Zerumbone Protects Rats from Collagen-Induced Arthritis by Inhibiting Oxidative Outbursts and Inflammatory Cytokine Levels

Abstract

Rheumatoid arthritis (RA) is an immunocompromised disorder characterized by a marked increase in the synthesis of inflammatory molecules that stimulates the destruction of bones and cartilage. The conventional treatment modalities for RA are associated with adverse side effects and lack sensitivity, suggesting an immediate demand for alternate beneficial therapeutic remedies. The current study sought to understand more about zerumbone’s anti-inflammatory properties in diagnosing collagen-induced arthritis (CIA) in experimental animals. The current study observed that zerumbone reduced clinical severity in CIA-induced animals compared to healthy animals. Zerumbone administration significantly decreased (p < 0.001) the concentration of SOD, CAT, GR, and GSH in treatment groups. Zerumbone administration drove down significantly (p < 0.001) the concentration of inflammatory cytokine molecules. Zerumbone was effective in bringing significant changes in levels of MPO, NO, LDH, MMP-8, and ELA. The therapeutic potential of zerumbone was found to be associated with reduced joint destruction and restored normal histology in the cartilage and tissue. Adsorption, distribution, metabolism, excretion, and toxicity studies were used to determine the druglike properties of zerumbone. ProTox-II studies revealed that zerumbone did not possess toxic properties like hepatotoxicity, immunotoxicity, carcinogenicity, mutagenicity, and cytotoxicity. Therefore, the present study evaluated the therapeutic properties of zerumbone in CIA animal models.

1. Introduction

Rheumatoid arthritis (RA) is a severe immune disorder marked by progressive deterioration of the cartilage and associated bones and severe swelling and pain, eventually resulting in joint destruction.¹ The predominant features of RA include deformity and the presence of autoantibodies. RA occurs in approximately 1% of the global population.² RA mainly occurs in people aged 40–70 years, although it may occur at any age.³ RA appears more frequently in women than in men (3:1).⁴ In the onset of RA, genetic composition and smoking are the predominant risk factors, and RA is diagnosed by clinical and laboratory assessments. In the diagnosis of RA, a combination of autoantibodies, i.e., rheumatoid factors (RF), anticitrullinated protein/peptide antibody (ACPA), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and X-ray imaging are frequently used.⁵⁻⁷ Research studies have reported that inflamed synovial fibroblasts play a major role in RA pathogenesis by stimulating the synthesis of proinflammatory mediator molecules.⁸⁻¹⁰ Mitogen-activated protein kinases (MAPK) and nuclear factor-κB are the main proinflammatory signalling pathways that mediate the expression of inflammatory molecules.¹¹

Collagen-induced arthritis (CIA) is an excellent model for RA as it has helped to know and reveal pathogenic perspectives of autoimmune disease. It is critical to know about the distinct types of cells and their roles in the development of CIA and to test and develop novel therapeutic approaches. This model shares various pathological characteristics with the disease, such as cartilage degradation, synovial hyperplasia, and infiltration of mononuclear cells. The CIA model is best suited for assessing the potential of anti-inflammatory prescriptions in treating and managing RA.¹² In comparison to other disease models of arthritis that only cause inflammatory manifestations without prior pathophysiology, a better suited model for studying immune function changes is the CIA model.¹³ Traditionally, RA patients have been treated by using conventional immunosuppressive medications, including corticosteroids, methotrexate, cyclooxygenase-2 (COX-2) inhibitors, and anti-inflammatory drugs.¹⁴ However, such medications may provide short-term relief, but their long-term use causes adverse effects such as cardiovascular diseases and gastrointestinal bleeding.¹⁴ Therefore, in such a scenario, an alternate is provided by plant-derived natural compounds or their bioactive agents that could possess anti-inflammatory properties with minimal side effects.

