Nitazoxanide alleviates CFA-induced rheumatoid arthritis in Wistar rats by modulating the STAT-3 and NF-κB pathways

Chemical and Drugs

Nitazoxanide was provided as a gift sample by Nirali Life Sciences Pvt. Ltd., Gujarat, India. Prednisolone was also received as a gift sample from Avik Pharmaceutical Ltd., Gujarat, India, while CFA was sourced from Sigma Aldrich, St. Louis, Missouri, USA. All additional reagents utilized in the experiment were of analytical grade. Enzyme-linked immunosorbent assay (ELISA) kits for assessing serum cytokine levels were supplied by Allianz Bioinnovation Pvt. Ltd.

Animals

The National Institute of Biosciences in Pune, India provided healthy Wistar rats weighing between 150 and 180 grams. The rats were accommodated in plastic Perspex cages under controlled conditions, with the temperature set at 25°C, relative humidity maintained between 45%–55%, and a 12-hour light-dark cycle. They were provided with unrestricted access to chow pellets and purified water. Before commencing the experiments, the rats were allowed a one-week acclimatization period in their new environment. The experimental protocol was reviewed and approved by the Institutional Animal Ethics Committee (CCSEA/IAEC/P-67/2023), which is registered under the “Committee for Control and Supervision of Experiments on Laboratory Animals (CCSEA)” of the Ministry of Environment and Forests, Government of India.

CFA‑induced Arthritic Model

Subcutaneous injection of Complete Freund’s Adjuvant into the subplantar region of the left hind paw of rats caused inflamed swelling (primary lesion) within 30 min, with peak inflammation occurring within 3–4 days. Secondary lesions appeared at non-injected sites, including the non-injected hind paw, forepaws, ears, nose, and tail, between 12–14 days post-injection. The CFA-induced joint inflammation generally persisted for 25–28 days.

Experimental Design

On day 0, a total of 0.1 mL of CFA was administered into the subplantar area of the left hind paw for all experimental groups, except for the normal control group, which was administered paraffin oil in an amount of 0.1 mL. Carboxymethyl Cellulose (CMC) served as the medium for the preparation of Prednisolone acetate.^[30] All groups received treatments through oral administration for 28 days. Animals were divided into 6 groups with 6 rats in each group (n = 6): Group 1, Normal Control (Normal Saline); Group 2, Disease Control (CFA with no treatment); Group 3, Standard Control (Prednisolone 10 mg/kg); Group 4, Nitazoxanide (100 mg/kg); Group 5, Nitazoxanide (200 mg/kg); Group 6, Nitazoxanide (400 mg/kg).

On the 28^th day, blood samples were collected, and the animals were sacrificed using CO₂. Following standard research methods, their hind paw limbs were sectioned and preserved in a 10% neutral formalin solution for further study.^[30]

Determination of Paw Volume

The measurement of paw volume was performed with a ple-thysmometer (Ugo Basile, Italy). The initial paw volume was documented on day 0, with follow-up measurements conducted on the 1^st, 3^rd, 7^th, 11^th, 14^th, 21^st, and 28^th days after the CFA injection.^[30]

Determination of Body Weight

The initial body weight was measured on day 0 before the CFA injection was given, following a one-week acclimatization period. Weight measurements were then taken 7^th, 14^th, 21^st, and 28^th days after the CFA injection.^[30]

Total Arthritic Index

The total mean arthritic index was calculated by grading the secondary lesions using accepted scoring procedures (Supplementary Table 1). The tail, nose, ears, forepaws, and hind paws were examined at the end of the dosing period based on the following criteria.^[31]

Behavioural Parameters

Stair Climbing Test

A seven-day acclimatization phase was instituted before the experiment to evaluate the stair-climbing capabilities of the rats. The rats were kept in a wooden enclosure (30 cm high) with three varying elevation steps (5 cm, 10 cm, and 15 cm) during this phase. A water-filled petri dish was placed strategically on the second step, and food pellets were positioned on the third step to promote climbing behaviour. On the evaluation day, the rats experienced a 6-hour fasting interval, after which their climbing performance was evaluated using a standardized scoring system: Score 0, No climb of animal; Score 1, One stair climbing by the animal; Score 2, First and second stair climbing by the animal; Score 3, Three stairs climbing by the animal.^[32,33]

Motility Test

The animals were acclimated for seven days before the administration of CFA. During this period, each rat was kept separately in a 100 cm by 50 cm by 50 cm wooden box with a climbing chamber. Motility assessments were conducted on the 1^st, 3^rd, 7^th, 14^th, 21^st, and 28^th days. The motility of each rat was scored based on predefined criteria during these observations: Score 0, The rat is noted to be in a stationary posture; Score 1, By crawling, the rat demonstrates movement; Score 2: By walking, the rat demonstrates ambulation activities; Score 3: The rat showcases a climbing activity; Score 4: The rat engages in climbing and running repetitively; Score 5, The rat performs climbing and running with expertise.

