The Antioxidant tempol delays the MIA-induced osteoarthritis progression in rats via modulation of signaling pathways involving TGF-β1/SMAD3/NOX4 axis


 Osteoarthritis (OA) is a complex disease characterized by structural, functional, and metabolic deteriorations of the whole joint and periarticular tissues. In the current study, we aimed to investigate the possible effects of tempol on knee OA induced by the chemical chondrotoxic monosodium iodoacetate (MIA) which closely mimics both the pain and structural changes associated with human OA. Rats were administrated oral tempol (100 mg/kg) one week post-MIA injection (3 mg/ 50 μL saline) at the right knee joints for 21 consecutive days. Tempol improved motor performance and debilitated the MIA-related radiological and histological alterations. Besides, it subsided the knee joint swelling. Tempol decreased the cartilage degradation-related biomarkers as matrix metalloproteinase-13, cartilage oligomeric matrix protein, and fibulin-3. The superoxide dismutase mimetic effect of tempol was accompanied by decreased NADPH oxidase 4 (NOX4), inflammatory mediators, nuclear factor kappa-B (NF-κB), over-released insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1). Tempol decreased the expression of chemotactic cytokine ligand 2 (CCL2), dickkopf‑related protein-1 (DKK-1), and protein kinase C (PKC). On the molecular level, tempol reduced the phosphorylated protein levels of p38 mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), and small mother against decapentaplegic 3 homologs (SMAD3). These findings suggest the promising role of tempol in ameliorating MIA-induced knee OA in rats via collateral suppression of the catabolic signaling cascades including TGF-β1/SMAD3/NOX4, NOX4/p38MAPK/NF-κB, and PI3K/Akt/NF-κB and therefore modulation of oxidative stress, catabolic inflammatory cascades, chondrocyte metabolic homeostasis, autophagy, and cell survival.