Plant-derived extracts have been known to possess many polyphenols that can decrease inflammation and cause minimal side effects compared to synthetic drugs. Natural bioactives isolated from plants have the potential ability to be used as antirheumatic drug molecules. Natural compounds including cardamonin, β-elemene, curcumin, daphnetin, polyphenols (olive oil), brazilin, celastrol, nobiletin thymoquinone, tamaractam, and lapachol are some of the used bioactives for treatment of RA.¹⁵⁻¹⁸

Zerumbone is a naturally occurring monocyclic sesquiterpenoid obtained from Zingiber zerumbet and is known to possess several pharmacological properties.¹⁹⁻²¹ Zerumbone is reported to exhibit anti-inflammatory, antioxidant, anticarcinogenic, and antiallergic properties.²²⁻²⁵ In addition, zerumbone has been known to have potential to treat many diseases such as atherosclerosis,²⁶ osteoarthritis,^27,28 cancer,^29,30 diabetic complications,^31,32 neuropathic pain,³³ and hepatic diseases (nonalcoholic fatty liver and chronic liver fibrosis).^34,35 Zerumbone inhibits the expression of nitric oxide synthase, tumor necrosis factor-alpha (TNF-α), and COX-2.³⁶ Zerumbone is also reported to suppress the generation of toxic free radicals and tumor initiation. Therefore, this study focused on revealing the therapeutic potential of zerumbone against CIA-prompted inflammation in animals by determining oxidative and inflammatory disease markers.

2. Material and Methods

2.1. Study Animals

Male Wistar albino rats were purchased from Indian Institute of Integrated Medicine (IIIM) Jammu, J&K and housed in an institutional animal house facility at FVSC & AH, SKUAST-Kashmir, Shuhama, Alusteng, J&K, India. The animals were free to access a standard pelleted diet with clean water. The animals were provided with a temperature-controlled room with a constant temperature of 22 ± 2 °C, a relative humidity of 40–45%, and a 12h/12h light–dark cycle. The rats were given a week to acclimate before being examined. The Institutional Animal Ethical Committee approved the study (IAEC approval no. AU/FVSc/VCC/1-3/19/815-16 dated 23-11-2019).

2.2. Induction of CIA

Arthritis was induced in male Wistar rats by immunization in the proximal third of the tail with collagen type II (CII) emulsified with complete Freund’s adjuvant (CFA), as previously described.^37,38 CII was initially dissolved in 0.1 M acetic acid (2 mg/ml) and emulsified in an equal volume of CFA fortified with 4 mg of a mycobacterium strain to create the inducing effect. About 100 μL of the emulsion was injected subcutaneously at the base of the tail of each rat. After 14 days of immunization, a booster dose was given to stabilize the disease symptoms.

2.3. Treatment Schedule

The experimental animals were arranged into 4 groups (6 rats per group). The treatment in animals was started on the 25^th day and was given continuously for the next 20 days. Depending upon the preliminary study, the dose was selected for the animals.

Group I: control (the animals in this group were given normal saline).

Group II: diseased group (collagen-induced arthritis (CIA) was induced in animals).

Group III: treatment group I (in this group, CIA + zerumbone 25 mg/kg body weight was given orally for 20 days).

Group IV: treatment group II (in this group, CIA + zerumbone 50 mg/kg body weight was given orally for 20 days).

2.4. Measurement of Arthritis

In the affected paw, the arthritis intensity was assessed using a macroscopic scoring and grading system.³⁹ On a subjective scale (0–4), arthritis severity in each affected paw was measured and given scores and grades. The measurement of paw thickness by a caliper depicted the progress of arthritis. The arthritic index is illustrated in Table 1.

Table 1. Arthritic Score and Grading System.

score	description
0	An arthritic index of 0 signifies healthy animals without any disease (normal/control group).
1	A score of 1 depicts mild swelling of the ankle/wrist. In this group, the swelling is limited to a single joint or a few joints.
2	In this, there is moderate swelling that is confined to a single ankle/wrist and not involving other interphalangeal joints.
3	Animals with an arthritic index of 3 possess severe swelling in the ankle/wrist. The foot dorsum along metatarsophalangeal and metacarpophalangeal joints is affected by severe inflammation.
4	The animals in this group show maximum inflammation with multiple joints involved.

2.5. Sample Collection

After the experiments were concluded, each of the animals was sacrificed under light ether anesthesia. From each animal, blood was collected by cardiac puncture and subjected to centrifugation at 3000 rpm for 10 min at 4 °C for serum harvesting. The homogenate was prepared from knee joints, and the liver was removed.

2.6. Preparation of the Liver Homogenate and the Postmitochondrial Supernatant (PMS)

In all the animals, after the liver was aseptically removed, it was then homogenized in chilled phosphate-buffered saline (PBS, 0.1M pH 7.4) and 1.17% (w/v) potassium chloride. The homogenate prepared was subsequently used for lipid peroxidation (LPO) studies. The nuclear debris was removed by centrifuging the homogenate at 800g at 4 °C for 5 min. To obtain the PMS for studying the antioxidant profile, the supernatant was subjected to centrifugation at 10,000g for 20 min at 4 °C.

