Nerolidol: a potential approach in rheumatoid arthritis through reduction of TNF-α, IL-1β, IL-6, NF-Kβ, COX-2 and anti-oxidant effect in CFA-induced arthritic model

Rheumatoid arthritis an autoimmune infectious disorder, is categorized by inammation and increased level of pro-inammatory cytokines which are released by immune cells, macrophages or activation of arachidonic acid metabolism. The expression of these cytokines, oxidative free radicals and the activation of COX-2 enzymes are crucial targets for chronic inammation. On the basis of established anti-inammatory ecacy of Nerolidol, the primary study was further appraised to determine its ecacy against Freund’s complete adjuvant (CFA) rheumatoid model. Arthritis was persuaded by inoculation of 0.1mL CFA injection into left hind footpad of rats. Anti-arthritic potential of nerolidol (at 200, 400 and 800mg/kg doses) was assessed by measuring the paw volume, body weight, serum analysis, histopathological and radio-graphics of ankle joints. Expressions of cytokine’s panels like IL-10, IL-4, COX-2, NF- K β, TNF-α, IL-6, PGE-2 and IL-1β were determined by real time qPCR. Antioxidant enzyme analyses was calculated by measuring the SOD, POD and catalase activity from serum and equated with arthritic control group. Nerolidol prevented the body weight loss, stabilized the biochemical and haematological homeostasis and signicantly reduced the paw volume. Furthermore, X-ray and histopathological assessment of ankle joints showed an improvement in the joint structure of rats treated with nerolidol. Besides that, over expression of gene pointers like TNF-α, IL-1β, IL-6, NF- K β, PGE-2 and COX-2 in CFA treated control rats were also reversed with nerolidol. This antiarthritic mechanism was further supported by the increased level of IL-10, IL-4 and serum anti-oxidant activity. The present ndings demonstrate that nerolidol reduce the adjuvant arthritis by down-regulating the proinammatory cytokines and up-regulating the aforementioned anti-inammatory cytokines and may be used as a therapeutic substance for the management of human rheumatoid


Introduction
Cytokines are involved in many biological processes including activation, differentiation and growth of cells, as well as in ammation (Feldmann et al., 1996). Chronic in ammatory disorders are caused by dysfunctional effects of adoptive and innate immune responses that lead to the induction of cytokines and cause in ammation (Agonia et al., 2020). In ammatory reaction produces a protective appliance against pathogens and results in the release of chemokine's, cytokines and in ammatory mediators (Lin et al., 2017). Arthritis is an autoimmune in ammatory syndrome that disturbs 1% of whole population and it is 2-3 times surplus abundant in womens than mens (Hegen et al., 2008). The augmented level of proin ammatory cytokines like IL-1β, TNF-α, NF-K β and interleukins-6 in acute phase ultimately leads towards bone deformity (Arya et al., 2011). Apart from augmented level of proin ammatory cytokines, increased level of oxidative strain is also an important factor that damages the joints during pathogenesis of RA. These factors increase the production of in ammatory cells especially neutrophils and macrophages, accelerates the release of reactive oxygen sorts in synovial uid, and cause further tissue destruction (Uttra et al., 2018). Phagocytic macrophages and granulocytes produce hydrogen peroxide and superoxide in excessive amounts. Which degrade lipids by the process known as lipid peroxidation (Biemond et al., 1984). Antioxidants defend the cells against oxidative stress on deoxyribonucleic acid, lipids and protein that has been associated with diseases like RA, diabetes, cancer and Alzheimer, and thus, involve in the improvement of treatment (De Carvalho et al., 2018). Antioxidants act through the inhibition of reactive oxygen species formation in cells. Glutathione peroxidase, catalase and superoxide dismutase are the enzymes that catalysis the hydrogen peroxides and hydro peroxides into harmless molecules including metal ion binding proteins such as caeruloplasmin and transferrin ( that chelates, sesquter iron) and prevent the production of free radicals (Ighodaro and Akinloye, 2018).
Conventional treatment of rheumatoid arthritis includes NSAIDs, corticosteroids, DMARDs that are aimed to decrease the joint in ammation and pain; are not pure anti-arthritic because they do not reduce the B cells and T cells mediated responses (Agarwal, 2010). So there is a need to introduce new novel herbal compound which is most effective and economical against B and T cells mediated response. Phytoconstituents derived from plants that inhibit the expression of cytokines have prospective against arthritis.
Henceforth, efforts should be organized to seek out novel bene cial and e cient phytoconstituents which can be used for long standing treatment of RA (Arya et al., 2011).
Nerolidol is derived from Peperomia serpens (Da Silva et al., 2006). Despite that, Peperomia serpens were used to treat pain and in ammation and reduces the expression of IL-1β and TNF-α in mouse model for pain (Fonsêca et al., 2016). Nano encapsulated nerolidol has also been evaluated for its anti-in ammatory role in zymogen induced arthritis in mice and they have also shown the gastro protective effect of nerolidol against ulcer (Trindade et al., 2020). Anti-oxidant effect of nerolidol on hippocampus of mice shows that nerolidol is bene cial against oxidative stress (Neto et al., 2013). Therefore, the aim of current investigation was to appraise the effect of nerolidol in standard model of arthritis (chronic immunological Freund's complete adjuvant rheumatoid arthritis).
The study was also intended to support the possible mechanism of action of nerolidol at molecular levels and to discover it's e cacy against joint infection.

