Marham‐Mafasel decrease joint inflammation and IL‐1β gene expression in rheumatoid arthritis animal model

Abstract Background Rheumatoid arthritis (RA) is a systemic chronic disease with synovial membrane, tendon and articular tissue inflammation. Current treatments of RA have many side effects and are quite expensive. Today, new treatments procedures and inexpensive herbal drugs are developed. Marham‐Mafasel is mainly made out of two traditional herbs (Arnebia euchroma and Martricaria chamomilla). Objective In this study, for the first time, the impact of Marham‐Mafasel on joint inflammation, histopathological changes and IL‐1β gene expression was evaluated in RA animal model. Methods The RA was induced by a single s.c. injection of 0.1 ml Freund's complete adjuvant into the left hind footpad. In continuous, 15 RA male Wistar rats were used in three groups: I: Control; II: Treatment I (Piroxicam) and III: Treatment II (Marham‐Mafasel). The volume of the hind paw was measured every day from 0 to 19 using water changed volume approach. The inflammation in the joint was evaluated using histopathology assay and gene expression of IL‐1β was evaluated with use of Real‐Time PCR. Results Hind paw swelling of Marham‐Mafasel at days 10th and 19th was reduced compared with the control group (p < 0.05). There was no statistically difference in histological degrading and changes index in three groups (p ≥ 0.05). Relative expression of IL‐1β in Marham‐Mafasel group was significantly decreased compared with other groups. Conclusion The co‐administration of M. Chamomile and A. euchroma, called Marham‐Mafasel, decreases IL‐1β gene expression that leads to a reduction in inflammation in rheumatoid arthritis (RA) animal model.


| INTRODUC TI ON
Rheumatoid arthritis (RA) is an autoimmune disease characterized by systemic complications, progressive disability, pain, progressive course affecting articular, mortality and socioeconomic costs (Combe, 2009;El Miedany et al., 2008). RA is the consequence of a disorder of immune system function and its reason is still controversial (Entezami et al., 2011). RA is found to affect up to 1% of individuals in developed countries (Suresh & Shetty, 2018). The most prevalent involved joints are feet and hands such as knees, ankles, shoulders, elbows and wrists. As a type of chronic autoimmune disease, RA causes erosive joint damage, functional impairment, progressive bone and cartilage destruction in the vast majority of the patients (Combe, 2009;El Miedany et al., 2008;Venables & Maini, 2014). Antibodies are found as an important driver in the pathogenesis of RA. Anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are proved useful for RA diagnosis and reported to be present in nearly 70% of the patients (Ursum et al., 2009). The IL-1β is an inflammatory cytokine that has association with elevated level of RA (Firestein & Malnnes, 2017).
Proteolytic degradation of joint cartilage and bone is generally irreversible and a characteristic feature of joint destruction. In RA, inflammatory cells infiltrate the synovial membrane and induce bone and cartilage degradation. Histologic observation has shown the stage of RA lesion and the therapeutic effects of the drugs (Fassbender, 1983;Shegarfi et al., 2012). RA is characterized by synovial inflammation in which Fibroblast-like synoviocytes (FLS) in synovial joint play a central role in the production of proinflammatory cytokines and chemokine (Bartok & Firestein, 2010;Bottini & Firestein, 2013). The course of the disease is varied according to the presence or absence of autoantibodies, frequency of swollen joints, the severity of inflammatory process and genetic background (Gossec et al., 2010;Heidari, 2011). Therapeutics available against RA can help symptomatic relief (no steroidal antiinflammatory drugs; NSAIDs) or modifies the disease progression (disease-modifying anti-rheumatic drugs; DMARDs). The prolonged use of drugs left the patients susceptible to gastrointestinal, anaemia, renal impairment and cardiovascular diseases (Guo et al., 2018;Wong, 2019). Today, the natural agents with therapeutic potential have increased attention and tested for the treatment of arthritis (Dudics et al., 2018;Zhang & Dai, 2012). Interestingly, about 59% of Pharmacopoeia is obtained from herbal plants (Swerdlow, 2000). In the history, Persian Medicine (PM) dates back to 10,000 years ago which was one of the burgeoning schools of medicine (Ghaffari et al., 2014(Ghaffari et al., , 2017. PM with several thousands of manuscripts, famous scientists and verbal sources in different languages can be helpful in the treatment of various diseases and in the development of new drugs by using modern technology (Ahmadi et al., 2009;Goshtasebi et al., 2015;Pasalar et al., 2013 (Soltanian et al., 2010). A. euchroma is a traditional herbal medicine that has some functions, including immune-modulatory, antifungal and anti-inflammatory (Siavash et al., 2016). M. chamomilla was previously administered in the USA, Europe, Western Asia, Australia and other countries (Mehmood et al., 2015). Also, previous studies on herbal medicines have revealed a significant decrease in the inflammation of oral mucositis and atopic eczema in the rat animal model (Ferreira et al., 2015;Srivastava et al., 2010;Tavakoli Ardakani et al., 2016). So, these herbal medicines have the potential to reduce the inflammation and inflammatory factors in RA patients. However, there is no study on anti-inflammatory activities of co-administration of these herbs on RA animal model. Therefore, in the present study, the effects of Marham-Mafasel (an herbal ointment) on the reduction in joint inflammation, histopathological changes and IL-1β gene expression in the hind paw of adjuvant-induced RA rat model was evaluated.

