Anti-Inflammatory and Antioxidant Properties of Malapterurus electricus Skin Fish Methanolic Extract in Arthritic Rats: Therapeutic and Protective Effects

The protective and therapeutic anti-inflammatory and antioxidant potency of Malapterurus electricus (F. Malapteruridae) skin fish methanolic extract (FE) (300 mg/kg.b.wt/day for 7 days, orally) was tested in monosodium urate(MSU)-induced arthritic Wistar albino male rats’ joints. Serum uric acid, TNF-α, IL-1β, NF-𝜅B, MDA, GSH, catalase, SOD, and glutathione reductase levels were all measured. According to the findings, FE significantly reduced uric acid levels and ankle swelling in both protective and therapeutic groups. Furthermore, it has anti-inflammatory effects by downregulating inflammatory cytokines, primarily through decreased oxidative stress and increased antioxidant status. All the aforementioned lesions were significantly improved in protected and treated rats with FE, according to histopathological findings. iNOS immunostaining revealed that protected and treated arthritic rats with FE had weak positive immune-reactive cells. Phytochemical analysis revealed that FE was high in fatty and amino acids. The most abundant compounds were vaccenic (24.52%), 9-octadecenoic (11.66%), palmitic (34.66%), stearic acids (14.63%), glycine (0.813 mg/100 mg), and alanine (1.645 mg/100 mg). Extensive molecular modelling and dynamics simulation experiments revealed that compound 4 has the potential to target and inhibit COX isoforms with a higher affinity for COX-2. As a result, we contend that FE could be a promising protective and therapeutic option for arthritis, aiding in the prevention and progression of this chronic inflammatory disease.


Introduction
Gout is regarded as an inflammatory response caused by the deposition of monosodiumurate (MSU) crystals around the joints [1]. When it comes to the progression of gout, the inflammatory response comes first. The underlying process is that MSU-crystals B, MDA, GSH, catalase, SOD, and glutathione reductase levels were all measured. According findings, FE significantly reduced uric acid levels and ankle swelling in both protective and peutic groups. Furthermore, it has anti-inflammatory effects by downregulating inflammat tokines, primarily through decreased oxidative stress and increased antioxidant status. A aforementioned lesions were significantly improved in protected and treated rats with FE, a ing to histopathological findings. iNOS immunostaining revealed that protected and trea thritic rats with FE had weak positive immune-reactive cells. Phytochemical analysis reveal  Anti-Inflammatory and Antioxidant P electricus Skin Fish Methanolic Extrac Therapeutic and Protective Effects B, MDA, GSH, catalase, SOD, and glutathione reductase levels were all measured. According to the findings, FE significantly reduced uric acid levels and ankle swelling in both protective and therapeutic groups. Furthermore, it has anti-inflammatory effects by downregulating inflammatory cytokines, primarily through decreased oxidative stress and increased antioxidant status. All the aforementioned lesions were significantly improved in protected and treated rats with FE, according to histopathological findings. iNOS immunostaining revealed that protected and treated arthritic rats with FE had weak positive immune-reactive cells. Phytochemical analysis revealed that