Animals
Sprague Dawley rats (150-250g) of either sex were used for investigational procedure. Animals were housed at animal house of Department of Pharmacology, College of Pharmacy, University of Sargodha with recommended housing conditions. All animals fed on water and standard diet and they were controlled by following the guidance in accordance with National Research Council. All tests were approved by animal ethics and review committee at University of Sargodha (Approval NO. SU/Pharm/Animal Ethics Approval/2019/215).

Complete Freund's adjuvant induced rheumatoid arthritis model
Animals were separated randomly into six different groups (n=5). The 1 st group designated as normal control group and 2 nd group (arthritic control group) received 2% tween 80 (3mL/kg). The 3 rd group (standard group) was given naproxen 20mg/kg while 4 th , 5 th and 6 th group served as treatment groups and received 200, 400 and 800mg/kg of nerolidol respectively. Arthritis was persuaded by inoculation of 0.1mL of CFA injection (containing 1mg/mL of heat killed M. tuberculosis in 0.15mL mono-oleate and 0.85mL para n oil) into left hind footpad of each rats except normal control group. The day of CFA shot was served as 0 day. The oral administration of different doses to treatment groups was continued for 28 days consecutively. Arthritis in all groups were evaluated by recording various factors (Mahdi et al., 2018).

Evaluations of arthritis from body weights and paw volume of rats
During the period of treatments, body weight of each rat was observed at every 7 th day. Paw size/oedema was measured using digital plathysmometer. Percent inhibition in paw oedema /size was calculated by following this equation.
"VC" and "VT" are the paw volume of arthritic control and treatment group.

