Fractionated whole body γ-irradiation aggravates arthritic severity via boosting NLRP3 and RANKL expression in adjuvant-induced arthritis model: the mitigative potential of ebselen

The Wistar female albino adult rats (weighing 175–190 gm) used in this study were attained from the animal breeding unit of the National Center for Radiation Research and Technology (NCRRT) (Cairo, Egypt). Rats had free access to standard pellets and water ad libitum and were acclimatized for one week, at least before the beginning of experimental procedures.

Experimental rats were handled by following the recommendations of the National Institute of Health (NIH No 85:23, revised 1996) for the care and use of laboratory animals and in accordance with the regulations of Ethical Committee of the NCRRT, Atomic Energy Authority, Cairo, Egypt (Approval No. 35A/22).

Adjuvant-induced arthritis (AIA) was developed in rats employing procedures proposed previously by Bao et al. (2019). Eight sham rats were chosen at random and received 0.1 mL of physiological saline subcutaneously into the plantar area of the footpad in the right hind paw just before induction. To induce AIA, rats received a single intradermal injection of 100 µL of dried and heat-killed (1 mg/mL) M. tuberculosis suspended in mineral oil (Freund's completed adjuvant; FCA). Shortly after the FCA injection, classic symptoms of inflammation were observed and culminated on day 12, with the day of FCA immunization being considered as day zero.

FCA-challenged AIA rats were exposed to a whole body γ-irradiation at a dose level of 2 Gy/fraction once per week for 3 consecutive weeks, for a total dose of 6 Gy, delivered at a dose rate of 0.401 Gy/min at the time of the experiment, following the guidelines of the Protection and Dosimetery Department, NCRRT. Rats were subjected to a whole body irradiation protocol, as previously demonstrated by Nylander et al. (2016) and Khalil and Al-Daoude (2019) utilizing total body irradiation approach with simultaneous pathological manifestations. Rats were irradiated at the NCRRT using Gamma Cell-40 biological irradiator with a Cesium-137 (Cs¹³⁷) source (Atomic Energy of Canada Limited; Sheridan Science and Technology Park, Mississauga, Ontario, Canada). Rats were inserted into the Gamma cell-40 plastic sample tray that has exhaust vents that correspond to ventilation parts across the principle shield, to enable a process for uniform irradiation for small animals for every requisite irradiation exposure and kept for a sufficient amount of time to accomplish the exposure level.

Rats were randomly grouped into 7 groups (eight rats/group) as follows: Group I (Sham group): normal rats just received a 5% CMC-Na solution vehicle. Group II (A group): rats were inoculated with 0.1 mL FCA and were orally gavaged with 5% CMC-Na solution. Group III (A + MTX group): FCA immunized rats were administered methotrxate (MTX), a reference anti-arthritic drug, at dose of (0.5 mg/kg; twice weekly, i.p) dissolved in physiological saline (Zhou et al. 2019). Group IV (A + EB group): AIA rats were given EB intragastrically at a dose of 20 mg/kg (Cheng et al. 2019), daily for two successive weeks. Group V (A + R group): AIA rats that were exposed to whole body γ-irradiation at a dose level of 2 Gy/fraction once per week for 3 consecutive weeks, up to a total dose of 6 Gy at a specific time points each week (middle of the week). Group VI (A + R + MTX group): AIA rats were exposed to fractionated whole body γ-irradiation and treated with MTX. Group VII (A + R + EB group): AIA rats were subjected to fractionated whole body γ-irradiation and treated with EB.

