Amelioration of rheumatoid arthritis in rats by 4-allylanisole through modulation of inflammatory mediator

doi:10.21203/rs.3.rs-3235676/v1

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Amelioration of rheumatoid arthritis in rats by 4-allylanisole through modulation of inflammatory mediator

https://doi.org/10.21203/rs.3.rs-3235676/v1

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Background 4-Allylanisole, also known as Estragole (EST), is an important chemical constituent of many aromatic plants found in nature and possesses anti-inflammatory properties. Aim: Arthritic rat model induced by Freund's complete adjuvant was used to determine the anti-arthritic potential of EST in present study.

Method: It was given to three groups which were administered low dose (10 mg/kg b.w.), medium dose (30 mg/kg b.w.), and of high dose (60 mg/kg b.w.). Piroxicam was used as reference drug. Arthritic score was evaluated macroscopically and through histopathological evaluation, while paw edema was evaluated using Vernier caliper. The RT-qPCR, (Real time reverse transcription polymerase chain reaction) was used to measure expression levels of pro-inflammatory mediators including interleukins (-1β) and (-6) and tumor necrosis factor. Hematological indices i.e. differential leukocyte count (DLC) and total leukocyte count (TLC), along with biochemical indices were also determined.

Result: All evaluated hematological, biochemical, and histopathological parameter, as well as, mRNA expression levels of pro-inflammatory cytokines were found raised in disease control group. 4-Allylanisole significantly attenuated development of arthritis and paw edema. These results were validated by histopathological evaluation which also demonstrated amelioration of arthritis in treated groups. DLC and TLC were also nearly normalized in treatment groups. 4-allyanisole significantly attenuated the raised levels of AST, ALT, urea and creatinine. RT-qPCR analysis showed that treatment with 4-allylanisole significantly reduced TNF-α, IL-1β, and IL-6 levels.

Conclusion: The results concluded that the phytochemical 4-allylanisole possesses significant anti-arthritic activity which may be attributed to down-regulation of TNF-α, IL-1β, and IL-6 levels.

4-Allylanisole

Estragole

Rheumatoid arthritis

Pro-inflammatory cytokines

Anti-arthritic

A chronic inflammatory autoimmune illness, rheumatoid arthritis (RA), affects around 1% of the global population. Joint stiffness, discomfort, synovitis, cartilage destruction, bone erosion, pannus development, and decreased functioning are all indications of arthritis [1]. Chronic inflammation caused by RA increases the risk of cardiovascular disease and other systemic illnesses, making it the 31st biggest cause of years year lived with disability worldwide [2]. As a result of these processes, joint swelling, deformity, and systemic inflammation develops [3]. Joint inflammation in rheumatoid arthritis causes cartilage and bone deterioration, disability, and sometimes even systemic consequences with increased morbidity and death. The causative factors of RA includes several variables such as genetics, the environment, and autoimmune [4]. Damage to synovia, articular cartilage, and subchondral bone can be traced back to the immune system's activation due to the aforementioned factors. This activation leads to autoantigen presentation, antigen-specific T- and B-cell activation, and the production of aberrant inflammatory cytokines [5]. Rheumatoid arthritis causes synovitis to grow and damage cartilage and bone in numerous joints. In RA, a T cell-mediated immune response drives cytokine release and antibody production, causing joint degeneration. Specifically, TNF-𝛼 causes IL-1β and IL-6 to be generated [6].

As a result, pro-inflammatory cytokines are crucial in the development of arthritis (especially TNF-α, IL-1β and IL-6) [7], while inhibitors of these cytokines are effective in controlling chronic inflammation [8]. Increases in tumors necrosis factor cause regional heat, swelling, discomfort, and redness. Systemic increase in TNF-α reduces cardiac output, cause microvascular thrombosis, and cause capillary leakage syndrome. TNF-α promotes and maintains inflammation by boosting inflammatory cytokines like interleukin 1β [9].

During an invasion, TNF-α is required for full-fledged inflammation to manifest itself, and TNF-α activity often decreases during self-limited inflammation. In a normal situation, the acute inflammatory response is restricted by highly conserved, counter-regulatory mechanisms that stop inflammatory mediators from entering the bloodstream. TNF-α receptor fragments, which bind and counteract its inflammatory and potentially hazardous activities, are released by activated immunologically competent cells [10].

