differentiation using alkaline phosphatase staining (ALP) and alizarin red S staining (ARS) staining. The expression levels of osteocalcin (OCN), RUNT-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), and receptor activator of the nuclear factor kappa ligand (RANKL) were detected by real-time quantitative PCR (RT-qPCR). The effects of ART on NF-κB p65 and Notch1 protein expression were analyzed by Western blot (WB) and immunofluorescence (IF). In the in vivo experiments, a postmenopausal osteoporosis rat model was established via ovariectomy. Bone tissue pathological injury was evaluated using hematoxylin eosin (HE) staining. Serum ALP levels were measured using a kit, bone density was determined by dual-energy X-ray absorptiometry, and serum levels of bone gla protein (BGP), OPG, RANKL, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and IL-1ß were measured by enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of NF-κB p65 and Notch1 in tissues was assessed by immunohistochemistry. RESULTS: In vitro experiments revealed that compared to the control group, ART dose-dependently promoted BMSCs proliferation and enhanced their osteogenic differentiation capability. The expression of OCN, RUNX2, and OPG significantly increased in the ART-treated group, while RANKL expression decreased significantly (p < 0.05). ART significantly inhibited the expression of NF-κB p65 and Notch1/Hes1 signaling pathway proteins (p < 0.05). Compared to ART treatment alone, combined treatment with ART and phorbol myristate acetate (PMA) or valproic acid (VPA) resulted in increased expression of NF-κB p65 and Notch1 proteins and decreased osteogenic differentiation capability (p < 0.05). In vivo experiments showed that in rats treated with ART, bone damage was significantly reduced, bone density and mineral content were restored considerably, and the expression of inflammatory factors (TNF-α, IL-6, IL-1ß) decreased significantly (p < 0.05). Additionally, ART treatment significantly reduced the expression of NF-κB p65 and Notch1 proteins, increased OPG expression, and decreased BGP and RANKL levels (p < 0.05). CONCLUSION: In summary, ART facilitates the osteogenic differentiation of BMSCs by inhibiting the NF-κB and Notch1/Hes1 signaling pathways, thereby exerting significant protective effects against osteoporosis.