Seven-week-old male Wistar rats weighing 180~250 g at the start of the experiment were purchased from Central Lab Animal Inc. (Seoul, South Korea). A maximum of three animals per cage were housed in a room with controlled temperature (20–26°C) and light (12-h light-dark cycle) conditions. The rats had free access to a gamma-ray-sterilized diet (TD 2018S, Harlan Laboratories, Inct / America) and autoclaved R/O water. All animal research procedures were conducted in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Experiments provided by the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine, The Catholic University of Korea. The IACUC and Department of Laboratory Animals of the Catholic University of Korea, Songeui Campus, accredited the Korean Excellence Animal Laboratory Facility in accordance with the Korean Food and Drug Administration in 2017 and full accreditation by the AAALAC International was acquired in 2018.
Induction of osteoarthritis and treatment with metformin
Animals were randomly assigned to the treatment or control groups before the study began. After anesthetization with isoflurane, the rats were injected with 3 mg of monosodium iodoacetate (MIA) (Sigma, St. Louis, MO, USA) dissolved in a 50-ml volume, using a 26.5-G needle inserted through the patellar ligament into the intra-articular space of the right knee. Metformin 100 mg/kg and the NSAID celecoxib 80 mg/kg were administered orally to the MIA-induced rats. In addition Metformin & celecoxib complex was administered at a concentration of metformin 50 mg/kg.
Assessment of pain behavior
Nociception in MIA-treated rats randomized to the different experimental groups was tested using a dynamic plantar aesthesiometer (Ugo Basile, Gemonio, Italy). The device is an automated version of the von Frey hair assessment procedure and is used to assess mechanical sensitivity. The assessment was conducted by placing the rats on a metal mesh surface in an acrylic chamber in a temperature-controlled room (20–26°C), where they rested for 10 min before the touch stimulator unit was positioned under each animal. An adjustable angled mirror was used to place the stimulating microfilament (0.5-mm diameter) below the plantar surface of the hind paw. When the instrument was activated, a fine plastic monofilament advanced at a constant speed and touched the paw in the proximal metatarsal region. The filament exerted a gradually increasing force on the plantar surface, starting below the threshold of detection and increasing until the stimulus became painful, as indicated by the rat’s withdrawal of its paw. The force required to elicit a paw-withdrawal reflex was recorded automatically and measured in g. A maximum force of 50 g and a ramp speed of 25 s were used for all aesthesiometer tests.
Assessment of weight bearing
Weight balance in MIA treated rat was analysed to incapacitance meter (IITC Life Science, CA, USA). The rats are allowed to acclimate for 5 minutes in an acrylic holder. After 5 minutes, fix both feet of the rat to the pad and measure the weight balance for 5 seconds. Repeat the three measurements in the same manner. Determine the weight of the unguided and guided legs and substitute them in the formula to find the% value. The% value is obtained by comparing the leg with and without osteoarthritis.
Knee joints were collected from each group at 2 weeks post-MIA induction. The tissues were fixed in 10% formalin solution, decalcified using Decalcifying Solution-Lite (Sigma, St. Louis, MO, USA), and embedded in paraffin. Sections of 4- to 5-μm thickness were cut, dewaxed using xylene, dehydrated through an alcohol gradient, and then stained with hematoxylin and eosin (H&E) and safranin O.
Paraffin-embedded sections were incubated at 4°C with the following primary monoclonal antibodies: anti-transient receptor potential cation channel subfamily V member (TRPV)-1 (R&D Systems), anti-calcitonin gene-related peptide (CGRP), anti-IL-1β, anti-IL-17, anti-MMP-3, anti-inducible nitric oxide synthase (iNOS), anti-caspase-1, anti-phospho-MLKL (Abcam, UK), and anti-phospho-AMPK (Cell Signaling Technologies, MA, USA). The samples were then incubated with the respective secondary biotinylated antibodies, followed by a 30-min incubation with a streptavidin-peroxidase complex. The reaction product was developed using 3, 3-diaminobenzidine chromogen (Dako, USA).