2.7. Evaluation of Malondialdehyde (MDA)

LPO was assessed in the liver homogenate and joint cell-free extract as described by Wright et al.⁴⁰ LPO was determined as n mol of MDA formed per g of tissue.

2.8. Assessment of Superoxide Dismutase (SOD)

The SOD activity was measured in liver PMS and joint cell-free extracts using the Marklund and Marklund method.⁴¹ The activity of SOD was assessed in units per mg of protein.

2.9. Estimation of Hydrogen Peroxide (H₂O₂)

The Pick and Mizel method was used for determination of H₂O₂.⁴²

2.10. Estimation of Catalase (CAT)

The Claiborne method was used to estimate the CAT activity in the liver PMS and joint cell-free extract. The enzyme’s activity was measured in n mol of H₂O₂ consumed/min/mg protein.⁴³

2.11. Evaluation of Glutathione Reductase (GR)

The assessment of GR was carried out as mentioned by Rashid et al.⁴⁴ The reaction mixture consisted of phosphate-buffered saline (825 μL of 1.0 M, pH 7.6), oxidized glutathione (25 μL of 1.0 mM), 50 μL of NADPH (0.1 mM), 50 μL of EDTA (0.5 mM), and 25 μL of PMS (5%) in a final volume of 1.0 mL.

2.12. Assessment of Reduced Glutathione (GSH)

The estimation of GSH was determined as described by Rashid et al.⁴⁴ In a ratio of 1:1 (v/v), the mixture of PMS was mixed with 4% sulfosalicylic acid. For a time duration of 60 min, the mixture was incubated at 4 °C and then subjected to centrifugation at 1200g for 15 min at 4 °C.

2.13. ELISA-Based Assays

NF-κB (cat. no. EMSNO), TNF-α, (cat. no. KRC3011), IL-6 (cat. no. BMS625), IL-10 (cat. no. ERA23RB), and IL-1β (cat. no. sBMS630) concentrations were assessed by using standard ELISA kits (Thermo Scientific, USA). Prostaglandin-E2 (PGE-2) was estimated via a standard ELISA kit (CUSABIO, cat. no. CSB-E07967r).

2.14. Myeloperoxidase (MPO) Assay

The MPO assay measures the intrusion of neutrophils in the synovial tissue as it directly correlates with the neutrophil number in affected tissues. In the synovial joints, levels of MPO were estimated using the ELISA-based assay (RayBiotech, USA, accession no. P11247).

2.15. Evaluation of Nitric Oxide (NO)

NO levels were estimated by using commercially available kits (Thermo Scientific, USA, cat. no. EMSNO).

2.16. Estimation of Lactate Dehydrogenase (LDH)

Commercially available kits (RayBiotech, USA, accession no. P06151) measured the LDH concentration.

2.17. Estimation of MMP-8

In a joint homogenate, specific matrix-metalloproteinase (MMP-8) rat assay kits (RayBiotech, USA, accession no. O88766) estimated MMP-8 levels.

2.18. Evaluation of Articular Elastase (ELA)

The ELA concentration in joints is an indicator of polymorphonuclear leukocyte (PMN) deposition and activation in swollen tissues.¹¹

2.19. Histopathological Studies

After the experiment completion, joint tissues were removed for histological studies. Immediately after the joint samples were removed, fixation was done in 10% formalin buffer, and subsequently, after some weeks, 10% EDTA solution was used for decalcification of these samples. Soon after the process of decalcification, the processing of samples was done into sections of 3–4 μm thickness, and further staining was carried out by using eosin and hematoxylin. From each group, four ankles were sectioned, and from each section, at least 4–5 pictures were taken for scoring and analysis.

2.20. ADMET Studies

ADMET (adsorption, distribution, metabolism, excretion, and toxicity) studies were used for evaluating pharmacodynamic properties of zerumbone. SWISSADME, a web-based online server, was used for determining ADMET properties of zerumbone.^45,46 PubChem was used to retrieve ligand smiles that were subsequently uploaded in SWISSADME. Lipinski’s rule of five parameters (molecular weight less than 500 g mol⁻¹, AloP should not be >5, the no. of hydrogen bond acceptors should be <10, and the no. of hydrogen bond donors should not be more than 5) was used to assess the ligand’s drug-likeness.

