Tumor necrosis factor-α (TNF-α) is a key proinflammatory cytokine that drives rheumatoid arthritis (RA) pathogenesis by sustaining synovial inflammation and joint destruction via binding to its receptors (TNFR1 and TNFR2), making this interaction a core therapeutic target. Ellagic acid (EA), a dietary polyphenol naturally abundant in pomegranates, berries, and nuts, is well known for its anti-inflammatory and antioxidant properties. In this study, we investigated whether EA targets the TNF-α pathway using a combination of molecular interaction analyses and cellular assays. Surface plasmon resonance (SPR) revealed that EA binds directly to TNF-α (K_D = 3.588 × 10^-6 M), TNFR1 (K_D = 6.488 × 10^-6 M), and TNFR2 (K_D = 7.952 × 10^-6 M) with high affinity. Molecular dynamics simulations and competitive SPR assays demonstrated that EA disrupts the TNF-α–TNFR interaction. Functionally, EA (0.625–2.5 μM) suppressed TNF-α-induced apoptosis in L929 cells and inhibited TNF-α-triggered NF-κB activation in 293-TNF-α Res (NF-κB) cells. In RA-relevant cell models using human synovial fibroblasts (HFLS and MH7A), EA attenuated TNF-α-stimulated NF-κB signaling by reducing phosphorylation of IKKα/β, IκBα, and p65, and by blocking p65 nuclear translocation. Notably, EA did not inhibit LPS-induced NF-κB activation, indicating pathway selectivity. These findings demonstrate that EA inhibits NF-κB activation by directly targeting the TNF-α–TNFR interaction, highlighting its potential as a food-derived functional ingredient for managing RA and related inflammatory conditions.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder that has long been hampered by limited treatment efficacy and significant side effects. Tumor necrosis factor-α (TNF-α) plays a pivotal role in RA pathogenesis by binding to its receptor (TNFR) and activating the downstream nuclear factor-kappa B (NF-κB) signaling pathway, which promotes the transcription of pro-inflammatory genes and perpetuates disease progression. Blocking the TNF-α–TNFR interaction thus represents a promising therapeutic strategy for RA. In this study, we identified a natural compound, 1-norbetulonic acid (DOCA), that disrupts the binding between TNF-α and TNFR, leading to therapeutic benefits in RA. Our results show that DOCA inhibits TNF-α-induced activation of the NF-κB pathway in human fibroblast-like synoviocytes and MH7A cells, and prevents nuclear translocation of the p65 subunit. Notably, DOCA demonstrated significant therapeutic efficacy in a mouse model of RA. Together, these findings support the hypothesis that DOCA alleviates RA by blocking TNF-α–TNFR signaling, underscoring its potential as a natural product-derived inhibitor of this interaction and highlighting a viable approach for the discovery of TNF-α/TNFR-targeted natural therapeutics.

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.

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 mol/L. Additionally, SA at a concentration of 10 μmol/L effectively hindered the binding of TNF-α to its receptors tumor necrosis factor receptor 1 (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 human fibroblast-like synoviocytes (HFLS) and human RA fibroblast-like synoviocytes (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 receptor activator of nuclear factor (NF) -κB (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 c-telopeptide of type I collagen (CTX-I) and tartrate-resistant acid phosphatase 5b (TRACP-5b) and strengthened bone gla protein (BGP) and procollagen I N-terminal peptide (PINP) activities in OVX mice. Mechanistically, EGCG significantly downregulated the expression of osteoclastogenesis-related marker genes and proteins, including nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), c-Fos, tartrate-resistant acid phosphatase (TRAP), c-Src, and cathepsin K. In addition, the phosphorylation levels of p65, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and protein kinase B (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, mitogen-activated protein kinase (MAPK), and AKT signaling pathways. EGCG has the potential to prevent and treat osteoclast-related diseases such as osteoporosis.