Bone destruction and articular cartilage damage indicated by erosions and narrowing of joint space in RA. Osteoclasts are key cells involved in stimulating erosions in inflammatory arthritis [11]. Osteoclast recruitment increases by IL-6 acting on hematopoietic stem cells from the granulocyte-macrophage lineage [12]. The destructive effects of IL-6 and their stimulation of receptor activator of NF-kB ligand, receptor activator of NF-kB, and osteoprotegerin (OPG) were studied. Where IL6 enhanced the expression of RANKL and induced bone resorption [13].

Methotrexate, hydroxychloroquine, and sulfasalazine are examples of disease-modifying anti-rheumatic medications (DMARDs) used in the treatment of RA [14]. However, these medications come with the risk of major side effects such nausea, vomiting, diarrhea, pneumonitis, and changes in liver function. Biological medicines, like antibodies against IL-1β, IL-6, and TNF-α, as well as, soluble receptors have lately emerged as important tools in the fight against RA [15]. As of yet, there is scarcity of knowledge regarding the long-term impacts and advantages of these drugs, despite the fact that they can reduce inflammation and joint damage [16]. These medications don't always work and often come with dangerous side effects including new infections or an elevated chance of developing cancer [14]. In addition, immunosuppressive and cytotoxic medicines (such as leflunomide, azathioprine, cyclosporine, and cyclophosphamide etc.) may be administered, however these come with their own set of potentially fatal adverse effects [17].

Essential oils are volatile molecules that ensure the survival of plants within their environments due to their crucial function in defense against microbes and predators. Terpene chemicals and phenylpropanoids make up the bulk of essential oils' chemical makeup, however their relative abundance might shift depending on where the oil was harvested and how it was extracted. These ephemeral components are stored in unique plant anatomical structures across all plant species. 4-Allylanisole, also known as Estragole, is a phenyl propene found in nature with anti-inflammatory and antioxidant properties. It is found in turpentine (pine oil), anise, fennel, bay, tarragon, and basil, among other trees and plants. 4-Allylanisole's anti-inflammatory anti-oxidant, anti-convulsant, anesthetics, vasoactive, bradycardic, and antibacterial activities are all well-documented [18]. The aromatic plants contain many essential oils. 4-allylanisole is an important chemical constituent of theses aromatic plants e.g. Ocimumbasilicum (Lamiaceae), Artemisia dracunculus (Asteraceae), Pimpinellaanisum (Apiaceae), Illiciumanisatum (Illiciaceae), Foeniculum vulgare (Apiaceae) [19] and Croton zehntneriPax et Hoffm (Euphorbiaceae) [20]. The essential oils obtained from different plants such as Feniculum vulgare, (at fully mature and flowering stage) and Oscimum basilicum (green and purple varities), Rosmarinus officinalis, Salvia officinalis, is noted to have 4-allylanisole as major component. 4-allylanisole was separated and tested for its antiviral activity against Herpes simplex type-1. Most of the tested compounds of plants and the components of essential oils manifested good anti-viral, antibacterial and antifungal effects [21]. A lot of work is done on 4-allylanisole recently showing its potential anti-inflammatory effects on a variety of diseases and conditions, including inflammatory ulcers and carrageenan-induced paw edema. Therefore, it was hypothesized that 4-allylanisole may also modulate levels of pro-inflammatory markers in animal model of rheumatoid arthritis [22]. 4-Allylanisole has been shown to possess antioxidant, anti-inflammatory, immunomodulatory, tranquilizing, anticonvulsant, antimicrobial, and anesthetic activity using in vivo and in vitro models [23]. The present study was carried out to evaluate the anti-arthritic activity of the 4-allylanisole using (Freund’s Complete Adjuvant) FCA-induced arthritic rat model.

2.1. Phytochemical used

4-Allylanisole was purchased commercially form Glentham Life Sciences from UK. Glentham code for product is GK6464.

2.2. Animal model and ethical approval

6–8 weeks old albino rats of either sex, 150–250g in weight, were retained at animal house, University of veterinary and animal sciences, Lahore (UVAS). Standard pellet diet ad libitum and water were provided to rats daily. The standard conditions including 12 h dark/light cycles, humidity (60–70%), and temperature (28 ± 2°C) were maintained where animals were kept for complete duration of study. Before the start of an experiment all the animals were acclimatized to the environment for a duration of one week. Approval for all the experiments was taken by Research Ethics Institutional Review Board, LCWU, Lahore (ORIC/LCWU/22–298)[24].

