Bergamottin (Ber) (purity >98%) and Phosphate buffered saline (PBS) was obtained from ChemeGen BioTechnology (Shanghai, China). Anti-Lamin B1 and anti-IκB were acquired from Proteintech (Wuhan, China). Anti-iNOS, anti-Sirt1, anti-P65, anti-GAPDH, Anti-Sirt1, and anti-COX-2 were purchased from Bioss (Beijing, China). EX-527 and MTT were purchased from Sigma-Aldrich (CA, USA).

The chondrocyte extraction and culture were performed according to the previous studies (Xian et al., 2022). Chondrocytes were isolated from the 5-day-old mice. Knee joint cartilage was exposed under sterile conditions and collected into a PBS solution. Then, treat cartilage tissue with type II collagenase. Finally, chondrocytes were cultured in DMEM/F12 medium (10% FBS and 1% penicillin/streptomycin) in an incubator with 5% CO₂ at 37°C.

C57BL/6 mice were obtained from Wenzhou Medical University. The model of OA was established by meniscus injury surgery. Mice were randomly separated into three groups (n = 6 in each): a control group (sham-operated), OA group (OA), and OA treated with Ber group (Ber). Mice were intraperitoneally injected with Ber (20 mg/kg) once a day for eight consecutive weeks. The mice were reared in specific pathogen-free cages, given unrestricted movement, and provided autoclaved food and water. All animal experiments were carried out and approved by the Animal Administration Committee of Wenzhou Medical University.

The Cell counting Kit 8 (CCK-8) reagent is a commonly used method for measuring cell viability. The chondrocytes were seeded into 96-well plates and treated with concentrations of IL-1β (10 ng/mL) and Ber (0–40 μM) for 24 or 48 h, respectively. The effect of Ber on cells was measured by using a spectrophotometer (Synergy HT, Bio-Tek, United States).

Chondrocytes (3 × 10⁵ cells per mL) were seeded in 6-well plates and treated with Ber(5, 10, or 20 μM) 24 h prior to the addition of IL-1β (10 ng mL⁻¹). They were incubated for 24 h and then the concentration of NO was measured using the Griess reaction as previously described (Au et al., 2007). The optical density of each sample was measured at a wavelength of 543 nm. The culture medium supernatant from each sample was collected, the levels of PGE2, Aggrecan, ADAMTS-5, and IL-6 in the culture medium were measured with an ELISA kit (R&D Systems, Minneapolis, MN, United States) according to the manufacturer’s instructions.

The cell proteins were isolated using RIPA lysis. The concentration of proteins was measured by the BCA Assay Kit. Protein samples (20 μM) were separated by SDS-PAGE electrophoresis and electrotransferred to the PVDF membrane (Millipore, MA, United States). The protein sample was transferred to a membrane and sealed in 5% nonfat milk for 1 h. The primary antibody (1:1000) was incubated overnight at 4°C. After being washed with TBST, the membranes were incubated with secondary antibodies for 60 min. Odyssey Infrared Imaging System (LI-COR, United States) was used to obtain images. The blot images were detected by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher)

First, we mapped the molecular structure of SA using ChemBioDraw and minimized its energy using ChemBio3D. Based on the requirements of current molecular docking experiments, we downloaded the corresponding Sirt1 from the PDB website (https://www.rcsb.org/). After being processed in PyMoL (version 1.7.6), the lowest energy conformations for docking were decided based on default parameters. When the molecular structures of Ber and Sirt1 required for docking analysis were finalized, subsequent molecular docking analysis was performed by AutoDockTools (version 1.5.6). The final 3D views of the images were obtained via PyMoL.

The chondrocytes were seeded into 6-well plates and treated with Ber(20 μM). Then chondrocytes were fixed with 4% paraformaldehyde (PFA, Servicebio, Wuhan, China) for 10 min and permeabilized with 0.1% Triton X-100 (BioFroxx, Germany) for 15 min. The sections were blocked with 5% bovine serum albumin. The samples were incubated with the primary antibody overnight at 4°C, followed by incubation with the fluorescent secondary antibody for 60 min. The next day, samples were treated with secondary antibodies for 45 min and stained with DAPI for 15 min. Finally, the samples were examined using an inverted fluorescence microscope (Leica DMIL, Germany).

The cartilage tissues of mice were fixed with 10% paraformaldehyde, then dehydrated, embedded, sectioned, and stained with hematoxylin-Eosin and Safranin O/Fast Green staining. The bright-field images were captured using a microscope (Thermo Fisher) and processed with ImageJ software (v1.8.0).

GraphPad Prism 7.0 software was used to execute statistical analyses and create the statistical charts. One-way ANOVA followed by Tukey’s t-tests multiple comparisons was used to compare the groups. p < 0.05 indicates a statistically significant difference.

Figure 1A illustrates the molecular structure of Ber. The Safranin O staining results for cellular proteoglycans are presented in Figure 1B. The cytotoxic effect of Ber on chondrocytes was assessed using the CCK-8 assay. First, the chondrocytes were treated with different concentrations of Ber (0, 2.5, 5, 10, 20, 30, and 40 μM) for 24 and 48 h. Subsequently, the toxicity of Ber was assessed using the CCK-8 reagent. As presented in the results in Figures 1C,D, Ber exerted no toxic effects on chondrocytes within the concentration range of 5–20 μM, while the Ber concentrations 30 μM resulted in evident cytotoxicity. Therefore, Ber concentrations of 5, 10, and 20 μM were selected for the subsequent cell experiments to be conducted in the present study.