| Animals and RA induction
The study procedures were confirmed by the Research Ethics

| Drug preparation
Marham-Mafasel is a mixture of two herb extracts, namely, A. euchroma and M. chamomilla (Soltanian et al., 2010), and purchased from special drugstore. Piroxicam was purchased from Razak laboratory of Iran.

| Evaluation of hind paws oedema and body weight
The volume of the hind paw was measured on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 19. The change in a water volume of the graduated cylinder was used for the evaluation of paws oedema (Fereidoni et al., 2000). The paw swelling indexes (%) was calculated by subtraction of each day volume from day 0 and divides the equal from each day (Gupta et al., 2014).

| Radiography
On the day 19, all the rats were sacrificed. Lateral and anterior radiographic images from left hind paws were taken to evaluate the cartilage and bone damages with a 55 kVp exposure for 6.4 mAs (Siemens Multiphos 15, Mumbai, and Maharashtra, India).

| RNA Isolation and cDNA synthesis
The one sample of cartilage and bone tissue of talus from each group were dissected and kept at -80°C until the RNA isolation. The total RNA was obtained with use of RNAqueous®-Micro Kit according to the manufacturer's instruction (Ambion). The genomic DNA was removed with use of DNase (Qiagen) at 37°C for 15 min. The RNA concentration was quantified by the A260/A280 ratio with use of a Nano Drop ND-2000. All the extracted RNAs, from three groups, were stored at −80°C. cDNA was synthesized with the use of PrimeScript RT reagent Kit (TaKaRa). Briefly, a mixture of total RNA (2.5 ng), oligo (dT), RT enzyme, random hexamer and buffer was heated at 37°C for 15 min. The cDNAs were stored at −20°C until Real-Time PCR was perform.

| Real-time PCR
The gene expression was evaluated with the use of SYBR Green PCR Kit (TaKaRa) and a Step One instrument (Applied Biosystem). All reactions were carried out in duplicate and in a total volume of 20 μl for 50 cycles. In this study, quantitative real-time PCR was used to assess the expression of the IL-1β gene (Forward: 5' -TCATTGTGGCTGTGGAGAAG -3' Reverse: 5' -CACTAGCAGGTCGTCATCATC -3'). IL-1β was an inflammatory cytokine that was used for evaluation of drug effects and beta-actin gene (Forward: 5' -GTCCACCTTCCAGCAGATG -3' Reverse: 5' -GCTCAGTAACAGTCCGCCTAG -3') used as endogenous control. Three different software were used for primer design including Beacon Designer, Gene Runner and Primer Express. The condition of each cycle was as follows: 95°C for 30 s (holding time), 95°C for 5 s (denaturing) and 60°C for 30 s (annealing and extension). The melting curve analysis was performed by 95°C in the first step then cooled to 60°C and stepwise heated to 95°C with a ramp rate of 0.3°C. Finally, the relative gene expression of IL-1β was calculated with use of ∆∆Ct method.

| Histopathology
On the day 19, all the rats were sacrificed, tibio-femoral joints were removed, fixed immediately in 10% neutral buffered formalin and decalcified in 10% formic acid. The samples were then dehydrated, embedded in paraffin wax and serially sectioned at a thickness of 5 μm. The sections were stained with Hematoxylin and Eosin to evaluate for structural changes and cellular infiltration. Histological changes and degrading index of sections were scored as described previously (Srivastava et al., 2010).