2.3. Induction of Rheumatoid Arthritis

Arthritic induction in sub plantar region was performed by subcutaneous injection of Freund’s complete adjuvant 0.1 ml (Dried and killed Mycobacterium tuberculosis was suspended in mineral oi) in left hind paw of rats at day 0 by keeping right hind paw as control [25]. Treatment was started on 8th day of arthritic induction. All treatments were given for 20 consecutive days [26]. Arthritis was evaluated by recording several parameters clinically. Vernier scale was used to measure paw edema on day 0, 8, 13, 18, 23 and 28. Percentage inhibition was calculated at different days of equal gap of 5 days.

2.4. Animals treatment

Animals were alienated into 6 groups. Each group contained six rats. Total 36 albino rats were used for experimental purpose. Group-I: Negative control (NC); A group of healthy six rats that was only given tween 80 (5%) which is used as vehicle (day 8–28). 5ml tween 80 (Polysorbate 80) was mixed with 95ml water to make 5% solution. Group-II: Positive control (PC); The group was induced arthritis with FCA and was given the tween 80 (5%) vehicle only (day 8–28). Group-III: Low dose group (LD); 4-allylanisole at the dose of 10 mg/kg per oral, b.w., dissolved in tween 80 (5%) was selected to treat the animal and denoted as low dose (day 8–28).Group-IV: medium dose group (MD); 4-allylanisole at the dose of 30 mg/kg per oral, b.w., dissolved in tween 80 (5%) was given to animals and labelled as medium dose (day 8–28). Group-V: High dose group (HD);4-allylanisole at the dose of 60 mg/kg per oral, b.w., dissolved in tween 80 (5%) was given to animals and labelled as high dose (day 8–28) [22].Group-VI: reference drug group (RD); Piroxicam at a dose of 10 mg/kg b.w. dissolved in 0.1N NaOH was given to the reference drug group (day 8–28) [27].

All the groups were given euthanized at day 28 by administering ketamine/ xylazine cocktail ratio 40 mg/kg: 10 mg/kg (Anesthesia (Guideline) | Vertebrate Animal Research, n.d.). Cardiac puncture technique was used to collect blood sample in purple vacutainer (EDTA tubes) for TLC and DLC and yellow vacutainer (gel vacutainer) for biochemical testing. The ankle joints were separated for histopathology and fixed in buffered formalin (10%) solution. The blood samples were mixed with TRIzol for RNA extraction and RT-PCR analysis.

2.5. Determination of Arthritic Development and Paw volume

The arthritic development was scored on a scale from 0 (no inflammation) to 4 (severe inflammation) with 0 indicating no pathology change, 1 indicating mild alterations, 2 indicating moderate, and 4 indicating severe changes. Vernier caliper was used to determine the presence of paw edema. Paw volume was also semi-quantified on a scale of 0–4 as mentioned earlier. The scoring for determination of both arthritic development and paw volume was conducted on different days i.e. 8th, 13th, 18th, 23thand 28th day [28].

2.6. Histopathological examination

Histopathological examination was conducted to examine the recruitment of inflammatory cells, bone erosion and development of pannus. Hematoxylin & Eosin staining method were performed for this purpose. Ankle joint samples were immersed in 10% nitric acid for decalcification. Sections of paraffin wax, 5 µm thick, were cut from the embedded tissues [26]. Examination was performed by a blinded histopathologist for presence of inflammation, pannus development, and bone degradation. The results were categorized using a scoring scale of 0 to 3, where 0 indicates no pathological change and scores 1 to 3 indicate mild, moderate, and severe changes, respectively.

2.7. Hematological and Biochemical examination

Total leukocyte count (TLC) and the proportion of different types of WBCs i.e. differential leukocyte count (DLC) were determined using an automated hematology analyzer (BT-pro; AH traders) to study blood hematology (DLC) [29]. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, and creatinine levels were measured using an automated chemistry analyzer (Human health care).