Control Group
89.90 ± 9.00 a 17. 55   Arthritic rats (G2, Photomicrographs 4-6, Figure 1) showed articular surface irregularity and deformity with necrosis of a significant number of chondrocytes, synovial membrane and subcut tissue showed heavy infiltration of inflammatory cells with edoema and congestion of blood vessels, and bone trabeculae showed widening of trabecular space compared to control (Photomicrographs 1-3, Figure 1). All of the aforementioned lesions were significantly improved in arthritic rats treated with both FE and a standard drug (G4, G5, Photomicrographs 7-10, 15,16, Figure 1). The articular surface and synovial membrane of protective groups injected with MSU and co-administered with FE (G3, Photomicrographs 11-14, Figure 1) showed nearly normal structure.
Arthritic rats (G2, Photomicrographs 4-6, Figure 1) showed articular surface irregularity and deformity with necrosis of a significant number of chondrocytes, synovial membrane and subcut tissue showed heavy infiltration of inflammatory cells with edoema and congestion of blood vessels, and bone trabeculae showed widening of trabecular space compared to control (Photomicrographs 1-3, Figure 1). All of the aforementioned lesions were significantly improved in arthritic rats treated with both FE and a standard drug (G4, G5, Photomicrographs 7-10, 15,16, Figure 1). The articular surface and synovial membrane of protective groups injected with MSU and co-administered with FE (G3, Photomicrographs 11-14, Figure 1) showed nearly normal structure.   (1), of control rats showed synovial membrane showing normal histological structure (arrow) (H&EX400), Photomicrograph (2) of control rats showed articular surface showing normal histological structure (arrow) (H&EX200), Photomicrograph (3) of control rats showed showing normal histological structure (H&EX400), Photomicrograph (4) of Arthritic rats showed synovial membrane with heavy infiltration of inflammatory cells (arrow) (H&EX200), Photomicrograph (5) of Arthritic rats showed articular surface showing irregularity of articular surface (arrow) with widening of trabecular space and necrosis (H&EX400), Photomicrograph (6) of Arthritic rats showed synovial membrane showing heavy infiltration of inflammatory cells (arrow) (H&EX400), Photomicrograph (7) of Arthritic rats treated with FE showed articular surface with smooth articular surface (arrow) with normal trabecular space and few necrosed chondrocytes (H&EX400), Photomicrograph (8) of Arthritic rats treated with FE showed synovial membrane with few inflammatory cells and mild edema (arrow) (H&EX200), Photomicrograph (9) of Arthritic rats treated with FE showed articular surface with smooth articular surface (arrow) with normal trabecular space and few necrosed chondrocytes (H&EX200), Photomicrograph (10), of Arthritic rats treated with FE showed synovial membrane with mild edema (H&EX400), Photomicrograph (11) of Arthritic-protective rats articular surface showing nearly normal and smooth articular surface with mild edema of synovial membrane (H&EX200), Photomicrograph (12) of Arthritic-protective rats showed synovial membrane showing mild edema and congestion of synovial membrane (H&EX200). Photomicrograph (13), of Arthritic -protective rats' articular surface showing normal articular surface and synovial membrane (H&EX400). Photomicrograph (14), of Arthritic -protective rats showing normal articular surface and synovial membrane (H&EX400). Photomicrograph (15) of Arthritic rats treated with standard drug showing smooth articular surface (arrow) with normal trabecular space and few necrosed chondrocytes (H&EX400). Photomicrograph (16) of Arthritic rats treated with standard drug showed, articular surface with normal and smooth articular surface and few necrosed chondrocytes (H&EX400). All lesions found in bone tissue were graded based on their severity. Table 4 shows a significant reduction in score lesion in either the treated or protective groups when compared to the Arthritic group. In standard drug-treated Arthritic rats, similar results were obtained. Table 5 also showed a significant reduction in the percentage expression of iNOS in the treated and protected groups when compared to the Arthritic group. The immunostaining expression of iNOS% area in different treated groups. iNOS immunostaining revealed no immune-reactive cells in the control group (Photomicrographs 17, 18; Figure 2). Sections from arthritic rats (G2, Photomicrographs 19-21; Figure 2) revealed a high level of iNOS expression. In contrast, arthritic rats treated with FE as well as standard drug (G4, G5) showed weak positive immune-reactive cells (Photomicrographs 22, 23, and 26; Figure 2) with percentages of iNOS reduction amounted to 31.87 and 48.46 in articular-surface and synovial membrane and for group 4 respectively. While the percentages of iNOS reduction for the articular surface, synovial membrane, and group 5 were 24.57 and 43.47%, respectively. However, the protective group (G3, Photomicrographs 24, 25; Figure 2) showed very few to no positive immune-reactive cells, with iNOS reduction percentages of 31.87 and 48.46% for the articular surface and synovial membrane, respectively. Widening of trabecular space The score process was composed as: score 0 = lack of the lesion in all rats of the group (n = 5), score 1 = (<30%), score 2 = (<30%-50%), score 3 = (>50%). G1: control rats, G2: Arthritic rats, G3: Arthritic rats co-administered with extract, G4 and G5: Arthritic rats treated with fish extract and standard drug respectively. Each value was expressed as mean ± SEM. Different letters in the same column are significantly different (p ≤ 0.05). G1: control rats, G2: Arthritic rats, G3: Arthritic rats co-administered with extract, G4 and G5: Arthritic rats treated with fish extract and standard drug respectively. % Reduction is calculated according to arthritic rats using the equation: (mean of disease rats − mean of treated rats/Mean of disease rats) × 100.

