Animals and experimental design
All experiments in this study were approved by the Committee on the Ethics of Animal Experiments of Xiangya Hospital, Central South University and carried out in strict accordance with the approved guidelines for the care and use of laboratory animals.
Eighty, 10-week-old, male C57BL/6 mice (mean weight: 27.3 g) were randomly assigned into 6 groups as the following:
Non-operation group: no special treatment without operation.
Sham-operation group: no special treatment with sham operation.
Intragastric saline (IGS) group: normal saline (10 ml/kg) was administered intragastrically 3 days after the DMM surgery; once daily for 8 weeks.
Intragastric metformin (IGM) group: metformin (200 mg/kg) was given 3 days after the DMM surgery; once daily for 8 weeks.
Intraarticular saline (IAS) group: normal saline (1 ml/kg) was injected into the knee joint cavity 3 days after the DMM surgery; twice a week for 8 weeks.
Intraarticular metformin (IAM) group: metformin (0.1 mmol/kg) was injected into the knee joint cavity 3 days after the DMM surgery; twice a week for 8 weeks.
The animals were housed in groups (four to five per cage) under controlled temperature on a 12-h light/dark cycle. Food and water were provided ad libitum.
After 1 week of acclimation, OA was induced by DMM as previously described . Briefly, mice were anesthetized with intraperitoneal injection with 4% chloral hydrate (10 ml/kg body weight), and after being shaved and disinfected, the right knee joint was exposed through a medial parapatellar approach. The patella was dislocated laterally, and the knee was placed in full flexion followed by transection of anterior medial meniscotibial ligament with a microsurgical knife. Complete disruption of the ligament was confirmed visually by manually displacing the medial meniscus with fine forceps. The joint cavity was washed with normal saline solution. The articular capsule sutured with 6–0 absorbable PGA sutures, and the skin closed with 5–0 medical silk braided sutures.
Sham operation was performed on the right knee of a separate group of mice. It consisted of a skin incision and medial capsulotomy only, followed by capsule and skin closure as described above. Eleven mice died before the intervention ended. Among them, 3 mice died due to severe injury caused by fighting after DMM-operation (before intervention); 4 injured mice also caused by fighting were removed with euthanasia to prevent pain or stress (before intervention); 2 mice died of anesthetic accident (before intervention); and the rest two mice died with unknown reason after intervention (one in IGM group and the other in IAS group). At 8 weeks post-DMM surgery or sham operation, the remaining mice were euthanized with cervical dislocation after isoflurane anesthesia. Thirteen mice in non-operation group, 12 mice in sham-operation group, 11 mice in IGS group, 10 mice in IGM group, 12 mice in IAS group, and 11 mice in IAM group were included for further analysis.
Scanning electron microscopy
Scanning electron microscopy (SEM) was performed to evaluate the surface ultrastructural characteristics of cartilage. After the mice were killed under anesthesia, the knee joints were isolated with scalpels and dissecting scissors and washed in 0.1 M phosphate buffer. Then the joints were fixed in 2.5% glutaraldehyde for 24 h and a second fixation step was performed with 1% osmic acid for 2 h. The specimens were washed in double-distilled water and dehydrated in a graded series of ethanol, then transferred into isoamyl acetate and dried with a critical point dryer (Hitachi High Technologies, Tokyo, Japan). The dried specimens were mounted on stages, coated with platinum/palladium (EiKO IB-5, Shawnee, USA) and observed using a HITACHI S-3400 N electron microscope (Hitachi High Technologies, Tokyo, Japan). We observed the alterations of the cartilage surface in the tibia regions.
Histological analysis and OA scoring
Each dissected knee was fixed in 4% paraformaldehyde for over 24 h and decalcified in 15% EDTA, which was changed every 5 days for 20 days. The decalcified knee was dehydrated in a graded series of ethanol and embedded in paraffin (HistoCore Arcadia H, Leica, Nussloch, Germany). Serial frontal knee sections of the exact 5 μm thickness were obtained by using a Leica RM2255 microtome (Nussloch, Germany) across the entire knee joint. Then, the slices were stained with Safranin O/Fast Green to evaluate the entire articular cartilage of the knee. All images were taken using the same settings on a Nikon Eclipse Ti-S microscope (Melville, USA). Semi-quantitative histopathological scoring system recommended by Osteoarthritis Research Society International (OARSI) was performed for grading mouse cartilage degeneration (on a scale of 0–6) . The severity of cartilage destruction was expressed as an average score of the three highest scores in all slides. The images were blinded-scored by two experienced scorers. If there was a disagreement on the score of cartilage destruction, the reading was adjudicated by a panel of three readers including the two who read the images. A consensus reading was reached when at least two of the three readers agreed.