Assessment of arthritis from serum and blood
At 28 th day, all rats were sacri ced and through cardiac puncture blood was collected for the assessment of biochemical and haematological markers comprising WBCs, RBCs, Hb, c-Reactive Proteins, RF, ESR, Platelets, ALP, SGOT, SGPT, Creatinine and urea. Collected blood samples were used for appraisal of expression of mRNA TNF-α, IL-1β, IL-6, COX-2, NF-Kβ, IL-4, and IL-10. TRIzol method was used for the extraction of total RNA from blood. According to this method, into 500uL of blood, 700uL of trizole solution was added. It was mixed gently and incubated it for 10 minutes. Then 200uL of chloroform was added into it. Shake thoroughly and placed it in the centrifuge machine for 15 minutes at 12000 rpm and 4 o C. The aqueous layer was taken and then added 500uL of isopropanol into it and again mixed vigorously. Samples were incubated for 10 minutes and retained it in the centrifuge machine at 12000 rpm and 4 ºC for 15 minutes. Discarded the supernatant solution and the RNA pellet was washed with absolute ethanol. Air dried the RNA pellet and added 30uL of puri ed water into it and then quanti ed the RNA from Nano drop reader. After this, cDNA was synthesized by following the kit manufacturer procedure (Gene-Direx). In brief, 1uL of RNA solution, 1uL of oligo (dT) 20, 1uL of dNTP Mix and then RNA free water was added into it. The reaction mixture was heated at 65°C for 3-5 minutes, spin it and placed promptly on frost. Then added 4uL of 1 st strand buffer, 1uL DTT, 1uL of Script RTase and nally made volume up to 20uL. Incubated it for 30-60 minutes at 50°C and the enzyme was inactivated at 70°C for 15 minutes.
Real time quantitative PCR was used to intensify and quantify the reaction by using Bio-Rad scheme in Pharmacology department, University of Health Sciences, Lahore. Afterwards, templates of cDNA were mixed with qPCR master mix and added the speci c primers of genes, nuclease free water, and then placed it in a thermal cycler for 45 cycles with denaturation temperature at 95°C, annealing at 56°C, extension at 72°C and then terminated the reaction at 72°C. Various markers of genes were nominated from Ensemble Genome Browser for determination of speci c gene primers physically by using Input primer 3 (v. 0.4.0.) which is available online software. The sequences of primers are provided in Table1 (Shabbir et al., 2016, Lim et al., 2017. ELISA (Enzyme linked immuno-sorbent assay) for Prostaglandin E 2 ELISA test was performed for quantitative identi cation of rat Prostaglandin E 2 in serum samples according to kit manufacturer procedures (rat Prostaglandin E 2 ELISA kit , Bio-assay technology laboratory having Cat No. E0504Ra, standard curve range = 0.05ng/ml-15ng/ml, size = 96 wells, sensitivity= 0.026ng/ml). By adding acidic solution, reaction was terminated and absorbance was measured at 450nm (micro-plate reader with 450 ± 10nm).

Estimation of peroxidase antioxidant enzyme activity
Peroxidase activity was measured by determining its capability to decrease hydrogen peroxide at wavelength of 470nm (Zia et al., 2011). The 0.06mL of enzyme extract was added in 3mL of buffer substrate solution that comprised of 47mL of phosphate buffer (0.2M), 0.7mL of guaiacol and 0.32mL of H 2 O 2 . After three minutes of enzyme reaction, optical density was measured at 470 nm spectrophotometer against blank (phosphate buffer guaiacol). Peroxidase activity was measured by using the undermentioned formula.
A = Absorbance at 470nm, 26.6= extinction coe cient of guaiacol (Mm -1 cm -1 , 0.06= volume of enzyme extract (mL), 3.0 = volume of phosphate buffer (mL) Estimation of catalase antioxidant enzyme activity Assay was performed for determining the catalase activity of antioxidant enzymes and its ability was checked to reduce H 2 O 2 at 240nm. Reaction mixture was comprised of 3.0mL of K 2 PO 4 buffer (50Mm, pH 7), 0.1mL of hydrogen peroxide (30Mm) and 0.1mL of enzyme extract. Absorbance was observed after 3 minutes of reaction time at 240nm (Chance and Maehly, 1955).Catalase activity was determined by given formula A3= Absorbance at 240nm, 0.04= Extinction coe cient for H2O2 (M -1 CM -1 )

Evaluation of superoxide dismutase
The SOD bustle was performed to check its ability to inhibit the photo reduction of nitro-blue tetrazolium. This test was performed by adopting the protocol with slight modi cation as discussed by Worthington 1988. Assay mixture was contained 1mL of 0.0067M potassium phosphate buffer (7.8pH), 0.05mL extract of enzyme and 0.016mL of 0.012mM solution of ribo avin. The reaction mixture was incubated in a light box for 12 minutes. After that, 0.067mL of EDTA/NaCN solution and 0.033mL of nitroblue tetrazolium solution was added into the reaction mix. After 30 second of reaction time, the absorbance was observed against blank through spectrophotometer at wavelength of 560nm. The activity of SOD was calculated by under mentioned formula: Histopathological assessment of ankle joints At the end of the treatment, ankle joints of arthritic control and treated rat paws were collected and static in 10% solution of formalin for the assessment of histopathology of joints (Shabbir et al., 2014).

Radio graphical assessment of joints
The legs were removed at knee joints and were subjected for radio graphical assessment with computerized radio graphical system (Toshiba 630 M) (Uttra and Hasan, 2017).

