Evaluation of Immunomodulatory and Antiarthritic Potential of Trigonella gharuensis Extracts

The genus of Trigonella has long been used for the treatment of arthritis and inflammatory disorders. This study was aimed to investigate the immunomodulatory activities of ethanol and n-hexane extracts of T. gharuensis in the rat model of rheumatoid arthritis. Freund's complete adjuvant (FCA) model was used to induce arthritis in rats. Arthritis was induced on day 0, while treatment which was started on day 8 continued for twenty days. Arthritic development and paw edema were determined using an arthritic scoring index and plethysmometer, respectively. Histopathology was evaluated using H&E staining. RNA extraction, reverse transcription, and polymerase chain reaction (RT-PCR) were performed to determine expression levels of proinflammatory markers such as TNF-α, NF-ĸB, IL-6, IL-1β, COX2, and anti-inflammatory cytokine IL-4. Prostaglandin E2 level (PGE2) was evaluated using ELISA. Blood analysis and biochemical parameters were also determined. The significance level was set as P < 0.05. Treatment with extracts reduced paw edema, arthritic progression, and histopathological parameters. Expression levels of abovementioned proinflammatory cytokines and COX2 were downregulated, while IL-4 was upregulated. PGE2 levels were found reduced with extract treatment. Blood parameters were nearly normalized in treatment groups. Extract treatment did not alter biochemical parameters. Both extracts had effects comparable with piroxicam. In conclusion, extracts of T. gharuensis ameliorated experimentally induced arthritis that may be ascribed to its immunomodulatory effects.


Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory and autoimmune disorder that presents with bone erosion, cartilage damage, and inflammation. Major symptoms include varying degrees of joint stiffness, pain, and swelling, while systemic symptoms include fever, fatigue, and weight loss [1].
Various cytokines and proinflammatory mediators are involved in the pathogenesis of RA. ese cytokines are released by activated macrophages and fibroblasts. Tumor necrosis factor (TNF-α) stimulates IL-6 which is responsible for activation of chondrocytes and fibroblasts in articular cartilage. is leads to erosion of collagen and proteoglycans resulting in joint destruction. IL-1β in diseased synovial membrane is considered responsible for pannus formation and bone erosion. IL-6 stimulates production of nuclear factor kappaB (NF-ĸB) which, in turn, activates osteoclasts, abnormal apoptosis, and proliferation of synovial cells [2]. Likewise, augmented levels of COX2 activate PGE2 production which leads to angiogenesis and articular cartilage destruction along with pain and inflammation [3].
Nonsteroidal anti-inflammatory drugs (NSAID) and opiates have been extensively used for the symptomatic treatment of arthritis [4]. Adverse effects of NSAIDs include gastric perforation and bleeding due to reduced prostaglandin production [5]. Continued use of opiates poses a serious risk of respiratory depression, dependence, and tolerance [6]. Corticosteroids are associated with peptic ulcer, osteoporosis, precipitation of diabetes, and higher susceptibility to infections due to immunosuppressive properties [7]. Other drugs used to treat autoimmune disorders, such as DMARDs and cytokines antagonist, have been associated with increased susceptibility to infections seen in the patients [8].
Due to many adverse effects of synthetic drugs, modern research focuses on the use of medicinal plants for treatment of inflammation and arthritis due to their easy availability and lower cost value [9]. Trigonella gharuensis is an herb of worldwide distribution [10]. It belongs to the second largest family of flowering plants, i.e., fabeaceae [11]. Our previous study has established anti-inflammatory properties of T. gharuensis using different paw edema models of acute inflammation such as carrageenan, histamine, serotonininduced paw edema models, and xylene-induced ear edema model. Moreover, GCMS analysis in our study showed that both extracts possessed considerable constituents responsible for anti-inflammatory and antioxidant properties [12]. Our current study focuses on antiarthritic potential of T. gharuensis using FCA-induced chronic model of inflammation in arthritic rats.

Plant Collection and
Identification. T. gharuensis was collected from Quetta district of Balochistan. It was identified by Department of Botany, University of Balochistan (UOB), Quetta, Pakistan. e voucher specimen (TG-RBT-05) was kept in the herbarium of university [12].

Preparation of Extracts.
Herb was dried under shade. It was then chopped followed by grinding. Maceration was done by soaking powder into ethanol and n-hexane, respectively. e mixtures were kept at 25°C. e mixtures were filtered and then concentrated at 37°C in a rotary evaporator (IKA Germany) under reduced pressure. After that, extracts were dried in an incubator at 40°C. e EETG (ethanolic extract of T. gharuensis) and NHTG (n-hexane extract of T. gharuensis) were dissolved in 1% Tween 80 before administration.

