The Protective Effects of Chronic Intermittent Hypobaric Hypoxia Against Osteoarthritis in Rats - Role of Nitric Oxide


 The study explored the effect of chronic intermittent hypobaric hypoxia (CIHH) on osteoarthritis (OA). CIHH conditioning was realized by exposing rats to hypobaric hypoxia environment mimicking 5,000 m high-altitude (PB=404 mmHg, PO2=84 mmHg) 6 h per day, once daily for 28 days. OA model was induced by surgically-induced medial meniscal tear. Male Wistar rats were randomly assigned 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 expression iNOS, nitric oxide (NO) content levels in the joint fluid were measured at 1, 2, 3 weeks after the OA modelling. Results revealed that OA modelling induced cartilage degeneration, up-regulated iNOS expression, increased joint fluid NO content. CIHH preconditioning and postconditioning reduced cartilage degeneration, prevented the NO production. Inhibitor groups showed alleviated joint degeneration than control group, but not as effective as CIHH condition. These results suggest that both CIHH preconditioning and postconditioning plays a protective role on OA, one of the mechanism was inhibiting the overexpression of iNOS and NO production.


Introduction
Osteoarthritis OA is a degenerative joint disease characterized by articular cartilage destruction, synovial membrane in ammation, and subchondral bone remodeling [1]. OA is the leading cause of lower extremity disability amongst older adults the estimated lifetime risk for knee OA is approximately 40% in men and 47% in women [2]. As life expectancy increases, the prevalence of osteoarthritis is projected to increase further, resulting in a more signi cant healthcare burden.
Chronic Intermittent hypobaric hypoxia (CIHH) has been proved to be bene cial to the cardiovascular system [12,13], central nervous system [14], and the immune system [15]. It was reported that CIHH helps protect against reperfusion injuries [16]. Furthermore, CIHH has been widely used in sports training to enhance the organs and tissue's tolerance to anoxia [17]. Previous studies have shown that intermittent hypobaric hypoxia preconditioning has a protective effect against rheumatoid arthritis [18], as osteoarthritis has similar pathological changes with rheumatoid arthritis, We hypothesize that hypotension has a protective effect on osteoarthritis.
NO plays an essential role in the adaption process of CIHH. For example, CIHH preconditioning protect brain ischemia by inhibiting the overexpression of NO [19]. Exposure of rats to acute hypoxia improved papillary muscle's tolerance to hypoxia by increasing cytosolic inducible nitric oxide synthase expression and NO production in left ventricle [20]. Kanika and Geetha prove that CIHH tolerance is mediated by nitric oxide pathway [21]. NO has long been thought to be related with OA. A high NO level has been shown in patients' serum and cartilage with arthritis than in normal cases [22,23]. NO is reported as a predominant mediator in the progression of OA and chondrocyte apoptosis [24,25].
Taken together, the Nitrergic system plays an important role both in the pathological process of osteoarthritis and the physiological response to hypobaric hypoxia [19,20]. This study aims to verify the protective effect of CIHH on OA and assess the role of the NO pathway in this process.

Materials And Methods
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 xed 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 in ow 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 nal 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 rst, and then CIHH induction. The control group receive OA surgery only. The inhibitor group received OA surgery, after the surgery rats received iNOS NGnitro-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 sacri ced through atlantoaxial dislocation at 1, 2 and 3 weeks after OA surgery modelling. For each group, the joint uid of rats was obtained before the execution. 10 rats of each group were killed and para n 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 xed in 4% paraformaldehyde for 48 h, decalci ed with 10% ethylenediaminetetraacetic acid (EDTA) solution for 15 days, and then embedded in para n. 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 uid 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 uid and serum were frozen at -80°C for biochemical determination. The concentration of joint uid and serum NO level was measured using the Griess reaction, the Griess reaction was rst 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 sulfatepolyacrylamide gel electrophoresis (SDS PAGE) loading buffer and were subsequently transferred to polyvinylidene di uoride (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 Scienti c, 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 signi cant.