2.21. Toxicity Analysis

ProTox-II, an online-based tool, was used to determine toxicity prediction (https://ox-new.charite.de/protox_II/; last accessed on 16 April 2022).

2.22. Statistical Analysis

Sigma plot (version 8) software was used for statistical analysis, and the values were presented as the mean ± standard deviation (SD). The differences among the groups were examined by ANOVA (one-way analysis of variance). The Tukey–Kramer test was used for multiple data comparisons across the groups with a difference of p < 0.05 presumed as statistically significant.

3. Results

3.1. Arthritic Index and Swelling of the Foot Pad

In this study, a significant increase in the foot pad thickness and arthritis index was observed in CIA animals in comparison to healthy rats. In all the animals, arthritis induction was successful as evident with swelling in hind paws, macroscopic evidence of erythema, synovial membrane hyperplasia, and a substantial increase in swelling after day 14 when compared to control animals. A group of CIA animals showed evident signals of arthritis at day 21 after immunization. In the animals, development of arthritic signs in the paws characterized by the extent of edema and erythema was the major criterion used for clinical evaluation of CIA model development. The arthritic index and swelling of the foot pad provided insights into the severity of disease. After the 25^th day of arthritic symptoms, the animals were administered with zerumbone until the 45^th day, and during that period, secondary symptoms improved to varying degrees. The onset of paw swelling indicates the antiarthritic activity of different drugs. Zerumbone controls the arthritic index and swelling of the foot pad that are regarded as symptomatic disease markers. The arthritic index of different groups of animals is illustrated in Table 2.

Table 2. Arthritic Index and Effect of Zerumbone Treatment on Different Groups^a.

variables	groups	mean ± SD	P-value
arthritic index	control (group I)	0 ± 0
	CIA (group II)	6.11 ± 0.71	0.001*
	treatment group I (group III)	2.85 ± 0.36	0.001*
	treatment group II (group IV)	1.48 ± 0.34	0.001*

^aAll values are represented in mean ± SD values between groups (n = 6/group) before and after treatment. *The mean difference is significant at the level of 0.05.

3.2. Antioxidant Profile

In the present study, a significant increase (p < 0.001) in lipid peroxidation (LPO) and H₂O₂ was observed in group II as compared to the control group. Similarly, a significant decrease (p < 0.001) in the concentration of SOD, CAT, GR, and GSH was observed in group II (CIA) in comparison to the control group. However, after the administration of zerumbone, the levels of LPO and H₂O₂ significantly decreased in the treatment groups (III and IV) in comparison to the CIA animals (group II). Moreover, the concentrations of SOD, CAT, GR, and GSH showed a substantial elevation in treatment groups (III and IV) after the intake of zerumbone (Table 3). The mean ± SD of lipid peroxidation and antioxidant profiles is shown in Table 3.

Table 3. Zerumbone Treatment Effects on Antioxidant Enzymes and Lipid Peroxidation^a.

	group I	group II	group III	group IV
LPO (n mol MDA formed/g tissue)	2.031 ± 0.19	4.927 ± 0.49***	3.945 ± 0.67##	3.16 ± 0.39###
SOD (U/mg protein)	49.63 ± 5.62	10.96 ± 1.13***	19.68 ± 2.32#	23.16 ± 2.32###
H2O2 (n mol of H2O2/g tissue)	152.7 ± 16.3	363.7 ± 23.1***	295.3 ± 25.7#	219.3 ± 22.0###
CAT (n mol/min/mg protein)	89.19 ± 7.44	27.13 ± 3.26***	39.04 ± 4.61#	51.43 ± 5.16##
GR (n mol/min/mg protein)	232.4 ± 18.2	109.8 ± 14.0***	165.5 ± 22.7#	201.8 ± 21.5###
GSH (n mol/mg protein)	260.7 ± 26.1	133.3 ± 14.4***	201.6 ± 19.2##	238.3 ± 22.4###

^aThe values were represented statistically as mean ± SD (n = 6). When compared to group II, the differences were significant, as indicated by ^***p < 0.001. Similarly, when compared to group II, ^#p < 0.01, ^##p < 0.01, and ^###p < 0.001 values were considered significant. Normal saline (10 mL/kg bw), collagen-induced arthritis (CIA), CIA + zerumbone (25 mg/kg bw), CIA + zerumbone (50 mg/kg bw), and CIA + zerumbone (50 mg/kg bw).