Introduction
Osteoarthritis (OA) is a chronic degenerative disease that is characterized by articular cartilage degeneration, subchondral bone sclerosis, and osteophyte formation. OA can arise in different sites, mainly affecting the knees, hips, hands, and spine 1,2 . The chief clinical symptoms include chronic pain, joint instability, stiffness, and radiographic alterations 3 .
The intra-articular injection of monosodium iodoacetate (MIA) is considered a convenient model for knee OA 4 . MIA directly affects the metabolic balance activity of chondrocytes by disrupting glycolysis as a result of inhibiting the activity of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzyme, which eventually leads to cartilage degeneration 5 . MIA is a fast-progressing chemically induced animal model of OA which is mainly used to observe and evaluate pain behavior as it causes loss of articular cartilage with structural lesions and pain, that resembles human OA 6,7 .
OA pathogenesis involves activation of signal pathways, regulators, and effectors such as mitogenactivated protein kinases (MAPKs), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (Akt) 8 . In ammatory mediators and reactive oxygen species (ROS) are released in response to OA injury, which further leads to exacerbation of cartilage tissue breakdown via up-regulation of the catalytic degrading matrix metalloproteinases (MMPs) 9 . Oxidative stress is elicited by an increased ROS release in response to OA injury. This results in mitochondrial membrane damage, which leads to the release of caspases and pro-apoptotic molecules 7 . Also, ROS mediate the effect of pro-in ammatory cytokines; tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6), which prompt the synthesis of MMPs, in ammatory cytokines, and chemokines 10 . This in consequence leads to disturbance in cartilage homeostasis and promotes catabolism via induction of cell death 11,12 . In January 2020, the American College of Rheumatology (ACR) and the Arthritis Foundation (AF) have been updated the treatment guidelines of knee OA. The treatment options are categorized into: strongly recommended options which include exercise, weight loss, oral and topical nonsteroidal antiin ammatory drugs (NSAIDs), and intra-articular corticosteroids. Besides, the conditionally recommended treatment options include duloxetine, cognitive behavioral therapy, and topical capsaicin. Moreover, the guidelines recommended against the use of glucosamine and chondroitin, hyaluronic acid injections, bisphosphonates, hydroxychloroquine, methotrexate, platelet-rich plasma injections, stem cell injections, tumor necrosis factor (TNF) inhibitors, and interleukin-1 receptor antagonists 13 .
Tempol (4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a membrane-permeable radical scavenger that possesses an anti-oxidant e cacy which is emphasized through its ability to facilitate the metabolism of a wide range of ROS and reactive nitrogen species (RNS). Tempol reaction with superoxide anion (O 2˙ ) forms hydrogen peroxides (H 2 O 2 ) which accounts for its superoxide dismutase (SOD) mimetic action.
Besides, tempol prevents the generation of hydroxyl radical (·OH) and hydrogen peroxide (H 2 O 2 ) by Fenton reaction which is responsible for its catalase-like action. Tempol also improves nitric oxide (NO) bioavailability and catalytically removes the highly reactive peroxynitrite (ONOO ) species. Accordingly, tempol is considered as a general-purpose redox cycling agent rather than a speci c SOD mimetic agent 14 . Currently, tempol is subjected to many clinical trials sponsored by Matrix Biomed, Inc. (Los Angeles, CA, USA) such as a phase II clinical trial of treatment of radiation and cisplatin-induced toxicities with tempol, and a clinical trial on a single-patient compassionate use of tempol for the treatment of prostate cancer.
Since the pharmacologic treatments have several limitations in the management of OA, researchers have been motivated to nd alternative therapeutic agents. To the best of our knowledge, this is the rst study to focus on the In-Vivo therapeutic effects and mechanisms of tempol on the knee OA model in rats. In 2000, Cuzzocrea et al., 15 demonstrated the bene cial effects of tempol on collagen-induced arthritis rodent model as an intracellular radical scavenger which may be useful in the treatment of conditions associated with local or systemic in ammation.
Based on these considerations, the present experimental study aims to explicate the therapeutic outcomes of tempol on the different pathological aspects of MIA-induced knee OA, including; a] functional evaluation of motor balance and pain, b] structural alterations (using physical assessment of knee joint edema, X-ray imaging, and histopathology), and c] knee joint degradation-related. In addition, we aimed to investigate the potential mechanisms and signaling pathways of tempol in ameliorating MIA-induced OA in rats involving; a] oxidative phosphorylation status, b] oxidative stress biomarkers, c] in ammatory and pain mediators, d] OA-overexpressed growth, e] intracellular signaling proteins (PKC, SMAD3, p38MAPK, PI3K, and Akt), and f] autophagy-related protein (beclin-1).

Ethical statement
All procedures for handling, use, and euthanasia of animals were reviewed and approved by the Ethical Committee for Animal Experimentation of the Faculty of Pharmacy, Cairo University, with the permit number PT 2389, and were performed following the guides for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8 th edition, NIH Publication, 2011).

Experimental animals
A total of 60 adult male Wistar albino rats (180-200 g; 8-10 weeks old) were obtained from the Animal House Colony at the National Research Centre (NRC, Giza, Egypt). Rats were randomly assigned to four groups (4-5/cage) in a room with a controlled temperature (22°C±1) on a 12/12 light-dark cycle. All animals were provided with standard rat chow with free access to water. Animals were adapted to the same laboratory environment for 2 weeks before the experimentation. Adequate non-pharmacological measures were taken to minimize animal pain or discomfort according to the National Centre for the Replacement, Re nement and Reduction of Animals in Research (NC3Rs) strategy and Animal Research: Reporting of In-vivo Experiments (ARRIVE) guidelines.

Induction of Osteoarthritis
Single unilateral intra-articular injection of monosodium iodoacetate (MIA) was used to induce knee osteoarthritis in rats. It was purchased from Sigma-Aldrich Chemical Co. (Lot# SLBZ7569, St. Louis, USA). On day zero, rats were anesthetized with 4% iso urane inhalation. Then MIA (3 mg dissolved in 50 μL of sterile saline) was injected through the patellar ligament into the joint space of the right knee using a sterile 100-U insulin syringe 11,12,[16][17][18][19] . The sham groups were injected with 50 μL physiologic saline in their right knees instead of MIA 20 .

