Tumor necrosis factor-α (TNF-α) is a key player in the pathogenesis of rheumatoid arthritis (RA) and considered a promising target for therapeutic drug development. Activation of the nuclear factor-kappa B (NF-κB) pathway upon TNF-α binding to its receptor is crucial for progression of RA. Stephanine (SA), an isoquinoline aporphine-type alkaloid recently identified in Stephania plants, exhibits anti-inflammatory properties, but its underlying mechanisms of action are unknown at present. In this study, we explored whether SA could ameliorate RA through inhibition of the NF-κB signaling pathway in association with TNF-α activity. Our experiments revealed a binding affinity (K_D) of SA for TNF-α of 2.934×10^-6 M. Additionally, SA at a concentration of 10 μmol/L effectively hindered the binding of TNF-α to its receptors TNFR1 and TNFR2. In vitro, SA prevented TNF-α-induced death of L929 cells and blocked NF-κB activation triggered by TNF-α in 293-TNF-α responsive, as well as HFLS and MH7A cell lines. Furthermore, in a collagen-induced arthritis (CIA) mouse model, SA alleviated the symptoms of RA through suppression of NF-κB signaling. Our collective findings support the therapeutic efficacy of SA, a natural compound targeting TNF-α, in the management of RA.

Excessive osteoclastogenesis-mediated osteoporosis has been recognized as a global health concern. Candidate compounds derived from medicinal plants or functional foods are promising to treat osteoporosis due to their high safety and efficiency. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active polyphenol in green tea. It can inhibit osteoclastogenesis in vitro by blocking RANK signaling pathways. This study used the ovariectomized (OVX) mouse model to estimate the therapeutic effect of EGCG on osteoporosis and verified the molecular mechanism in vivo. The results revealed that EGCG significantly inhibited the OVX-induced body weight gain. Moreover, no adverse effects were observed on blood glucose, histomorphological features, weights, as well as indices of liver and kidney in OVX mice. EGCG could significantly ameliorate bone loss in OVX mice by inhibiting osteoclastogenesis. This effect was evidenced by the reduced number of osteoclasts and the increased trabecular bone area in the femurs. Moreover, EGCG inhibited the activities of CTX-Ⅰ and TRACP-5b and strengthened BGP and PⅠNP activities in OVX mice. Mechanistically, EGCG significantly downregulated the expression of osteoclastogenesis-related marker genes and proteins, including NFATc1, c-Fos, TRAP, c-Src, and cathepsin K. In addition, the phosphorylation levels of p65, JNK, ERK1/2, p38, and AKT were significantly suppressed in OVX mice. It was found that EGCG could alleviate OVX-induced bone loss in mice by suppressing osteoclastogenesis by blocking the NF-κB, MAPK, and AKT signaling pathways. EGCG has the potential to prevent and treat osteoclast-related diseases such as osteoporosis.

Steroidal alkaloids are the main active components in many medicinal plants and exhibit diverse biological activities. Axillaridine A (AA) is a newly discovered steroidal alkaloid. However, whether AA could suppress osteoclastogenesis and alleviate ovariectomy-induced bone loss in mice remains unknown. In vitro, AA significantly suppressed the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclast differentiation via downregulating the expression of osteoclastogenesis-related marker genes, proteins, and transcriptional regulators, including tartrate-resistant acid phosphatase (TRAP), c-Src, matrix metallopeptidase-9 (MMP-9), cathepsin K, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and c-Fos. This was achieved by blocking RANKL-RANK interaction and inhibiting RANKL-mediated RANK signaling pathways, including NF-κB, AKT, and mitogen-activated protein kinases (MAPKs) in osteoclast precursors. In vivo, AA significantly inhibited the ovariectomized (OVX)-induced body weight gain and blood glucose increase in mice. AA did not adversely affect the histomorphologies, weights, and indices of the kidney and liver in OVX mice. AA effectively ameliorated bone loss in OVX mice by inhibiting osteoclastogenesis. AA significantly inhibited the serum levels of tartrate-resistant acid phosphatase 5b (TRACP-5b) and C-telopeptide of type Ⅰ collagen (CTX-Ⅰ). AA significantly inhibited the OVX-induced expression of osteoclastogenesis-related marker genes and proteins in the femur. In summary, AA alleviates ovariectomy-induced bone loss in mice by suppressing osteoclastogenesis via inhibition of RANKL-mediated RANK signaling pathways and could be potentially used for the prevention and treatment of osteoclast-related diseases such as osteoporosis.