Effect of nerolidol on morphological deviations and paw volume
The results speci ed in Fig. 1 describe that treated groups revealed a signi cant increase p<0.01) in paw oedema on day-1 but paw volume decreased signi cantly (p<0.001) throughout the period. A signi cant reduction (75.99, 79.97, and 81.54%) in paw volume was detected on 28 day of treatment for the dose of 200, 400 and 800mg/kg, respectively. Moreover, primary marks of chronic in ammation like arthralgia, redness, immobility and swelling of joints were substantially low in rats treated with nerolidol and naproxen sodium, respectively, compared to arthritic control. There was a noteworthy reduction in body weight from 207.00 ± 13.20 to 152.60±15.29g that was observed in arthritic rats during the treatment period. However, signi cant upsurge in weight was observed in rats treated with nerolidol at 200, 400 and 800mg/kg on 28 th day of treatment. Major difference was found in weight gain when dose doubled from 200mg/kg as described in Fig. 2 on day 7.

Effect of nerolidol on biochemical and haematological parameters
Haematologic alteration was observed in injected CFA arthritic control rats. Among them signi cant decline in hemoglobin RBCs count, increase in value of platelets, WBCs and ESR , ALP, SGPT, SGOT, creatinine, urea and C-reactive proteins had documented. Oral administration of nerolidol signi cantly increased the RBCs and hemoglobin while noticeably reduction in the liver enzymes, WBCs, C reactive proteins, ESR, Platelets, creatinine and urea was observed equated to the arthritic control rats as revealed in Table 2. These results showed protective effects against the signs of nephrotoxicity and hepatotoxicity at doses of 200, 400 and 800mg/kg. Moreover, nerolidol signi cantly decreased the RF values in CFA injected rats. Nerolidol inhibits the mRNA expression level of TNF-α, IL-6, IL-1β, COX-2, NF-K β, and induces the expression of IL-10 and IL-4 On 28 th day of treatment, blood was collected and RT-qPCR was performed for the evaluation of different in ammatory cytokines. Augmented manifestation of TNF-α was identi ed in diseased control rats (6.98 ± 0.26) and marked reduction in TNF-α was noticed in nerolidol treated rats at 200, 400 and 800mg/kg (3.09 ±0.28, 2.84 ±0.23 and 2.67 ± 0.16) respectively and overall percentage inhibition was 16.6%. Treated rats with nerolidol and naproxen revealed (p<0.001) signi cant decline in IL-1β (3.14±0.204, 2.93±0.22, 1.97±0.04 and 3.14±0.17) normalized with adjuvant control rats. Similarly, a noticeable decrease in IL-6 was 2.87 ± 0.08, 2.48 ± 0.18 and 1.92 ±0.12 in rats treated with nerolidol 200,400 and 800mg/kg respectively as compared to arthritic control rats (3.5 ± 0.32). We found a prominent decrease (p<0.001) in the expression of COX-2 in rats treated with nerolidol 200mg/kg (3.72±0.15), 400mg/kg (3.26±0.09) and 800mg/kg (3.12±0.097) as compared to expression of COX-2 in arthritic rats (7.58±0.408). Besides, signi cant reduction in NF-kβ (2.6 ± 0.17, 2.56 ± 0.08, 2.0 ± 0.07, 3.0 ±0.09) was found in nerolidol and naproxen treated rats as equated to arthritic rats (5.50 ± 0.17). The level of IL-4 and IL-10 was increased in rats treated with nerolidol (800mg/kg) and naproxen verses arthritic rats as shown in Figure 3