Experimental design
The experimental protocol is summarized in (Figure. 1). Sixty adult male rats were randomly allocated into four groups (N=15/group). In the rst sham group, the animals were subjected to a single intraarticular injection of normal saline in their right knee joints on day 0 and then administered a daily oral dose of the vehicle (1 ml of distilled water), one week after saline injection for 21 days. In the second group (sham+tempol), rats were exposed to intra-articular injection of normal saline in their right knee joints on day 0 and then administered tempol (100 mg/kg/day) by oral gavage starting from the 7 th day of the experiment for 21 consecutive days. In the third MIA-induced OA group, OA was induced by a single unilateral intra-articular injection of MIA on day 0 in a dose of 3 mg dissolved in 50 μL saline. In the fourth MIA+tempol group, osteoarthritic rats were administered tempol (Sigma-Aldrich Chemical Co., St. Louis, USA) starting from the 7 th day of the experiment in a dose of (100 mg/kg/day) by oral gavage for 21 consecutive days. Tempol was freshly prepared and daily dissolved in pure distilled water in a concentration of 0.4 g/20 ml. The choice of tempol dose was on a pilot trial guided by previously published studies to gure out the appropriate method of administration [21][22][23][24] (Supplementary le 1). In this study, we did not compare with a pharmacological control since the existing approved pharmacological treatments for OA are limited for the relief of OA symptoms (e.g. NSAIDs and corticosteroids), and there is a lack of licensed disease-modifying osteoarthritis drugs (DMOADs) that could slow the narrowing of joint space and provide symptomatic relief.

Assessment of knee joint edema
The diameter (in mm) of the right knee of each rat in the studied groups (N=8/group) was measured using a calibrated digital caliper (SL-1112, INSIZE Co., USA), at the corresponding days 0, 1, 3, 10, 17, and 24 25 .
2.6. Evaluation of motor performance using accelerating rotarod test Motor stability, coordination, and pain were evaluated using an accelerating rotarod apparatus (Model 7750, Ugo Basile, Italy). Each rat was trained through 3 different sessions for three consecutive days (one session/day) just before the experimentation. Hence, rats were randomly placed on the rotating rod forcing them to walk continuously with increasing speed (from 4 to 40 rpm) for 5 min (300 s) 26 .
Throughout the experiment, rats were tested for their persistence on the accelerating rotarod according to the speci c schedule of the different groups on study days 1, 7, 14, 21, and 28. The duration that each rat was able to maintain on the rod was recorded as latency time to fall in seconds (N=8/group) 27,28 .

Radiographical examination
On day 28, rats were anesthetized by ketamine/xylazine (100/10 mg/kg, i.p.). Afterward, the rat's right knee was exed in the anterior to posterior position (AP view, angle 10°), and lateral position (angle 30°) with an appropriate focal length to ensure clear X-ray imaging 29 . The X-ray lms were captured by 50 mA mobile X-ray camera apparatus (Model F50-100II 50mA, Perlong Medical Equipment Co., China). All X-ray images were blindly examined by two investigators.

Quantitative real-time PCR (qRT-PCR) measurements for gene expression
The messenger RNA (mRNA) expression levels of CCL2, DKK-1, DDR2, bulin-3, and PKC were analyzed by quantitative real-time PCR. About 30 mg of knee joint tissue was homogenized in RNA lysis solution supplied by RNeasy mini kit (QIAGEN, Maryland, CA, USA) and centrifuged at 10,000×g for 10 min, the supernatant was used for RNA extraction according to the manufacturer's protocol. The concentrations of the isolated RNA were obtained using ultraviolet spectrophotometry, and RNA purity was assessed based on the A260/A280 absorption ratio. Then, RNA was reverse-transcribed into cDNA as described in the manufacturer's using High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time PCR was performed with a PCR mixture containing 1 µmol/l of each primer and SYBR Green Master Mix Applied Biosystems, Foster City, CA, USA) using StepOne™ PCR system (version 3.1, Applied Biosystems, Foster City, CA, USA). The sequences of the primers used are listed in (Table 1). All primer sets had a calculated annealing temperature of 60°C. Ampli cation conditions were: 95°C for 10 min, and then 40 cycles of denaturation for 15 s and annealing/extension at 60°C for 10 min. All values were normalized to the β-actin which was used as the endogenous control (reference gene). The relative expression of the target genes was obtained using the comparative threshold cycle C T (ΔΔC T ) method.
The relative expression was calculated from the 2 −ΔΔCT formula 31 .