3.3. Inflammatory Markers

In the present study, ELISA was performed to assess the levels of inflammatory markers, and it was found that NF-κB, TNF-α, IL-6, IL-10, IL-1β, and PGE-2 (pg/mL) were significantly increased (p < 0.001) in group II (CIA) as compared to the healthy animals. However, after treatment, the cytokine levels were driven down significantly. The concentration of inflammatory cytokine molecules is shown in Table 4.

Table 4. Effect of Zerumbone on Inflammatory Markers in Collagen-Induced Arthritis^a.

	group I	group II	group III	group IV
NF-κB (pg/mL)	401.3 ± 51.72	994.4 ± 112.6***	898.0 ± 72.09#	601.4 ± 58.22###
TNF-α (pg/mL)	209.43 ± 22.1	634.3 ± 72.4***	590.4 ± 53.4#	301.47 ± 37.2###
IL-6 (pg/mL)	710.64 ± 90.3	1410.7 ± 105.4***	1210.3 ± 98.2##	934.43 ± 91.4###
IL-10 (pg/mL)	698.09 ± 71.4	1210.4 ± 132.1***	1004.2 ± 90.2##	897.4 ± 110.4###
IL-1β (pg/mL)	893.12 ± 80.7	1486.2 ± 112.4***	1122.0 ± 94.9##	999.6 ± 89.61###
PGE-2 (pg/mL)	943.65 ± 92.9	1486.2 ± 112.4***	1122.0 ± 94.9##	999.6 ± 89.61###

^aThe values were statistically expressed as mean ± SD (n = 6). The differences were significant as indicated by ^***p < 0.001 when compared with group II. Similarly, ^#p < 0.01, ^##p < 0.01, and ^###p < 0.001 values were considered significant when compared with group II. Group I: normal saline (10 mL/kg bw), group II: collagen-induced arthritis (CIA), group III: CIA + zerumbone (25 mg/kg bw), and group IV: CIA + zerumbone (50 mg/kg bw).

3.4. Estimation of Myeloperoxidase

In present study, a significant increase (p < 0.001) in MPO levels was found in group II (CIA) in comparison to the healthy group (Table 5). In treatment groups (III and IV), the levels decreased significantly after zerumbone administration (Table 5).

Table 5. Effect of Zerumbone on MPO, NO, LDH, MMP-8, and ELA Profiles^a.

	group I	group II	group III	group IV
MPO	0.12 ± 0.04	0.47 ± 0.05***	0.35 ± 0.05#	0.21 ± 0.04###
NO	21.56 ± 2.97	70.83 ± 3.93***	52.15 ± 4.54#	29.67 ± 2.84###
LDH	214.2 ± 3.63	457.5 ± 12.71***	351.5 ± 11.26##	272.9 ± 14.72###
MMP-8	989.54 ± 87.88	1764.3 ± 177.4***	1587.3 ± 139.5##	1024.6 ± 100.75###
ELA	60.43 ± 5.42	229.3 ± 3.12***	170.4.2 ± 18.83##	100.6 ± 9.12###

^aThe values were expressed as mean ± SD (n = 6). The differences were significant as represented by ^***p < 0.001 when compared with group II. Similarly, ^#p < 0.01, ^##p < 0.01, and ^###p < 0.001 values were significant when compared with group II. Group I: normal Saline (10 mL/kg bw), group II: collagen-induced arthritis (CIA), group III: CIA + zerumbone (25 mg/kg bw), and group IV: CIA + zerumbone (50 mg/kg bw).

3.5. Nitric Oxide

A significant increase (p < 0.001) in levels of NO was found in the CIA group as compared to the healthy group, whereas a significant reduction was observed in treatment groups (III and IV) (Table 5).

3.6. LDH

In this study, LDH levels showed a significant increase (p < 0.001) in the CIA group, whereas after administration of zerumbone, a significant decrease was observed in treatment groups (III and IV) (Table 5).

3.7. MMP-8

In the present study, MMP-8 levels were significantly increased in group II (CIA) as compared to the healthy group. The levels of MMP-8 significantly decreased in treatment groups (III and IV) (Table 5).

3.8. Articular Elastase

In this study, ELA levels were significantly increased in group II in comparison to animals of the healthy group (Table 5). After treatment, a significant decrease was observed in treated groups (III and IV) (Table 5).