Results
The effect of CIHH on articular cartilage in rats OA model established successfully. After 1 week of OA modeling, Mild cartilage degeneration of control group was detected characterized by super cial cellular clusters. After 2 week of OA modeling, Moderate degeneration of cartilage of control group was detected characterized by super cial cellular clusters and the less cartilage matrix staining. After 3 weeks of OA modelling, cartilage degeneration of control group extends into the deep zone and ssures into calci ed cartilage layer appear. After 1 2 and 3 weeks of OA modeling, markin scores of each group were recorded ( Table 1). Rats received both precondition and postcondition got better markin score than control group. Markin score of inhibitor group was lower than control group, but higher than preconditioning and postconditioning group (Fig. 1-5).
The effect of CIHH on joint lavage uid NO As shown in Table 2 and Fig. 6-8, at all 1, 2, and 3 week time point, joint lavage uid NO of preconditioning group, postconditioning group and inhibitor group were lower than control group. The joint lavage uid NO in both preconditioning group and postconditioning group rats were signi cantly higher than the inhibitor group.
The effect of CIHH on the expression of iNOS In order to detect the effect of CIHH on NO, western blotting was used to analyze the expression of iNOS, as shown in Table 3 and Fig. 9-11. At each time point, there were signi cant differences between the ve groups (P < 0.001). Further pairwise comparison showed CIHH downregulated the expression of iNOS in cartilage, while l-name The inhibitor had no effect on iNOS in cartilage compared with control group.

Discussion
Animal experiments show that CIHH tolerance is mediated by the heat shock response and nitric oxide pathway [21]. Many studies have shown that hypobaric hypoxia reduces NO concentration in vivo. Since oxygen is the substrate for NO production by the L-arginine pathway, the cell oxygen concentration is considered to regulate the enzymatic reaction of NO [33]. Hypoxia limits the endogenous production of no, considered an essential mechanism of hypobaric hypoxia in regulating NO concentration in vivo. However, many studies have shown that the response of NO to CIHH is different in different organs, which indicates that the effect of hypobaric hypoxia environment on NO concentration in vivo is more complicated.
CIHH reduce the concentration of NO in brain tissue by inhibiting the overexpression of Neuronal nitric oxide synthase (nNOS and iNOS) induced by cerebral ischemia in rats to improve brain tissue tolerance to ischemia [19]. This is the rst study to investigate the protective effect of CIHH on osteoarthritis. We found that both CIHH preconditioning and postconditioning signi cantly reduce the pathological damage of osteoarthritis. Rats received CIHH conduction got better markin score, and CIHH conduction decrease iNOS and NO production. NO is an essential pathway for hypoxic tolerance [21], and NO has long been considered as a pathogenic factor of osteoarthritis. The present study proved that the protective effect of CIHH on OA is related to the NO pathway, and this is the rst study to determine the impact of CIHH on NO of the cartilage.
CIHH alleviated the pathological damage of osteoarthritis and downregulated the NO pathway. To determine if there a causal relationship between them, we added the inhibitor group. Rats in the inhibitor group got better markin scores than the control group, but worse than the CIHH group. It proved No is involved in the protective effect of CIHH on osteoarthritis, but there must other pathways. Follow up studies are needed to determine the mechanism of CIHH on OA.
In conclusion, NO is an essential molecule in adapting to CIHH, and it also participates in the pathological process of OA. We believe that NO can protect osteoarthritis by regulating the production of no and the release of in ammatory factors in articular cartilage. This study proved that CIHH has protective effect on OA. One of the mechanisms is the NO pathway. Other mechanisms are waiting for us to explore, and it's meaningful for clinical OA treatment. At all 1, 2, and 3 week time point, the markin score of preconditioning group, postconditioning group and inhibitor group were lower than control group, the difference was signi cant (P 0.05). the markin score of preconditioning group and postconditioning group were lower than inhibitor group, the difference was signi cant (P 0.05). At all 1, 2, and 3 week time point, there were signi cant differences in joint lavage uid NO among the ve groups (P < 0.05), The order was as follows: inhibitor group blank control group preconditioning group postconditioning group control group. At all 1, 2, and 3 week time point, iNOS of both preconditioning group and postconditioning group were lower than control group and inhibitor group, the difference was signi cant (P 0.05). There was no difference between inhibitor group and control group.