Histopathological Examinations
Autopsy samples were taken from the rats' right knee joint of different groups (N=3/group) and xed in 10% neutral buffered formalin for 48 hr. Followed by decalci cation by using Cal-Ex™ II Fixative/Decalci er (Fisher Chemical™ Scienti c, USA) for 20 days. Joints were trimmed and sectioned at the mid-sagittal point then serial step sections were obtained and examined at different levels. Subsequently, samples were processed using serial dilutions of ethanol and cleared in xylene followed by in ltration and embedding in paraplast tissue embedding media. Tissue sections (5 μm thickness) were made by rotatory microtome and mounted on glass slides for hematoxylin and eosin (H&E) staining for general histological examination of joint samples, and also for histochemical assessment by alcian blue stain (AB; pH 2.5) for assessment of cartilage extracellular matrix and proteoglycans reactivity. Nuclear fast red was used as a standard counterstain for alcian blue staining. All guidelines for sample xation, processing, and staining were done according to Culling et al., 33 . Morphological assessment of tibiofemoral articular cartilage was conducted according to the modi ed Mankin scoring system 34,35 with 0 indicating normal cartilage and 13 indicating the maximal score of osteoarthritis.

Statistical analysis
Statistical comparisons between different means of the biochemical parameters were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Furthermore, knee joint diameter and rotarod latency time were tabulated and analyzed statistically using repeated measures ANOVA test for factors time and group, followed by the Bonferroni post-hoc test. Statistical analysis was performed using GraphPad Prism (version 5.0, GraphPad Software, Inc., San Diego, USA). All data points are presented as the means ± SD. A difference was considered to be statistically signi cant at P<0.05.
In the same context, MIA-OA rats exhibited a spike increase in the serum levels of CTX-II, and COMP by 15.0 and 4.4 folds, respectively, as compared to the sham group (P<0.001). On the other hand, the tempol post-treated group showed marked reductions in serum CTX-II (4.5 folds, P<0.001) and serum COMP (2.5 folds, P<0.001) relative to the MIA group (Fig. 2e&f).
3.2. Effect of tempol on mitochondrial complex IV oxidase and oxidative stress biomarkers MIA disrupted the knee joint activity of CcO (complex IV or the terminal enzyme of the respiratory chain of the mitochondria) by 39.2% (P<0.001) as compared to the sham group (Fig. 3a). Besides, SOD was analyzed calorimetrically and its activity was diminished in the diseased knee joints by MIA by 79.9% (P<0.001) as compared to the sham group (Fig. 3b). In contrast, NOX4 which constitutively produces H2O2 was signi cantly elevated in the MIA-induced OA group by four folds (P<0.001) compared to the sham group (Fig. 3c). However, tempol post-treatment increased CcO (53.3%) (P<0.001) and SOD (218.7%) (P<0.001) signi cantly, along with a noticeable decline in NOX4 by 52.0% (P<0.001) as relative to the MIA-OA group (Fig. 3a-c). Symptoms of the joint swelling were observed after the intra-articular injection of MIA especially at the rst 10 days of the experiment schedule, where, the diameter of the diseased right knee joints showed marked increases on day 1 (73.05%, P<0.001), day 3 (62.04%, P<0.001), and day 10 (41.38%, P<0.001) as compared to the sham group. In contrast, relief in joint swelling by (20.88%, P<0.001) was observed starting from day 10 of the experiment (the 4th day of treatment) compared to the MIA-OA group. On the other hand, tempol post-treated rats exhibited normal knee diameter measures at days 17 and 24, relative to sham groups.

