Surgical method of OA model
The surgical method of OA modelling was previously described [26, 27]. Rats were anesthetized via intraperitoneal injections of pentobarbital sodium (50 mg/kg ip) and fixed in supine position. After shaving the knee joint, the skin was disinfected with iodine. A skin incision was made over the medial aspect of the right knee, the medial collateral ligament exposed by blunt dissection, and then transected. A full-thickness cut was made through the medial meniscus to simulate a complete tear. The skin and subcutis were closed with 4-0 Vicryl suture by using a subcuticular pattern. The rats were injected with penicillin for 3 days (80000 U / 100g, once a day) after the surgery.
Induction of CIHH
Animals were placed in a hypobaric chamber. The air pressure inside was controlled using a vacuum pump and an adjustable inflow valve. The chamber was also provided with a manometer to check the experimental altitude during the process. Hypoxia was induced by down-regulating the environmental pressure to a final barometric pressure of 404 mm Hg,The temperature of the chamber was kept at 28 °C. These conditions simulate an altitude of 5000 m. Animals were placed in the chamber 5 h/day (9 AM–2 PM), 5 days/week for 5 weeks.
Animals Grouping and Management
Healthy male Sprague–Dawley rats (weighing 180–190g) were provided by Hebei Province Laboratory Animal Center (Shijiazhuang, China), and housed in a temperature and light-controlled room (24 ± 1°C, 12 h light/dark cycle), with access to food and water. All experiments were conducted accoeding to the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). All animal procedure were approved by the ethics committee of The Third Hospital of Hebei Medical University (Z2018-015-1). 100 rats were randomly divided into 5 groups: preconditioning group (CIHH + OA), postconditioning group (OA + CIHH), control group, inhibitor group [OA + inducible nitric oxide synthase (iNOS) inhibitor], blank control group. The preconditioning group received CIHH induction before OA surgery. The postconditioning group received OA surgery first, and then CIHH induction. The control group receive OA surgery only. The inhibitor group received OA surgery, after the surgery rats received iNOS NG-nitro-L-arginine methyl ester (MedChemExpress. America. New Jersey) by intraperitoneal injection 50 mg/kg per day for 7 days. Sham group receive the same incision as surgery modelling of OA, but with medial collateral ligament and medial meniscus intact.
Rats were sacrificed through atlantoaxial dislocation at 1, 2 and 3 weeks after OA surgery modelling. For each group, the joint fluid of rats was obtained before the execution. 10 rats of each group were killed and paraffin sections of the tibial plateau were prepared for OA evaluation by Hematoxylin and Eosin (H&E) staining and safranin O/fast green staining. The other 10 rats were killed and the tibial plateau cartilage was prepared for detecting inducible nitric oxide synthase (iNOS) expression through western blot.
Histology
After the mice were executed, the knee joint were fixed in 4% paraformaldehyde for 48 h, decalcified with 10% ethylenediaminetetraacetic acid (EDTA) solution for 15 days, and then embedded in paraffin. The samples were cut to a 5 µm thickness sagittal sections from the joint medial compartment using a microtome (Leica biosystems, USA). Afterwards, the sections were stained with safranin O/fast green or hematoxylin-eosin. The markin scoring [28] was used to evaluate the severity of the OA changes. Scoring was done by two independent investigators who are blinded to the grouping.
NO detection
1, 2, 3 weeks after OA modelling, Normal saline (1 mL) was injected into the knee joint cavity of all the studied rats. The fluid from the joints was then collected into a test tube. The rats were killed and blood samples were collected. After centrifugation we collected the serum, the joint fluid and serum were frozen at -80°C for biochemical determination. The concentration of joint fluid and serum NO level was measured using the Griess reaction, the Griess reaction was first presented in 1879 as a colorimetric test for nitrite detection [29, 30, 31, 32]. Serum and an equal volume of Griess reagent were thoroughly mixed. The mixture was incubated for 10 min at room temperature and absorbance was recorded at 540 nm. Blank and standards were also run in parallel.
Western blot
Rats were decapitated in the indicated time as points previously mentioned and the articular cartilage of the tibial plateau was rapidly isolated, placed on ice, the tissue blocks were washed with cold phosphate buffer saline for 2-3 times to remove the blood stain, and cut into small pieces and placed in the homogenization tube. Add 2 magnetic beads, add 10 times of tissue volume lysis buffer, add protease inhibitor within minutes before use. Select the procedure of complete homogenization, 100s, and cool down intermittently. After homogenization, take out the sample tube, ice bath for 30min, shake every 5min to ensure complete tissue lysis. After centrifugation at 12000 rpm for 10 min, the supernatant was collected, which was the total protein solution. Samples were loaded onto 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) loading buffer and were subsequently transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with 5% skim milk for 1 hour at room temperature, followed by incubation with the primary antibodies and Glyceraldehyde triphosphate dehydrogenase (GAPDH) overnight at 4 ℃. Primary antibodies are produced by Hangzhou Xianzhi biology limited liability company, after washing twice with tris buffered saline tween (TBST), the membranes were incubated with secondary antibodies (BOSTER biological technology. Wuhan China) for 1 hour at room temperature. Protein bands were detected using an electrochemiluminescence (ECL) detection kit (Thermo Scientific, 34077) and imaged.
Statistical analysis
The measurement data in accordance with normal distribution was expressed as Mean ± standard deviation. Monofactoria analysis of variance was used for comparison between groups. When there was statistical difference, Student-Newman-Keuls (SNK) method was used for pairwise comparison. The measurement data that did not conform to normal distribution was expressed as Median and quartile. Rank sum test was used for comparison between groups. Bonferroni method was used for pairwise comparison when there was statistical difference, All data were analyzed by spss25.0 statistical software. Probability values of P < 0.05 were considered statistically significant.