2.8. mRNA expression and level of inflammatory mediators

Total RNA was isolated from blood samples using the TRIzol procedure. Blood was added in TRIzol reagent and incubated at room temperature [28]. Then chilled chloroform was mixed and centrifuged for 15 min which generated three layers in each sample. The clear supernatant layer containing RNA was separated from other layers and an equivalent volume of isopropanol was added and then this mixture was centrifuged for 10 minutes. The supernatant layer was gently discarded and pallet was obtained. Ethanol (75%) was used to wash RNA and subsequently centrifuged for 5 min. The ethanol was discarded and the RNA pellet was air dried. At the end of the procedure RNase free water was added in samples. Total RNA 1000 ng per reaction was used to performed reverse transcription with (Revert Aid First Strand Kit, Thermo Scientific, USA), and synthesis of cDNA was performed in thermal cycler [30]. The cDNA prepared was used as template, amplified and quantified by real time PCR using targeted primers. SYBER Green ROX (25X) (0.8µl), reverse primer (0.5µl), forward primer (0.5µl), Phire buffer (10µl), Phire 2Ⅱ enzyme (0.25µl) and nuclease free water (5.95µl) was used in qPCR master mix. In PCR tubes 2µl of cDNA sample, and 18µl PCR master mix was added to make volume 20µl. Products were amplified in qPCR using a Qiagen system and subjected to real-time polymerase chain reaction (qPCR) for quantification. The qPCR protocol consisted of 35 cycles of denaturation (95°C for 5min), annealing (60°C for 20 s), and extension (72°C for 30 s). Using the following sequence derived from the aforementioned articles (Table 1), the TNF-α IL-1β and IL-6 genes were constructed. It was decided to utilize GAPDH as an internal reference gene.

Table 1

Primer sequence and their product size
Primers	F/R	Sequence	Amplified bands	Reference
TNF-α	Forward Reverse	5′-CCTCTTCTCATTCCTGCTCGT-3′ 5′-TGAGATCCATGCCATTGGCC-3′	266	[27]
IL-1β	Forward Revers	5′-GCTGTCCAGATGAGAGCATC-3′ 5′-GTCAGACAGCACGAGGCATT-3′	293	[31]
IL-6	Forward Reverse	5′-AGACTTCCAGCCAGT TGCCT-3′ 5′-CTGACAGTGCATCATCGC TG-3′	233	[31]
GAPDH	Forward Reverse	5′-TCTCTGCTCCTCCCTGTTCT-3′ 5′-CTTGCCGTGGGTAGAGTCAT-3′	229	[27]

Statistical analysis

Data has been presented as Mean ± SD. The groups were compared by using one-way ANOVA (Analysis of variance) followed by post hoc Tukey’s test/t-test, where applicable. Graph Pad 8 version (8.0.2) software was used for statistical analysis. The results of the variables calculated for each group by P value < 0.05 for significant results. Where ****=P < 0.0001, ***=P < 0.001, **=P < 0.01 and *=P < 0.05 denotes comparison of treatment groups with arthritic control group, while ### denotes comparison of negative control group with arthritic control group. ns = non-significant.

3.1. Effects of 4-allyanisole on arthritic score

Arthritic development was observed at different days at equal gap of 5 days where a significant change in arthritic score was noted. Table 2 showed that 4-allyanisole significantly reduced arthritic score at medium and high doses when compared with arthritic control. Reference drug also decreased arthritic score.

Table 2

Arthritic inhibition by 4-allylanisole at different days
Treatment Groups	Day 0	Day 8	Day 13	Day 18	Day 23	Day 28
Normal control	4.987 ± 0.410	6.180 ± 0.444	6.168 ± 0.424	6.215 ± 0.439	6.205 ± 0.415	6.707 ± 0.682
Arthritic control	5.027 ± 0.336	8.170 ± 0.280***	8.330 ± 0.299^###	8.598 ± 0.252^###	8.697 ± 0.197^###	8.807 ± 0.152^###
Low Dose	5.202 ± 0.434	8.433 ± 0.597***	7.420 ± 0.756^ns	7.895 ± 0.306^ns	8.025 ± 0.204^ns	7.705 ± 0.707^ns
Medium Dose	5.162 ± 0.176	8.128 ± 0.456***	7.122 ± 0.672*	7.443 ± 0.464**	7.732 ± 0.385**	7.203 ± 1.171**
High Dose	5.408 ± 0.183	7.922 ± 0.415***	6.255 ± 0.356***	7.477 ± 0.666**	7.668 ± 0.638**	7.460 ± 0.667*
Reference Drug	5.237 ± 0.283	8.020 ± 0.480***	6.835 ± 0.957**	7.672 ± 0.424*	7.213 ± 0.543***	6.377 ± 0.484***
P < 0.05, p < 0.01 and *=P < 0.001 denotes comparison of treatment groups with arthritic control group, while ### denotes comparison of negative control group with arthritic control group. ns = non-significant (Low dose)

3.2. Effects of 4-allyanisole on paw edema

Arthritic development was confirmed by comparing all the groups with healthy group at day 8. The values were compared with normal control (6.180 ± 0.440) and were found significantly raised in positive (8.170 ± 0.280) control, low dose (8.433 ± 0.597), medium dose (8.128 ± 0.456) and high dose (7.922 ± 0.415), as well as in reference drug (8.020 ± 0.480) groups.

