Evaluation of Antiarthritic and Antinociceptive Effects of Cedrol in a Rat Model of Arthritis

Pharmacological studies revealed that cedrol, a natural sesquiterpene, has antioxidant, anti-inflammatory, and analgesic properties. This study is aimed at evaluating the potential antiarthritic activity of cedrol in a rat experimental model of arthritis induced by using complete Freund's adjuvant (CFA). Arthritis was induced in Wistar rats by CFA (0.1 ml) injection. Cedrol (10 and 20 mg/kg) and indomethacin (5 mg/kg) were orally administered from day one and continued for 21 days. The antiarthritic activity was assessed through mechanical allodynia and thermal hyperalgesia responses, paw edema assessment, and arthritis scores. Serum TNF-α and IL-1β levels were measured for the evaluation of inflammation. Furthermore, serum oxidative stress markers, including malondialdehyde (MDA) and thiol levels, as well as superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, were also assessed. Oral administration of cedrol and indomethacin significantly decreased paw edema and arthritis score. Besides, cedrol and indomethacin significantly decreased pain responses. In the serum of the CFA group, TNF-α, IL-1β, and MDA were higher, while thiol and SOD and GPx were lower than the control group. Treatment by cedrol and indomethacin corrected the biochemical parameters in the serum. In this study, cedrol offers potential antiarthritic properties through its anti-inflammatory and antioxidant effects.


Introduction
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease described by multijoint inflammation, pain, and destruction of cartilage and bone [1,2]. The disease may progress to a severe disability with negative consequences on the quality of life and increased mortality rate [3]. Autoimmunity and chronic inflammation are activated by an imbalance between pro-and anti-inflammatory cytokines, resulting in joint damage in RA [4]. Although nonsteroidal anti-inflammatory drugs effectively reduce symptoms, they do not prevent the disease from progressing and do not protect the tissue or joint against erosion. Also, they have severe side effects or cardiovascular complications; growing interest in herbal remedies in RA patients indicates the need to find new agents from herbal medicines [5]. Indeed, safe and effective agents for the treatment of RA are urgently required. Natural plant compounds are suggested to prevent or treat RA [6]. Terpenes, also known as terpenoids, are the largest and most diverse group of natural products. They are classified according to the number of isoprene units to mono, di, tri, tetra, and sesquiterpenes [7]. Sesquiterpenes (C15) are composed of 3 units of isoprenoid, which are secondary metabolites produced mostly in plants [8]. Recent studies showed that sesquiterpenes have various therapeutic effects, such as being effective in pain management [9], protection against stroke [10], and RA [11]. Cedrol, a natural sesquiterpene, is known to carry many pharmacological effects, including antioxidant [12], anti-inflammatory [13], antibacterial [14], analgesic [12], sedative [15], hair growth inducer [16], platelet-activating factor (PAF) antagonist [17], and antitumor effects [18]. The present investigation

Induction of Arthritis and Treatment Protocol
Arthritis was induced on day 0 by a 0.1 ml subcutaneous injection of CFA into the right hind footpad of the rat. Rats were randomly divided into five experimental groups.
(i) Group I. For the control group, rats were administered with 0. Nociceptive behavioral examines, paw volume evaluation, and arthritis score were examined on days 0, 7, 14, and 21. The hind footpad's paw thickness (right) was measured in each rat using a caliper.

Arthritis
Score. The severity of arthritis was assessed as follows [1]: 0 = no change, 1 = mild erythema or swelling of the digits, 2 = moderate swelling and erythema, 3 = severe swelling and erythema involving the ankle, and 4 = ankylosis and inability to bend the ankle.
4.3. Thermal Hyperalgesia. The hot plate was used to assess thermal hyperalgesia. In this test, rats were individually placed on a hot plate maintained at 55°C. The latency to the first sign of paw licking or jump response to avoid the heat nociception was taken as an index of the pain threshold; the cut-off time was set at 10 s to prevent any injury to the tissues of the paws [1].

Mechanical
Allodynia. Animals were placed on an elevated box (30 × 30 × 30 cm) with a metal wire floor, and an ascending series of von Frey filaments (Bioseb) were applied in ascending style to the plantar surface. The cut-off threshold was set at 60 g to prevent tissue damage. Each filament was tested five times. A positive response is if the animal responded to at least three withdrawals out of five consecutive trials. That gram force was considered as the paw withdrawal threshold [19].