Effect of tempol on rotarod performance test
The accelerating rotarod was used for the functional assessment of motor balance and pain as presented in (Fig. 4b). Progressive motor dysfunction was observed after MIA insult, where, MIA-OA rats displayed considerable reductions in the latency time to fall at the corresponding day 1 (1.98 folds, P<0.001), day 7 (2.02 folds, P<0.001), day 14 (2.36 folds, P<0.001), day 21 (3.43 folds, P<0.001), and day 28 (3.16 folds, P<0.001) when compared to the sham group. Similar ndings were exhibited by tempoltreated rats at days 1, 7, and 14 as related to the MIA-OA group. On the contrary, tempol post-treatment revealed pronounced improvements in the motor coordination through signi cant increases of latency time to fall at day 21 (2.55 folds, P<0.001), and day 28 (2.86 folds, P<0.001) as compared to the MIA-OA group.
3.6. Effect of tempol on DKK-1 and beclin-1 Elevated mRNA expression levels of DKK-1 (4.9 folds, P<0.001) were noticed in the MIA-induced OA group corresponding to the sham group, but conversely, the mRNA expression level of DKK-1 was reduced by 67.5% in the tempol-treated group as compared to MIA-OA ones (P<0.001) (Fig. 4g). On the other hand, the autophagy-related biomarker, beclin-1 tissue contents were decreased (70.9%, P<0.001) in the MIA-OA group as compared to the sham group, and in contrast, tempol post-treatment increased beclin-1 contents (28.9%, P<0.001) in the osteoarthritic rats (Fig. 4h).

Effect of tempol on growth factors and PKC expression
A metabolic shift has occurred following MIA injection toward catabolic cascade which is characterized by the increased production and activation of different anabolic and catabolic factors that act as mediators of cartilage loss sequence as shown in (Figure 5). This may be considered as a compensative mechanism from chondrocyte to counteract the MIA destructive effect, where signi cant increments in tissue TGF-β1 (3.3 folds, P<0.001), serum IGF-1 (8.3 folds, P<0.001), and mRNA expression levels of PKC (6.2 folds, P<0.001) were pronounced in MIA-OA group when compared to the sham group (Fig. 5a-c). On the contrary, these effects were mitigated by tempol post-treatment by reducing TGF-β1 (2.1 folds, P<0.001), and PKC (51.8%, P<0.001) as well as restoring serum IGF-1 to its normal value, relative to the MIA-induced OA group (Fig. 5a-c).

Effect of tempol on the radiographic changes
The experiment was conducted for X-ray evaluation on day 28. The X-ray images of the right knee joints of the sham groups (Fig. 6a&b) showed normal joint structure, normal joint spaces, normal radio-density, and normal surfaces of the femoral condyles and proximal tibia. On the other hand, the osteoarthritic changes of the MIA-induced group (Fig. 6c) revealed increased joint opacity corresponding to joint effusion and osteophytes formation. Besides, rough surfaces of the femoral condyles and proximal tibia were also observed corresponding to the articular surfaces lysis. However, the radiographs related to tempol post-treated rats showed almost normal joint space with little lysis of the femoral articular surface, and few osteophytes (Fig. 6d).

Effect of tempol on the histopathological alterations
The sham groups showed normal histological structures of articular cartilage layers with apparent intact isogenous chondrocytes. Regular smooth articular surfaces and intact synovial membrane were also observed. Besides, well-organized cartilaginous matrices were recorded with strong high density and reactivity of proteoglycans to alcian blue stain (Fig. 7a&b). Severe degenerative and necrotic changes with signi cant loss of chondrocytes were found in MIA-induced OA photomicrographs accompanied by focal erosions and ssures of articular cartilage super cial zones (Fig. 7c1), in addition to occasional subchondral extravasation of blood (Fig. 7c2), edema with in ammatory cell in ltrations of synovial membranes (Fig. 7c3). Signi cant loss of proteoglycans reactivity to alcian blue was recorded all over articular surfaces in the MIA-OA group (Fig. 7c4). On the other hand, photomicrographs of the tempol post-treated group demonstrated distinguished improvements in the chondrocytes (Fig. 7d1&2) with mild in ammatory cells synovial membrane in ltrates (Fig. 7d3). Post-treatment with tempol was able to retain the higher reactivity of proteoglycan (Fig. 7D4). Modi ed Mankin score was recorded in the histogram panel (Fig. 7e). MIA-induced OA group attained the maximal mean of the modi ed Mankin score of (10±1.2, P<0.001) as compared to the sham group. However, the mean of modi ed Mankin score of tempol post-treated group was found to be (2.5±0.5, P<0.001) relative to the MIA-OA group.