The paw edema was found ameliorated at day 13 in all treated groups. The comparison with normal (6.168 ± 0.424) control group showed elevated paw volume in positive (8.330 ± 0.299) control group. Treatment with 4-allylanisole reduced arthritic score in low dose (7.420 ± 0.756), medium dose (7.122 ± 0.672), and high dose (6.255 ± 0.356) groups. Reference drug group (6.835 ± 0.957) also showed amelioration in paw volume.

The paw edema was decreased in all treatment groups at day 18 in contrast to the arthritic control group (8.598 ± 0.252). 4-Allylanisole treatment decreased paw edema in low dose group (7.895 ± 0.306), Medium dose group (7.443 ± 0.464) and high dose group (7.477 ± 0.666). The reference group (7.672 ± 0.424) also ameliorated the paw edema.

4-allylanisole treated groups were compared in contrast to positive group (8.697 ± 0.197) at day 23. The groups showed reduced paw edema in low dose (8.025 ± 0.204), medium dose (7.732 ± 0.385), and high dose (7.668 ± 0.638) groups treated with 4-allylanisole. Piroxicam treatment (7.213 ± 0.543) also reduced paw edema.

The data showed significant reduction in paw edema in comparison to arthritic control group (8.807 ± 0. 152) at day 28. Treatment with low dose (7.705 ± 0.707), medium dose (7.203 ± 1.171), and high dose (7.460 ± 0.667) of 4-allylanisole reduced paw edema, similar to reference drug (6.377 ± 0.484).

Figure 1. Effect of 4-allylanisole on paw edema at day (A) 18, (B) 13, (C) 18, (D) 23 and (E) 28. *P < 0.05, **p < 0.01 and ***P < 0.001 denotes comparison of treatment groups with arthritic control group, while ### denotes comparison of negative control group with arthritic control group. ns = non-significant.

3.3. Effects of 4-allyanisole on Hematological parameters

The results showed elevated TLC in arthritic control group (11.44 ± 0.477) which was effectively decreased after treatment with of 4–allylanisole at low dose (8.333 ± 0.864), medium dose (7.400 ± 0.5292), and high dose (5.683 ± 1.235). Piroxicam (6.867 ± 1.540) also decreased the TLC significantly.

Neutrophils count was raised in positive group (25.58 ± 6.499) in comparison to negative control group (8.333 ± 2.251) and the infiltration of neutrophils was reduced after treatment with 4-allylanisole at low dose (67.83 ± 4.401), medium dose (6.917 ± 1.960), high dose (9.833 ± 3.710) and reference dose (18.41 ± 1.984).

In contrast to healthy group (74.05 ± 3.585) lymphocytes percentage was highly raised in positive group (89.22 ± 4.427) and was reduced once treated with 4-allylanisole at low dose (67.83 ± 4.401) for medium dose (76.07 ± 3.788) for high dose (72.92 ± 2.815) and for reference dose (63.33 ± 8.641).

The outcomes for combined cells (eosinophils, basophils and monocytes) revealed significantly reduced number of all three types of cells after treatment with 4-allylanisole at low dose (7.167 ± 2.483), medium dose (7.000 ± 2.828), and high dose (7.000 ± 1.549) as well as with reference drug (5.167 ± 2.317) in contrast to the arthritic control group (11.50 ± 2.881).

3.4. Effects of 4-allyanisole on biochemical parameters

Effects of 4-allylanisole on biochemical parameters i.e. AST, ALT, urea, and creatinine on liver and kidney were evaluated with changed blood profile. The biochemical indices levels which were increased in all groups except normal group were significantly decreased when treated with 4-allylanisole and piroxicam when compared to the arthritic control group.

ALT levels were found decreased significantly in treatment groups of low (46.50 ± 4.764), medium (49.00 ± 5.329) and high doses (39.50 ± 3.507) as well as in reference drug group (44.67 ± 14.69 in comparison with arthritic control group (63.67 ± 5.922).

Levels of AST were decreased in the treatment groups of low (51.17 ± 11.86), medium (17.17 ± 19.34), and high doses (76.00 ± 28.92), as well as in reference drug group (69.83 ± 17.75) in contrast to arthritic control group (63.67 ± 5.922).

Urea levels were decreased significantly in treatment groups of low (34.67 ± 5.317), medium (22.83 ± 9.390), and high doses (20.50 ± 11.50) respectively in comparison with arthritic control group (47.00 ± 5.514). While in reference drug group (41.33 ± 21.04) urea levels were not decreased significantly in contrast to the arthritic control group.