3.9. Histology

In CIA animals, the development of arthritis was found to be associated with an increased number of inflammatory macrophage cells and inflammation, thereby leading to destruction of joints and bones (Figure 1). However, after treatment with zerumbone, these inflammatory processes were suppressed in addition to reduction in cell influx and joint destruction events (Figure 1).

Figure 1.

(A–D) Group (A) photomicrograph reveals normal cartilage and tissue histology, with no signs of cellular infiltration. Group (B) photomicrograph indicates severe necrosis with cellular infiltration in the section of the articular cartilage. Group (C) treated with a lower dose of zerumbone (CIA + zerumbone (25 mg/kg bw) shows lesser regions of necrosis and infiltration. Group (D) with a higher dose of zerumbone (CIA + zerumbone 50 mg/kg bw) revealed normal histology in the cartilage and tissue section. Double arrows in red represent cellular infiltration, and the yellow stars represent areas of necrosis in groups (C) and (D).

3.10. ADMET Analysis

In the present study, physicochemical and pharmacodynamic (PD) properties of zerumbone were determined. In describing the pharmacokinetics of a drug, ADMET properties are particularly significant. Different properties like high gastrointestinal absorption (GI absorption), good LOGP scores/lead-likeness/bioavailability, better synthetic accessibility, and blood–brain barrier (BBB) permeability were selected for this study, and the compound yielded good results. Lipinski’s rule of five parameters was followed by the zerumbone compound with a molecular weight within the acceptable range and XLOGP (lipophilicity value between 0–5). The compound passed the ADMET test and was subjected to toxicity analysis. The number of hydrogen bond acceptors and donors was found within the acceptable range as well. The molecules with a TPSA score of less than 140 Å signify a substantial permeability in the plasma membrane. Zerumbone is not a P-gp substrate (p-glycoprotein), thereby suggesting a potential anticancer agent. The MR of the compound was found within the acceptable range (40–130 for most cases) and had good skin permeability (log Kp of −4.8 cm/s). In addition, Ghose, Veber, Egan, and Muegge rules were within the acceptable limits. The bioavailability score of 0.55 is considered respectable that describes good pharmacokinetic properties of a molecule. The ADMET results are shown in Table 6.

Table 6. ADMET Analysis of Zerumbone^a.

ADMET properties	zerumbone
molecular weight (g/mol)	218.33
topological polar surface area (TPSA) (Å)	17.07
num. of H-bond acceptors	1
num. of H-bond donors	0
molar refractivity (MR)	70.62
XLOGP	3.94
iLOGP	2.72
MLOGP	3.37
WLOGP	4.21
Lipinski	yes
Ghose	yes
Veber	yes
Egan	yes
Muegge	no
bioavailability score	0.55
GI absorption	high
BBB permeability	yes
P-gp substrate	no
CYP1A2 inhibitor	no
CYP2C19 inhibitor	no
CYP2C9 inhibitor	yes
CYP2D6 inhibitor	no
CYP3A4 inhibitor	no
log Kp (skin permeation) cm/s	–4.83
pan assay interference compounds (PAINS)	0
Brenk	1
lead-likeness	2
synthetic accessibility	3.47

^aThe above table shows the physiochemical properties, pharmacokinetics, drug-likeness, and medicinal chemistry of zerumbone. The rules of 5 (Lipinski’s rules) evaluated drug-likeness depending upon the molecular mass ≤ 500 g mol^–1, log P (≤5), and the number of hydrogen bond acceptors (≤10) and donors (≤5).

3.11. Toxicity Analysis

In this study, different factors were considered for toxicity analysis that included the lethal dose (LD₅₀ (4590 mg/kg) predicted toxicity class, hepatotoxicity, immunotoxicity, carcinogenicity, mutagenicity, and cytotoxicity. Levels of toxicity were classified as class 1 and 2 (fatal if swallowed), class 3 (toxic if swallowed), class 4 (harmful if swallowed), class 5 (may be harmful if swallowed), and class 6 (nontoxic). The zerumbone compound belonged to class 5, showed no toxicity, and was found to be inactive. During clinical trials, toxicity leads to unfavorable effects that cause drug breakdown. The probabilities of predicted toxicity as calculated by the ProTox-II tool are shown in Table 7.