| Statistical analysis
Histological and degrading Index was analysed using Chi-squared test. The relative levels of mRNA derived in different groups were analysed by REST 2009 Software (Qiagen, Hilden, Germany). The normality of hind paw swelling was analysed by Kolmogorov-Smirnov (KS) test and their significance was analysed by one-way ANOVA (Tukey's post-hoc test) and expressed as mean ± SD. The analyses were performed with use of the SPSS Statistical program, version 22 (SPSS). The comparison tests were considered to be significantly different at p < 0.05.

| Measurement of arthritis swelling
As shown in Figure 1, the hind paw swelling was showed in three groups from days 0 to 19 (Figure 1) with the control group (p < 0.05). Also, the mean of the inflammation index in the Piroxicam group was more than Marham-Mafasel, but the difference was not statistically significant (p ≥ 0.05) (Figure 1).

| Radiography
On the 19-day of experiment, all the rats were sacrificed then immediately lateral and anterior radiographic images were taken from left hind paw. The radiological changes in the bone and cartilage were evaluated in which there was no statistically difference between the groups (p ≥ 0.05) (Figure 2).

| Histological index
In this study, the grading of histological index, including synovial hyperplasia, pannus formation, mild cell migration, mononuclear cell infiltration and granulation tissue, in all groups was analysed. Our results demonstrated that pannus formation and granulation tissue occurred in Piroxicam and Marham-Mafasel groups but not observed in the control group. Mild cell migration index occurred in all of the samples in Piroxicam groups compared with other groups. Our results showed that mononuclear cell infiltration occurred identically in all groups (p > 0.05). Taken together, there was no significantly difference in histological changes in all three groups (p > 0.05) ( Table. 1 and Figure 3).

| Histological degrading index
In this study, the histological degrading index including thinning of the cartilage layer, low erosion, extreme erosion and breaking of cartilage in all groups was analysed. Our results showed that the thinning of the cartilage layer, low and extreme erosion were not observed in Marham-Mafasel samples but observed in Piroxicam group. The extreme erosion was not observed in all samples of three groups. There was no statistically significant difference in histological changes in all three groups (p > 0.05) (Table. 2 and Figure 3).

| IL-1β gene expression
This study revealed that the relative gene expression of IL-1β in

Marham-Mafasel was decreased compared with Piroxicam and in
Piroxicam was decreased compared with control (p < 0.05) (Figure 4).

| D ISCUSS I ON
RA is a chronic systemic disease which can cause multi-joints damages, chronic joint inflammation, pain, joint deformity and disability (Kim et al., 2017). Rat Adjuvant-Induced Arthritis (AIA) model is a reliable, reproducible and easy animal model of arthritis with a limited duration. AIA has been widely used to study the F I G U R E 2 On the day 19, all the rats were sacrificed. Radiography Images were taken from lateral and anterior aspect of left hind paws. There was no statistically significant difference between the three groups -control, Piroxicam and Marham-Mafasel. (L; Lateral view, A; Anterior view)

F I G U R E 1
The mean of hind paw swelling index has been shown for the three groups of control, Piroxicam and Marham-Mafasel. Hind paw swelling of Marham-Mafasel at day 10th and 19th was reduced compared with the control group (p < 0.05). *The significant days between the groups TA B L E 1 The grading of histological index, including synovial hyperplasia, pannus formation, mild cell migration, mononuclear cell infiltration and granulation tissue, was performed in all three groups Note: There was no significant difference in histological changes between groups (p > 0.05). N; the numbers of Wistar rat in each groups.

F I G U R E 3
Histopathological degrading index including thinning of the cartilage layer, low erosion, extreme erosion and breaking of cartilage was analysed in three groups of control, Piroxicam and Marham-Mafasel. There was no statistically significant difference in histological changes in all three groups (p > 0.05) ( Table. 2  Note: There was no statistically significant difference in histological changes between groups (p > 0.05). N; the numbers of Wistar rat in each groups.

