Danshensu inhibits the IL-1β-induced inflammatory response in chondrocytes and osteoarthritis possibly via suppressing NF-κB signaling pathway

Abstract

Purpose: Osteoarthritis (OA) is the most common inflammatory disease associated with pain and cartilage destruction. Interleukin (IL)-1β is widely used to induce inflammatory response in OA models. This study aimed to explore the role of Danshensu (DSS) in IL-1β-induced inflammatory responses in OA.

Methods: IL-1β was used to induce chondrocyte inflammation. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assay. IL-6, COX-2, TNF-α, and iNOS mRNA levels were detected by qRT-PCR. MMP3, MMP13, ADAMTS4, ADAMTS5, Aggrecan, Collagen, p-IκBα, and p-p65 protein levels were detected by Western blot. An OA mouse model was established by surgical destabilization of the medial meniscus (DMM), and the Osteoarthritis Research Society International (OARSI) score was evaluated by H&E staining.

Results: DSS did not affect the levels of inflammatory indicators including IL-6, COX-2, TNF-α, iNOS, PEG2, and NO but suppressed COX-2 and iNOS protein expression in IL-1β treated chondrocytes. In addition, DSS downregulated IL-1β-enhanced expression of MMP3, MMP13, ADAMTS4, and ADAMTS5 and upregulated aggrecan and collagen expression. Moreover, DSS significantly inhibited IL-1β-induced phosphorylation of p-IκBα and p-p65 in a dose-dependent manner in chondrocytes, suggesting it plays a role in the NF-κB signaling pathway. Furthermore, DSS significantly reduced DMM-induced cartilage OARSI score in mice, further demonstrating its protective role in OA progression in vivo.

Conclusions: Our study revealed the protective role of DSS in OA, suggesting that DSS might act as a potential treatment for OA.

Keywords: Danshensu; IL-1β; Inflammatory response; NF-κB; Osteoarthritis.

Fig. 1

The cytotoxicity of DSS on chondrocytes. A The structure of DSS. B The morphology of primary chondrocytes. Scale bar = 200 μm. C, D Chondrocyte viability was detected by the CCK-8 kit after treatment with different concentrations of DSS for 24 h (C) and 48 h (D), respectively. ***p < 0.001

Fig. 2

DSS inhibited IL-1β-induced chondrocytes inflammation. The chondrocytes were pretreated with 2.5, 5, and 10 μM DSS for 24 h and stimulated with 10 ng/ml IL-1β for 2 h. A The mRNA levels of inflammation-related markers IL-6, COX-2, TNF-α, and iNOS were evaluated by qRT-PCR. B The protein levels of iNOS and COX-2 was detected by Western blot with β-actin as the internal reference. C The production of PGE2, TNF-α, NO, and IL-6 were detected by ELISA. **p < 0.01; ***p < 0.001

Fig. 3

DSS protected chondrocytes against IL-1β-induced ECM degradation. The chondrocytes were treated with 2.5, 5, and 10 μM DSS for 24 h and then stimulated by 10 ng/ml IL-1β for 2 h. A, B Protein levels of MMP3, MMP13, ADAMTS4, ADAMTS5, aggrecan, and collagen II in chondrocytes (A), and their quantification by the Quantity ONE software (B). C, D Representative fluorescence images of collagen II and MMP3 detected by immunofluorescent staining assay (C) and the percentage of cell immunofluorescence in chondrocytes that were pretreated with 10 μM DSS for 24 h and stimulated by 10 ng/ml IL-1β for 2 h (D). Scale bar = 50 μm. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

DSS inhibited IL-1β-induced activation of the NF-κB pathway in chondrocytes. The chondrocytes were treated 2.5, 5 and 10 μM DSS for 24 h and then induced with or without 10 ng/ml IL-1β for 2 h. The protein levels of p65, p-p65, IκBα, and p-IκBα were detected by Western blot (A) and quantitative analysis (B). The protein levels of IκBα in the cytoplasm and p65 in the nucleus of chondrocytes were detected by Western blot (C) and quantitative analysis (D). *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 5

DSS attenuated OA progression in mice model. A Representative images of safranin O and H&E staining of cartilage sections from different groups. B The cartilage OARSI scores of different groups. Scale bar = 50 μm. Ten mice in each group, ***p < 0.001