Test Animals.
Sprague Dawley rats (6-8 weeks old) were placed in animal house of e University of Lahore, Lahore. ey were acclimatized with environment for one week under controlled humidity conditions (60-70%) and temperature 25°C ± 2, respectively. Food and water access was freely provided. Light and dark cycles were maintained. Approval from the Institutional Research Ethics Committee, e University of Lahore (IREC-2017-23), was taken for the experiment.
2.5. Arthritic Score Measurement. Different parameters such as inflammation, redness, and erythema were observed on different day intervals of 8, 15, 22, and 28 using macroscopic criteria. Score 0 was given to normal, score 1 to minimal, score 2 to mild, score 3 to moderate, and score 4 to severe changes [16].
2.6. Paw Volume Assessment. Digital water displacement plethysmometer was used to measure paw edema of all groups at days 0, 8, 15, 22, and 28.

Histopathological Evaluation.
On day 28, all rats were sacrificed. Ankle joints were cut longitudinally and placed in formalin (10%) for fixation. Formic acid was used for decalcification of samples. Tissues were cut into thin slices, and paraffin wax was used for embedding the tissues. H&E (hematoxylin and eosin) staining was used. e slides were examined by a blinded histopathologist for presence of pannus formation, bone erosion, and inflammation. Scoring criteria were followed as mentioned previously [15].

Evaluation of Hematological
Parameters. Blood samples were collected through intracardiac puncture. Automated hematology analyzer (Sysmex XT-1800i) was used to evaluate hemoglobin content and levels of RBCs, WBCs, and platelets.

Statistical Analysis.
All values were expressed as mean ± SEM. Data were analyzed using Graph Pad Prism (v 6.0). One-way ANOVA followed by Tukey's post hoc test was used for comparison. Significance level was observed as P < 0.05.

T. gharuensis Inhibited Arthritic Development.
e arthritic progression was induced by using FCA, and it was measured through macroscopic criteria. e increased trend of arthritic development was continued in the diseased control group because no treatment was provided to this group. Table 2 shows that (P < 0.001) arthritic score was reduced at days 15, 22, and 28, respectively, with extract treatment as compared to the positive control group. e piroxicam also significantly reduced (P < 0.001) arthritic score at different day intervals which is comparable to EETG and NHTG groups.

T. gharuensis Prevented Paw Edema.
Remarkable edema was observed in the FCA-induced model, and it was measured through digital water plethysmometer on different day intervals such as 15, 22, and 28. e values of vehicle control group were considered zero because there was no induction of disease in this group. Table 3 shows increased trend in inflammation, and score was continued until the 28th day of this experimental study in the positive control group. EETG, NHTG, and piroxicam treatment significantly (P < 0.001) reduced paw edema when compared with the arthritic control group. e results of extracts treatment were comparable to the piroxicam group.

T. gharuensis Significantly Reduced Histopathological
Parameters. EETG, NHTG, and piroxicam showed significant inhibition of inflammation, bone erosion, and pannus formation in comparison to the arthritic control group ( Table 4). H&E staining picture revealed that aggregates of inflammatory cells presented in the arthritic control group were markedly reduced in piroxicam-and extract-treated groups.

T. gharuensis Downregulated Proinflammatory and Upregulated Anti-Inflammatory Cytokines.
e collected blood sample on the 28th day was processed for mRNA expression levels. Table 5 shows that significant levels of proinflammatory markers such as TNF-α, IL-6, IL-1β, NF-ĸB, and COX2 were increased in the positive control group. e augmented levels of these cytokines were markedly reduced (P < 0.001) in EETG and NHTG extract-and piroxicam-treated groups in comparison to the positive control group. Levels of anti-inflammatory cytokines such as IL-4 decreased in the positive control group. IL-4 levels were increased in extract-and piroxicam-treated group in contrast to the positive control group.

T. gharuensis Significantly Reduced PGE2 Levels.
Increased (P < 0.001) serum PGE2 levels were noticed in the arthritic control group as compared to the vehicle control group. Significant reduction (P < 0.001) in PGE2 levels was observed in piroxicam-, EETG-, and NHTG-treated groups, as presented in Figure 1.

T. gharuensis Modulated Hematological Parameters.
e reduction in hematological parameters, such as RBC and Hb levels, were seen in the positive control group. ese parameters were nearly normalized in treatment groups as compared to the positive control group. Similarly, elevated WBC and platelets levels were observed in the arthritic control groups which were nearly normalized after treatment with extracts and the reference drug in contrast to the positive control group (Table 6).