These evaluations were performed following the established research methodology.^[32,33]

Assessment of Red Blood Cells (RBC), White Blood Cells (WBC), Platelets, and Haemoglobin (Hb)

On day 28, the rodents were euthanized, and blood samples were collected via retro-orbital puncture. These specimens were subsequently analyzed to determine platelet count, WBC count, RBC count, and Hb levels.^[32,33]

Assessment of STAT-3, NF-κB, TNF-α and IL-6 Levels

On day 28, the rats were euthanized, and blood samples were collected via retro-orbital puncture. The serum concentrations of TNF-α and IL-6 were measured. Additionally, the hind paws were excised, and the soft tissues were separated. Using a tissue homogenizer, a homogenate of phosphate-buffered saline (PBS) (pH 7.4) was prepared. The homogenate was centrifuged at 12, 000 rpm for 10 min, and the supernatant was analyzed for STAT-3 and NF-κB concentrations. These concentrations were quantified using kits obtained from Allianz Bio Innovation Pvt. Ltd., following the manufacturer’s protocol.^[34,35]

Histopathological Examination

Hind paw tissues from the rats were fixed in 10% buffered formalin, trimmed, and decalcified in 5% formic acid. The samples were then processed, embedded in paraffin, and sectioned into 5 μm slices for hematoxylin and eosin staining. The stained sections were examined under a light microscope at 100× magnification to observe any inflammatory changes resulting from normal control as well as in treatment groups.^[36]

Radiological Analysis

On the 28^th day, a radiographic analysis of the ankle joints was conducted. Anaesthetized rats were placed on radiography equipment, about 90 cm away from the X-ray source. The left hind paws were examined utilizing 40 kW exposures lasting 0.01 seconds. Radiographic scoring was based on articular cartilage injury, soft tissue swelling, and osteophyte growth. The following criteria were used to determine the scores: Score 0 indicated no harm; score 1 indicated mild damage; score 2 indicated considerable damage; and score 3 indicated serious damage.^[37]

Statistical Analysis

Data were expressed as mean ± standard error of the mean (SEM) and analysed using One-way ANOVA to determine statistical significance. Tukey’s post hoc test was conducted to identify the significant differences among groups, and Dunnett’s test was used for specific comparisons to the disease control group. Statistical analysis was performed using GraphPad Prism (9.5.1), with a P-value of < 0.05 was considered significant.

Paw Volume

The measurement of paw volume in CFA-induced arthritic rats indicated significant results. A progressive increase in paw volume was observed, reflecting the advancement of arthritis over time. Prednisolone treatment demonstrated robust anti-inflammatory effects, leading to a notable reduction in paw volume. On day 14, nitazoxanide at a dose of 200 mg/kg exhibited measurable anti-inflammatory activity compared to the disease control group (P < 0.01). Furthermore, Nitazoxanide at 400 mg/kg showed significant anti-inflammatory effects across multiple time points, including days 7, 11, 14, 21, and 28 (P < 0.01; P < 0.001), with F = 8.186, as depicted in Figure 1a. The study’s results elucidated the progressive characteristics of CFA-induced arthritis and highlighted the clinical effectiveness of prednisolone and nitazoxanide in alleviating inflammation associated with rheumatoid arthritis.

Figure 1

A, NTZ effect on left hind paw volume in CFA-induced arthritic rats. B, Average body weight in CFA-induced arthritic rats. C, Mean stair-climbing performance in CFA-induced arthritic rats and D Mean motility score in CFA-induced arthritic rats. The values are expressed as mean ± SEM. Data were analysed by one-way ANOVA followed by a post-hoc Tukey HSD test. (^###P < 0.001 vs. Normal control, ***P < 0.001 vs. disease control, **P < 0.01 vs. disease control, and *P < 0.1 vs. disease control)

Body Weight

CFA-induced arthritis significantly affected body weight, with a pronounced decrease observed from days 7 to 28 compared to the control group (P < 0.01, P < 0.001), as shown in Figure 1b. In the initial phases, no notable alterations were observed; however, by the seventh day, variations in weight became apparent across all treatment cohorts. The administration of Prednisolone did not result in a statistically significant alteration in body weight when compared to the arthritic control group. Conversely, Nitazoxanide at dosages of 200 mg/kg and 400 mg/kg demonstrated substantial enhancements in body weight, with significant increases recorded on days 14 (P < 0.01), 21 (P < 0.01), and 28 (P < 0.001). By day 28, all treatment groups, including those receiving Prednisolone, exhibited significant and persistent increases in body weight (P < 0.001, F = 1.323). These findings highlight the severity of arthritis and suggest the promising potential of Nitazoxanide as a therapeutic agent for managing weight loss and inflammation associated with rheumatoid arthritis.