Biochemical
Assays. The serum interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α) levels were detected using a commercial ELISA kit (Karmania Pars Gene Company, Kerman, Iran). The levels of malondialdehyde (MDA), as the final product of the lipid peroxidation process, superoxide dismutase (SOD) activity, and thiol concentration in serum, were assessed based on the methods reported by previous publication [20]. Glutathione peroxidase (GPX) activity was detected using a commercial ELISA GPX assay kit (ZellBio, German). 4.6. Statistical Analysis. GraphPad Prism (version 6.0) was used for analyzing the data. All data were shown as mean ± SEM. Behavioral parameters were measured by two-way analysis of variance (ANOVA) followed by Bonferroni's test. Biochemical parameters were measured by one-way ANOVA followed by Tukey's test. p values lower than 0.05 were statistically significant.

Effect of Cedrol on the Arthritis Score in CFA-Induced
Arthritic Rats. As shown in Figure 1, the arthritis score in all groups on day 0 was 0. The arthritis score in the cedrol (10 mg/kg) group was lower than that detected in the CFA group on day 21 (p < 0:05). The arthritis score in the cedrol (20 mg/kg) group was lower than that detected in the CFA group on days 7, 14, and 21 (p < 0:05, p < 0:001, and p < 0:001, respectively). A significant decrease in arthritic index was recorded in the indomethacin group compared to the CFA group on days 7, 14, and 21 (p < 0:05, p < 0:01, and p < 0:001, respectively).

Effect of Cedrol on Paw Volume in CFA-Induced
Arthritic Rats. As shown in Figure 2, there was no significant difference in paw volume between groups on day 0. The paw  Oxidative Medicine and Cellular Longevity volume in the CFA group significantly increased on days 7, 14, and 21 compared to the control group (p < 0:001). The paw volume in the indomethacin (5 mg/kg) group was lower than that detected in the CFA group on day 7 (p < 0:01). The paw volume in the cedrol (20 mg/kg) group and indomethacin (5 mg/kg) group was lower than that detected in the CFA group on day 14 (p < 0:05 and p < 0:01, respectively). Over time, the paw volume decreased further in the treatment groups; the paw volume in the cedrol (20 mg/kg) and indomethacin (5 mg/kg) group was lower than that detected in the CFA group on day 21 (p < 0:01).

Effect of
As displayed in Figure 5(b), administration of CFA significantly increased the serum TNF-α level (p < 0:001) in the arthritic (CFA group) compared to the control group. The treatment with indomethacin was effective to decrease TNF-α level in serum of CFA-induced rats (p < 0:01 versus CFA group), besides the treatment with cedrol (10 and 20 mg/kg) reduced TNF-α level in serum of CFA-induced rats compared to the CFA group (p < 0:05 and p < 0:01, respectively).

Effect of Cedrol on the Oxidative Stress in CFA-Induced
Arthritic Rats. MDA level was significantly higher (p < 0:001) in the arthritic (CFA group) compared to the control group. The treatment with indomethacin was effective in decreasing MDA level in serum of CFA-induced rats compared to the CFA group (p < 0:05), besides the treatment with cedrol (20 mg/kg) reduced MDA level in serum of CFA-induced rats compared to the CFA-control group (p < 0:05) (Figure 6(a)).
As shown in Figure 6(b), administration of CFA markedly decreased serum levels of thiol as compared to the control group (p < 0:01). The treatment with cedrol (20 mg/kg) and indomethacin significantly increased thiol level in serum of CFAinduced rats compared to the CFA-control group (p < 0:01).
SOD activity was significantly lower (p < 0:001) in the arthritic (CFA group) compared to the control group. In the treatment groups (cedrol (20 mg/kg) and indomethacin), SOD activity elevated significantly compared to the CFA group (p < 0:01) (Figure 6(c)).
Gpx activity was significantly lower (p < 0:01) in the arthritic (CFA group) compared to the control group. The treatment with indomethacin was effective to increase Gpx activity in serum of CFA-induced rats compared to the CFA-control group (p < 0:05), besides the treatment with cedrol (20 mg/kg) increased Gpx activity in serum of CFA-induced rats compared to the CFA group (p < 0:05) (Figure 6(d)).