The severity of AIA was evaluated each three days post induction using body weight, paw swelling, polyarthritis index, and global arthritis assessment scores, as described earlier (Chang et al. 2016). At 3 day intervals, the body weights of the sham, A, and A + R groups, whether treated or untreated, were recorded. The body weights were recorded on day zero, before the FCA immunization. This was known as the initial body weight, although the value measured on other days has been known as the terminal body weight. The right hind paw volumes (mL) of every rat were estimated on day zero just before FCA immunization employing water displacement technique with a plethysmometer (UGO Basile, Italy) (Patil et al. 2012), and were observed each 3 days till the 21st day as primary swelling. The change in paw edema for each group is determined by subtracting the initial paw volume (basal) out from volume measured at each time point using following formula: (ml) = V_t‐V₀, where V₀ is the paw volume prior to FCA injection (ml) and V_t is the volume at (t) day after FCA immunization (ml) (Tong et al. 2018). The polyarthritis index scoring method spanned from 0 to 4 using a formerly reported macroscopic scoring technique (Bao et al. 2019), where 0 indicating no evidence of hyperemia; 1 denoting mild erythema and edema of ankle or wrist joints; 2 implying erythema and swelling of paws; 3 inferring severe inflammation of the whole paw; and 4 demonstrating entire limb and digits with substantial swelling and malformation. The polyarthritis index score was determined by adding the cumulative scores from each rat's four paws, with a peak value of 16. The global arthritis assessment score is obtained using macroscopic examination of clinical symptoms in multiple aspects of AIA rats, where 0 = no nodule and erythema; 1 = nodule and redness in one ear; 2 = nodule and redness across both ears, Nose: 0 if there is no connective tissue swelling and erythema; 1 if there is visible connective tissue swelling and erythema. Tail: 0 if there is no nodule and no redness, 1 if there is a noticeable nodule and erythema. Paws: 0 if there is no swelling and redness, 1 denoting one paw with swelling, 2 inferring mild swelling and erythema in two paws, 3 implying severe swelling and erythema in three paws, and 4 indicating redness, intense swelling, and malformation in four paws (Xiu et al. 2015).

The experimental procedures started after the development of arthritis on day 9, then exposed to radiation on day 12 and certain time points in each week successively. Under gentle anesthesia, 1 h immediately following the last drug dosing, blood samples were gathered by cardiac perforation, permitted to coagulate at ambient temperature, and then centrifuged at 4000 rpm for 15 min using a centrifuge (Hettich Universal 32A, Germany). The collected sera was further separated and kept at − 80 °C until use. Ankle joins and synovial tissues were excised. A portion was then washed in ice-cold saline solution and homogenates were prepared for biochemical estimation, while the remaining part was used for histopathological investigation. The total protein content in the tissue aliquots was determined using the Bradford method (Bradford 1976).

The ankle joints were excised out after sacrifice on the last day of the experiment and promptly fixed in 4% paraformaldehyde and decalcified in 10% ethylenediaminetetraacetic acid (EDTA) for 30 days till the complete decalcification at 4 °C. Tissue samples were cleared up in xylene and enclosed in paraffin for 24 h at 56 degrees in a hot air oven. To prepare paraffin beeswax tissue blocks for slicing at 5 µm thicknesses, a sledge microtome was used. The tissue sections were obtained, deparaffinized, and stained with hematoxylin and eosin (H&E) stain for examination under a light electric microscope (Bancroft et al. 1996). Tissue sections were graded for inflammatory cell infiltration, synovial proliferation, pannus formation, and cartilage damage on a scale of 0–3 (0 = none, 1 = mild, 2 = moderate, and 3 = severe), and the average score was calculated (Wei et al. 2013; Xiang et al. 2016). An unbiased pathologist who was unaware of the therapeutic regimen accomplished the distinctive pathological debilitating lesions of the tibiotarsal joints blindly and at random using an Olympus CX41 light microscope (Olympus, Tokyo, Japan) equipped with a high resolution digital camera system.

