Aucubin alleviates knee osteoarthritis in mice by suppressing the NF‑κB signaling pathway

doi:10.12122/j.issn.1673-4254.2025.10.06

Abstract

Abstract:

Objective To assess the therapeutic effect of aucubin in mice with knee osteoarthritis (KOA) and investigate the underlying mechanism. Methods Sixty C57BL/6J mice were randomized equally into sham operation group, KOA model group, glucosamine (positive control) treatment group, and low-, medium-, and high-dose aucubin treatment groups (2, 4, and 8 mg/kg, respectively). KOA mouse models were established by transection of the anterior cruciate ligament (ACL), and the treatment was initiated on day 1 postoperatively and administered weekly for 8 weeks. Safranin O-fast green staining, immunohistochemistry, and microCT were used to evaluate the changes in cartilage pathology, inflammatory protein expression, and subchondral bone volume fraction (BV/TV). The expression levesl of COL2, SOX9, p-P65, IL-1β and MMP13 proteins in the cartilage tissues were detected using Western blotting. In a chondrocyte model with IL-1β treatment for mimicking KOA, the effect of aucubin on chondrogenic differentiation was observed with Alcian blue and Safranin O staining, and cellular COL2, SOX9 and TNF‑α mRNA expressions were detected with RT-qPCR. Results Compared with those in the model group, the mouse models receiving aucubin treatment showed significantly upregulated COL2 and SOX9 protein levels and downregulated p-P65, IL-1β and MMP13 expressions in the cartilage tissues. In the IL-1β-induced chondrocyte model, aucubin treatment significantly upregulated the mRNA expressions of SOX9 and COL2 but lowered the mRNA expression of TNF-α. Alcian blue and Safranin O staining confirmed that aucubin promoted the synthesis of cartilage extracellular matrix and enhanced chondrogenic differentiation of the cells. Conclusion Aucubin can effectively alleviate KOA in mice by inhibiting NF‑κB-mediated cartilage inflammation, promoting cartilage matrix synthesis, and improving subchondral bone microstructure.

Key words: aucubin, osteoarthritis, nuclear factor-κB, cartilage repair, matrix metalloproteinases

Yongxin MAI, Shuting ZHOU, Ruijia WEN, Jinfang ZHANG, Dongxiang ZHAN. Aucubin alleviates knee osteoarthritis in mice by suppressing the NF‑κB signaling pathway[J]. Journal of Southern Medical University, 2025, 45(10): 2104-2110.

Figures/Tables 6

Tab.1 Primer sequences for RT-qPCR for amplifying mRNAs associated with cartilage matrix synthesis and inflammatory response

Gene	Primer sequences
COL2A1-Forward	GGCAATAGCAGGTTCACGTACA
COL2A1-Reverse	CGATAACAGTCTTGCCCCACTT
SOX9-Forward	TCCTCAGGCTTTGCGATTT
SOX9-Reverse	TGCTCGGGCACTTATTGG
TNF-α-Forward	CTGACGAATTACAGGGCCAAT
TNF-α-Reverse	TGCGTGAACCTCTTGAACAGT
18S-Forward	TGGTTGCAAAGCTGAAACTTAAAG
18S-Reverse	AGTCAAATTAAGCCGCAGGC

Fig.1 Morphological analysis of knee joint cartilage in mice. A: HE staining. B: Safranin-O/Fast Green staining.

Fig.2 MicroCT 3D reconstruction and bone mineral density analysis of the subchondral bone in the mouse models. *P<0.05, ***P<0.001 vs Model group.

Fig.3 Immunohistochemical staining for inflammatory proteins in the cartilage tissues of the mouse models. A: p65 staining. B: TNF-α staining. C, D: Quantitative analysis of p65 and TNF-α expression levels. *P<0.05, **P<0.01, ***P<0.001 vs Model group.

Fig.4 Western blotting of protein expressions in the chondrocyte model. A: Representative protein blots. B-F: Quantitative analysis of p-p65/p65, MMP-13, Collagen II, SOX9, and IL-1 expression levels. *P<0.05, **P<0.01, ***P<0.001 vs Sham group; ##P<0.01, ###P<0.001 vs Model group.

Fig.5 Alcian blue-Safranin O staining (A, B; Original magnification: ×100) and expressions levels of SOX9, COL2, and TNF-α mRNAs (C, D) in the chondrocyte model. *P<0.05, **P<0.005, ***P<0.001 vs 0 μmol/L.

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