Discussion
The present study demonstrates the potential role of tempol as a membrane-permeable radical scavenger in attenuating the related physical, structural, functional, and biochemical deleterious alterations of the knee osteoarthritis rodent model (Figure 8). Accordingly, OA was induced by a single unilateral intraarticular injection of monosodium iodoacetate (MIA) in the rat's right knee joint. MIA is a simple induced and non-invasive experimental model for OA 36 . MIA prompts chondrocyte death with functional, radiological, histological, and biochemical alterations that resemble human OA in the early phases 17,37,38 . In the current investigation, the degenerative effects of MIA on cartilage are demonstrated biochemically by predominant elevations of MMP-13 contents and the cell surface DDR2 expression levels in the MIA-OA group, our results are consistent with previous studies 25,26 .
A variety of MMPs are produced by OA chondrocytes, among which is MMP-13 or collagenase-3 which contributes mostly to degrade collagen type II besides proteoglycans and other ECM components.
Accordingly, MMP-13 is considered the major catabolic effector in OA 39 . The proteoglycans depletion consequently augments the exposure and interaction of collagen type II with DDR2 resulting in its activation and further upregulation of MMP-13 40 . Consequently, the chondrocytes undergo hypertrophy and lose their ability to form new cartilage matrix constituents. Hereafter, the subchondral bone undergoes abnormal remodeling, leading to osteophytes formation as an attempt to correct the resulted joint deformity 41 .
In the present investigation, the destructive effect of MIA on the cartilage is presented by MIA-related structural alterations, where, the radiographic ndings reveal joint opacity and osteophytes formation associated with rough surfaces of the femoral condyles and proximal tibia. Besides, the histopathological ndings show severe degeneration in the chondrocytes and severe loss of proteoglycans reactivity in response to alcian blue staining. Similar ndings are mentioned before 6,17,26 .
In addition, further biochemical investigations of cartilage turnover biomarkers indicated elevations in bulin-3 expression, bone ALP activity, and serum levels of CTX-II and COMP, which may be subordinate to MIA-induced MMP13 upregulation. Fibulin-3 is a member of the ECM proteins that provides organization and stabilization to ECM structure 42 . Bone ALP is expressed in injured cartilage tissues on the cell surface and within matrix vesicles 43 . Furthermore, CTX-II is a product of collagen type II denaturation, Likewise, COMP, or called thrombospondin-5 is one of the non-collagen ECM components 43 . They are prognostic biomarkers for cartilage breakdown as their levels are highly increased in serum and synovial uid following MIA insult 44,45 .
Contrariwise, the post-treatment of tempol repressed all MIA-induced elevations of knee joint degradationrelated biomarkers. Moreover, tempol showed a little lysis of the femoral articular surface and few osteophytes in the related radiographs, and also retained the higher proteoglycan reactivity in response to alcian blue staining. Thus, these outcomes suggest the potential role of tempol in reversing the MIAdestructive effect on the cartilage and further highlight the anti-osteoarthritic e cacy of tempol.
MIA disrupts glycolysis and aerobic cellular respiration via inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 6,46 . As a consequence, MIA instigated oxidative phosphorylation disruption, ROS elevation, the release of cytochrome c from the mitochondrion, activation of caspase-3, and nally provoking chondrocytes apoptosis in rats 47 . In the present study, cytochrome c oxidase (CcO, complex IV) activity was diminished in the MIA-OA group. CcO is the terminal electron acceptor in the electron transport chain (ETC) and the regulation site for mitochondrial oxidative phosphorylation sequel 48 . Thus, our ndings indicated that oxidative phosphorylation and mitochondrial energy homeostasis were disrupted following MIA insult. On the contrary, CcO was replenished in the tempol-treated rats. Likewise, a previous study has demonstrated that tempol maintained the mitochondrial respiratory function in cisplatin-induced nephrotoxicity in mice 21 .
In the current investigation, MIA insult accompanied the perturbed oxidative phosphorylation by oxidative stress status of lower SOD activity and elevated NOX4 contents, these results are consistent with previous studies 11,16  Inversely, tempol post-treatment mitigated MIA-induced oxidative stress via replenishing SOD and diminishing NOX4 activity. These ndings reasonably rely on the anti-oxidant e cacy of tempol.
Interestingly, these results can collectively support the linkage between the anti-oxidant effect of tempol and its ability to maintain mitochondrial function dynamics and cellular redox balance which primarily emphasize the potential role of tempol in alleviating cartilage degeneration.
In this study, MIA prompted an in ammatory response that was noticeably presented by joint swelling especially during the rst 10 days of the experimental period, and then subsided gradually, accompanied by edema and in ammatory cells in ltration of the synovial membrane in the related histological photomicrographs. Likewise, the MIA-induced biochemical alterations showed signi cant elevations of the pro-in ammatory mediators including serum IL-1β, tissue contents of IL-6 and NF-κB p65 , and CCL2 expressions in OA rats, these outcomes were previously mentioned 6,20,57 .
It has been noted that the abnormally generated ROS exacerbates the in ammatory process in OA and aggravates ECM and joint degradation 10  At the early stage of OA, chondrocytes stimulate TGF-β to activate the canonical signal transducer SMAD3 directly as a repair attempt in response to cartilage degeneration 75 , which subsequently activates the target transcription of NOX4 76 . Henceforward, the expression of NOX4 derives excessive ROS generation (especially H 2 O 2 ), which activates the downstream p38MAPK phosphorylation thus resulting in deleterious signaling events 77,78 . Moreover, it has been documented that TGF-β activates the non-canonical p38MAPK and NF-κB signaling by the upstream activator TGF-β-activated kinase-1 (TAK1) 79 .
The stress kinase, p38MAPK is activated in response to the in ammatory cytokines, oxidative stress, and growth factors activation 77 . Its activation is mediated by PKC signaling 80 . p38MAPK is a member of the family of serine/threonine kinases that consists of p38 kinase, c-Jun N-terminal kinase (JNK), and the extracellular signal-regulated kinases (ERKs) 81 . Subsequently, activation of p38MAPK triggers NF-κB signaling activity and its sequels 82 . Besides, it has been noted that oxidative stress-induced the phosphorylation of p38MAPK which is correlated to the activation of caspase 9 mediated apoptotic pathways 77 .
Hereby, our ndings demonstrate that MIA injection resulted in elevations of anabolic growth factor TGF-β1, PKC, p-SMAD3, and p-p38MAPK, and in contrast, tempol suppressed all these elevations. Relying on these ndings, the anti-oxidant mechanism of tempol is not related only to its intracellular redox cycling negotiation, but it is also related to the ability of tempol to suppress the TGF-β1/SMAD3/NOX4 pathway, which further diminishing NOX4-derived ROS generation sequels via suppression of p-p38MAPK/NF-κB p65 damaging signaling cascades. These outcomes support the advance probable anti-osteoarthritic effect of tempol in promoting cartilage regeneration and opposing catabolic cascades interlinked with its anti-oxidant e cacy.
Furthermore, chondrocytes potentiate TGF-β to secrete more IGF-1 to boost the production of ECM synthesis proteins as an additional repair attempt to OA insult 74  In this study, the autophagy-associated protein, beclin-1 contents were downregulated in MIA-induced OA rats. It has been suggested that beclin-1 expression was signi cantly downregulated in OA cartilage tissue through PI3K/Akt/mTOR signaling 91 Figure 1 Schematic diagram of the experimental protocol. Rats were randomly assigned into four groups. In group (1): normal rats were subjected to a single intra-articular injection of normal saline in their right knee joints on day 0 and then subjected to a daily 1 ml of distilled water by oral gavage, one week after saline injection for 21 days. In group 2: rats were exposed to intra-articular injection of normal saline in their right knee joints on day 0 and then administered tempol (100 mg/kg/day) by oral gavage starting from the 7th day of the experiment for 21 consecutive days. In group 3: OA was induced by a single unilateral intra-articular injection of MIA on day 0 in a dose of 3 mg dissolved in 50 μL saline. In group 4:

