Visfatin

Human OA chondrocytes, at the first passage, were plated in 6-well dishes at a starting density of 1 × 10⁵ cells/well until they became confluent. Human recombinant visfatin (Sigma–Aldrich, Milan, Italy) was first dissolved in phosphate buffered saline (PBS) (Euroclone, Milan, Italy), according to the manufacturer’s instructions and then it was diluted in the culture medium immediately before the treatment to reach the final concentration required.
The cells were cultured for 24 h in DMEM with 0.5% FBS and visfatin at concentration of 5 μg/mL and 10 μg/mL. The concentrations of the adipokine used in this in vitro study were selected according to those used by other authors [13 (link),18 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
Afterwards, cells were pre-incubated for 2 h with 1 μM BAY 11-7082 (NF-κB inhibitor, IKKα/β, Sigma–Aldrich, Milan, Italy) and then treated 24 h with the tested concentrations of visfatin.
After the treatment, the media were removed, centrifuged and stored at −80 °C, while the cells were immediately processed to carry out flow cytometry analysis and quantitative real-time PCR.

Mice were anesthetized with an intraperitoneal (ip) injection of tribromoethanol (250 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) and placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). The cannula (26 gauge) was implanted into the right lateral ventricle (coordinates of 0.1 mm lateral, 0.03 mm posterior, and 2.4 mm ventral to the bregma) and secured to the skull with dental cement. Animals were kept warm until they recovered from the anesthesia and then placed in individual cages. After surgery, a recovery period of 7 days was allowed before starting the experiments. Mice were sacrificed 90 min after icv injection of the recombinant mouse visfatin (2 μg, Lugen Sci, Bucheon, Korea) to prepare the sections for immunohistochemistry (IHC). The changes of food intake and body weight were measured every day after visfatin administration for 7 days. To evaluate whether the microglia activation is required for visfatin-induced anorexia and body weight loss, mice were injected icv with minocycline (10 μg/2 μl/day, Sigma-Aldrich) or 0.9% saline once a day for 3 consecutive days. On the fourth day, mice were injected icv with saline or visfatin and measured their food intake and body weight change 24 h after injection.

T/C-28a2 cell line and human OA chondrocytes, at the first passage, were plated in 6-well dishes at a starting density of 6 × 10⁶ cells/well until they became confluent. Human recombinant visfatin (Sigma–Aldrich, Milan, Italy) and human recombinant resistin (BioVendor, Rome, Italy) were first dissolved in phosphate buffered saline (PBS) (Euroclone, Milan, Italy), according to the manufacturer’s instructions, and then they were diluted in the culture medium immediately before the treatment to reach the final concentration required.
The cells were treated for 24 h with visfatin at concentration of 5 μg/mL and 10 μg/mL or resistin 50 ng/mL and 100 ng/mL. The concentrations of the adipokines used in our in vitro study were selected according to those used by other authors [12 (link),39 (link)]; the final concentrations were chosen based on the best results obtained in terms of viability.
After the treatment, the media were removed, centrifuged, and stored at −80 °C; the cells were immediately processed to carry out cell viability assay, flow cytometry analysis, and quantitative real-time PCR.
Afterwards, the cells were pre-incubated for 2 h with 1 μM BAY 11-7082 (NF-κB inhibitor, IKKα/β, Sigma–Aldrich, Milan, Italy) and then treated 24 h with the tested concentrations of visfatin and resistin. Subsequently, the gene expression of MMP-1, MMP-13, and Col2a1 was evaluated.

Cells were stimulated using mouse recombinant resistin (Cat. N°: SRP4560, Sigma‐Aldrich), visfatin (Cat. N°: SRP4908, Sigma‐Aldrich) and ghrelin (Cat. N°: 494127, Sigma‐Aldrich) at a concentration of 100 ng/ml.
The 1.0 μg/ml ERK1/2 inhibitor (FR180204) (#328007, Calbiochem) and 3.67 μg/ml HIF‐1α inhibitor (IDF‐11774) (HY‐111387, MedChemExpress) were used. 1.0 μg/ml DimethyIsulphoxide (DMSO) (D8418‐50ML, Sigma‐Aldrich) was used as negative‐control group.