Isonardosinone attenuates osteoclastogenesis and OVX-induced bone loss via the MAPK/NF-κB pathway

Abstract

Osteoporosis is a globally prevalent metabolic bone disease that manifests itself as a decrease in bone mineral density and deterioration of bone structure, which reduces overall bone strength and increases fracture risk. However, the effect of anti-inflammatory isonardosinone (the active ingredient in Nardostachys chinensis) on osteoclastogenesis is unknown. We first predicted the main pathways and targets of ISO action in osteoporosis by network pharmacology. CCK-8 was used to test whether ISO affects cell proliferation of BMMs (osteoclast precursor cells) and to determine the safe action concentration. TRAcP and F-actin staining were used to characterise the inhibitory effect on osteoclast differentiation. RT-PCR and WB were used to examine changes in the relative expression of genes and proteins generated by osteoclasts under isopinacolone treatment, and we examined its effects on the RANKL-activated MAPK and NF-κB signaling pathways. An ovariectomy-induced osteoporosis model was constructed to assess the in vivo therapeutic effects of ISO. CCK-8 results showed that ISO had no cytotoxic or proliferative effects on BMMs at concentrations below 30 μM; TRAcP staining showed that ISO suppressed osteoclastogenesis in a concentration- and time-gradient-dependent manner; and F-actin staining showed that ISO suppressed osteoblast skeleton formation and expansion; RT-PCR and Western Blot assays showed that ISO suppressed the expression of CTSK, NFATC1, MMP9, C-Fos, and ACP5, inhibited the phosphorylation of JNK, P38, and ERK, and reversed the degradation of IκB-α, especially within 15 min. The in vivo results indicated that ISO has therapeutic effects on osteoporosis by improving bone microstructure to rescue bone loss. Taken together, these results lead to the conclusion that ISO is an attractive drug development strategy for the treatment of osteoporosis by effectively suppressing osteoclastogenesis through the MAPK/NF-κB signaling pathway, thereby reversing the bone loss associated with ovariectomy in vivo.