Figures
osteoarthritic rats were administered tempol starting from the 7th day of the experiment in a dose of (100 mg/kg/day) by oral gavage for 21 consecutive days. Where, MIA: monosodium iodoacetate, mm: millimeter, OA: osteoarthritis, and rpm: rotations per minute.   Effect of tempol on the catabolic in ammatory cascade on MIA-OA rats. Rats were subjected to a single intra-articular injection of 3 mg MIA/50 μL saline in their right knees, and then tempol was administered starting from the 7th day of the experiment in a dose of (100 mg/kg/day) by oral gavage for 21 consecutive days. Values of (a) knee joint diameter and (b) latency time to fall are expressed as mean ± SD (N=8). Statistical analysis was carried out using repeated measures ANOVA test for factors time and group, followed by the Bonferroni test. Whereas, values of (c) The serum levels of Interleukin-1β (IL-1β), (d) Tissue contents of Interleukin-6 (IL-6), (e) Tissue contents of nuclear factor-kappa B (NF-κBp65), (f) The mRNA expression of chemotactic cytokine ligand 2 (CCL2), (g) The mRNA expression of dickkopf  Representative X-ray radiographs. Normal radiographs were revealed in the sham group (a) and Sham+Tempol group (b) along the hip-knee-ankle (HKA) axis. However, the images of the MIA-induced OA group (c) showed increased joint opacity (black arrow), and osteophyte formation (red arrow) associated with rough surfaces of the femoral condyles and proximal tibia (red and black dashed arrow). On the other hand, the X-ray images of the tempol post-treated group (d) showed almost normal joint space with little lysis of the femoral articular surface (red dashed arrow) and few osteophytes (red arrow). Where, AP view: anterior-posterior view (Flexion angle 10°), and lateral view (Flexion angle 30°).