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Synovial membrane 0 60.15 ± 3.00 b 19.00 ± 1.00 a 31.00 ± 1.00 c 34.00 ± 2.00 c % Reduction to arthritic rats --68. 41 48.46 43.47 Each value was expressed as mean ± SEM. Different letters in the same column are significantly different (p ≤ 0.05). G1: control rats, G2: Arthritic rats, G3: Arthritic rats co-administered with extract, G4 and G5: Arthritic rats treated with fish extract and standard drug respectively. % Reduction is calculated according to arthritic rats using the equation: (mean of disease rats − mean of treated rats/Mean of disease rats) × 100.

Amino Acid Content
Amino acids are required for the synthesis of a wide range of proteins, including carriers of CO 2 , oxygen, structural proteins, and vitamins [21]. Table 7 and Figure S10 show the amino acid content of M. electricus extract. The total amino acid content of the M. electricus extract was 4.584 mg/100 mg, with a high concentration of glycine (0.813 mg/100 mg) and alanine (1.645 mg/100 mg). M. electricus extract contained 1.213 mg/100 mg of essential amino acids such as threonine, valine, isoleucine, leucine, phenylalanine, histidine, and lysine (0.144, 0.206, 0.108, 0.247, 0.124, 0.142, and 0.242 mg/100 mg, respectively).

