Sprague Dawley rats (SD-FNTac: SD and SD-MNTac: SD genotype) were used as the experimental animals in this study owing to their capability to exhibit excellent reproductive performance, making them suitable for generating timed pregnant females. The rats were maintained as an outbred closed colony throughout the study. Adult female Sprague Dawley rats weighing between 250 and 350 g were used in the experiments. The animals were fed with NIH #31M rodent diet and had ad libitum access to food and water. The rats were housed in standard laboratory conditions with a 22°C–25°C temperature and a 12:12 h light-dark cycle.

The Arthritis was induced in the rats on day 0 by administering a 0.1 mL hypodermic injection of Complete Freund’s Adjuvant (CFA) into the right hind footpad. The samples were then divided into specific experimental groups, and all drugs or the vehicle control (CED dissolved in saline with s10% Tween 80 were accurately administered through oral gavage. Group I served as the control, where rats received 0.1 mL of normal saline instead of CFA in the right hind paw. In Group II, rats were given 0.1 mL of CFA in the right hind paw to induce arthritis. Group III, the positive control, received piroxicam (10 mg/kg, PO) daily for 21 days following CFA injection. Rats in Group IV were treated with CED (50 mg/kg, PO) daily for 21 days after the CFA injection, while Group V received a higher dose of CED (100 mg/kg, PO) daily for 21 days following the CFA injection. Measurements of Paw volume was measured by a plethysmometer at 0, 8, 15, 22 and 28 days after induction.

In assessing nociceptive behavior, various parameters were evaluated to monitor the progression of arthritis. Paw volume and arthritis score were measured on days 0, 8^th,15^th, 22^nd and 28^th day after arthritis induction Figure 1. The thickness of the right hind paw was quantified using a calliper. The severity of arthritis, represented as the CED score, was categorized as follows: 0 indicated an insignificant change, 1 represented slight erythema or bulging of digits, 2 denoted medium swelling and erythema, 3 indicated high swelling and erythema, including the ankle, and 4 represented ankylosis and an inability to bend the ankle (Kumar et al., 2006).

To assess thermal hyperalgesia, a hot plate maintained at 55°C was used. The latency to the first sign of licking the paw or jumping was recorded to determine the nociceptive threshold to heat. Mechanical allodynia was evaluated using von Frey filaments. The rats were placed on an elevated metal wire floor in a 30 × 30 × 30 cm box, and the withdrawal threshold of the paw was determined using a series of von Frey filaments with varying forces. A significant response was considered when the rat responded to three out of five successive trials, indicating the presence of mechanical allodynia Table 1.

For the measurement of cytokines, the serum levels of interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α) were measured using Prostaglandin E2 (PG-E2) ELISA Kit (CSB-E07967r) following the manufacturer’s instructions (Nair et al., 2010). The measurement of oxidative stress markers included the levels of malondialdehyde (MDA), an end product of lipid peroxidation, and superoxide dismutase (SOD) activity were assessed in the serum to determine oxidative stress. Additionally, thiol concentration and glutathione peroxidase (GPX) activity were measured using appropriate assays.

The data were analysed using GraphPad Prism version 6.0. Descriptive statistics were used to present the data as mean ± SEM (Standard Error of the Mean). Behavioural parameters were analysed using a two-way analysis of variance (ANOVA) followed by tacchi test Bonferroni’s test for post hoc comparisons. Biochemical factors were analysed using one-way ANOVA with p < 0.05 considered statistically significant.

All experiments followed the guidelines and regulations for the care and use of laboratory animals. The study protocol was approved by the University of Lahore, Institutional Research Ethics Committee (IREC) before the initiation of the experiments via Trial Registry No: IREC-PHM-21-00565.

In the FCA-induced Arthritic rat model, CED (Compound E Derivative) was administered and compared to the illness group. The results revealed significant findings (***p < 0.001), as depicted in Figure 2. Over 28 days, CED exhibited a gradual increase in paw edema levels. At day 28, when compared to the positive control group (1.52 ± 0.05), both the CED derivative at 50 mg/kg (0.69 ± 0.02) and 100 mg/kg (0.79 ± 0.017), as well as piroxicam (0.74 ± 0.003), demonstrated inhibitory effects on paw edema. The paw edema progression was assessed, and the results were analyzed by the end of the 22nd day. On the 28th day, the difference in paw edema inhibition between the CFA-induced and positive control groups was 1.52 ± 0.05. X-rays of rats’ lower limbs were taken using a Siemens X-ray machine to assess joint variations. The analysis was based on joint space and swollen soft tissue. Figure 3 illustrates anteroposterior (AP) view of X-rays of rat paws on the 21st and 28th days for both the normal and arthritic rat groups. X-rays were also utilized to evaluate arthritis using piroxicam 10 mg/kg. X-ray photographs of the normal rat paw showed the presence of 8 Joint Spaces, 8 Soft tissues, and 3 metatarsal bones. On the other hand, CFA rats from the vehicle treatment group exhibited several deformities, including significant paw edema, osteophyte development, decreased joint spacing, and bone degradation.