Table 7. Toxicity Analysis of Zerumbone^a.

		prediction probability
compounds	predicted toxicity class	hepatotoxicity	carcinogenicity	immunotoxicity	mutagenicity	cytotoxicity
zerumbone	5	0.69	0.73	0.99	0.91	0.92

^aThe acute toxicity model of compounds based on the validation approach of leave-one-out cross-validation with the prediction type (lethal dose value in mg/kg body weight). The prediction model involves 33 models with respective confidence scores, and prediction scores are provided as an overall toxicity radar chart. Zerumbone is predicted to be inactive for 5 endpoints, connecting different layers of the ProTox-II classification scheme. The above table indicates the positive toxicity results in comparison to the average of its class.

4. Discussion

The general hallmarks of arthritis are inflammation and joint destruction. CIA is an immune hyperfunctional animal model. However, the exact mechanism of the occurrence of destruction is not well-known. Monitoring paw swelling is a dependable and straightforward method for determining the severity of inflammation.^47,48 In this study, zerumbone treatment decreased the paw thickness and joint diameter by interfering with inflammatory mediator molecules depicting its anti-inflammatory potential in CIA. In the current study, we investigated the role of the cellular inflammatory cascade and oxidative stress in CIA animal models. Elevated oxidative stress contributes to the emergence of autoimmune conditions through the activation of the cellular inflammatory cascade, apoptosis, and the breakdown of immunological tolerance.⁴⁹ The findings of this study are in concordance with the reports of many studies that found MDA levels to be significantly higher in RA patients compared to healthy controls.⁵⁰⁻⁵³ Accordingly, higher levels of MDA or lipid peroxides were found in animals immunized with FCA, which were substantially reduced with zerumbone intervention; these findings agree with previous research studies.^54,55 The increase in the MDA concentration might be because of elevated levels of reactive oxygen species (ROS) that result in a pro-oxidation environment.

In the present study, SOD was decreased significantly in the CIA group compared to controls; however, the levels were replenished in treatment groups. SOD catalyzes the conversion of the superoxide anion into H₂O₂ and subsequently serves as the first line of defense against ROS. Various authors have reported similar findings of lowered SOD activity in RA individuals than controls.⁵⁶⁻⁵⁸ The decrease in the SOD activity in RA patients could be due to the degradation process in which free radicals degrade SOD during the detoxifying process.⁵⁰

In the present study, a significant increase in H₂O₂ was observed in CIA animals compared to the healthy group. However, after the administration of zerumbone, the levels significantly decreased in treatment groups. Veselinovic et al.⁵⁹ have reported similar findings. The increased levels of H₂O₂ could be due to more oxidative stress and decreased antioxidant enzyme systems.

The present study observed a significant decrease in the CAT activity in the CIA group compared to the controls. The findings of this study agree with the previous reports.^60,61 As reported previously, the production of H₂O₂ inactivates the CAT activity in RA patients, suggesting that oxidative enzymes may play a significant role in increasing oxidative stress and the rheumatic process. In the present study, a significant decrease in the GR and GSH activity in the CIA group was found compared to the control group; however, after the zerumbone administration, the levels were enhanced in treatment groups. Previous research studies have reported similar findings.⁶²⁻⁶⁹ The decrease in the antioxidant enzyme activity could be due to enzymatic inhibition and saturation of antioxidant enzyme systems.

Inflammatory molecules play a critical role in bone and articular cartilage destruction during RA. In the current study, the CIA group had a considerable increase in the levels of inflammatory markers (NF-κB, TNF-α, IL-6, IL-10, 1L-1β, and PGE-2) when compared to the controls. However, after intake of zerumbone, the levels of inflammatory molecules were significantly reduced in the treatment groups. The findings of this study agree with previous reports.^65,70−73 Similar results have reported a significant increase in TNF-α, IL-6, IL-1β, PGE-2, and IL-10 in CIA animals as compared to healthy ones.⁷⁴⁻⁷⁹ NF-κB is a key player in RA that initiates and exacerbates inflammation.⁸⁰⁻⁸² At the disease site, TNF-α triggers the synthesis of IL-6 and IL-1β by macrophages that further facilitates leukocyte infiltration and vasodilation.⁸² In this study, IL-10 levels were significantly increased in CIA animals compared to healthy ones, but the exact mechanisms of elevated release of this anti-inflammatory cytokine remain unclear. However, it is suggested that more IL-10 is produced in RA as a compensatory mechanism to inhibit continuous proinflammatory cytokine production and inflammatory cascade, thus resulting in elevated IL-10 levels.⁸³ In the present study, higher levels of PGE-2 were reported in CIA rats as compared with controls, and this could be associated with the inflammatory processes that suppress B and T cell proliferation and cytokine synthesis as well.⁸⁴

We observed a substantial elevation in MPO, NO, LDH, MMP-8, and ELA levels in the CIA group as compared to healthy animals; however, zerumbone intake significantly decreased their expression in the treatment groups. Increased MPO levels in RA in the synovial fluid^85,86 and serum^87,88 have been reported. The elevated concentration of MPO may be associated with increased cardiovascular complications in RA individuals.