The level of reactive oxygen species (ROS) was quantified using a commercially available rats’ enzyme linked immunosorbent (ELISA) kit purchased from MyBioSource (San Diego, CA, USA; CAT# MBS039665). Lipid peroxidation, in terms of malondialdehyde (MDA), was estimated according to the method of Yoshioka et al. (1979), and the reduced glutathione (GSH) content was measured as described by Ellman (1959). The activity of glutathione peroxidise (GSH-Px) was determined according to the method of Gross et al. (1967). The antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) activities were assayed by the methods of Kakkar et al. (1984) and Bergmeyer et al. (1988), respectively. The levels of pro- and anti-inflammatory mediators such as IL-1β, TNF-α, IL-4, and IL-10 were measured using the corresponding specific rat ELISA kits from Abcam (Cambridge, UK; CAT# ab100768, CAT# ab100785, CAT# ab100770, and CAT# ab214566, respectively), while the rat caspase-1 ELISA kit from MyBioSource (CAT# MBS765838) was utilized to estimate caspase-1 level. These biochemical indices were measured in the serum and synovial tissues of arthritic and arthritic irradiated rats, whether treated or untreated, according to the manufacturers’ instructions. For the detection of NF-κB p65 in synovial tissue, synovial specimens were sliced into small pieces and incubated for 24 h in 2 ml of serum-free RPMI-1640 (Thermo Fischer Scientific, Rockford, IL, USA) containing 0.25% lactalbumin hydrolysate in a 5% CO2 incubator.. Tissue samples were homogenized on ice in a 50 mM Tris–HCl buffer with a pH of 7.5 (Chang et al. 2015). Tissue homogenates were used to prepare nuclear extracts as previously illustrated (Lee et al. 2006). In the yielded nuclear extracts, the level of NF-κB p65 was assessed using an ELISA kit (MyBiosource; CAT# MBS015549) as per the manufacturers’ instructions.

Immunohistochemical detection of RANKL, NLRP3, Collagen II, and Caspase-3 expression was performed on 4 µm thick demineralized ankle joint sections. All sections were dewaxed by xylene, dehydrated, and thoroughly washed three times in PBS for 5 min before being incubated with 3% H₂O₂ for ten min at 37 °C. They were then rinsed 3 times in PBS before being blocked with goat serum blocking solution. Excess fluid was removed, diluted primary anti-RANKL (1:50, Cell Signaling, CAT# 4816), anti-NLRP3 (1:300, Abcam, CAT# ab214185), anti-Collagen II (1:200, Abcam, CAT# ab34712), and anti-Caspase-3 (1:100, MyBiosource, CAT # MBS9700318) were placed drop by drop, and sections were preserved in a humidified cabinet at 4 °C for 24 h. After 20 min of stability and shaking at 37 °C, the sections were washed thoroughly and incubated for 20 min with a streptavidin–horseradish peroxidase-labeled secondary antibody. The yellow 3, 3′-diaminobenzidine (DAB) staining and hematoxylin counterstaining were used to view the expression of biomarkers. Phosphate-buffered saline (PBS) served as a negative control. To ascertain immunostaining, immunohistochemical analysis was carried out using Image-Pro Plus 6.0 software.

Statistical analysis was accomplished by one-way analysis of variance (ANOVA) then followed by Tukey–Kramer multiple comparison tests, except for analysis of changes in body weight, arthritis score, edema volume, and global polyarthritis assessment which used two-way ANOVA followed by the post hoc Dunnette’s test for multiple comparisons. The scored data of selected histopathological parameters were presented as median and range, and the difference between all tested groups was analyzed using Mann–Whitney U test for non-parametric analysis. The Kolmogorov–Smirnov (KS, P > 0.10) test was used to confirm data normality, and the proper test was used when needed. Graph Pad prism 8 was used for statistical analysis (Graph Pad Software Inc, San Diego, California, USA). Data were expressed as mean values ± standard error of the mean (SEM) and differences between values are considered significance at P ˂ 0.05.