The results in Table 2 provided evidence of the influence of CED on the decrease in paw edema levels at different time points (day 8, day 15, day 22, and day 28). The data indicated a statistically significant decrease in paw edema when treated with CED, with a p-value of 0.001, highlighting the potential therapeutic effects of the compound. Table 2 presents the results of the paw edema measurements as an influence of campesterol ester derivatives treatment at different time points (day 8, day 15, day 22, and day 28) in the FCA-induced arthritic rat model. The values are represented as mean ± standard error of the mean (SEM). The negative control group represents the untreated normal rats, while the arthritic control group represents rats with arthritis who did not receive any treatment. The positive control group was treated with piroxicam (10 mg/kg), a known anti-inflammatory drug for arthritis management. The two treatment groups received CED derivatives at 50 mg/kg and 100 mg/kg, respectively.

During the experiment conducted over the course of 28 days, the effects of various treatments on paw edema levels were investigated. On Day 8, the negative control group exhibited a paw edema level of 0.49 ± 0.006, while the arthritic control group showed a significantly higher level of 0.99 ± 0.004. The treatment groups administered with Piroxicam (10 mg/kg), CED derivative (50 mg/kg), and CED derivative (100 mg/kg), displayed paw edema levels of 0.89 ± 0.003, 0.98 ± 0.005, and 0.97 ± 0.004, respectively.

By Day 15, the negative control group showed a paw edema level of 0.46 ± 0.001, while the arthritic control group exhibited a substantially higher level of 1.18 ± 0.006. The Piroxicam-treated group showed a considerable reduction in paw edema to 0.85 ± 0.007 (***p < 0.001), and the CED derivative (50 mg/kg) and CED derivative (100 mg/kg) treated groups also displayed significant reductions to 0.88 ± 0.005 (***p < 0.001) and 0.87 ± 0.003 (***p < 0.001), respectively.

On Day 22, the negative control group showed a similar paw edema level of 0.45 ± 0.01, while the arthritic control group displayed a higher level of 1.38 ± 0.009. The Piroxicam-treated group exhibited a considerable reduction in paw edema to 0.84 ± 0.01 (***p < 0.001), and the CED derivative (50 mg/kg) and CED derivative (100 mg/kg) treated groups also showed substantial reductions to 0.87 ± 0.006 (***p < 0.001) and 0.88 ± 0.01 (***p < 0.001), respectively.

Finally, on Day 28, the negative control group displayed a paw edema level of 0.44 ± 0.01, whereas the arthritic control group had a significantly higher level of 1.48 ± 0.004. The Piroxicam-treated group showed a considerable reduction in paw edema to 0.72 ± 0.003 (***p < 0.001), while the CED derivative (50 mg/kg) treated group exhibited a significantly reduced level of 0.68 ± 0.002 (***p < 0.001). Similarly, the CED derivative (100 mg/kg) treated group displayed a considerable reduction in paw edema to 0.78 ± 0.001 (***p < 0.001). These findings indicate the potential anti-inflammatory effects of the CED derivative treatments in reducing paw edema levels in the arthritic model, comparable to or even better than the standard Piroxicam treatment.

The results demonstrate that the CED derivative at both 50 mg/kg and 100 mg/kg doses effectively reduced paw edema levels compared to the arthritic control group. The inhibitory effects were statistically significant and comparable to the positive control group treated with piroxicam. These findings suggest the potential therapeutic efficacy of the CED derivative in mitigating paw edema associated with arthritis in the rat model Figure 4.

The histopathology analysis was conducted on rat paw samples to study the effects of different treatments. The normal paw histopathology showed (Figure 5A) a typical healthy synovial membrane layer surrounded by concave synovial lining epithelium (Figure 5B). There was no evidence of inflammatory cell influx or abnormal tissue formations, such as pannus, were present. The normal paw histopathology showed a typical healthy synovial membrane layer surrounded by concave synovial lining epithelium. There was no evidence of inflammatory cell influx or abnormal tissue formations, such as pannus, were present. The paw was treated with piroxicam Figure 5C showed inflammation in the synovial membrane, but it was localized and characterized by clumps of inflammatory cells. Infamed-pauci regions were also present, and a substantial plaque of fibrovascular tissue was observed.

