Bach1 deficiency reduces severity of osteoarthritis through upregulation of heme oxygenase-1

Bach1^-/- mice on C57BL/6 background were described previously [12]. Only male mice were used in this study. All animal experiments were performed according to protocols approved by Hiroshima University Animal care and Use Committee. Knee joints were harvested at 6 months (n = 7), 12 months (n = 11), and 22 months (n = 14) to monitor spontaneous age-related OA. Experimental OA was induced in 10 week-old, wild-type mice (n = 13) and Bach1^-/- mice (n = 11) by transection of the medial meniscotibial ligament (MMTL) and the medial collateral ligament (MCL) in the right knees [17]. Mice were sacrificed 8 weeks after surgery, and the knee joints were collected for histological analysis.

All knee joints were embedded intact in paraffin after fixation in 4 % Paraformaldehyde Phosphate Buffer Solution and decalcification in K-CX (FALMA, Tokyo, Japan). Knee joints sectioned (4.5 μm) in the sagittal plane through the central weight-bearing region of the medial and lateral femorotibial joint. The sections were stained with Safranin O/fast green and at least two different sections per sample were analyzed microscopically. In this study, we applied multiple separate scoring systems for articular cartilage, meniscus, synovitis, osteophyte formation and subchondral bone thickening. Osteoarthritic damage of articular cartilage was scored using a modified Mankin system [18, 19]. Meniscus degradation was evaluated using a scoring system [20] which included the following 6 criteria, meniscus integrity (0 = smooth surface, 1 = irregularity of superficial layer or slight fibrillation, 2 = moderate fibrillation, 3 = severe fraying, tear or disruption), collagen structure (0 = normal, 1 = slight disturbance, 2 = moderate disturbance, 3 = severe disturbance or mucoid substances), cellular abnormalities (0 = normal, 1 = hypercellularity, 2 = cloning tendency, 3 = hypocellularity), stainability of safranin O staining (0 = normal, 1 = slight reduction, 2 = moderate reduction, 3 = severe reduction), calcification and cyst formation (0 = normal, 1 = slight, 2 = moderate, 3 = severe). The maximum possible score per meniscus was 18. Osteophyte formation and subchondral bone thickening were scored on a scale of 0–3, where 0 = normal, 1 = mild, 2 = moderate and 3 = severe changes, and the average scores for tibia and femur were recorded. The severity of synovitis was evaluated according to a previously described histopathological classification system [21]. The parameters of synovitis included hyperplasia/enlargement of synovial lining layer, degree of inflammatory cell infiltration and activation of resident cells/synovial stroma. All parameters were graded from 0 (absent), 1 (slight), 2 (moderate) to 3 (strongly positive) and summarized ranging from 0–9, where 0–1 corresponds to no synovitis (grade = 0), 2–3 to a slight synovitis (grade 1), 4–6 to moderate synovitis (grade 2), and 7–9 to severe synovitis (grade 3).

Knee joint sections were immunostained with anti-HO-1 antibody (1:75, ab52947, Abcam, Austin, TX, USA) using Vectastain ABC-AP alkaline phosphatase (Vector Laboratories, Burlingame, CA, USA) as described previously [22]. For anti-microtubule-associated protein 1 light chain 3 (LC3) antibody (1:100, AP1801a, ABGENT, San Diego, CA, USA), anti-manganese superoxide dismutase (MnSOD) antibody (1:100, SPC-117, StressMarq, Victoria, BC, Canada), sections in Immunoactive pH 6.0 (Matsunami Glass, Osaka, Japan) were heated in a microwave oven and kept at 85 °C for 1.5 minutes. Slides were cooled for 20 minutes at room temperature after antigen unmasking. After washing with PBS, 3 % H₂O₂ treated for 10 minutes, sections were blocked with 10 % serum for 20 minutes at room temperature. Antibodies were applied and incubated overnight at 4 °C. After washing with PBS, sections were incubated with biotinylated secondary antibody for 30 minutes at room temperature and then incubated using the peroxidase based Elite ABC system (Vector Laboratories) for 30 minutes. Slides were washed, and sections were incubated with 3,3 -diaminobenzidine (DAB) substrate.

Primary articular chondrocytes were dissected from the femoral heads of 1-month-old Bach1^-/- and wild-type mice by digestion with 0.3 % collagenase Type 2 (Worthington, Lakewood, NJ, USA) in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) for 2 h. Isolated chondrocytes were cultured in DMEM with 10 % fetal bovine serum.

Articular chondrocytes from wild-type mice and Bach1^-/- mice were seeded at 5 × 10⁴ cells/well on a 24-well plate and were transfected with small interfering RNA (siRNA) for HO-1 using Lipofectamine RNAiMax Reagent (Invitrogen, Carlsbad, CA, USA). The sequences of the siHO-1 were: (sense) 5′- CAACAGUGGCAGUGGGAAUTT -3′ and (antisense) 5′- AUUCCCACUGCCACUGUUGTT-3′ (Hokkaido System Sciences, Hokkaido, Japan). Control siRNAs were also prepared for the control group (siRNA negative control; siNega #1, Invitrogen). At 24 h after transfection, articular chondrocytes were treated with IL-1β (1 ng/ml; PeproTech, Rocky Hill, NJ, USA) for an additional 24 h.

Total RNA was extracted from chondrocytes using TRIzol Reagent (Invitrogen). Complementary DNA (cDNA) was synthesized using 500 ng of total RNA with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). A real-time PCR assay was performed using TaqMan Gene Expression Assay probes (Applied Biosystems, Foster City, CA, USA) to amplify the Bach1 (Mm01344527), Hmox-1 (Mm00516005), Col2a1 (Mm01309565_m1), Acan (Mm00545807), Mmp-13 (Mm01168713), Adamts-5 (Mm01344182_m1), and Sod2 (Mm01313000_m1), and Gapdh (Mm99999915_g1) was used as the internal control to normalize the sample differences. Relative expression was calculated using the ΔΔCt values, and results were expressed as 2-ΔΔCt.

DNA microarray (TORAY, Tokyo, Japan, 3D-Gene, Mouse Oligo chip 24 k) analysis was performed using total RNA from chondrocytes from wild-type mice and Bach1^-/- mice.

For immunoblotting, proteins were extracted from cultured chondrocytes using M-PER^TM protein extraction reagent including protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Anti-HO-1 antibody (diluted 1:2000), anti-LC3 antibody (diluted 1:1000), and anti-MnSOD antibody (diluted 1:1000) were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (sc-2030; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-mouse IgG antibody (sc-2005; Santa Cruz Biotechnology) were used as secondary antibody. The signal was detected with chemiluminescence of enhanced immuno-enhancer (Wako, Osaka, Japan) using the ImageQuant LAS 4000 system (GE Healthcare, Uppsala, Sweden).

Articular chondrocytes were seeded at 1.5 × 10⁴ cells/well on 96-well plates and transfected with siHO-1 or siRNA negative control. Articular chondrocytes were treated with tert-butyl hydroperoxide (t-BHP) (200 uM; Wako) for 5 h at 24 h after transfection. Apoptotic chondrocytes were quantitated by counting the numbers of cell nuclei stained with Cell Event Caspase-3/7 Green Detection Reagent^TM and NucBlue Live cell stain ReadyProbes^TM (Invitrogen) in three random fields on each duplicate well at a magnification of × 10 under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).

The data were analyzed using the Mann–Whitney U test, Steel or Steel–Dwass to determine statistical differences. Differences were considered statistically significant at P <0.05 (*) and P <0.01 (**).