Increased NO levels were measured in the CIA group during this study, similar to previous research conducted in RA patients.^89,90 NO is a strong oxidant produced by macrophagic cells during inflammation and plays a crucial role in cell signaling pathways.^91,92 These inflammatory substances keep the inflammation going by allowing chemotactic factors to be produced at the local site.⁹³

LDH is an enzyme that is present in every tissue and converts sugar into energy to be used by cells. Arafah et al.⁶⁵ have reported similar findings. In the present study, an increased concentration of LDH might be associated with myocardial infarction or cardiac injury and therefore gets released into the bloodstream. The elevated levels of LDH might be related to oxidative stress and consequent inflammatory reactions.⁹⁴

MMPs are activated by TNF-α and are known to be associated with extracellular matrix degradation. In the present study, analysis of MMP-8 levels was increased significantly in CIA animals compared to controls. This agrees with the previous findings.⁷⁶ The rise in MMP-8 could be linked explicitly to collagen type II breakdown, suggesting MMP-8’s matrix-degrading ability during the development of RA.⁹⁴

This study shows a considerable rise in ELA in CIA rats compared to healthy animals in the current study. This is consistent with the previous findings.^74,90 Elastase levels have risen due to a large influx of activated PMNs.⁹⁵ There is a rapid decrease in ELA after zerumbone administration, which could be attributed to the inhibition of lipid peroxidation and the subsequent reduction in chemotactic peroxide.³⁷

The druglike properties of zerumbone were determined by Lipinski’s rule. These factors are particularly essential as they are associated with permeability and dissolution and often surpass toxicity described by ADMET properties.^96,97 Natural products can be used as an alternative to treat RA and other ailments owing to their minimal side effects and less cost.¹⁶⁻³⁵ Natural bioactives can control inflammation associated with RA through multiple pathways involving induction of IL-10 and IL-4 (anti-inflammatory mediators), Th17/Treg balance, and auto-immune cross-talk modulation.¹⁶⁻³⁵ Thus, plant bioactives can serve as potential molecules for disease treatment, and using computational approaches can decrease the time frame.

5. Conclusions

As manifested from the findings in the present study, zerumbone administration decreased lipid peroxidation, increased the enzymic antioxidant functions, and regulated the concentration of proinflammatory and inflammatory molecules. The results obtained from biochemical and molecular studies revealed the potential anti-inflammatory and antioxidant characteristics of zerumbone against CIA-induced rheumatoid arthritis, thereby suggesting that the protective properties of zerumbone can be due to attenuation of inflammation and oxidative stress. Thus, based on the present study’s findings, the combination of both anti-inflammatory and antioxidant properties of zerumbone might prove significant as a remedial measure for the treatment and prevention of joint diseases.

Glossary

Abbreviations

ACPA

anticitrullinated protein/peptide antibody

CRP

C-reactive protein

ESR

erythrocyte sedimentation rate

MAPK

mitogen-activated protein kinases

COX-2

cyclooxygenase-2

CII

collagen type II

LPO

lipid peroxidation

H₂O₂

hydrogen peroxide

GR

glutathione reductase

GSH

reduced glutathione

PGE-2

prostaglandin-E2

RA

rheumatoid arthritis

CFA

complete Freund’s adjuvant

RF

rheumatoid factor

CIA

collagen-induced arthritis

TNF-α

tumor necrosis factor-alpha

PMS

postmitochondrial supernatant

MDA

malondialdehyde

SOD

superoxide dismutase

MPO

myeloperoxidase

MMPs

matrix metalloproteinases

NO

nitric oxide

ELA

articular elastase

CAT

catalase

ELISA

enzyme-linked immunosorbent assay

IL

interleukin

NF-κB

nuclear factor-kappa B

LDH

lactate dehydrogenase

SD

standard deviation

ANOVA

one-way analysis of variance

BBB

blood–brain barrier

PMNs

polymorphonuclear leukocytes

The authors declare no competing financial interest.