Figure 7
Representative H&E and alcian blue histopathological photomicrographs. The micrographs of the sham groups (a&b) revealed normal histological structures of articular cartilage layers with apparent intact isogenous chondrocytes (black arrow) and regular smooth articular surfaces. Intact synovial membranes and normal blood vessels are recorded as a blue arrow in the sections of (a3&b3). Higher proteoglycans reactivity to alcian blue staining was also observed in (a4&b4). The worst distortion is observed in MIAinduced OA sections, where severe degenerative and necrotic changes with signi cant loss of many chondrocytes (red arrow) are accompanied by many focal erosions and ssures of articular cartilage super cial zones (black star) in (c1) photomicrograph with occasional subchondral extravasation of blood (red star) in (c2). Signi cant edema of synovial membranes with many in ammatory cells in ltrates (red dashed arrow) were recorded in (c3). The MIA-induced OA demonstrated severe loss of proteoglycans reactivity to alcian blue staining all over the articular surfaces (c4). Photomicrographs of the tempol post-treated group (d1&d2) showed more intact chondrocytes (black arrows) and regular smooth articular surfaces. Mild in ammatory cell in ltrates (red dashed arrow) were also observed in synovial membranes (d3). Post-treatment with tempol (d4) retained the higher proteoglycan reactivity.
Panel (e) represents the means of the modi ed Mankin scoring system (Score range: 0-13, from normal cartilage to the maximal score of osteoarthritis), where the statistical analysis was carried out using oneway ANOVA followed by Tukey's multiple comparison test. Results are expressed as mean ± SD (N=3). † † †P<0.001 vs. the sham group, and ‡ ‡ ‡P<0.001 vs. MIA-induced OA group.