Total Arthritic Index

The total mean arthritic index was evaluated by individually assessing arthritic scores for the nose, ears, tail, hind paws, and fore paws. CFA-induced disease group rats exhibited the highest total mean arthritic index at 5.16 ± 0.51. In contrast, treatment with NTZ and prednisolone significantly reduced the total mean arthritic index. Specifically, NTZ at 400 mg/kg resulted in a score of 3.66 ± 0.30 (P < 0.05), pred-nisolone achieved 2.83 ± 0.35, NTZ at 200 mg/kg recorded 3.91 ± 0.42 (P < 0.05), and NTZ at 100 mg/kg showed 4.5 ± 0.47. The percentage reductions in the total mean arthritic index compared to CFA-induced disease group rats were calculated as 30.0% for NTZ (400 mg/kg), 45.1% for pred-nisolone, 24.2% for NTZ (200 mg/kg), and 12.7% for NTZ (100 mg/kg). These results highlight the efficacy of NTZ, particularly at 400 mg/kg, in mitigating inflammation in arthritic rats (Supplementary Table 2).

Behavioral Parameters

STAT-3, NF-κB, TNF-α and IL-6 Levels

In our comprehensive study, we looked closely at how different therapies affected important inflammatory indicators in RA. Significant variations were noted in the concentrations of several important cytokines. Notably, NF-κB activity was inhibited only in the NTZ 400 mg/kg treatment group, indicating the potent anti-inflammatory effect of NTZ in this context (P < 0.001, F = 3.218). This suggests NTZ’s potential to modulate NF-κB, a crucial transcription factor in inflammation. In addition, the NTZ 400 mg/kg, NTZ 200 mg/kg, and prednisolone-treated groups had significantly lower levels of TNF-α, a key pro-inflammatory cytokine (P < 0.001, F = 0.6944). This reduction highlights NTZ’s ability to suppress TNF-α, a key mediator in arthritis inflammation. Similarly, significant reductions in IL-6 levels were observed in both the NTZ 400 mg/kg and prednis-olone-treated groups (P < 0.001, F = 5.236), reinforcing NTZ’s capacity to regulate this inflammatory cytokine. Additionally, NTZ at the 400 mg/kg dose significantly decreased STAT-3 levels (P < 0.001, F = 2.299), suggesting that NTZ may influence inflammatory signalling pathways involving STAT-3, further enhancing its anti-inflammatory profile. Collectively, these findings indicate that NTZ, particularly at the 400 mg/kg dose, effectively modulates several key inflammatory cyto-kines, including IL-6, TNF-α, NF-κB, and STAT-3, positioning it as a promising candidate for targeting immune responses in rheumatoid arthritis, as shown in Figure 2.

Figure 2

NTZ effect on pro-inflammatory cytokines in CFA-induced arthritic rats. (a) interleukin (IL-6) levels, (b) TNF-alpha levels, (c) STAT3 levels, and (d) NF-κB levels. The values are expressed as mean ± SEM. Data were analysed by one-way ANOVA followed by a post-hoc Tukey HSD test. (^###P < 0.001 vs. Normal control, ***P < 0.001 vs. disease control, **P < 0.01 vs. disease control, and *P < 0.1 vs. disease control)

