Mmp 8

XZK powder was kindly provided by the WBL Peking University Biotech Co., Luye Pharma Group (Beijing, China). 7-ketocholesterol (7-KC), Oil red O and sirius red were purchased from Sigma-Aldrich (St. Louis, MO, USA). Atorvastatin was obtained from Pfizer Ltd. (New York, NY, USA). The antibodies against α-actin, β-actin, MMP8, MMP13, TNFα, phosphorylated IRE1α (p-IRE1α), IRE1α, eIF2α, PERK and IκBα were from Abcam (Cambridge, MA, USA); antibodies against BiP, phosphorylated PERK (p-PERK), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), spliced x-box binding protein 1 (s-XBP1), cleaved PARP, and active caspase-3 were from Cell Signaling Technology (Beverly, MA, USA); and antibodies against CCAAT-enhancer-binding protein homologous protein (CHOP) and ATF6 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Secondary antibodies including Alexa Fluor 488-labeled donkey anti-rabbit antibody, Alexa Fluor 647-labeled goat anti-rat antibody, and Alexa Fluor 555-labeled donkey anti-mouse antibody were from Invitrogen (Carlsbad, CA, USA). The TUNEL assay kit (In Situ Cell Death Detection kit) was from Roche (Mannheim, Germany), real-time PCR (qPCR) reagent kits were from TAKARA Biotechnology (Dalian, China), and 6-diamino-2-phenylindole (DAPI) was from Beyotime Biotechnology (Shanghai, China).

The freshly isolated neutrophils were resuspended in dHBSS at a concentration of 5 × 10⁶ cell/ml. The indicated reagents were added to the cells and incubated for 2 h at 37 °C. After the incubation, 1 ml supernatant was collected and concentrated by centrifugation through Amicon Ultra-0.5 (Millipore) to the same final volume of 50 μl. The residual neutrophil pellet was lysed and protein samples were prepared. Antibodies to phospho-p38 MAPK (Thr 180/Tyr 182; Cell Signaling), p38 MAPK (Cell Signaling) and MMP8 (Abcam, 1:1 000 for all) were used.

Cell lysates were obtained after the addition of radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) containing a cocktail of protease inhibitors (KangChen, China) to the cells. Proteins were quantified using a bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific, Lafayette, United States). Equal amounts of protein were separated using 10% SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA). Membranes were blocked in 5% skim milk for 1 h and subsequently incubated with primary antibodies against MMP-9 (Cell Signaling Technology, Boston, MA), MMP-1 (Proteintech Group, United States), MMP-8 (Abcam), vascular cell adhesion molecule-1 (VCAM-1) (Abcam), intercellular adhesion molecule 1 (ICAM-1) (Abcam), and GAPDH (KangChen, China) at 4°C overnight. Membranes were washed thrice with TBS-T, and then incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein signals were detected using an ECL detection system.

A lentiviral vector containing the coding sequence of the visfatin gene was commercially sourced from Invitrogen (Shanghai, China). Recombinant human visfatin was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal anti-GAPDH was purchased from Cell Signaling Technology Inc. (Danvers, MA). Rabbit monoclonal anti-visfatin was purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibodies to α-smooth muscle cell actin and MMP-8 were both purchased from Abcam (Cambridge, UK). Rat anti–mouse monoclonal antibody for macrophages was purchased from Abcam (Cambridge, UK). Rabbit polyclonal antibodies to phospho-NF-κB (p65) and phospho-IκBα were both purchased from Cell Signaling Technology Inc. (Danvers, MA). BAY11-7082 and SC-514, selective inhibitors of NF-κB, were both purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies to smooth muscle-myosin heavy chain (SM-MHC), 22 kDa smooth muscle protein (SM22α), smooth muscle calponin (SM-Calponin), smooth muscle myosin light chain kinase (SM-MLCK), h-Caldesmon (h-CALD), Ki-67 and Osteopontin were all purchased from Bioss (Beijing, China). Rabbit polyclonal antibodies to MMP-1, MMP-2 and MMP-9 were also purchased from Bioss (Beijing, China).

Protein levels of matrix metalloprotease (MMP) and receptor activator of nuclear factor-κB ligand (RANKL) were assessed by WB analysis. The gingival tissue around the maxillary second molar was carefully removed and homogenized. After quantitative determination of protein concentration with the tissue BCA method, we executed WB analysis of the target protein and internal reference protein as previously described [27 (link)]. The exposed film was scanned directly, and the image format was converted with Image J software (NIH, Bethesda, MD, USA). The integrated optical density (IOD) values of the bands were read and analyzed with Total Lab Quant V11.5 (Newcastle upon Tyne, UK). Primary antibodies against β-actin, MMP-8, MMP-9, and RANKL were purchased from Abcam (Cambridge, MA, USA).