Discussion
Medicinal plants have proved to be safer and cheaper in comparison to synthetic medicines for the treatment of arthritis. Rheumatoid arthritis is characterized by bone damage, deformity, hyperplasia, pannus formation, and inflammation in the joints [20,21]. e preferred model used for this study is FCA-induced model due to its similarities with arthritic disorder seen in human. Ethanolic and n-hexane extracts of T. gharuensis extracts are shown to reduce paw edema and arthritic progression. ese results are further supported by the histopathological investigations which showed clear amelioration of hallmarks of RA.
After FCA immunization, different cytokines are released by activated macrophages and monocytes which, in turn, release various cytokines such as TNF-α, IL-6, and IL-1β. ese cytokines aggravate inflammation, bone erosion, and cartilage destruction in joint tissues [22]. ese cytokines also stimulate NF-ĸB, a transcriptional factor, which promotes bone resorption by activating osteoclasts and proliferation of synovial cells in joints.
is factor also worsens the symptoms of RA by favoring 1 response [17]. Hence, NF-ĸB inhibitors might be useful therapeutically and efficacious in ameliorating symptoms of RA. Our study shows marked decreased in the levels of TNF-α, IL-6, NF-ĸB, and IL-1β in extract-treated groups in comparison to the arthritic group. e COX2 and prostaglandins are crucial mediators which are involved in pain and swelling. TNF-α and IL-1β increase levels of COX2 and PGE2 in activated synovial cells. COX2 also activates process of bone erosion in juxta-articular cartilage. e extracts in current study reduced the levels of COX2 and PGE2 which might be responsible for the amelioration of inflammation and cartilage damage found in the study.
Immunomodulatory potential of T. gharuensis is further validated by augmented levels of IL-4 in EETG-, NHTG-, and piroxicam-treated groups in contrast to the arthritic group. One of the reasons for the development of RA is imbalance between the levels of proinflammatory and anti-inflammatory cytokines. Elevation in the levels of proinflammatory cytokines or reduction in the levels of anti-inflammatory cytokines might lead to imbalance and result in development of RA. IL-4 mediates an anti-inflammatory response in RA. IL-4 inhibits 1 response and favors 2 immunomodulatory cells. erapy with recombinant IL-4 has proved to inhibit cytokine production [17,23]. is study showed significant elevation in the expression levels of IL-4 in extract-treated groups, probably in an attempt to ameliorate 1 response.
Arthritic patients were found to have altered hematological picture with increased levels of WBCs and platelets, while reduced RBC count and Hb levels. e anemia may be attributed to inadequate erythropoiesis by the bone marrow and disorganized deposition of iron in synovial tissue and reticuloendothelial system [24,25]. Increased WBC and platelet counts may be ascribed to overactive immune response. Treatment with T. gharuensis extracts led to normalisation of these values in the blood picture. In order to determine the safety of the plant extracts, biochemical parameters such as AST, ALT, urea, and creatinine were measured. e increased levels of these markers are indicative of liver injury and kidney dysfunction, respectively. e results showed no significant differences among all the groups which deemed that extracts were safe to use. e inferences of current study are in line with the study of Shabbir et al. [18].
Previously, we published the identification of phytochemical constituents in both plant extracts using GCMS analysis. e data showed the presence of different antiinflammatory compounds in the extracts, e.g., ethylpalmitate, phytol, n-hexadecanoic acid, ethyl linoleate, nanocosane, and coumarin. Presence of these constituents might be responsible for anti-inflammatory activities of both the extracts [12]. EETG, ethanolic extract of T. gharuensis (400 mg/kg); NHTG, n-hexane extract of T. gharuensis (400 mg/kg); piroxicam (10 mg/kg). Values were denoted as mean ± SEM. * * P < 0.01 and * * * P < 0.001 when compared with the arthritic control group. ###Difference between vehicle control group and positive control group. Evidence-Based Complementary and Alternative Medicine 5

Conclusion
Amelioration of joint inflammation confirmed that T. gharuensis extracts possessed significant immunomodulatory and antiarthritic potential in the FCA-induced arthritic rat model. T. gharuensis extracts significantly reduced paw edema, arthritic progression, and histopathological parameters. e attenuation of RA might be ascribed to downregulation of proinflammatory markers such as TNFα, IL-1β, IL-6, NF-ĸB, and COX2 and upregulation of antiinflammatory IL-4. Moreover, PGE2 levels were also found reduced after treatment with plant extracts.

Data Availability
Data used to support the findings of this study are available on request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.