Pain-related behavior assessment
Mechanical allodynia and hind paw weight distribution were performed to assess pain-related behavior once a week from on day 0 (pre-operation) to day 56 post-surgery.
Mechanical allodynia was measured using an electronic von Frey anesthesiometer (IITC, Woodland Hills, CA, USA). Briefly, the plantar surface of the hind paw was stimulated with ascending force intensities of von Frey filaments. A brisk lifting of the foot was recorded as a positive response, and the number of positive responses for each stimulus was automatically recorded by the instrument. For each mouse, this test was performed three times with a time interval of 10 min between two adjacent stimuli. The mean value of the three readings was calculated as the final threshold value [24, 25].
Changes in hind paw weight distribution between the right (osteoarthritic) and left (control) limbs were measured as an index of joint discomfort in the osteoarthritic knee as previously described . An incapacitance meter tester (IITC, Woodland Hills, CA, USA) was employed to evaluate hind paw weight distribution. Mice were placed in an angled plexiglass chamber positioned so that each hind paw rested on a separate force plate. The force exerted by each hind limb (measured in grams) is averaged over a 5-s period. Each data point is the mean of three, 5-s readings. The change in hind paw weight distribution was calculated by determining the difference in the amount of weight (g) between the left and right limbs.
To obtain consistent results, animals were allowed to adapt to the grid environment for 30 min. All behavioral tests were performed by the same technician who was blinded to the study groups and identification of animals in order to avoid subjective differences in interpretation, which could occur with different observers.
Culture of articular chondrocytes and cartilage explants
To obtain mouse primary chondrocytes, we harvested the knee joints from the femoral condyles and tibial plateaus of postnatal day 3–4 C57BL/6 mice, and digested with 0.1% collagenase (Biosharp) overnight, as described previously . A 2-mm biopsy punch was used to harvest macroscopically intact human cartilage explants from femoral condyles of total knee arthroplasty patients as described elsewhere . Written informed consent was obtained from all participants.
Treatments of chondrocytes and cartilage explants with metformin and AMPK inhibitor
Chondrocytes and cartilage explants were grown in culture medium with 10 ng/ml recombinant interleukin-1β (IL-1β) (R&D Systems, USA) and metformin (1, 10, and 20 mM, Sigma-Aldrich, USA). Chondrocytes and cartilage explants were also cultured in the presence of 10 ng/ml recombinant IL-1β alone. A control sample of chondrocytes and cartilage explants cultured in the absence of metformin and IL-1β was also evaluated. Finally, the effect of the addition of metformin was evaluated in the presence of 10 mM metformin and IL-1β samples, with or without dorsomorphin (10 uM, Sigma-Aldrich, USA), which is an AMPK inhibitor. Dimethyl sulfoxide (DMSO) was used as a vehicle of dorsomorphin. The chondrocytes in each group were respectively treated for 24 h by the corresponding intervention methods, then RNA and protein extraction were performed and the medium was collected. The cartilage explants were treated for 48 h. The medium was collected. All in vitro experiments and assays were repeated three times.
Cell counting Kit-8 assay
The cell viability was assessed by Cell counting Kit-8 (CCK8) (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s protocol. The experiments were performed in sextuplicate.