Discussion
The CFA-induced experimental model of arthritis is known to have numerous human RA features, such as robust hypersensitivity to mechanical and heat stimuli, polyarticular inflammation, and deterioration of the joint structures. This model is widely used to study RA's pathogenesis and assess potential therapeutic targets useful for the treatment of RA [1,21,22]. Following intraplantar injection of CFA, the mycobacterial constituents in the CFA cause Tlymphocytes to provoke a robust immune response in the rat paws. The T-lymphocytes interact with dendritic cells, monocytes, and macrophages to produce the major proinflammatory cytokine participating in the pathogenesis of RA, including TNF-α, IL-1β, and IL-6 in the synovial membrane [23][24][25][26]. The interaction between these proinflammatory mediators causes synovial inflammation and cartilage/ bone destruction [27]. It has been revealed that TNF-α blockade reduces the symptoms of RA [28]. The inflammatory cytokines IL-1β and TNF-α are the therapeutic targets in RA treatment strategies [29,30].
On the other hand, arthritis can cause chronic inflammatory pain. Moreover, chronic inflammatory pain is modeled via CFA attributed to provoke inflammatory cytokines, which sensitizes nociceptive neurons and reduces the pain threshold [31]. Previous studies have highlighted that sesquiterpenes show anti-inflammatory, antioxidant [32], and analgesic properties [33]. Consequently, in the present study, the potential protective properties of cedrol were examined against CFA-induced arthritis in rats. According to the present study, cedrol improved the nociception behaviors of the rats exposed to CFA. Anti-inflammatory and antioxidant effects also accompanied it. Cedrol reduced the paw's thickness by inhibiting the release of inflammatory mediators (IL-1B and TNF-α), indicating its antiinflammatory potential in CFA-induced arthritis. As a nonsteroidal anti-inflammatory drug, indomethacin inhibits the production of prostaglandins by inhibiting the activity of cyclooxygenase [34]. In this study, indomethacin reduced the thickness of the paw and exerted antinociceptive behaviors. Besides, indomethacin reduced serum levels of TNF-α and IL-1β as well. Antioxidant therapy has thus represented an effective treatment for oxidative stress/inflammationrelated diseases. Oxidative stress occurs due to an imbalance between prooxidants and antioxidants and consequent excessive production of reactive oxygen species (ROS) [35]. Several defense systems, including enzymatic (SOD and GPx) and nonenzymatic antioxidants (GSH), have been involved in the cells preventing uncontrolled ROS. Indeed, these antioxidants' impairments have been reported in active RA patients [36]. ROS production inside the joints has an essential role in arthritis pathogenesis since oxidants by direct action or indirect activation of latent collagenases degrade matrix ingredients [37]. Besides, ROS are positively linked with the severity of RA [38]. ROS participate in the signaling of inflammation. Mitochondrial ROS stimulate proinflammatory cytokine production, IL-1B, IL-6, and TNF-α.
On the other hand, the inflammation process also causes oxidative stress as host immune cells, like neutrophils, also known as polymorphonuclear neutrophils, release large amounts of ROS via the NADPH oxidase enzyme pathway [38]. In the current study, cedrol or indomethacin showed antioxidant properties, as it decreased the MDA levels while antioxidant molecules (thiol, SOD, and GPx) increased. These results are in accordance with the previous study reported that administration of cedrol (20 and 40 mg/kg, i.p.) once a day for 14 days postchronic constriction injury model of neuropathic pain attenuated nociception pain behaviors in rats by inhibition of inflammatory response and reduction of oxidative stress markers [12]. Another study showed cedrol reduced collagen-induced arthritis in mice and modulates the inflammatory response in lipopolysaccharide-mediated fibroblast-like synoviocytes [39]. Treatment with Budlein A, a sesquiterpene lactone from Viguiera robusta, showed anti-inflammatory and 8 Oxidative Medicine and Cellular Longevity analgesic effects in antigen-induced arthritis in mice [40]. Arthritis score and swelling of the paw are indexes to access the antiarthritic action of numerous medicines [5], and these indexes were used to evaluate the effect of cedrol in this study. The cedrol groups noteworthy attenuated paw thickness and arthritis scores. In the current investigation, cedrol modified different parameters of arthritis in rats, including arthritic score, paw volume, nociception behaviors, oxidative stress markers, and inflammation. Thus, cedrol may be effective as a long-term antiarthritis agent to overcome the distressing manifestation of RA.
In conclusion, we conclude that cedrol exhibits antiarthritic properties presumably through inhibition of oxidative stress and inflammation. Overall, these results provided supportive evidence for the therapeutic potential of cedrol in RA treatment.

Data Availability
The dataset that supports the results and findings of this research is available from the corresponding author upon request.