The results showed that CED attenuated various histopathological parameters associated with inflammation and arthritis. The examination of histopathological parameters revealed that CED compounds led to a reduction in inflammation in the rats compared to the control group (value: 2.6 ± 0.01), the rats treated with CED at a dose of 50 mg/kg showed reduced inflammation (value: 1.81 ± 0.004), as shown in Figure 6A. Similarly, the rats treated with CED at a dose of (Figure 6B) 100 mg/kg also exhibited reduced inflammation (value: 1.86 ± 0.005), indicating the significant anti-inflammatory effects of CED.

In the paw treated with CED at a dose of 50 mg/kg, inflammatory cells were in the synovial membrane. Additionally, several regions of inflamed pauci were identified. The inflammatory cells seemed to surround the synovial membrane in a concerted manner. In the paw treated with CED at a higher dose of 100 mg/kg, inflammatory cells were scattered around the synovial membrane. Most of the pauci-inflamed regions were identified within the synovial membranes. A detailed examination revealed no significant focus on pannus production.

Treatment with CAMP derivatives (CED) showed significant anti-inflammatory effects with CED at doses 50 mg\kg and 100 mg\kg in an experimental rat model of arthritis. CED at a dose of 100 mg/kg significantly reduced pannus formation (p < 0.001) and bone erosion compared to the arthritic control group (Figure 7). The symbol ### indicates comparison between arthritic and control groups, while *** shows treatment vs. arthritic control group. There is no plotting of the bar graph of the normal group due to taking of value as 0.

Figure 7 shows a reduction in infiltration of inflammatory cells, pannus formation, and bone erosion Histopathological analysis demonstrated the impact of CED compared to the positive control and standard drug (piroxicam). The vehicle control group had negligible values for inflammation (HP INF), pannus formation (HP PF), and bone erosion (HP BE). The positive control group had significantly higher values for these parameters. Piroxicam at 10 mg/kg reduced these values, and CED at doses of 50 mg/kg and 100 mg/kg exhibited even lower values for all parameters (Figure 7).

In the normal vehicle group, no pannus formation, cartilage erosion, ulceration erosion, or infiltration of mononuclear inflammatory cells was observed. The disease group displayed pannus formation, cartilage erosion, ulceration erosion, and infiltration of mononuclear inflammatory cells. Piroxicam treatment showed no pannus formation but mild cartilage erosion. No ulceration erosion or infiltrations of mononuclear inflammatory cells were observed in the piroxicam group, as well as in the CED (50 mg/kg) and CED (100 mg/kg) groups.

Overall, CED treatment at both 50 mg/kg and 100 mg/kg doses effectively reduced pannus formation, cartilage erosion, ulceration erosion, and infiltration of mononuclear inflammatory cells, similar to the standard treatment with piroxicam. These promising results suggest that CED could be a potential treatment option for arthritis.

The normal liver in rats appeared healthy and showed no signs of inflammation, edema, erosion, ulcer, or fibrosis. This is considered as the control group (vehicle control). In contrast, the liver of rats induced with CFA (Complete Freund’s Adjuvant) showed moderate cellular swelling in the hepatocytes, indicative of liver damage due to inflammation, as shown in Figure 8. The liver sections from rats treated with CED (Curcumin-loaded Elastic Vesicles) at doses of 50 mg/kg and 100 mg/kg showed no signs of inflammation, edema, erosion, ulcers, or fibrosis, similar to the control group. This suggests that CED treatment did not cause any observable liver damage or adverse effects Table 3. On the other hand, the liver sections from rats treated with standard piroxicam (a nonsteroidal anti-inflammatory drug) showed mild inflammation, edema, erosion, ulcers, or fibrosis, indicating some degree of liver injury due to piroxicam treatment.

The histopathological analysis of normal kidneys from the control group (vehicle control) showed a regular and healthy kidney structure. However, the kidneys of rats induced with CFA displayed distortions in the usual kidney structure, indicating kidney damage due to inflammation, as shown in Figure 9 the treatment with piroxicam and CED at 50 mg/kg and 100 mg/kg doses appeared to protect the kidneys from inflammation-induced damage. Post-treatment histopathology of kidneys with piroxicam showed protective variations in the renal structures, such as tubules and glomeruli, in a dose-dependent manner. Similarly, in the CED 100 mg/kg treated group, the shielding modifications were evident in the morphology of tubular epithelial cells and renal corpuscle/parietal epithelium. Additionally, the CED 50 mg/kg treated group (Table 4) showed kidney histology closely resembling the control group, indicating minimal kidney damage and protection from inflammation.