Creatinine level were found decreased significantly in treatment groups of low (0.733 ± 0.081), medium (0.6833 ± 0.116), high dose (0.600 ± 0.109) similar to the reference drug group (0.700 ± 0.126) respectively, in comparison to the arthritic control group (0.983 ± 0.132).

3.5. Effects of 4-allyanisole on expressions of pro-inflammatory mediators

The mRNA expression levels of TNF-α, IL-6, and IL-1β in different treatment groups were found significantly suppressed in comparison with arthritic control group. Furthermore, similar results for TNF-α, IL-6 and IL-1β were demonstrated in animals treated with piroxicam. 4-allylanisole anti-arthritic potential at different doses (10, 30, and 60 mg/kg) can be attributed to its ability to down-regulate the pro-inflammatory genes (TNF-α, IL-6and IL-1β).

Rats were treated with a phytochemical 4-allylanisloe and showed significant attenuation in the levels of TNF-α in all treated groups, in contrast to the arthritic control group (2.004 ± 0.866). 4-allylanisole showed decrease in expressions of TNF-α at test doses i.e. medium dose (1.015 ± 0.358) and high dose (1.004 ± 0.555). Low dose (1.237 ± 0.338), showed non-significant decrease in comparison to the arthritic control group.

The data showed that treatment with medium dose (1.875 ± 0.381), and high dose (1.460 ± 0.253) of 4-allylanisole ameliorated IL-6 expression levels in all treatment groups, similar to the effect of piroxicam (1.532 ± 0.299) in comparison to the arthritic control group (3.093 ± 0.802). Low dose (2.358 ± 0.538) showed non-significant decrease in comparison to the arthritic control group.

Treatment with 4-allylanisole at low dose (1.036 ± 0.198), medium dose (1.010 ± 0.203), high dose (1.004 ± 0.145) and reference drug (1.017 ± 0.064) demonstrated significantly decreased levels of IL-1β in comparison with arthritic control group (1.599 ± 0.308).

3.6. Effects of 4-allyanisole on histopathological parameters

Treatment with 4-allylanisole significantly attenuated the arthritic score at medium dose and high dose as compared to arthritic control group. Piroxicam also demonstrated significant attenuation in comparison to the arthritic control group. Low dose group did not significantly reduce arthritic score (Table 3).

In Fig. 5, Normal control group (A) showed the normal histology of the ankle joint or synovial joint by H&E staining and no pathological changes were seen in synovial cavity, bone and cartilage. Arrows represent; Synovial membrane (intimal layer) Rectangle; Sub-intimal layer Star; Bone, Diamond; Bone marrow. Arthritic control group (B) showed increased inflammation, pannus formation and bone erosion in contrast to normal group. Rectangle represents; Inflammation and pannus formation, Diamond; bone marrow and Star; Bone. The arthritic parameters i.e. inflammation, pannus formation and bone erosion by histological staining technique that were found increased in arthritic control group were evaluated in contrast to the normal control group. Low dose of 4-allylanisole (C) shows non-significant results in contrast to the arthritic control group and the histopathological parameters were highlighted as Rectangle; inflammation and pannus formation Arrows; Intimal layer Star; Bone. Medium dose of 4-allylanisole (D) shows significant reduction in inflammation and pannus formation. Rectangle; Mild Inflammation with focal pannus formation, Star; Bone. 4-allylanisole at high dose (E) shows reduced arthritic score by decreasing inflammation and pannus formation in comparison to the arthritic control group. Rectangle highlights the Inflammation and pannus formation, Star; Bone and Diamond; Bone marrow. Piroxicam (F) shows significant *=P < 0.05 anti-arthritic potential by decreasing inflammation and pannus formation and bone erosions. Rectangle; Inflammation and pannus formation Star; Bone, Diamond; Bone marrow.

Table 3

Effects of 4-allylanisole on histopathological markers
Histopathological parameters	Arthritic Score Mean ± SD values
Negative control (Tween 80)	0.000 ± 0.000
Arthritic control (Tween 80)	2.167 ± 0.2582###
4-Allylanisole (10 mg/kg)	1.933 ± 0.163ns
4-Allylanisole (30 mg/kg)	1.583 ± 0.5601*
4-Allylanisole (60 mg/kg)	1.633 ± 0.3141**
Reference drug Piroxicam	1.700 ± 0.3464 *
P < 0.05, p < 0.01 and **=P < 0.001 denotes comparison of treatment groups with arthritic control group, while ### denotes comparison of negative control group with arthritic control group. ns = non-significant (Low dose)

Rheumatoid arthritis (RA) is an inflammatory immune-mediated illness that can last for years. Hypertrophy, a rise in synovial fluid volume, and synovial membrane hyperplasia are the telltale alterations [32]. If rheumatoid arthritis isn't treated in a timely manner, it might destroy cartilage and bone around the affected joint. Inflammatory indicators, such as cytokines, TNF-α, IL-1β, and IL-6, which enhance damage, are stimulated by interactions between inflammatory cells and local cells [33].