Every 3 days after AIA induction, rats' body weight gain, ankle circumferences, and inflammatory clinical arthritic scores, including paw volume, arthritis index score, and global polyarthritis assessment, were measured (Fig. 1A–E). Body weight gain in animals is widely regarded as a reliable predictor of the anti-inflammatory and immunomodulatory outcomes of the medications assessed in the AIA model. Over the first six days after FCA immunization, the average body weight of rats in sham, A, A + MTX, and A + EB groups increased (Fig. 1A). In the A + R and A + R + MTX groups, a steady decline in mean body weight of rats begins within the initial three days after FCA challenge. While, as secondary arthritis progressed, the mean body weights of rats immunized with FCA dropped drastically as revealed in A group. Conversely, the average body weights of rats in the sham group rose over time. Notably, the A + R and A + R + MTX groups demonstrated a significant reduction (P ˂ 0.05) in mean body weight beginning on day 9 and clearly diminished continuously to the last day of experiment when compared to sham, A, A + MTX, A + EB, and A + R + EB groups. However, in the A + MTX group, a noticeable reduction (P ˂ 0.05) in mean body weight was only observed on day 15 and peaked at day 21. Oral administration of EB at a dose of 20 mg/kg daily to either arthritic or arthritic irradiated rats (A + EB and A + R + EB groups) efficiently maintained the mean body weight of rats across all the stages of arthritis progression (primary and secondary arthritis) as compared to the A, A + MTX, A + R, and A + R + MTX groups (Fig. 1A). In Fig. 1B, the mean arthritic index score for the A and A + R groups implies that secondary arthritis has started on day 9 and increased substantially by day 12 when compared to the sham set, with the highest score recorded in the A + R group as compared to the respective A group. A significant improvement (P ˂ 0.05) was observed in the mean arthritis index score of the A + MTX group when compared to the A, A + R, and A + R + MTX groups (Fig. 1B). Intriguingly, the A + EB group exhibited a mean arthritic index score profoundly lower (P ˂ 0.05) than those recorded in the corresponding A, A + R, and A + R + MTX groups. On the other hand, in the A + R + EB group, treatment of arthritic irradiated rats with EB effectively reduced (P ˂ 0.05) the mean arthritis index score as compared to the A + R group, with a comparable score to that recorded in the A + R + MTX group, but with a better improvement. Macroscopic photographs and measurement of paw edema from FCA-inoculated hind paws were employed to evaluate the degree of inflammation among groups (Fig. 1C, D). An acute inflammatory and autoimmune phase of swelling was induced after day 6 of FCA inoculation in both inoculated and non-inoculated paws that surged at day 9 (P ˂ 0.05, 143%), and this increase in paw circumference was prolonged to day 21, with a steady decrement to 86% on the last day of experiment (day 21) in the A group compared to the sham rats (Fig. 1D). Whereas, in the A + R group, a remarkable rise (P ˂ 0.05, 168%) in paw volume started on day 9 after the FCA challenge and continued with an gradual lowering to reach 117% and 18% when compared to the sham and A groups, respectively, on day 21. When administered twicely/week at a dose of 0.05 mg/kg, MTX exerted a significant abolishment (P ˂ 0.05) in paw volume by 28% in A + MTX group as compared to A group and by 18% in the A + R + MTX group as compared to A + R group on day 21. Most importantly, the A + EB and A + R + EB groups showed a pronounced improvement (P ˂ 0.05) in paw swelling as perceived by a decrease in paw volume of 38% in the A + EB group as compared to the A group and by 32% in the A + R + EB group when compared to the A + R group on day 21 of the experimental course (Fig. 1D). As compared to the sham group, rats in the A and A + R groups had systemic inflammation and the evolution of arthritis was transient, as indicated by the emergence of nodules on the tail and non-inoculated paws, as well as redness of the ears and nose, with the utmost global polyarthritis assessment score (P ˂ 0.05) at days 12 and 15 following FCA immunization (Fig. 1E). The A + MTX, A + EB, A + R + MTX, and A + R + EB groups exhibited a profound improvement in their global polyarthritis assessment scores when compared to the A and A + R groups, with the most improvement observed in the A + EB and A + R + EB groups when compared to the A + MTX and A + R + MTX groups, respectively (Fig. 1E).

