healthmedicinet

Animal models of OA

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.

Histological assessments

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 ?m) 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).

Immunohistochemical analysis

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 % H
₂
O
₂
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.

Isolation and culture of mouse articular chondrocytes

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.

Transfection of small interfering RNA into mouse articular chondrocytes

Articular chondrocytes from wild-type mice and Bach1
^-/-
mice were seeded at 5?×?10
⁴
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-1? (1 ng/ml;
PeproTech, Rocky Hill, NJ, USA) for an additional 24 h.

Quantitative real-time polymerase chain reaction (PCR)

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 ??Ct values, and results were expressed
as 2-??Ct.

DNA microarray analysis

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.

Immunoblotting assay

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).

Apoptosis assay

Articular chondrocytes were seeded at 1.5?×?10
⁴
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).

Statistical analysis

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 (**).