Human chondrocytes were stained using FITC-conjugated anti-BODIPY (Sigma, St. Louis, MO, USA), PE-conjugated anti-LC3b, and APC-conjugated anti-LAMP1 (Santa Cruz Biotechnology, USA).
Real-time polymerase chain reaction (PCR)
Total RNA isolated from human chondrocytes using the TRIzol reagent (Molecular Research Center, USA) was used to synthesize cDNA. The relative expression of specific mRNA was quantified by real-time PCR using Sensil FAST SYBR (Bioline, USA) and the following sense and antisense primers: for MMP-1, 5’-CTG AAG GTG ATG AAG CAG CC-3’ (sense) and 5’-AGT CCA AGA GAA TGG CCG AG-3’ (anti-sense); for MMP-3, 5’-CTC ACA GAC CTG ACT CGG TT-3’ (sense) and 5’-CAC GCC TGA AGG AAG AGA TG-3’ (anti-sense); for MMP-13, 5’-CTA TGG TCC AGG AGA TGA AG-3’ (sense) and 5’-AGA GTC TTG CCT GTA TCC TC-3’ (anti-sense); for TIMP1, 5’-AAT TCC GAC CTC GTC ATC AG-3’ (sense) and 5’-TGC AGT TTT CCA GCA ATG AG-3’ (anti-sense); for TIMP3, 5’-CTG ACA GGT CGC GTC TAT GA-3’ (sense) and 5’-GGC GTA GTG TTT GGA CTG GT-3’ (anti-sense); for AMPKα1, 5’-AAC TGC AGA GAG CCA TTC ACT TT-3’ (sense) and 5’-GGT GAA ACT GAA GAC AAT GTG CTT-3’ (anti-sense).
Western blot analysis
Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Western blotting was performed using a SNAP i.d. protein detection system (Millipore). The hybridized bands were detected using an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific, MA. USA) and the following antibodies: anti-phospho-AMPK(Cell Signaling Technologies, MA, USA), anti-caspase-1, anti-caspase-3, anti-GAPDH (Abcam, UK), and anti-goat anti-rabbit IgG-HRP.
In vivo micro-computed tomography (CT) imaging and analysis
Micro-CT imaging and analysis were performed using a bench-top cone-beam type in vivo animal scanner (mCT 35; SCANCO Medical, Br¨ uttisellen, Switzerland). The animals were imaged at settings of 70 kVp and 141 μA using an aluminum 0.5-mm thick filter. The pixel size was 8.0 μm and the rotation step was 0.4°. Cross-sectional images were reconstructed using a filtered back-projection algorithm (NRecon software, Bruker micro CT, Belgium). For each scan, a stack of 286 cross-sections was reconstructed at 2,000 × 1,335 pixels. Bone volume and surface were analyzed at the femur.
Human articular chondrocyte differentiation
Articular cartilage for human was acquired from patients to Replacement arthroplasty or joint replacement surgery. cartilage obtained from the patient was digested and reacted with 0.5 mg/ml hyaluronidase, 5 mg/ml protease type ⅩⅣ, and 2 mg/ml collagenase type V. Finally, Chondrocytes were incubated Dulbecco’s Modified Eagle Medium (DMEM) including 10% fetal bovine serum.
KL grade analysis in OA patients
The radiographs of both knees of OA patients treated with NSAIDs for > 3 years were evaluated retrospectively. The data from 60 patients divided into two groups, diabetic patients who had taken metformin to control their blood glucose level and non-diabetic patients, were collected and analyzed (UC18RESI0038).
The results are expressed as the mean ± standard deviation (SD) and were obtained from three separate experiments. Statistical significance was determined according to the Mann-Whitney U-test or an ANOVA with Bonferroni’s post-hoc test, performed using GraphPad Prism (version 5.01, GraphPad Software, San Diego, CA). A p value <0.05 was considered to indicate statistical significance.