Current literature on RA portrays a lifelong patient-oriented therapy for the management and treatment and to increase the quality of life. NSAIDs, DMARDs, and other biological drugs like tumor inhibitors,IL-6-inhibitors, TNF-inhibitors, and B-cell depleting drugs are just some of the many drug classes available for the treatment and management of RA and other inflammatory disorders [34]. However, due to severe side effects to the patient, treatment has been shifted towards phytochemicals due to their relatively less adverse effects. The alternative therapies being marketed for RA and products having false claims about natural products requires rigorous effort in providing safe therapy to the patients with other comorbidities. There is need to explore new strategies for providing safe therapy with less side effect to the patients[35].Therefore, plants and their extracts are being tested to discover their curative effect in different disease. So many essential oils are now being used as medicinal agent due to their reportedly less side effects and multiple pharmacological activities. The current literature in pharmacology is abundant in anti-inflammatory and targeted treatment approaches for rheumatoid arthritis that particularly focus on specific genes involved in inflammatory pathway [36].

Essentials oils are volatile, occasionally colored, clear, liquid, lipophilic, and soluble in organic solvents. In addition to their widespread role as smells, it is crucial to highlight their biological achievement for use in human health. The chemical compound 4-allylanisole (Estragole) is a phenyl propene and hence a member of the phenylpropanoid family of chemicals [23]. Isomers of 4-allylanisole are found in numerous plants and are documented to generate response through antioxidants and immune-modulatory mode of actions along with down-regulation of different genes [37–39]. Anti-inflammatory medicinal drugs can be a valuable tool in the treatment of inflammatory illnesses because they inhibit the body's inflammatory response, therefore eliminating the harmful stimulus. Medicinal plants and their phytochemicals are widely employed in the treatment of inflammation-related illnesses around the globe. The hunt for novel bioactive natural compounds to combat inflammation is ongoing, and essential oils are increasingly being recognized as a surprising source [40]. Likewise, the 4-allylanisole anti-inflammatory potential have been demonstrated in NSAID induced ulcer and stress induced gastric ulcer models [41].

T cell-mediated immune response in RA involves the stimulation of pro-inflammatory cytokines and stimulates the antibody formation which leads to the extensive joint destruction. Specifically, TNF-α stimulates the of pro-inflammatory genes expression aggravation and up keeping of chronic inflammation. Therefore, pro-inflammatory cytokines principally IL-1β, IL6, and TNF-α are important in arthritis development. Hence, these cytokines inhibitors would be effective in controlling chronic inflammation [6]. It has been attributed that IL-1β strongly stimulates matrix metalloproteinases release and osteoclast genesis resulting in bone erosion evident from cartilage destruction by TNF-α. Production and function of pro-inflammatory mediator inhibition is supposed to be an effective technique to the RA treatment [42]. These are all recognized components of RA and describe the TNF-α inhibiting effects in patients. Moreover, TNF-α targeted treatments are recognized to be effective [31].

This study used FCA-induced arthritic model of rats for anti-arthritic evaluation. The diseased group showed a significant increase in the expressions of pro-inflammatory mediators (TNF-a, IL-6, IL-1β). IL-6 plays a crucial role in the local and systemic expressions of RA, and it is not just a pro-inflammatory cytokine but it also relates in complex mode with the cells participating in bone remodeling [43]. The cytokines were released by the signals generated by auto-immune activation of inflammatory pathway including macrophages activation and fibroblast activation as shown in previous studies on rheumatoid arthritis pathophysiology [2].

A previous study has shown that 4-allylanisole with distinct inflammatory response profiles in an acute inflammatory model of mouse paw edema was able to minimize carrageenan-induced paw edema. This study examined the anti-edematogenic effects 4-allylanisole and found it effective at preventing paw edema induced by tumor necrosis factor alpha (TNF-α), bradykinin, substance P, and histamine [22]. 4-Allylanisole reduced paw edema significantly. In, addition it is attributed that the induction of immune response against attacking pathogen causes Increase in WBC count. Increase in white blood cells (neutrophils, lymphocytes, basophils, eosinophils and monocytes) were noted in blood. Total and differential leukocyte counts that was increased in arthritic group reverted to near normal in the treatment groups treated with 4-allylanisole. This drop is suggestive of the fact that chemo-attraction and cellular differentiation processes were increased during the development of FCA-induced inflammation, possibly due to the increased expressions of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [44–46]. In a recent study, 4-allylanisole has shown inhibition in leukocyte migration and stimulate phagocytic activity of 4-allylaniaole at different doses in in vivo model [19].