TA B L E 2
The histological degrading index including thinning of the cartilage layer, low erosion, extreme erosion and breaking of cartilage was performed in all three groups anti-inflammatory activity of drugs because of the similarity of pathology with human RA (Bolon et al., 2010;Kannan et al., 2005). AIA is a classic experimental model of RA and shares many pathologic features with RA such as synovial hyperplasia, cartilage damage and excessive inflammation (Cai et al., 2017). The synovial fibroblasts are directly involved in cartilage and bone destruction by a regulatory network of cytokines that implicate inflammation (Choy, 2012;Guo et al., 2018). The common treatments of RA are typical with immunosuppressant that often cause adverse impacts, whereas herbal products are considered safe and effective (Swerdlow, 2000).
These drugs prevent arthritic joints from the tissue damage, bone breakdown and also be highly tolerated and convenient for the patients (Nanjundaiah et al., 2013). The results of hind paw oedema assay in this study suggested that the Marham-Mafasel treatment has anti-oedema activities. The effect of Marham-Mafasel on the reduction in pain on primary knee osteoarthritis has been identified (Soltanian et al., 2010). Our study was in one direction with The main pathological characteristics of the RA are synovial cell hyperplasia, infiltration of inflammatory cells, cartilage degeneration, bone erosion and pannus formation that also occurred in AIA rats (Amraei et al., 2015). The histopathological index in the articular structure was confirmed by H&E staining (Taranov et al., 2016).
In the present study, no obvious differences were noted among the Marham-Mafasel, Piroxicam and control groups. On this regard, Fan et al revealed that there was no improvement of cartilage and bone destruction in a low dose of the A. euchroma but the histopathological index was reduced after receiving a high dose (Fan et al., 2012).
It should also be noted that there are no histopathological index reports that show the impact of co-administration of M. chamomilla, M.chamomilla and A. euchroma on treatment of arthritis. We considered this result to the fact that we study an AIA model in which little histological injury had yet occurred. Our purpose was to evaluate the joint preservation effect in early stage of the disease and also determine the severity of the inflammation. Therefore, for future studies, we suggest the evaluation of different concentrations of the co-administered of these herbs on histopathological changes in AIA rat models.

F I G U R E 4
The relative quantitative gene expression of IL-1β (Mean ± SEM) was calculated with the use of ΔΔCT method. The relative expression of IL-1β in Marham-Mafasel was decreased compared with Piroxicam and in Piroxicam was decreased compared with control (p < 0.05); *p < 0.05, **p < 0.01, **p < 0.001 The pathophysiology of the RA is not completely understood yet (Imboden, 2009), But previous studies have confirmed that pro-inflammatory cytokines, such as TNFα, IL-1 and IL-6, mainly produced by increased numbers of macrophages, are responsible for the inflammation and joint destruction (Guo et al., 2018).
Disease-modifying anti-rheumatic drugs (DMARDs), NSAIDs and corticosteroids are commonly used in the treatment of the arthritis patients (Kemper et al., 2011). These drugs are first-line drug therapies which have an anti-inflammatory activity against the mentioned cytokines (Kemper et al., 2011). IL-1β is the initiating factor in inflammation to regulate a variety of cytokines, cell adhesion molecules and inflammatory mediators. Previous studies showed that high level of IL-1β was associated with bone erosion and cartilage destruction in RA and manifested additional characteristics of osteoarthritis (OA) which included increased osteophyte complex form (Guo et al., 2018;Wojdasiewicz et al., 2014).
Also, previous studies have demonstrated that the IL-1 has main role in the pathophysiology of the RA. Elevated concentrations of IL-1 in plasma and synovial fluids of the RA patients were detected compared with OA or non-inflammatory joint disease (Kay & Calabrese, 2004). Given all these facts, the gene expression of

| CON CLUS IONS
This study, for the first time, has observed that the co-administration of M. Chamomile and A. euchroma, called Marham-Mafasel, decrease the gene expression of IL-1β that leads to a reduction in the inflammation in (AIA) model.

ACK N OWLED G M ENTS
The authors would like to thank National Cell Bank Department, Pasteur Institute of Iran, Tehran, Iran and Shahed University, Tehran, Islamic Republic of Iran for providing the possibility of doing the study.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENT
The experimental animal's process was performed based on guidelines by the Research Ethics Committee of Shahed University of Medical Sciences and Pasteur Institute of Iran, Tehran, Iran.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.430.