As discerned in Fig. 2A, we then examined the ability of EB to mitigate the extent of injury in the tibiotarsal joints of AIA rats' ankles using histopathological analysis. The joint architecture in H&E stained sections of sham rats shows an average joint cavity with an intact normocellular synovial membrane (black arrow-i), average fibrous capsule (blue arrow-i), and an average surrounding soft tissue (red arrow-i) (H&E X 200). An intact normocellular superficial layer of synovial membrane (black arrow-ii) and average surrounding soft tissue (red arrow-ii) are visible at a high magnification power (H&E X 400). Contrarily, moderate degenerative lesions were observed in the examined joint sections of the A group as represented by pannus formation (black arrow-i), destructing articular cartilage (blue arrow-i), destructed fibrous capsule (red arrow-i), and moderate inflammatory infiltrate in the surrounding soft tissue (green arrow-i) (H&E X 200). In addition, an intact synovial membrane (black arrow-ii), with a large pannus showing a moderated inflammatory infiltrate composed mainly of lymphocytes (blue arrow-ii) and fibroblasts (red arrow-ii), was revealed on high power view (H&E X 400). Meanwhile, an improvement was perceived in the assessed joint sections of the A + MTX group as indicated by a focally hyperplastic synovial membrane (black arrow-i), intact normocellular articular cartilage (blue arrow-i), and a mild inflammatory infiltrate in the surrounding soft tissue (red arrow-i) (H&E X 200). Another view elucidating the narrow joint cavity with pannus formation (black arrow-ii) and intact underlying articular cartilage (blue arrow-ii), and mild inflammatory infiltrate in surrounding soft tissue (red arrow-ii) (H&E X 400). Along with that, but to a greater extent, the scrutinized joint sections of the A + EB group displayed minor joint damage as manifested by the formation of tiny pannus with underlying mild inflammatory infiltrate (black arrow-i), focally destructed articular cartilage (blue arrow-i), and mild edema in surrounding soft tissue (red arrow-i) (H&E X 200). Moreover, at high power magnification, a small pannus with an underlying mild inflammatory infiltrate composed mainly of lymphocytes (black arrow-ii) and thick-walled blood vessels (blue arrow), as well as intact normocellular articular cartilage (red arrow-ii) was noticed (H&E X 400). In A + R group examined sections, arthritic rats were exposed to fractionated whole body γ-irradiation, which exacerbated the severity of the joint pathological lesions as implied by the narrow joint cavity (black arrow-i) with diffuse synovial hyperplasia (blue arrow-i), accompanied by small pannus formation (red arrow-i), focally destructed articular cartilage (green arrow-i), and the surrounding soft tissue showing marked inflammatory infiltrate (yellow arrow-i) (H&E X 200). Another view showing diffuse synovial hyperplasia (black arrow-ii) with large pannus formation (blue arrow-ii), destructed articular cartilage (red arrow-ii), and a marked inflammatory infiltrate with congested blood vessels in surrounding soft tissue (green arrow-ii) (H&E X 400). Whereas, in the A + R + MTX group, the examined joint sections showed a reasonable improvement in the preceding lesions, as evidenced by an average articular cartilage (red arrow-i) with large pannus formation, as well as a marked inflammatory infiltrate composed primarily of lymphocytes (black arrow-i), and mildly congested blood vessels (blue arrow-i) (H&E X 200). A further view depicts diffuse hyperplastic synovial membrane (black arrow-ii), large pannus with markedly congested blood vessels (blue arrow-ii), and intact normocellular articular cartilage (red arrow-ii) (H&E X 400). Interestingly, a promising amelioration was observed in the examined joint sections from the A + R + EB group as represented by a focally destructed normocellular articular cartilage (red arrow-i) and diffuse synovial hyperplasia (black arrow-i) with a small pannus showing mild inflammatory infiltrate (blue arrow-i) (H&E X 200). Also, a high power view showing diffuse synovial hyperplasia (black arrow-i) with a small pannus showing moderated inflammatory infiltrate (blue arrow-ii), and intact normocellular articular cartilage (red arrow-ii) (H&E X 400). A summary of the quantified histopathological lesion scoring to discriminate the severity between groups is illustrated in Fig. 2B and C. Obviously, when compared to the sham set, a significant increase in the pathological lesion scoring was revealed in the A, A + R, A + R + MTX, A + R + EB, A + MTX, and A + EB groups, while these degenerative changes were attenuated considerably in the A + EB, A + MTX, A + R + EB, and A + R + MTX groups, as compared to the A and A + R groups (Fig. 2B and C).