To establish the safety of 4-allylanisole, serum levels of ALT, AST, creatinine and urea in all treatment groups were measured. Biochemical parameters were also improved, as AST, ALT, Urea and creatinine showed a marked reduction relative to their level in arthritic control group. These biochemical parameters are indication of liver injury [26]. Their rise in arthritic control group portrays arthritis associated liver injury whereas their fall in treatment groups imply there hepatoprotective and anti-oxidant potential [22].

Different studies on ankle joints in the arthritic rat model described superficial degradation of joint structure and decreased joint space, as determined by histological testing [42]. Histopathological examination also irregular hyperplasia of synovial membranes, collagen fiber deposition, bone degradation, and inflammatory cell infiltration in the arthritic control groups [47]. In this study, similar effects were found in arthritic control groups which were ameliorated by treatment with 4-allylanisole. These effects of 4-allylanisol on inflammatory on inflammation, pannus formation and bone erosion.

4-allylanisole exhibited a significant activity against arthritis at different doses in our study. These results demonstrated noticeable change as compared to arthritic control. The experimental substance is observed to have anti-inflammatory activity and subsequently reduce the influx of inflammatory cells in the synovial membrane. Since, Inflammation is documented to be a key mechanism through which arthritis induces injury. The anti-arthritic effect of 4-allyanisol is attributed to its anti-inflammatory activity [48].

Based on the results of this study, it may be concluded that 4-allylanisole possesses anti-arthritic property which was evident by attenuation of paw edema and histopathological parameters. This immunomodulatory and anti-inflammatory properties can be attributed in part to its ability to inhibit the mRNA expression levels of TNF-α and interleukins (IL-6 and IL-1β). Furthermore, 4-allyanisole also nearly normalized the levels of hematological and biochemical parameters which supports its overall anti-arthritic effects.

In future further targets can also be explored for example matrix metalloproteinases enzymes, cyclooxygenase enzymes, toll like receptors, NF-ĸB, IL-4 and IL-10 etc. Effects of 4-allylanisole on different antioxidant enzymes and different signaling pathways may also be explored.

FCA; Freund’s Complete Adjuvant, TNF-α; Tumor Necrosis Factor, IL-

6; Interleukin 6, IL-1β; Interleukin-1β.

Ethical approval

Approval for all the experiments was taken by Research Ethics Institutional Review Board, LCWU, Lahore (ORIC/LCWU/22-298).

Availability of data and materials

All data and material will be available on request.

Funding

The authors did not receive any financial support.

Competing Interests

The authors declared no conflict of interest.

Author Contributions

Sajida Parveen: Conceptualization, data acquisition, data analysis, writing manuscript; Arham Shabbir: Supervision, conceptualization, data analysis, editing of final draft; Adeel Masood Butt, Muhammad Imran and Anum Jamil: Data acquisition and data analysis; Ashna Asim and Kiran Mashaal: data analysis and data validation

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Amelioration of rheumatoid arthritis in rats by 4-allylanisole through modulation of inflammatory mediator

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Abstract

Figures

1. Introduction

2. Materials and Method

2.1. Phytochemical used

4-Allylanisole was purchased commercially form Glentham Life Sciences from UK. Glentham code for product is GK6464.

2.2. Animal model and ethical approval

2.3. Induction of Rheumatoid Arthritis

2.4. Animals treatment

2.5. Determination of Arthritic Development and Paw volume

2.6. Histopathological examination

2.7. Hematological and Biochemical examination

2.8. mRNA expression and level of inflammatory mediators

3. Results

3.1. Effects of 4-allyanisole on arthritic score

3.2. Effects of 4-allyanisole on paw edema

3.3. Effects of 4-allyanisole on Hematological parameters

3.4. Effects of 4-allyanisole on biochemical parameters

3.5. Effects of 4-allyanisole on expressions of pro-inflammatory mediators

3.6. Effects of 4-allyanisole on histopathological parameters

4. Discussion

5. Conclusion

Abbreviations

Declarations

References

Additional Declarations

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