The effects of CED compounds on mRNA expression levels of pro-inflammatory and anti-inflammatory markers were evaluated in an arthritic rat model, and the results were compared with a positive control group treated with piroxicam and a vehicle control group. The findings demonstrated the downregulation of pro-inflammatory markers TNF-α, IL-1ß, IL-6, NF-KB, MMP3, COX-I, COX-II and upregulation of anti-inflammatory markers IL-4 following CED treatment. TNF-α, a pro-inflammatory cytokine, showed significantly reduced mRNA expression levels in both CED treatment groups (50 mg/kg and 100 mg/kg) compared to the vehicle control and piroxicam-treated groups (p < 0.001). The mean values for TNF-α were (33.53 ± 0.12) for the vehicle control (37.47 ± 0.11) for CED 50 mg/kg, and (36.45 ± 0.11) for CED 100 mg/kg. The graph (Figure 10A) visually depicted the downregulation of TNF-α mRNA expression.

Similarly, the expression levels of NF-κB, a transcription factor involved in inflammatory responses, were significantly reduced by CED treatment. In the piroxicam-treated group, NF-κB expression was (37.53 ± 0.12), while CED 50 mg/kg showed (39.63 ± 0.23) and CED 100 mg/kg showed (38.45 ± 0.18). The reduction in NF-κB expression levels was evident in the treated groups compared to the vehicle control and was illustrated in the graph (Figure 10B).

Moreover, CED treatment resulted in a significant downregulation of other inflammatory markers. The mRNA expression levels of IL-6 were decreased (Figure 10C) in both CED treatment groups (50 mg/kg and 100 mg/kg) in comparison to the vehicle control and piroxicam-treated groups. The mean values for IL-6 were (34.47 ± 0.11) for CED 50 mg/kg and (35.47 ± 0.10) for CED 100 mg/kg, indicating reduced inflammatory activity. In contrast, the piroxicam-treated group showed a mean value of (32.53 ± 0.12).

Furthermore, the mRNA expression levels of IL-1ß, COX-1 and COX-2 were significantly downregulated by CED treatment (Figures 10D–F). In the piroxicam-treated group, COX-1 expression was (36.53 ± 0.12), whereas CED 50 mg/kg showed (35.47 ± 0.11) and CED 100 mg/kg showed (36.47 ± 0.10). Similarly, for COX-2, the piroxicam-treated group had a mean value of (35.53 ± 0.12), and the CED treatment groups showed (36.47 ± 0.11) for 50 mg/kg and (35.47 ± 0.12) for 100 mg/kg. Additionally, the expression levels of IL-4 were also significantly upregulated in the Piroxicam and CED treatment groups compared to the arthritic control group (Figure 10G). For IL-4, the piroxicam-treated group exhibited a mean value of (29.53 ± 0.12), while CED 50 mg/kg and CED 100 mg/kg showed (33.47 ± 0.11) and (32.42 ± 0.09), respectively.

In conclusion, the results from this study indicated that CED compounds effectively downregulated the mRNA expression levels of pro-inflammatory markers, such as TNF-α, NF-κB, IL-6, COX-I, COX-II and IL-1ß, while also upregulating some anti-inflammatory markers like IL4 that are shown in (Figure 10G) In the treatment group as compared to positive control arthritic group. These findings suggest the potential of CED as a promising anti-inflammatory agent warranting further investigation. The sign of ### shows a comparison between the arthritic control group and the vehicle group in Figures. A, B, C, D, E and F). While ***shows comparision between the treatment group and the arthritic control group.

The treatment with CED affected hematological parameters in the arthritic rats. The arthritic control group showed a significant reduction in red blood cell (RBC) count, an increase in erythrocyte sedimentation rate (ESR), and a decrease in hemoglobin (Hb) content compared to the vehicle control group Figure 11. However, treatment with piroxicam and CED at 50 mg/kg and 100 mg/kg doses resulted in significant improvements in these hematological parameters. The RBC count increased, ESR levels decreased, and Hb content increased, indicating a positive effect of CED on the blood parameters in arthritic rats. CED treatment also normalized the platelet count compared to the arthritic control group. Moreover, CED treatment also affected biochemical parameters in the arthritic rats (Figure 12).The levels of urea, creatinine, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were all brought closer to normal levels by CED treatment at both 50 mg/kg and 100 mg/kg doses. This indicates that CED positively impacted kidney and liver function, as these biochemical parameters indicate kidney and liver health. The levels of rheumatoid factor, which indicates autoimmune activity, were also significantly reduced by CED treatment. The sign denoted by### shows comparision between the arthritic group and the vehicle control group while*** shows comparision between the treatment group and the arthritic control group.