Introduction: In rheumatoid arthritis (RA), the autoimmune response against articular tissues is the first mechanism proposed for the pathogenesis of the disease (1). It has been suggested that genetic and environmental factors could develop the disease (2,3). Environmental factors including infections, diet, and lifestyle may activate the genetic drivers of RA to stimulate the innate and adaptive arms of the immune system in order to produce inflammatory mediators namely tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-6 (4). It has been declared that while environmental factors could propel innate immune cells to the articular space, genetic abnormalities affect T-lymphocytes repertoire selection and antigen presentation, which could shift the balance between the osteoblasts and osteoclasts, leading to bone erosion and joint deformity (5). These two important symptoms influence a patient’s quality of life (5). The results of previous study revealed that the prevalence of depressive symptoms and anxiety resulting from physical disability was high in RA patients, which could, in turn, influence their social lives (6). Although numerous decades have passed since the first description of RA and a progress has been made in the understanding of the disease pathogenesis, the diagnosis of this complication and the treatment strategies for the patients have not run into any changes. Nutrition bio-shield (NBS) supplement is a herbal dietary supplement derived from wheat grains (NBS Organic Company, Turkey). It has been stated that the wheat germ contains considerable amounts of tocopherol, policosanol, phytosterol, riboflavin, thiamin, and niacin (7). Furthermore, in another study, it was found that
the NBS supplement was able to stimulate the immune system upon changing the balance of neutrophils to lymphocytes (8). Since 2010, the European League Against Rheumatism and the American College of Rheumatology have elected rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), and C-reactive
protein (CRP) for serological diagnosis of RA (9). By making some modifications to the treatment, the treatment of this diseases begins shortly after its detection; to this end, immunosuppressive agents like non-steroidal antiinflammatory drugs and glucocorticoids are used (10). Recently, TNF blockers have found their way into the treatment protocol of RA, as either a single agent or in combination with immunosuppressive drugs. Despite their impressive success, several side effects, like the high risk of infections, have restricted the clinical usage of such agents in RA patients (11). IL-6 inhibitors, rituximab, and abatacept (T cell inhibitor) are other therapeutic agents that have been under evaluation in pre-clinical
studies (12). In addition to these synthetic drugs, herbal and natural medicines also consist a fertile ground for treating RA (13,14). Since many of these therapies have proven efficient in ameliorating the disease symptoms without serious toxicity, much attention has been drawn to this field so far. The current study attempts to assess the NBS supplement’s therapeutic potential in the case of
RA-induced rats.
Methods: Animal and Ethical Statements
Twenty-five male Wistar rats with the estimated weights of 200-250 g were purchased from Institute of Medicinal Plants, ACECR, Karaj, Iran. They were kept in special cages at 22-25ο C and provided with food and water in 12 hours of light and darkness. They were transferred to the laboratory one week before the experiment to adapt them to the new conditions. The protocol was investigated
and confirmed by the Institutional Review Board of Islamic Azad University of Mashhad (IR.IAU.MSHD.REC.1398.233).
Induction of Rheumatoid Arthritis (RA) in Rats
To induce the RA model, rats received xylazine ketamine for anesthesia and then, 0.2 cc Freud’s complete adjuvant (FCA) was injected into their knees (15). It should be noted that one group of animals consisting of 5 rats was only injected with 10 mg of normal saline and it remained as the control group. RA-induced rats were then divided into four groups (each having five rats) for further analysis.
Treatment of RA-Induced Rats with the NBS Supplement
In order to measure the therapeutic value of the supplement, three groups of RA-induced rats received oral treatment containing different NBS supplement concentrations (12.5, 25, and 50 mg/kg) in the form of gavage for 30 days after induction of RA. The ingredients of the NBS supplement are shown in Table 1. After 30 days, xylazine ketamine was used to anesthetize the rats and 3-5
mL of blood was sampled from their hearts to assess the serum levels of ESR using the Westergren method (16), CRP, and RF (each evaluated three times to ensure the results are exact). Of note, one of the rat groups did not receive any supplement concentrations and was considered as the negative control.
Statistical Analysis
The serological tests were performed in triplicate and the outcome was yielded in the form of the mean ± standard deviation. In addition, the KolmogorovSmirnov test was adopted to confirm the normality of data distribution. The One-Way ANOVA test via IBMSPSS software was also employed to gauge the data significance with a favorable probability level of p<0.05. The Tukey Post-hoc test was used to perform Post-hoc analysis with One-Way ANOVA
Results: To evaluate whether the application of the NBSsupplement treatment to RA-induced rats could ameliorate the inflammatory responses, we first evolved an RA model in rats by injecting FCA into their knees. Analysis of their blood samples revealed that the number of inflammatory parameters such as ESR, CRP, and RF increased in the RA-induced rats in comparison to the control group (FCA-untreated rats) (Table 2). Then, RA-induced rats were treated by the NBS supplement (12.5, 25, and 50 mg/kg) for a month. Our results demonstrated that the NBS supplement could not only robustly diminish the levels of ESR and CRP, but also significantly reduce the levels of RF in the treated rats (Table 2). Although the ESR and CRP levels of rats in group 1 (treated with 12.5 mg/kg of the NBS) reduced in comparison with the negative control group, they remained higher than normal (Figure 1). Maximum effect was observed in the group treated by the 50 mg/kg NBS supplement.
The conducted ANOVA test results point to a statistically significant difference among ESR, CRP, and RF levels of groups [(ESR: F(4.20)=88.92, p-value=0.00), (CRP: F(4.20)=121.88, p-value=0.00), (RF: F(4.20)=147.71, p-value=0.000]. ESR, CRP, and RF of RA-induced mice were statistically significantly lower after treatment by any dosage of the NBS supplement compared to the negative control (untreated) group.
Mean ESR in group 3 was statistically considerably lower than that in groups 1 and 2 (p<0.001 and p=0.002, respectively). However, no statistically significant
difference was found between group 1 (12.5 mg/kg of the NBS) and group 2 (25 mg/kg of the NBS) (p=0.172). The mean CRP in groups 2 and 3 was statistically and significantly lower than that in group 1 (p=0.001 and p=0.003, respectively). No statistically significant difference was found between group 2 (25 mg/kg of the
NBS) and group 3 (50 mg/kg of the NBS) (p=0.997). Finally, RF level was statistically significantly lower in group 3 than that in groups 1 and 2 (p<0.001 and p=0.001, respectively). However, no noticeable difference between groups 1 and 2 was observed (p=0.279). All results of the ANOVA and the Tukey Post-hoc tests are shown in Table 3.
Conclusion: In light of obtained findings and based on their interpretation, this study proposed NBS supplement as an herbal dietary supplement that could restore the blood RF of the studied rats to a normal level at any applied dosages. In addition, the supplement had significant restoring effect on the ESR and CRP levels at higher concentrations, especially at 50 mg/kg. To reduce both inflammatory markers and RF, the administration of 50 mg/kg of the NBS supplement managed to produce better anti-inflammatory outcome, meaning that it could be integrated into the treatment protocol of RA. Yet, further analysis is required to investigate its therapeutic potential. For future research, it is suggested that more clinical and histopathological data before and after the intervention of the NBS may open up a new frontier to ensure a better understanding of its therapeutic effects on the RA disease.