IL-1β induction leads to the production of various inflammatory cytokines in chondrocytes, ultimately causing inflammation in these cells. Whether Ber could inhibit IL-1β-induced inflammation in chondrocytes was assessed in the present study. ELISA and Western blotting were performed to detect the expression levels of various inflammatory cytokines, and the results are presented in Figures 2A–C. In comparison to the control group, the IL-1β group presented significantly increased expression levels of COX-2 and iNOS, while the Ber-treatment group exhibited reduced expression levels of IL-1β-induced COX-2 and iNOS in a dose-dependent manner. The ELISA results revealed a significant upregulation in IL-6, PGE2, TNF-α, and NO expression levels in the IL-1β-induced group. In contrast, the Ber group exhibited an inhibitory effect on the expression levels of IL-1β-induced NO, TNF-α, IL-6, and PGE2 (Figures 2D–G). The above results indicated that Ber could significantly inhibit IL-1β-induced inflammation in chondrocytes.

Type II collagen and proteoglycans are the major components within chondrocytes, and the expressions of these reflect the functionality of chondrocytes. IL-1β induction leads to the production of metalloproteinases, which could lead to in the degradation of the extracellular matrix of chondrocytes. Therefore, the impact of Ber on IL-1β-induced degradation of the extracellular matrix of chondrocytes, was investigated in the present study using Western blotting to determine the expressions of metalloproteinases. The results are presented in Figures 3A–D, IL-1β induction led to abnormal expressions of MMP-13 and ADAMTS-5, while Ber significantly inhibited their expressions of these metalloproteinases. Collagen II fluorescence staining results revealed that Ber effectively inhibited the IL-1β-induced degradation. These findings suggested that Ber could significantly inhibit the IL-1β-induced degradation of the extracellular matrix (Figures 3E,F).

NF-kB plays a significant regulatory role in inflammatory diseases, and its activation could further exacerbate inflammation. Therefore, the impact of Ber on NF-kB in IL-1β-induced chondrocytes was investigated in the present study. Western bloting was conducted to evaluate the expressions of IκBα and P65, and the results are presented in Figures 4A–D. It was observed that under normal conditions, P65 expression was low, and IκBα expression was high. IL-1β then induced an increase in P65 expression, and Ber inhibited the expression of P65 in a dose-dependent manner. The chondrocytes were subjected to p65 cellular immunofluorescence staining.The results revealed that IL-1β could cause P65 to translocate to the nucleus of the cell., and Ber could inhibit this phenomenon (Figure 4E). The above results suggested that Ber inhibited the activation of NF-kB within chondrocytes, thereby exerting an inhibitory effect on IL-1β-induced inflammation in chondrocytes.

Sirt1 effectively inhibits the activation of NF-kB. In the molecular docking analyses conducted in the present study, Ber could bind to Sirt1 (Figures 5A–D). The small molecule Ber could bind to the SIRT1 protein through hydrophobic interactions with three residues: PHE273 at the distances of 3.5Å, 3.7Å, and 3.8Å; ARG274 at the distances of 3.2Å and 3.9Å; and ILE411 at a distance of 3.9Å. In addition, Ber exhibited hydrophobic interactions with four residues: PHE297 at distances of 3.5Å, 3.5Å, 3.8Å, and 3.8Å. Accordingly, it was inferred that presence of these interactions allowed the small molecule Ber to stably bind to the receptor protein SIRT1. Subsequently, the impact of Ber on IL-1β-induced Sirt1 in chondrocytes was investigated by assessing the expression levels of Sirt1 using Western blotting. Ber was observed to increase the expression levels of Sirt1 in a dose-dependent manner (Figures 4A,B). This finding suggested that the inhibition of IL-1β-induced NF-kB activation caused by Ber could have occurred through the activation of Sirt1.

The potential mechanism underlying the activation of Sirt1 by Ber was investigated using EX-527 (an inhibitor of Sirt1). In the Western blotting analysis, EX-527 could significantly inhibits the expression of Sirt1, while the nuclear expression of p65 in chondrocytes was observed to increases (Figures 6A–C). In addition, EX-527 markedly eliminated the IL-1β-induced upregulation of COL-II expression in chondrocytes (Figures 6D–F). Furthermore, EX-527 eliminated the downregulation of MMP-13, NO, PGE2, TNF-α, and IL-6 in chondrocytes caused by treatment with Ber and IL-1β (Figures 6G–J).

The effects of Ber on osteoarthritis (OA) were also investigated in vivo, The OA mouse model was established through DMM surgery. Afterward, these mice were administered with 20 mg/kg of Ber orally every day for the next 8 weeks. Safranin O staining was performed to assess the severity of OA in these mice. The results revealed that compared to the sham surgery group, the surgery group exhibited significant cartilage degradation, loss of extracellular matrix glycosaminoglycans, and cellular degeneration (Figure 7A). On the other hand, the group treated with Ber exhibited evident cartilage repair. H&E staining also revealed similar results and indicated that Ber had significantly improved osteoarthritis (Figure 7B). The subsequent immunohistochemical staining revealed a marked upregulation of Sirt 1 in the Ber group (Figures 7C,D). The changes in OARSI scores were also consistent with the results of H&E and Safranin O staining analyses, with the Ber group presenting lower than scores compared to the DMM group (Figure 7E). In summary, the above results provided evidence that Ber mitigated the progression of OA by limiting cartilage degradation, cartilage sclerosis, and the abnormal formation of bone spurs.