RBC, WBC, Platelets, and Hb Levels

The effect of NTZ on haematological parameters was assessed using blood specimens collected on the 28th day. Significant changes were observed in comparison to the disease control group, particularly in the groups treated with NTZ and the Prednisolone 10 mg/kg. In the prednisolonetreated group, red blood cell (RBC) levels showed a notable increase, indicating enhanced erythropoiesis. Similarly, the NTZ 400 mg/kg treatment group exhibited a marked rise in RBC levels, reflecting improved hematopoietic activity. White blood cell (WBC) counts demonstrated a significant reduction in the NTZ 400 mg/kg group compared to the disease control. This decrease suggests that NTZ may have anti-inflammatory properties, as lower WBC counts often correlate with a reduction in systemic inflammation. Haemoglobin (Hb) levels also increased significantly in the NTZ 400 mg/kg group (P < 0.001, F = 2.144), indicating a positive effect on oxygen-carrying capacity and erythropoiesis. Platelet counts showed a substantial increase in the NTZ 400 mg/kg group compared to the disease control, suggesting that NTZ may influence platelet production or turnover. This increase in platelets, along with changes in other haematological parameters, points toward NTZ’s role in modulating both inflammatory responses and haematological functions. These findings suggest that NTZ, particularly at a dose of 400 mg/kg, and prednisolone significantly impacted haematological parameters, reflecting their potential to regulate inflammation and improve hematopoietic balance. The observed effects underscore NTZ’s therapeutic potential in inflammatory conditions such as rheumatoid arthritis (P < 0.001 vs. disease control, P < 0.01 vs. disease control, P < 0.1 vs. disease control; F = 1.146), as illustrated in Figure 3.

Figure 3

NTZ effect on haematological parameters across all groups, showing changes in (a) RBC levels, (b) WBC levels, (c) Hb levels, and (d) platelet levels. The values are expressed as mean ± SEM. Data were analyzed by one-way ANOVA followed by a post-hoc Tukey HSD test. ***P < 0.001 vs. disease control, **P < 0.01 vs. disease control, and *P<0.1 vs. disease control)

Histopathology Examination

Histopathological analysis using hematoxylin-eosin staining of ankle joints revealed distinct differences among treatment groups, allowing for the assessment of various therapeutic interventions on the progression of arthritis in experimental groups. Comparisons were made to both disease and normal control groups. The normal control group exhibited no abnormalities, maintaining a typical joint structure with smooth sy-novial cells arranged in a single layer. In contrast, the disease control group showed characteristic signs of arthritis, including pannus formation, mild synovial fibrosis, mild to moderate synovial inflammation, and minimal to mild cartilage erosion, resulting in a histopathological grading of 2 to 3. The disease control group (Figure 4b) demonstrated reduced joint space, significant infiltration of inflammatory cells, and pan-nus formation, which are key features of RA. In contrast, the therapeutic interventions led to notable improvements in joint structure. The prednisolone-treated group (c) exhibited partial recovery, with improved joint space, suggesting a reduction in synovial inflammation and cartilage erosion, though not complete restoration of normal joint morphology. Joints treated with NTZ at 100–200 mg/kg (d-e) displayed mild edema and inflammatory cell infiltration, with some residual inflammation and pannus, but also indicated a reduction in the severity of the condition. Remarkably, joints treated with NTZ at 400 mg/kg (f) showed near-normal joint morphology, characterized by restored joint space and smooth synovial lining. The severe pannus formation and synovial inflammation observed in the disease control group were significantly reduced in this high-dose NTZ group, highlighting the therapeutic efficacy of NTZ, especially at the 400 mg/kg dosage. The analysis demonstrated the progression of arthritis in the disease control group, where pannus formation and joint deterioration were prominent. Following treatment with NTZ and prednisolone, improvements in joint structure were observed. NTZ, particularly at the higher dosage, showed significant potential in reducing synovial inflammation and preserving joint integrity, suggesting promising therapeutic benefits for arthritis management (Supplementary Table 3).

Figure 4

Histopathological examination of experimental groups (Images (a–f) represent (a) normal control, (b) disease group, (c) predniso-lone-treated group, (d) NTZ 100 mg/kg, (e) NTZ 200 mg/kg, and (f) NTZ 400 mg/kg.

Radiological Analysis

Radiological evaluation of hind limb joints in CFA-induced arthritic rats revealed significant pathological changes in the disease control group, including joint space narrowing, cartilage loss, bone erosion, soft-tissue oedema, and joint malformations, indicative of severe arthritis progression. In contrast, the Normal Control group displayed intact joint architecture without signs of damage. NTZ treatment showed dose-dependent improvements. The 100 mg/kg group exhibited partial preservation of joint structure and reduced inflammation, while the 200 mg/kg group demonstrated moderate protection against bone erosion and joint degeneration. The 400 mg/kg group showed substantial recovery, with near-complete preservation of joint integrity and minimal arthritis-related changes. The Prednisolone 10 mg/kg exhibited reduced radiological damage. These findings highlight NTZ’s therapeutic potential in alleviating joint degeneration, promoting cartilage repair, and protecting against arthritis-induced bone damage, as depicted in Figure 5, where arrows indicate areas of bone erosion and disturbances in joint integrity.

Figure 5

Radiological analysis of rat hind limbs across different groups. Blue arrows indicate regions affected by arthritis. NTZ: Nitazoxanide.