2.1. Chemicals and reagents
Gigantol (purity ≥ 98%) was bought from Sigma-Aldrich (St Louis, MO, USA). Dimethylsulfoxide (DMSO), collagenase type II, and recombinant human IL-1β were purchased from Sigma Chemical Co. (St. Louis, MO, United States). The Cell-Counting Kit-8 (CCK-8) was bought from Dojindo (Kumano, Japan). Inducible nitric oxide synthase (iNOS) antibody was purchased from Sigma-Aldrich (St Louis, MO, United States). Goat anti-mouse and anti-rabbit IgG-HRP were obtained from Bioworld (OH, United States). Primary antibodies against GAPDH and Lamin B1 were bought from Abcam (CA, United Kingdom). 4′,6-diamidino-2-phenylindole (DAPI) was bought from Beyotime (Shanghai, China). Primary antibodies for P-Akt, P-PI3K, Akt, PI3K, IκBα, COX-2, as well as p65 were purchased from CST (MA, United States). Cell culture chemicals were obtained from Gibco (Grand Island, NY, United States). Secondary antibodies (Alexa Fluor®488 and Alexa Fluor®594 labeled Goat Anti-Rabbit IgG (H + L)) were acquired from Jackson ImmunoResearch (West Grove, PA, United States)
2.2. Primary osteoarthritis chondrocyte culture
In vitro, C57BL/6 mice were anestheticized using sodium pentobarbital and sacrificed to prepare chondrocytes. We extracted knee cartilage under aseptic conditions and dissolved them at 37°C in collagenase II (0.1%) for 4 hours, followed by centrifugation at 1000rpm for 5 min. Then, extracted cells were seeded in micro-well plates containing DMEM/F12 with fetal bovine serum (FBS, 10%) and streptomycin/penicillin (1%). Incubation was done in a 5% CO2 environment at 37°C. Next, to reach 80–90% confluence, cell passaging was done using Trypsin-EDTA (0.25%). For avoiding phenotype loss, only passages 1 to 3 were adopted.
2.3. Animal models
Sixty C57BL/6 wild-type (WT) male mice (ten-week-old) were obtained from the Animal Center of the Chinese Academy of Sciences, Shanghai, China. Study approval was obtained from the Animal Care and Use Committee of Wenzhou Medical University. As previously described, mice OA surgical induction was achieved by the destabilization of medial meniscus (DMM).30 First, mice were randomized into 3 treatment groups, vehicle, sham, and gigantol groups. After anestheticization through peritoneal injection of pentobarbital (3%; 1 mL/kg), joint capsules of mice right knees were incised and their medial meniscotibial ligaments transected using microsurgical scissors. An arthrotomy was also conducted in the sham group, but without medial meniscus ligament transection.
2.4. Experimental design
Cells were exposed to IL-1β (10 ng/ml) either alone or when combined with gigantol pretreatment at various concentrations (10, 20 or 40 µM). Apart from medium changes, the control group was untreated. Cell harvesting was done after 24 h of incubation. Inflammatory responses in chondrocytes were sufficiently induced by 24 h of exposure to IL-1β.
In vivo study, as described above, we performed surgical DMM in mice. Gigantol was dissolved in 0.1% DMSO. Then, the gigantol treatment groups were orally treated with gigantol (25 mg/kg) every day for 8 successive weeks. The DMM alone groups were treated with an equal volume of 0.1% DMSO (physiological saline). Cartilage tissue samples were collected after eight weeks post-surgery for iconographic as well as histological evaluations.
2.5. Cell viability assay
Gigantol cytotoxicity on chondrocyts was assessed by cell counting kit-8 (CCK-8; Dojindo Co, Kumamoto, Japan). First-passage chondrocytes were cultivated in 96-well plates (50000 cells/cm2), incubated for 24 h and treated for 24 and 48 h with varying gigantol doses (0, 10, 20, 40, 60 or 100 µM). At indicated timepoints, cells were washed using phosphate buffered saline (PBS), DMEM/ F12 (100 µl) containing the CCK-8 solution (10 µl) was supplemented to every well followed by 2 h of incubation at 37°C. Absorbance was spectrophotometrically (Thermofisher) read at 450 nm. Assays were done five times.
2.6. NO, PGE2, TNF-α, IL-6 measurements
Assessment of cell culture medium NO levels was done by the Griess reagent31 while cell culture supernatant collagen II, TNF-α, PGE2, IL-6, MMP13, aggrecan, and ADAMTS5 levels were assessed using ELISA kits (R&D Systems, Minneapolis, MN, United States). All assays were conducted five times.