When compared to the sham group, the data from the A group showed a significant upregulation (P ˂ 0.05) of ROS levels in serum by 3.7-folds and in synovial tissue by 3.5-folds, accompanied by a surge (P ˂ 0.05) of MDA levels in serum by 1.81-folds and in synovial tissue by 3.2-folds, as shown in Fig. 3A and B. These results were associated with a substantial decline (P ˂ 0.05) in the antioxidant defensive machinery of the A group. This was represented by a considerable decrement in SOD, CAT, and GPx activities as well as GSH levels in serum by 54.99%, 51.99%, 44.96%, and 48.62%, respectively, and in synovial tissue by 62.95%, 52.70%, 65.88%, and 58.17%, respectively, as compared to the respective sham set.

The data of the A + MTX group revealed a significant improvement (P ˂ 0.05) against arthritic status through diminishing ROS (by 38.36% and 36.87%) and MDA (by 31.13% and 39.95%) levels, along with a significant augmentation (P ˂ 0.05) in SOD (by 1.53- folds and 1.65-folds), CAT (by 1.60-folds and 1.65-folds), GPx (by 1.4-folds and 1.51-folds) activities as well as GSH level (by 1.81-folds and 1.8- folds) in serum and synovial tissue, respectively, when compared with the A group (Fig. 3A and B).

The administration of EB at a dose of 20 mg/kg daily to arthritic rats as perceived in the A + EB group efficiently counteracted (P ˂ 0.05) oxidative stress through decreasing levels of ROS (by 42% and 37.39%) and MDA (by 34.11% and 39.78%), coupled with boosted (P ˂ 0.05) enzymatic antioxidant biomarkers including SOD (by 1.5-folds and 1.6-folds), CAT (by 1.42-folds and 1.70-folds), GPx (by 1.46-folds and 2- folds) activities and GSH level (by 1.90-folds and 1.88-folds) in serum and synovial tissue, respectively, as compared to the A group (Fig. 3A and B).

When compared to the A group, exposure of arthritic rats to fractionated whole body γ-irradiation as shown in the A + R group resulted in a significant rise (P ˂ 0.05) of ROS and MDA levels in serum by 1.5-folds and 1.5-folds and synovial tissue by 1.35-folds and 1.45-folds, respectively. This elevation in oxidative stress indicators was correlated with a pronounced curtailment (P ˂ 0.05) in antioxidant markers of the A + R group, as implied by a noticeable decrement in SOD, CAT, and GPx activities as well as GSH level in serum by 42.17%, 36.05%, 39.05% and 61.33%, respectively, and in synovial tissue by 44.61%, 49.93%, 46.39% and 47.08%, respectively, as compared to the A group (Fig. 3A and B).

In the A + R + MTX group, the obtained data demonstrated a significant modulation (P ˂ 0.05) in the oxidative stress status through a decrease in the levels of ROS by 32.36% and 27.42% and MDA by 41.03% and 41.32%, paralleled by a significant reinforce (P ˂ 0.05) in the activities of SOD (by 1.83- folds and 2.4- folds), CAT (by 1.80 folds and 2.02-folds), GPx (by 1.63-folds and 1.73-folds) and level of GSH (by 2.8-folds and 2.4-folds) in serum and synovial tissue, respectively, when compared to the A + R group, as displayed in Fig. 3A and B.