Molecular Modeling Study
Fatty acids, particularly unsaturated ones, have been shown to have significant anti-inflammatory potential [22][23][24]. The COX pathway, along with its main product prostaglandin E2 (PGE-2) [25,26], is one of the primary inflammatory mediators implicated in the inflammatory phase of joints and its accompanying pain. It was recently discovered that inhibiting COX-2 specifically was associated with faster pain relief and decreased joint inflammation in experimental mice [27]. Based on their structural similarity to arachidonic acid (AA), the primary precursor of the COX enzyme, we investigated the isolated fatty acids' potential interactions with both COX-1 and COX-2. Each fatty acid's modelled structure was docked into the active sites of both COX-1 and COX-2 (PDP codes: 3KK6 and 3HS5, respectively). The convergent scores for all docked compounds (1-4) ranged from −7.34 to −8.75 kcal/mol.
To gain a better understanding of each isolated fatty acid's affinity for the active sites of COX-1 and COX-2, the best generated docking pose for each was subjected to MDS-based ∆Gbinding calculation using the Free Energy Perturbation method (FEP) [28]. As a result, compound 4 was found to have the highest affinity for both enzymes (COX-1 and COX-2) with a slightly higher affinity for COX-2 (Table 8). Compound 4 has the most double bonds of any compound and thus has the least flexible molecules. As a result of compound 4 s low flexibility in comparison to the remaining fatty acids, its entropic component in the binding free energy was relatively significant, and thus its affinity towards the enzymes' active site was the best (Table 8). According to this preliminary finding, the interactions of compound 4 inside the active sites of both enzymes were investigated further using 50 ns MDS experiments. Before beginning the simulations, the structural alignment of compound 4 and the co-crystalized ligands inside the active sites of COX-1 and COX-2 revealed perfect matches in terms of binding orientation and interactions ( Figure 4). In the case of COX-1, both compound 4 and the co-crystalized ligand exhibited the same hydrophobic and hydrophilic interactions ( Figure 4A). Except for H-bonding, compound 4 established a single H-bond with SER-530, whereas the shorter co-crystalized fatty acid (i.e., arachidonic acid) established a single H-bond with TYR-385 ( Figure 4B). Over the 50 ns MDS, compound 4 and each co-crystalized ligand inside the active site of both enzymes (i.e., COX-1 and COX-2) demonstrated nearly identical binding stability with average RMSDs of 2.2 for COX-1 and 1.3 for COX-2 ( Figure 4C).   Figure 5A. Furthermore, the carboxylate moiety formed three H-bonds with HIS-90, HIS-513, and SER-516. It is worth noting that, with the exception of the stable H-bond with SER-516, the binding mode of the co-crystalized ligand of COX-1 changed over the course of simulation and became different from that of compound 4 ( Figure 5B). Similarly, within COX-2′s active site, compound 4′ long hydrocarbon chain established multiple hydrophobic interactions with 6 amino acids (VAL-116, VAL-349, TYR-355, PHE-518, ALA-527, and  Figure 5A. Furthermore, the carboxylate moiety formed three H-bonds with HIS-90, HIS-513, and SER-516. It is worth noting that, with the exception of the stable H-bond with SER-516, the binding mode of the co-crystalized ligand of COX-1 changed over the course of simulation and became different from that of compound 4 ( Figure 5B). Similarly, within COX-2 s active site, compound 4 long hydrocarbon chain established multiple hydrophobic interactions with 6 amino acids (VAL-116, VAL-349, TYR-355, PHE-518, ALA-527, and LEU-531), whereas arachidonic acid (COX-2 s co-crystalized fatty acid) interacted with only three (i.e., VAL-116, TYR-355, and PHE-518). As a result, compound 4 s significant hydrophobic interactions within the active site of COX-2 explain its higher affinity in terms of Gbinding over arachidonic acid (Table 8). In terms of H-bonding, compound 4 formed very stable ones with SER-530 and TYR-385, whereas arachidonic acid formed fewer stable ones with ARD-120 and TYR-355, as well as a single water bridge with SER-119 ( Figure 5C,D).

Discussion
Gout is an auto-inflammatory condition associated with elevated blood urate levels due to the deposition of MSU-crystals in and around joints [29], which resulted in an increase in neutrophils. Neutrophils correlate with the elevation of IL-1β, IL-8, and TNF-α levels, which are necessary cytokines in inflammation and significant mediators implicated in the pathogenesis of gout [2]. A network that stimulates the interpretation of NF-κB regulates the expression of several cytokines. NF-κB is a key regulator of proinflammatory gene expression that can increase the expression of a variety of cytokines, including TNF-α, IL-1β, and IL-8 [30]. Many inflammatory genes, including cytokines, adhesion molecules, and chemokines, are activated by NF-κB [31]. As a result, downregulation of the NF-B signaling pathway may be an appropriate approach for gout treatment. In the current study, we measured the levels of TNF-α, IL-1β, and NF-κB in arthritic rats caused by MUS in a rat model [32][33][34][35], while management or protection by fish extract reduced the levels in the serum.
Furthermore, another inflammatory process associated with gout is oxidative stress.

Discussion
Gout is an auto-inflammatory condition associated with elevated blood urate levels due to the deposition of MSU-crystals in and around joints [29], which resulted in an increase in neutrophils. Neutrophils correlate with the elevation of IL-1β, IL-8, and TNF-α levels, which are necessary cytokines in inflammation and significant mediators implicated in the pathogenesis of gout [2]. A network that stimulates the interpretation of NF-κB regulates the expression of several cytokines. NF-κB is a key regulator of proinflammatory gene expression that can increase the expression of a variety of cytokines, including TNF-α, IL-1β, and IL-8 [30]. Many inflammatory genes, including cytokines, adhesion molecules, and chemokines, are activated by NF-κB [31]. As a result, downregulation of the NF- study, we measured the levels of TNF-α, IL-1β, and NF-κB in arthritic rats caused by MUS in a rat model [32][33][34][35], while management or protection by fish extract reduced the levels in the serum.