2.7. Real-time PCR
Extraction of total RNA from chondrocytes that had been exposed to 10 ng/ml IL-1β and gigantol was done using the TRIzol reagent (Invitrogen). Total RNA (1 µg) was utilized in cDNA synthesis (MBI Fermentas, Germany). Then, 10 µl of the reaction volume (0.25 µl of each primer, 2 × SYBR Master Mix (5 µl), and dilute cDNA (4.5 µl)) was used for quantitative real-time PCR (qPCR) analyses on a CFX96 Real-Time PCR System (Bio-Rad Laboratories, CA, United States). PCR parameters were: 10 min at 95°C, 40 cycles for 15 s at 95°C and 1 min at 60°C. The obtained cycle threshold (Ct) levels of target mRNA were normalized to GAPDH values. Determination of relative mRNA expression levels for every target gene was done by the 2-ΔΔCt method. The NCBI Primer-Blast Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) was used for designing IL-6, iNOS, COX-2, and TNF-α primers, whose sequences were: COX-2 (F) 5′-GAGAGATGTATCCTCCCACAGTCA-3′, (R) 5′-GACCAGGCACCAGACCAAAG-3′; IL-6, (F) 5′-GACAGCCACTCACCTCTTCA-3′, (R) 5′-TTCACCAGGCAAGTCTCCTC-3′; iNOS (F) 5′-CCTTACGAGGCGAAGAAGGACAG-3′, (R) 5′-CAGTTTGAGAGAGGAGGCTCCG-3′; TNF-α (F) 5′-GTCAGATCATCTTCTCGA ACC-3′,(R) 5′-CAGATAGATGGGCTCATACC-3′.
2.8. Western blot analysis
The RIPA lysis buffer and Phenylmethanesulfonyl fluoride (PMSF; 1 mM) were used for proteins extraction from chondrocytes. For 10 min, lysates were placed on ice, centrifuged for 15 min at 12000 rpm, 4°C, after which protein levels were assessed by the BCA protein assay kit (Beyotime). Then, proteins separation was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 40 ng of it transferred to polyvinylidene difluoride membranes (PVDF), blocked at room temperature (RT) for 2 h using non-fat milk (5%), and incubated overnight in the presence of primary antibodies for COX-2 (1:1000), IκB-α (1:1000), iNOS (1:1000), PI3K (1:1000), p65(1:1000), P-PI3K (1:1000), AKT (1:1000), Lamin-B (1:5000), GADPH (1:5000) and P-AKT (1:1000) at 4°C. This was followed by 2 h of incubation at RT with respective secondary antibodies. They were washed 3 times using TBST. Blot visualization was conducted using the electrochemiluminescence plus reagent (Invitrogen). The quantification of blot intensities was done using the image Lab 3.0 software (Bio-Rad).
To stain collagen II in vitro, cells were seeded in six-well plates, treated with either IL-1β (10 ng/ml) alone or with gigantol (40 µM) for 24 h followed by overnight incubation in serum-free medium. To stain p65, cells were exposed to gigantol and IL-1β for 2 h, rinsed thrice using PBS, fixed in paraformaldehyde (4%), and permeabilized using PBS-dissolved Triton X-100 (0.1%) for 15 min. Cell blocking was done at 37°C for 1 h using bovine serum albumin (5%), rinsed using PBS followed by incubation in the presence of primary antibodies for p65 (1:200) and collagen II (1:200) at 4°C overnight. Glass plates were washed after which chondrocytes were incubated for 1 h in the presence of secondary antibodies (1:400) at RT and DAPI-stained for 5 min. Samples were evaluated by fluorescence microscopy (Olympus Inc., Tokyo, Japan). Assessment of fluorescence intensities was done using the Image J software 2.1 (Bethesda, MD, United States).
2.10. X-ray imaging method
At 8 weeks post-surgery, mice underwent X-ray imaging to evaluate osteophyte formation, joint space as well as alterations in cartilage surface calcification. Imaging was done by a digitized X-ray machine (Kubtec Model XPERT.8; KUB Technologies Inc) whose settings were: 160µA and 50Kv.
2.11. Histopathologic analysis
Fast Green/Safranin-O staining was performed for slides for each joint. Morphologic changes in the chondrocytes as well as in surrounding tissues were microscopically observed and imaged. The OARSI scoring system was used to score medial femoral condyle as well as medial tibial plateau to assess destruction for the articular cartilage.32 Each group had a total of 15 mice.
Sections were deparaffinized, rehydrated and incubated overnight in the presence of primary antibodies for p-AKT (1: 200) and p-PI3K (1: 200) at 4°C. Sections were washed, incubated for 2 h at RT with secondary antibodies. After hematoxylin and DAB (Zsbio, China) staining, positive cells showed a brownish-yellow color gradient in the nucleus or cytoplasm. The abundance (percentage) of cells that were positively stained cells in the articular surface was determined. Histological assessment was done using 15 mice per group.
2.13. Statistical analysis
Data are shown as mean ± S.D. SPSS ver. 20.0 was used for statistical analyses. Comparisons of means among groups was conducted by one-way ANOVA followed by the Tukey’s post hoc test. Comparisons of non-parametric data were done by Kruskal–Wallis H test. Significance was determined at p ≤ 0.05.