After EB supplementation to the arthritic irradiated rats, as represented by the A + R + EB group, the results exhibited a remarkable protection (P ˂ 0.05) against oxidative stress status portrayed in the A + R group through restraining the levels of ROS (by 30.35% and 29.64%) and MDA (by 45.63% and 38.87%) concomitantly with a significant upregulation (P ˂ 0.05) in antioxidant system via augmenting the activities of SOD (by 1.93-folds and 2.1-folds), CAT (by 1.94-folds and 2.5-folds), and GPx (by 2-folds and 2.53-folds) activities as well as the level of GSH (by 3.26-folds and 2.8-folds) in serum and synovial tissue, respectively, as compared to the A + R group (Fig. 3A and B).

Figure 4A and B depicts the inflammatory status biomarkers, which include both pro-inflammatory mediators (TNF-α and IL-1β) as well as the anti-inflammatory cytokines (IL-4 and IL-10) concurrently with caspase-1. When compared with the sham group, the data of the A group showed a marked rise (P ˂ 0.05) in the levels of TNF-α (by 2.2-folds and 3.52-folds), IL-1β (by 2.21-folds and 3.35-folds), and caspase-1 (by 4.87- folds and 4.3-folds) concordantly with a noteworthy diminution (P ˂ 0.05) in the levels of IL-4 (by 58.52% and 63.87%) and IL-10 (by 46.98% and 63.26%) in serum and synovial tissue, respectively.

When compared to the A group, the A + MTX group had a significant alleviation (P ˂ 0.05) in the inflammatory status, with lower levels of TNF-α (by 35.46% and 40.42%), IL-1β (by 35.65% and 41.78%), and caspase-1 (by 47.83% and 39.89%), which was correlated with enhanced levels of IL-4 (by 1.6-folds and 1.5-folds) and IL-10 (by1.35-folds and 1.7-folds) in serum and synovial tissue, respectively (Fig. 4A and B).

The data presented in Fig. 4 A and B revealed a significant modulation (P ˂ 0.05) in inflammatory markers in the A + EB group, including a notable decrease in the levels of TNF-α (32.32% and 38.62%), IL-1β (44.74% and 40.28%), and caspase-1 (51.82% and 44.65%), as well as an elevation in the levels of IL-4 (by 1.75-folds and 1.65-folds) and IL-10 (by 1.46-folds and 1.71-folds) in serum and synovial tissue, respectively, when compared to the A group.

Exposure of arthritic rats to fractionated whole body γ-radiation (A + R group) markedly raised (P ˂ 0.05) the levels of TNF-α (by 1.78-folds and 1.5-folds), IL-1β (1.64-folds and 1.6-folds), and caspase-1 (by 1.33-folds and 1.5-folds), accompanied by a significant diminish (P ˂ 0.05) in the levels of IL-4 (by 55.92% and 54.02%) and IL-10 (by 34.30% and 44.12%) in serum and synovial tissue, respectively, when compared to the A group (Fig. 4A and B).

As demonstrated in the A + R + MTX group, MTX treatment of arthritic irradiated rats resulted in a significant modulation (P ˂ 0.05) in inflammatory status via reductions in the levels of TNF-α (by 49.22% and 43.05%), IL-1β (by 41.28% and 50.49%), and caspase-1 (by 46.33% and 28.48%), with an augment in the levels of IL-4 (by 2.42-folds and 3-folds) and IL-10 (by 1.57-folds and 2.5-folds) in serum and synovial tissue, respectively, as compared to the A + R group (Fig. 4A and B).