Anti-Inflammatory an electricus Skin Fish M Therapeutic and Prote
Furthermore, another inflammatory process associated with gout is oxidative stress. The current findings revealed a significant increase in MDA levels, as well as a distinct decrease in catalase, GSH reductase, GSH, and SOD. This oxidative state is caused by the production of ROS and pro-inflammatory cytokines [36], which results in hyaluronic acid depolymerization, degradation of proteoglycans and collagen, protein decomposition, and inhibition of proliferation. These cells are known to have low levels of antioxidants such as catalase and SOD [37]. In the state of gout, crystals increase the production of NO [38], and when it combines with O 2 , peroxy nitrite (ONOO) is formed, disrupting cellular proliferation, connective material degradation, and joint degeneration [39]. NO mediates the body's innate immune response. Extracellular catalysts can initiate iNOS expression by activating NO signaling pathways [40][41][42][43][44]. In the case of arthritis, NO now interferes with inflammation [40]. Similarly, as shown in our findings, gouty arthritis synovial tissue has elevated iNOS interpretation ( Figure 2). Furthermore, chondrocyte apoptosis occurs as a result of the activities of reactive nitrogen intermediaries and proinflammatory cytokines, which contribute to significant cartilage loss. Bone destruction is accelerated by the release of cytokines such as GM-CSF and the activation of NF-κB, which promotes osteoclast differentiation and cellular invasion of the cartilage-affected surface [42]. TNF-α, IL-1β, IL-6β, and GM-CSF can promote the management of adhesion molecules, other inflammatory cytokines, and chondrocyte and osteoclast activation, all of which contribute to joint loss [44].
The present study investigated the GC/MS composition, amino acids content, as well as the phytochemical composition of FE and evaluated its anti-inflammatory and antioxidant potency in treatment or protective arthritic Wistar albino male rats' joints. The phytochemical analysis revealed that FE was high in fatty acids (both saturated and unsaturated) and amino acids (essential, non-essential). The most abundant compounds were vaccenic (24.52%), 9-octadecenoic (11.66%), palmitic (34.66%), stearic acids (14.63%), glycine (0.813 mg/100 mg), and alanine (1.645 mg/100 mg). In addition, the current findings stated that either treatment or protective-arthritic inflamed rats with FE showed significant improvement in all mentioned parameters; additionally, the articular surface and synovial membrane showed nearly normal structure with low lesion score in irregularity and deformities of the articular surface, necrosis of chondrocytes and synovial membrane infiltrated with inflammatory cells, and widening of the trabecular space (Tables 1-5, Figures 1 and 2). Furthermore, there is little to no positive iNOS expression in inflamed immune-reactive cells. Furthermore, cytokine modulation was associated with improvements in inflammatory, clinical, and histological frameworks in animals treated with FE, indicating that FE is a promising treatment for arthritis. Overall, these findings are significant because, while anti-arthritic medications reduce inflammation, they do not consistently prevent or improve bone erosion and cartilage damage. The reduction of inflammatory infiltrates in the cartilage of animals treated with FE may be related to the modulatory activity of these inflammatory mediators, including the downregulation of TNF-α, IL-1, 2, 6, and GM-CSF, the reduction of NO, and the increase of IL-10, indicating that FE reduced inflammatory infiltrate, synovial hyperplasia, and bone erosion [20].