Total RNA extraction and quantificational real-time polymerase chain reaction
Total RNA was isolated using TRIzol reagent (Invitrogen). In brief, chondrocytes were washed with cold PBS and lysed directly in a dish by adding 1 ml of TRIzol reagent. After passing several times through a pipette, the homogenized samples were incubated for 5 min at room temperature, then transferred to a 1.5 ml RNase-free tube; 0.2 ml of chloroform was added to the lysate to extract RNA. The samples were centrifuged at 10,000×g for 15 min at 4 °C, and the upper aqueous phase was transferred into a fresh tube and mixed with 0.5 ml of isopropyl alcohol. Samples were incubated with ice-cold for 10 min and then centrifuged under 10,000×g for 10 min at 4 °C. After removing the supernatant, the RNA pellet was washed by adding 75% ethanol. The mixture was centrifuged under 10,000×g for 5 min at 4 °C before air-dry. The concentration of each sample was measured by NanoDrop 2000 (Thermo Scientific, USA). Complementary DNA (cDNA) synthesis was performed by 1 μg of total RNA using a cDNA synthesis kit (Trans Script, China) according to the manufacturer’s protocols. Gene expression assay primer pairs were ordered for the detection of matrix metalloproteinase 13 (mmp13) (primers: forward 5′-ACACTCAAATGGTCCCAAACG-3′, reverse 5′-TCATGATGTCAGCAGTGCCA-3′), type II collagen alpha 1 chain (col2a1) (primers: forward 5′-AGCGACTGTCCCTCGGAAAAAC-3′, reverse 5′-CCAGGTAGGCGATGCTGTTCTTAC-3′) and β-actin (primers: forward 5′- GGCTGTATTCCCCTCCATCG − 3′, reverse 5′- CCAGTTGGTAACAATGCCATGT − 3′). Quantitative analysis of the cDNA was performed using the ABI Quant Studio 3 (Applied Biosystems, USA) and All-in-one qPCR (Gene Copoecia, USA). The thermal cycling conditions were 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. β-actin was used as the housekeeping gene for internal control. mRNA levels were normalized by β-actin levels of each sample. Comparative quantification was determined using the 2−ΔΔCt method.
Protein extraction and western blot
Cells were washed twice with ice-cold PBS and extracted by 2× SDS reagent with protease inhibitor cocktail (Roche, USA). After treatment with an ultrasonic cell disruption system, the cell lysate was clarified by centrifugation at 11,000 rpm for 10 min at room temperature, protein content in the supernatant was collected and the protein concentration was determined by BCA assay (Pierce, USA). Aliquots (30 μg) of protein were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a poly (vinylidene difluoride) membrane (Millipore, USA). The membrane was blocked with 5% (w/v) skimmed milk in TBST (10 mM Tris-HCl, pH 7.8, 150 mM NaCl, and 0.1% Tween-20) for 1 h and then incubated with anti-tubulin primary antibody (1:2000, Abcam, USA) or anti-GAPDH primary antibody (1:2000, Santa Cruz Biotechnology, USA), anti-MMP13 primary antibody (1:3000, Abcam, USA) or anti-type II collagen primary antibody (1:5000, Abcam, USA) or anti-phosphorylated alpha subunit of AMPK (pAMPKα) primary antibody (1:2000, Cell Signal Technology, USA) or anti-AMPK primary antibody (1:1000, Abcam, USA), in TBST containing 5% (w/v) BSA overnight at 4 °C. After washing three times, the blots were treated with anti-mouse and anti-rabbit IgG, respectively (1:5000, Cell Signal Technology, USA) in TBST containing 5% (w/v) BSA for 60 min, and the immune complex was detected using an ECL plus detection kit (Cell Signaling Technology, USA). Densitometric analysis was performed using ImageJ software (National Institutes of Health, USA).
Enzyme-linked immunosorbent assay
Culture supernatant of chondrocytes and cartilage explants was collected after 24 h or 48 h of incubation respectively. The concentrations of MMP-13 were measured by enzyme-linked immunosorbent assay (ELISA) (mice chondrocytes: Cusabio, China; human cartilage explants: R&D Systems, UK) following the manufacturer’s instruction and were normalized to cell protein concentrations.
All quantitative data were presented as means ± standard deviation (SD) and analyzed by Program Graph Pad Prism version 6.0. Multiple comparisons were performed by one-way ANOVA with Tukey’s post hoc test or repeated measures ANOVA with Bonferroni’s post hoc test as appropriate. The interaction effect between time and groups was also assessed in the repeated measures ANOVA. p value < 0.05 was considered statistically significant for all tests.