Effect of nerolidol on antioxidant (SOD, POD and CAT) activities
The nerolidol by oral administration at the doses of 200, 400 and 800mg/kg, increased the antioxidant enzyme activity of SOD by 28.76, 33.66 and 44.76% respectively and induced the protective defense mechanism inside body. Catalase is also an alternative enzyme that work together with SOD and POD through antioxidant defense system of enzymes that breakdowns the H 2 O 2 into O 2 and water that protects the cell from O 2 toxicity and lipid peroxidation. Nerolidol at 800mg/kg shows signi cant increase (p<0.001) in enzyme activity of catalase (47.41%). Similarly, peroxidase activity was 67.17% at 200mg dose of nerolidol for 28 days as compared to arthritic control. It was found that the highest concentration of nerolidol was 1.5% more effective than naproxen sodium.
Effects of nerolidol on histopathological assessment of rats ankle joints Evaluation of histopathology of ankle joint was conceded at day 28 of treatment. Histopathology of normal control rats showed that they exhibited normal joint space with intact articular cartilage and synovial tissues having no signs of in ammation with compact arrangements of cells. Ankle joint of arthritic control rats showed noticeable synovial lining, distinct propagation of synoviocytes and incursion of in ammatory cells with granulous and pannus development. Similarly, the erosive changes in bone and cartilage as well as deposition of collagen bers were also observed in arthritic control groups ( Figure 5). However, naproxen (20mg/kg) showed mild incursion of in ammatory cells, cartilage destruction, and thickening of synovial intergalactic space and decreased pannus development. Also, oral administration of nerolidol at 800mg/kg showed considerable forti cation against proliferation of vascular lesions, limited space between joints, cartilage destruction and small number of in ammatory cells with no pannus development that resemble to a very much normal architecture of joints. Whereas, the 400mg/kg dose of nerolidol showed a moderate cartilage destruction with little cellular invasion and pannus formation. Alternatively, nerolidol treated rats with 200mg/kg exhibited little reduction in cartilage with minor invasion of in ammatory cells and no formation of pannus.

Effect of nerolidol on radio graphical changes
The radio graphical examination is useful for the analysis of rheumatoid arthritis for estimation of severity and progression of disease. It has been found that reduction in joint space results in loss of cartilage which initiates the variety of irrational mechanism. X-rays of normal rats showed normal morphology and architecture of cartilage with no swelling around joints. While examination of x-rays of arthritic control rats showed that they possessed narrowing of joint space, severe swelling of tissues and bone erosion. In contrast, rats treated with nerolidol 800mg/kg exhibited good inhibition of tissue in ammation, bone injury and narrowing of joint space and joint distortion. Likewise, rats treated with nerolidol 400mg/kg showed moderate protection against swelling, bone erosion and joint deformity. Similarly, moderate changes occurred in soft tissues of joints with distinct reduction in joint space were also observed in naproxen treated rats as illustrated in Figure 6. Rheumatoid cachexia was observed in rheumatism characterized by appetite and weight loss that is due to increased production of cytokines, which accelerates the proteolysis and resting metabolism (Adeneye et al., 2014). Proin ammatory cytokines TNF-α, IL-6 and IL-1β are key elements of synovitis and also stimulate the NF-kβ; causes the proteolysis of muscles by activating the proteasome pathways. Overexpression of these pro-in ammatory cytokines induces the anorexia and ultimately muscle loss. Besides that, consumption of glucocorticoids as pharmacological symptomatic treatment of RA might aggravate the rheumatoid cachexia (Masuko, 2014). The observed cachexia may also be due to muscle proteolysis induced by lysosomal protease that is mediated by decrease absorption of 14 C-glucose and 14 C-leucine and also prostaglandin-E 2 in the intestines of rats. Anti-in ammatory medications have the capacity to recover the damage caused by the disease (Alamgeer et al., 2017). Similarly they also decreased the expression of proin ammatory cytokines and also resolved the intestinal absorption of rats (Ahsan et al., 2021). In addition, in CFA persuaded arthritis, we also evaluated the biochemical and haematological tests. Anemia is the most common haematological abnormality in rheumatoid arthritis. From the ndings of this study, it was apparent that decrease in haemoglobin level and RBCs count in arthritic rats, signify the anemia, that may be due to the destruction of premature RBCs, decreased level of erythropoietin and decreased iron loading in the synovial tissue of reticuloendothelial system and synovial tissues (Alamgeer et al., 2017). Nerolidol caused the notable increase in hemoglobin and RBCs possibly due to the decreased manifestation of NF-Kβ. So it normalized the invasion of in ammatory cells in the synovial uid and ultimately inhibited the cascade of in ammation (Iqubal et al., 2019). Besides, the level of platelets and WBCs were augmented in arthritic rats due to the excretion of TNF-α and IL-6, (Fig. 5A, 5B) these cytokines initiates acute phase reaction in the rheumatoid arthritis. Though, nerolidol signi cantly decreased the level of platelets and WBCs in rats by preventing the manifestation of IL-6 and TNF-α, it is might be due to its immunomodulatory effect.

