Interleukin 6 (il 6)

The tissue was homogenized in RIPA buffer containing a protease inhibitor cocktail (Beyotime Biotechnology). A BCA protein assay (Beyotime Biotechnology) was used to determine the protein concentrations. Equal amounts of protein were separated by SDS-PAGE and then transferred electrophoretically to polyvinylidene fluoride membranes (Bio-Rad) [29 (link), 30 (link)]. The membranes were incubated with the following primary antibodies for 2 h at room temperature: IL-6 (1:1000, Abcam, England), Gp130 (1:1000, Santa, America), pSTAT3 (1:1000, Bioss, China), and NMDA receptors (1:1000, Bioss, China). The membranes were then incubated with GAPDH (1:1000, Solarbio, China), goat anti-mouse IgG (Bioss, China, Gp130, 1:1000 and GAD67, 1:1000) or goat anti-rabbit IgG (Bioss, China, pSTAT3, 1:1000, NMDA receptors, 1:3000 and IL-6, 1:1000) secondary antibodies for 2 h at room temperature. Finally, the membranes were placed in a gel imaging analysis system for exposure and analysis (AlphaView FluorChem FC3).

Brain protein (40 mg) was subjected to SDS-PAGE under reducing conditions following the transfer to polyvinylidene difluoride membranes. The membranes were incubated with 5% nonfat milk, followed by incubation using a specific antibody. The following primary antibodies (dilutions) were used: CD11b (1:1000; Bioss Antibodies, Woburn, MA, USA), phospho-ERK1/2 and total ERK1/2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), IL6 (1:1000, Bioss Antibodies, Woburn, MA, USA), TNFα (1:1000, LSBio, Seattle, WA, USA), postsynaptic density-95 (PSD-95, 1:1000; Cell Signaling, Danvers, MA, USA), brain-derived neurotrophic factor (BDNF, 1:1000; Millipore Sigma, St. Louis, MO, USA), GPBAR1 (1:3000, LSBio, Seattle, WA, USA) and β-Actin (1:10,000; Millipore Sigma, St. Louis, MO, USA). Then, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. The signals were detected using Pierce Super Signal West Pico chemiluminescent substrates (Thermo Fisher Scientific, Rockford, IL, USA).

After the brain tissue was embedded in paraffin, the part between the optic chiasm and mammillary body was resected in the rostro-caudal direction. The tissue was serially sectioned on a paraffin slicer, and sections that were approximately 1.50 mm from the bregma were obtained [25 (link), 26 (link)]. After dewaxing the slices, 3% H₂O₂ was used to block endogenous peroxidase activity, and 0.01 M citric acid was used to retrieve the antigens prior to antibody incubation. Then, the slices were incubated with primary antibodies overnight at 4 °C [27 , 28 (link)]. The sections were immunohistochemically labelled to identify IL-6 (Bioss, China, 1:100), Gp130 (Santa Cruz, America, 1:20), pSTAT3 (Bioss, China, 1:50), NMDA receptors (Bioss, China, 1:50), and GAD67 (Abcam, England, 1:2000) and then incubated with secondary antibodies (anti-mouse for Gp130 and GAD67; anti-rabbit for NMDA receptors, pSTAT3 and IL-6) for 20 min at room temperature. For each rat, the positive neurons within the bilateral borders of the PVN were manually counted in three consecutive sections, and the average value is reported.

FaDu and FaDu/DDP cells were lysed by RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) with protease inhibitor cocktail and phosphatase inhibitor (Thermo Fisher Scientific) and kept on ice for 15 min. After centrifugation at 15,000g for 25 min at 4°C, the concentration of protein was tested by BCA Protein Assay Kit (Beyotime Institute of Biotechnology). Samples with 25 μg protein were separated on 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). Membranes were blocked by 5% BSA (Sigma-Aldrich, Shanghai, China) in tris-buffered saline (TBS, 10 mmol/L Tris, 10 mmol/L NaCl) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Primary antibodies were shown as follows: GAPDH (Proteintech Group, Wuhan, China), S100A9 (Proteintech Group), CEACAM6 (Bioss, Beijing, China), IVL (Proteintech Group), IL6 (Bioss, Beijing, China), E-cadherin, and vimentin (Cell Signaling Technology, MA, USA).

The sections were stained with the following primary antibodies for the IHC analyses: IL-1β, IL-6, RANKL, and Cathepsin K (Bioss, Woburn, MA, USA) (1:400 dilution). After deparaffinization and rehydration, sections were treated with 3% hydrogen peroxide (Abcam) for 10 min to quench the endogenous peroxidase activity, followed by the incubation with normal goat serum to block the non-specific binding for 30 min at room temperature. Primary antibodies with different specific concentrations were applied to the sections and incubated overnight at 4 °C. The following day, the slides were incubated with the biotinylated secondary antibody for 30 min using the VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Labs, Burlingame, CA, USA). Subsequently, the prepared VECTASTAIN ABC reagent was applied to the slides and incubated for another 30 min. Sections were stained with 3,3’-Diaminobenzidine (DAB) (Abcam) and counterstained with hematoxylin.

Proteins were extracted from RAW264.7 cells using radioimmunoprecipitation (RIPA) buffer (Yazyme, Shanghai). Protein concentration was determined by BCA protein detection kit (Biyuntian, Shanghai). SDS-PAGE was used to separate proteins from different samples. After transferring the separated proteins to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA), the PVDF membranes were blocked with protein-free fast blocking solution (Yazyme, Shanghai) for 15 min. Blocked PVDF membranes were then incubated in diluted primary antibody for 12 h at 4°C. The dilution ratios of different antibodies (Boaosen, Beijing) were as follows: β-actin (1 : 1000), NF-κB-p65 (1 : 1000, Bioss), p-NF-κB-p65 (1 : 1000, Bioss), IL-1β (1 : 500, Bioss), IL-6 (1 : 500, Bioss), and TNF-α (1 : 500, Bioss). Next, the PVDF membrane and diluted secondary antibody were incubated for 1 h at room temperature. Finally, proteins on PVDF membranes were imaged using enhanced chemiluminescence (ECL) solution (GlpBio, USA), and protein bands were processed using ImageJ to detect protein expression levels.