When compared with the A + R group, the A + R + EB group exhibited profound amelioration (P ˂ 0.05) in inflammatory markers via a decrease in the levels of TNF-α (by 52.55% and 45.12%), IL-1β (by 45.76% and 43.58%), and caspase-1 (by 42.69% and 34.92%), as well as an increase in the levels of IL-4 (by 2.66-folds and 3.45-folds) and IL-10 (by 1.75-folds and 2.65-folds) in serum and synovial tissue, respectively, as shown in Fig. 4A and B.

The synovial NF-κBp65 level showed a marked elevation (P ˂ 0.05) in the A and A + R by 2.5-folds and 3.65-folds, respectively, as compared to the sham rats (Fig. 4B). When compared to the A group, a significant reduction (P ˂ 0.05) in synovial NF-κBp65 level by 37.5% and 45% was shown in the A + MTX and A + EB groups, respectively. The treatment of arthritic irradiated rats with MTX and EB produced a noticeable curtailment (P ˂ 0.05) in synovial NF-κBp65 level by 33% and 40%, as represented by the A + R + MTX and A + R + EB groups, respectively, when compared to the A + R group (Fig. 4B).

We further sought to determine the expression pattern of RANKL and NLRP3 proteins in the ankle joints of the arthritic and arthritic irradiated rats, whether treated or untreated, to highlight the mitigative potential of the investigated treatments. FCA inoculation to rats caused a considerable upregulation (P ˂ 0.05) in RANKL and NLRP3 expression by 14.2- and 23.6-folds, respectively, as shown in the A group versus the sham animals, as illustrated by Fig. 5A–D. Oppositely, when compared to the A group, MTX markedly diminished (P ˂ 0.05) RANKL and NLRP3 expression by 39.2% and 32.5%, respectively, as observed in the A + MTX group. Surprisingly, a comparable effect was noticed in the A + EB group, but to a greater extent, as EB administration to arthritic rats proficiently reduced (P ˂ 0.05) RANKL and NLRP3 expression by 48.6% and 49.2%, respectively, when compared to the A group (Fig. 5 A-D). Whereas, exposure of arthritic rats to fractionated whole body γ-radiation (A + R group) boosted (P ˂ 0.05) RANKL and NLRP3 expression by 1.22- and 1.3-folds, respectively, when compared to the A group (Fig. 5 A-D). However, in the A + R + MTX group, there was a significant abolishment (P ˂ 0.05) in RANKL and NLRP3 expression by 23.3% and 21%, respectively, when compared to the A + R group. Most importantly, administration of EB to arthritic irradiated rats in the A + R + EB group effectively curtailed (P ˂ 0.05) RANKL and NLRP3 expression by 33.3% and 46%, respectively, when compared to the A + R group (Fig. 5A–D).

FCA inoculation to rats induced a significant reduction (P ˂ 0.05) in collagen type II protein expression by 83% as well as substantially raised (P ˂ 0.05) caspase-3 by 15-fold, as noticed in the A group when compared to the sham set (Fig. 6A–D). Obviously, MTX and EB were proficiently capable of maintaining the structural integrity of the joints in arthritic rats, as indicated by markedly restoring (P ˂ 0.05) collagen type II protein expression by 4- and 5.3-folds, and significantly suppressing (P ˂ 0.05) caspase-3 protein expression by 35% and 56%, respectively, as observed in the A + MTX and A + EB groups when compared to the A group. In contrast, exposure of arthritic rats to fractionated whole body γ-radiation (A + R group) significantly limited (P ˂ 0.05) collagen type II protein expression by 96.3%, while inducing a remarkable enhancement (P ˂ 0.05) in caspase-3 protein expression by 21%, when compared to the A group (Fig. 6A–D). Noteworthy, treatment of arthritic irradiated rats with MTX or EB as indicated in A + R + MTX and A + R + EB groups significantly replenished (P ˂ 0.05) collagen type II protein expression by 10.6- and 18-folds, while profoundly suppressing (P ˂ 0.05) caspase-3 protein expression by 41% and 58%, respectively, when compared to the A + R group (Fig. 6A–D).