Discussion
The overexpression of the pro-in ammatory cytokine causes the accumulation and release of ROS. When the production of reactive species is higher than natural antioxidant defense system, oxidative stress destroys normal physiological function of the DNA, proteins and lipids (Valdivieso-Ugarte et al., 2019). ESR and C-reactive protein are the primary markers for the early analysis of RA. The augmentation of CRP and ESR in the arthritic rats showed the presence of infective proteins in the blood while these levels were considerably reduced (p < 0.001) in rats treated with naproxen and nerolidol. Since its values relate with the radiological progression of disease (Babu et al., 2014). Treatment with nerolidol prevented the alleviated reactions of acute phase, when equaled with arthritic control rats. A striking decrease in RF values in treatment groups ensured the protective effect of nerolidol against arthritis. The factor RF is formed against Fc portion of autoantibody and initiates the immune cascade towards the progression of RA. Moreover, excessive concentration of serum ALP in diseased control group may be due to increased bone erosion and integrity of lysosomal enzymes (Chakraborty et al., 2010). However, administration of different doses of nerolidol to respective groups signi cantly reduced the ALP, inhibited the atypical rise in SGPT and SGOT that is might be due to reduction in bone loss and improved steadiness of lysosomes.
Proliferating synovial cells, T lymphocytes and macrophages play a crucial role in the progression of arthritis (Goronzy and Weyand, 2001). Proin ammatory cytokines are produced by macrophages and stimulated T cells that is involved in the initiation and progression of rheumatoid arthritis (Vandooren et al., 2009). T cell mediated immune reaction activates the discharge of proin ammatory cytokines and accelerates the formation of antibodies which lead to joint destruction. Over expression of these cytokines causes the irreparable proliferation of tissues, tissue eradication, bone loss and automatic cell death . Hence, potential antidotes against these cytokines has ability to protect the cells from in ammation . Therefore, in current investigation effect of nerolidol on expression level of proin ammatory (TNF-α, IL-6, IL-1β), in ammatory cytokines (NF-kβ, COX-2) and anti-in ammatory cytokines (IL-4, IL-10) were studied by using RT-qPCR. It has been attributed that IL-1β induces the fabrication of (MMPs) matrix metalloproteinase and instigation of osteoclast and ultimately bone erosions (Barksby et al., 2007). In addition, it is described that endogenous inhibitors of MMPs are tissue inhibitors of MMPs (TIMPs). In the manifestation of soluble IL-6R, IL-6 induces the fabrication of TIMPs in synovial broblast and cultured chondrocytes. The protective catabolic response produced in tissues are important for extracellular matrix turnover (Silacci et al., 1998). From present ndings it was suggested that anticipatory effect of nerolidol on joint demolition was arbitrated to the prevention of induction of IL-6 induced RANKL and formation of MMPs via suppression of IL-6 followed by osteoclastogenesis. Therefore, therapeutic agents that reduced the IL-6, IL-1β and TNF-α categorized a major invention in the treatment of RA. Augmented level of these proin ammatory cytokines was observed in arthritic control rats, however, signi cant (p < 0.001) decrease in the expression of these cytokines were detected in rats treated with nerolidol at all doses as shown in gure (3B and 3C) which suggested its anti-arthritic and anti-in ammatory role. NF-kβ is involved in the activation and differentiation of osteoclasts which causes development of Th1 response and bone resorption. It has been already been shown that NF-kβ controls the manifestations of TNF-α, later, it works as an potent inducer for the stimulation of NF-kβ (Shabbir et al., 2016). The existing study elucidates that increased level of NF-kβ in diseased rats was signi cantly inhibited by nerolidol on last day of treatment.
The level of COX-2 is increased at the site of injection and they up-regulates the formation of prostaglandin-E2 that mediates the angiogenesis, vasodilation, extravasation of uid and vascularization in the synovial membrane (Shabbir et al., 2016). Overproduction of in ammatory cytokines causes the increased expression of PGE 2 . This augmented level of PGE 2 accelerates the differentiations of osteoclast and generating the degrading enzymes and hence stimulates the bone erosion, vasodilation and migration of leukocytes at site of in ammation. So, any substance that prevents the generations of aforementioned mediators carries great importance in the management of rheumatism (Manan et al., 2020). In current study, increased expression of COX-2 and PGE-2 were noted in diseased controlled rats, however, signi cant decrease in COX-2 and prostaglandin-E2 were observed in rats treated with nerolidol. Hence, we suggest that nerolidol protected the animals from joint in ammation, may be due to decreased production of prostaglandins and signi cant inhibition of COX-2 manifestations. Consequently, prevention of arachidonic acid metabolism might be another mechanism of action of nerolidol possessing anti-arthritic activity.
Moreover, IL-4 is an anti-in ammatory cytokine and maintains the production of Th2 cells and inhibits the autoimmune reaction mediated by Th1 cells (Schulze-Koops and Kalden, 2001). The IL-10 also has capability to inhibit the cellular defense mechanism by inactivating the macrophages in response and modi es the synovitis of rheumatoid arthritis (Bozkurt et al., 2006). In the progression of RA, IL-10 not only inhibit the Th1 mediated immune response but also down regulate the function of antigen presenting cells and protects the reliability of joint (Uttra et al., 2018) as same was observed with tested drug as shown in Fig. 3D.
In histopathological assessment, in amed joints exhibited the continuous migration of polymorph -nuclear leukocytes, macrophages and lymphocytes into joints and synovium. They all produce the in ammatory cytokines in the synovial uid and joints. Thus, inhibition of migration of macrophages, leukocytes decreased the accumulation of in amed debris in joints and may have bene cial effect for the protection of joints Issekutz, 1994, Wilder et al., 1989). Our histopathological examination suggested that the nerolidol treated groups inhibited the migration of these leukocytes and protects the joint from hyperplasia, joint deformity, pannus formation and destruction of cartilage as shown in Fig This increased the concentration of antioxidant enzymes in the synovial uid that diminished the destruction of cartilage and tissue deformity in the ankle joint of treated rats. Lipid peroxidation is the process in which poly unsaturated fatty acids undergo the oxidation process and cause functional abnormality in the cell membrane. However, nerolidol decreased the lipid peroxidation level, so they act as an in-vivo antioxidant agents and reduce oxidative stress.