The current study's findings agreed with those of Bahadori and colleagues, et al., in their 2010 report [45], which published the value of fish extract or n-3 PUFA in rheumatoid arthritis. These findings were consistent with those of Ruggiero et al. 2009 [46], who described more reliable data supporting the efficacy of omega-3 PUFAs in pain relief and the treatment of nonsteroidal anti-inflammatory drugs. Alam et al., 1993 [47] investigated the effects of dietary fats on inflammatory mediators in alveolar bone. They discovered that these dietary lipids had a significant impact on the formation of fatty acids in bone lipids. Arachidonic-acid accumulations in the overall phospholipids of the mandibles and maxillae were significantly lower in rats fed fish oil diets. Arachidonic acid is a precursor to prostaglandin-E2 and leukotriene C4. A significant decrease in its concentration could result in lower levels of eicosanoids in the alveolar bone. Furthermore, early research established that omega-3 fatty-acid-derived lipid-mediators, recognized as repairing and protecting, may play an important role in repairing inflammation due to their strong antiinflammatory activities. These mediators suppress inflammation even further, cause bone resorption, and directly influence osteoclast differentiation [48][49][50]. Several studies have found that n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) rich in fatty fish oils have significant anti-inflammatory properties and beneficial effects on bone metabolism, most likely by inhibiting proinflammatory mediators such as prostaglandin E2, IL-1, 6, and TNF-α [50,51].
Furthermore, Saxena et al., 1984 [52] stated that amino acids (L-phenylalanine, DLisoleucine, L-isoleucine, and L-leucine) had anti-inflammatory action by interfering with prostaglandin synthesis and/or action. Furthermore, Lee et al., 2017 [53] discovered that leucine, valine, and isoleucine inhibited NO production as well as inducible nitric oxide synthase mRNA expression. Glutamine has been shown to have an anti-inflammatory effect by inhibiting pro-inflammatory chemokine and cytokine production, which was achieved through excessive glutamine nutrient absorption, leading to the strengthening elimination of the inhibitor of nuclear-factor-kappa-B-kinase (Iκκ) action and the reduction of the inhibitor of nuclear-factor-kappa-B-kinase subunit-beta (IκB) degradation [54]. Glutamine is also known to inhibit NF-κB and P38 mito-gen-activated-protein-kinase (MAPK) pathway signaling factors [54,55]. Arginine has anti-inflammatory properties such as glutamine in that it inhibits chemokine re-ply of IL-8 production and inhibits Iκκ action [54,55]. Cysteine has been shown to inhibit IL-8 induced by TNF-α [56]. Cysteine inhibits NF-κB activation even further when combined with TNF-α [56]. Kynurenic acid is a byproduct of tryptophan metabolism [57]. Kynurenic acid can reduce IL-6 and TNF-α levels [58]. Furthermore, He et al., 2018 andLiu et al., 2017 [59,60] stated that the functions of amino acids (essential and non-essential) in inflammation are primarily associated with overcoming oxidative stress and inhibiting proinflammatory cytokines the expression. MAPK, NF-B, Nrf2, ACE2, iNOS, mTOR, CaSR, and GCN2 are among the signaling processes that carry out these functions.
Consequently, Omega-3 fatty acids and protein-rich FE have been shown to reduce inflammation which could be attributed to inhibitory and modulatory actions on the production and release of nitric oxide and cytokines, both of which are involved in disease pathogenesis. According to molecular modelling and dynamics simulation, one of the major components of the crude extract (compound 4) has the potential to target and inhibit COX isoforms with a higher affinity for COX-2.

