Monosodium iodoacetate mia

Anterior cruciate ligament transection (ACLT) was done for surgically induced OA group on the right stifle with a scalpel, under surgically sterile conditions, as described by Vignon et al. (1987) . The animals were anesthetized with Zoletil^® (Virbac, Milperra, Australia) at 3 mg/kg via intramuscular route and maintained with isoflurane (3%) (Piramal Healthcare, India). In order to expose the anterior cruciate ligament, medial patella dislocation was performed by lateral parapatellar arthrotomy approach with the stifle joint being fully flexed. Once visualized, irrigation with sterile saline was made onto the stifle joint. Subsequently, using 4-0 polydioxanone suture, closure of the joint capsule and subcutaneous tissue was done. Skin closure was done using 3-0 nylon suture. Tramadol (Duopharma, Kuala Lumpur, Malaysia) 2mg/kg was given twice daily for three days as painkiller. The animals were caged with unrestricted movement until anaesthetic recovery.
The OA chemical induction was administered with 0.32 ml of 25 mg/ml monosodium iodoacetate (MIA) (Sigma-Aldrich, Burlington, MA, USA) dissolved in saline, injected into the intraarticular space of the stifle joint, under general anaesthesia (Zoletil^®) at 2 mg/kg via intramuscular route.

The allosteric modulator LUF6000 (N-(3,4-dichloro-phenyl)-2-cyclohexyl-lH-imidazo [4,5-c]quinolin-4-amine) was synthesized for Can-Fite BioPharma at Leiden Academic Centre for Drug Research (Leiden, The Netherlands)/Haoyuan Chemexpress Co., Ltd (Shanghai, China). A stock solution of 10 mM was prepared in DMSO and further dilutions were prepared in PBS. ConA (Canavalia ensiformis, Jack Bean Hemagglutinin) was purchased from Calbiochem-EMD Millipore (San Diego, CA). Monosodium iodoacetate (MIA; Sigma, St. Louis, MO) was prepared in saline solution.
Rabbit polyclonal antibodies against rat A₃AR, phosphoinositide 3-kinase (PI3K), IκB kinase (IKK), IκB, nuclear factor kappa B (NF-κB), Janus kinase 2 (Jak-2), and signal transducer and activator transcription 1 (STAT-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Rat IL-1β protein (purity > 90%) was acquired from Sino Biological company (Beijing, China). RNA interference sequences for ADAM8 were purchased from GenePharma in Suzhou, China. Thermo Fisher Scientific (St. Louis, USA) provided DMEM/F12 for cell culture, as well as fetal bovine serum (FBS) and phosphate buffer saline (PBS). Monosodium iodoacetate (MIA) was obtained from Sigma in Gillingham, UK. Antibodies included anti-MMP-13, TNF-α, IL-6, COX2, P-JNK, JNK, P-ERK, ERK, P-P38, P38, and β-actin antibodies were obtained from Abcam, UK. The antibodies included anti-ADAM8 and MMP-1 were obtained from ThermoFisher, USA. BDP-13176, a FSCN1 inhibitor, was purchased from MedChemExpress (New Jersey, USA).