Conclusion
In a nutshell, oral administration of nerolidol in arthritic rats signi cantly reduced the paw volume, regained body weight, normalized the altered biochemical and haematological parameters and also inhibited mRNA expression level of the above-mentioned cytokines.
So anti-arthritic effect of nerolidol may be due to its capability to decrease the level of NF-K β, COX-2, PGE2, TNF-α, IL-1β, IL-6 and by increasing the level of IL-4 and IL-10 and antioxidant enzyme activity of SOD, POD and CAT. Hence, nerolidol may be recommended as a therapeutic substance for the treatment and management of severity of rheumatism for clinical trials.   Table 2: Effect of oral administration of nerolidol on biochemical and haematological parameter in CFA induced model

Declarations
Note: Values are expressed as mean ± SEM (n=5) followed by one-way ANOVA with Dunnet posttest. *** p<0.001, the values were significant as compared to arthritic control. Values are articulated as mean ± SEM followed by one-way ANOVA with Dunnett posttest ( *** p< 0.001) Figure 1 Effect of different doses of nerolidol given for 28-days on paw volume induced by CFA. Result was followed by Two way ANOVA using graph pad prism with Bonferroni posttest whereas ***p< 0.001, **p< 0.01

Figure 2
Effect of different doses of nerolidol given for 28-days on body weight of CFA-induced rats. Result was followed Two way ANOVA using graph pad prism with Bonferroni posttest whereas ***p< 0.001, **p< 0.01 and * p< 0.05.  Effect of daily oral administration of nerolidol (200, 400 and 800mg/kg) and Naproxen for 28 days on Prostaglandin E2. The results were analyzed by using Mean±SEM followed by One-way ANOVA was performed (n=5), where ***p<0.001.