Fish Collection
M. electricus fish-were-purchased-from-a-local-market in Beni-Suef, Egypt, in May 2022, and then identified using a fish-identification-key [61]. A voucher specimen (2022-BuPD-87) was archived-at Beni-Suef University's Department-of-Pharmacognosy, Faculty of Pharmacy. Following purchase, the fish was-stored in plastic-bags and-preserved with dry ice.

Sample Preparation and Extraction
The analyses of the fish samples were carried out in accordance with the AOAC procedure (2006) [62]. The fish sample (1000 g) was thawed, beheaded, de-skinned, and the skin (250 g) washed with water before being ground on an OC-60B/60B grinding-machine-(60-120 mesh, Luohe, China). Extraction was carried out using MeOH (2 L, 2×, three days each). The extract was then chilled, filtered, and dehydrated-by adding-sodium-sulphate anhydrous. Finally, the extract was concentrated by solvent evaporation for 24 h at room temperature, yielding 25 g of crude extract. Following that, 15 g of crude extract were used in the biological study, and 10 g of dried extract was re-suspended in 30 mL of distilled water and defatted with n-Hex. In each step, the organic phase was treated as before, and then evaporated under reduced pressure to yield fractions I (5.0 g), while the aqueous remaining mother liquor was also concentrated to yield fraction (II). All fractions were kept at 4 • C until further phytochemical studies were carried out.

Animal Preparation
In this case, 25 male Wistar rats (weight 200 ± 20 g) were obtained from the Animal House of the National Research Centre in Egypt. Animals were-placed in cages-under controlled-conditions (12 h of light/dark cycles, temperatures-of 22 ± 1 • C, and humidityof 40-60%), with ad libitum feeding. The-National Research Centre's Ethics Committee approved all animal treatments and experimental-procedures with ethical approval no: 5449102021.

MSU-Crystal Synthesis
Uric acid (0.8 g) was dissolved in 155 mL Aquabidest, which also contained 5 mL NaOH (1 M), and the-pH was adjusted-to 7.2 with HCl. Gout-solution was-cooled and stirred at room temperature before being stored overnight at 4 • C to form crystals. After filtering the precipitate from the solution, it was dried at 70 • C for 4 h, ground into a fine powder, sieved through a 200-mesh metal filter, sterilized by heating at 180 • C for 2 h, and stored in sterile conditions. MSU-crystals were suspended-in phosphate-buffered saline (pH 7.2) at a-concentration of 20 mg/mL prior to-administration [63].

Gouty-Arthritis-Animal Model
The development of arthritis was assessed by measuring the size of the joint immediately before the injection with an MK-550 volume meter. Subsequent measurements of the same ankle joint were taken 24 h after the injection and 7 days later.
For the experimental protocol, 25 rats were randomly divided into five groups of five rats each: Group 1: Negative control, each animal received 50 µL injection of saline into the left ankle joint cavity. The remain 20 rats were injected with 50 µL of MSU solution (20 mg/mL) for 7 days into the left ankle joint cavity to induce arthritis until the uric acid level reached 10 mg/dL (as determined by a blood sample taken from the retro-orbital plexus) and ankle swelling was measured at the end of 7 days [64]. Then the groups were classified into: Group 2: MSU-induced arthritic rats (positive control), Group 3: MSU + FE (300 mg/kg.b.wt/day; [65], protective group, where rats in this group received FE (300 mg/kg.b.wt/day) combined treatment at the same time of MSU-injection, daily for 7 days). Groups 4: MSU-induced arthritic rats for 7 days, then at the end of MSU injection, rats were treated with FE (300 mg/kg.b.wt/day) for another 7 days (Therapeutic group). Group 5: MSU-induced arthritic rats for 7 days, then post MSU-injection, rats were treated with reference drug indomethacin, 5 mg/kg.b.wt/day [63]) for another 7 days (Therapeutic reference group) (Figure 6). At the end of the experiment, the synovial tissue of the joint was evacuated and partially homogenized and centrifuged, yielding a supernatant that was stored at 20 • C for the examination. and ankle swelling was measured at the end of 7 days [64]. Then the groups were classified into: Group 2: MSU-induced arthritic rats (positive control), Group 3: MSU + FE (300 mg/kg.b.wt/day; [65], protective group, where rats in this group received FE (300 mg/kg.b.wt/day) combined treatment at the same time of MSU-injection, daily for 7 days). Groups 4: MSU-induced arthritic rats for 7 days, then at the end of MSU injection, rats were treated with FE (300 mg/kg.b.wt/day) for another 7 days (Therapeutic group). Group 5: MSU-induced arthritic rats for 7 days, then post MSU-injection, rats were treated with reference drug indomethacin, 5 mg/kg.b.wt/day [63]) for another 7 days (Therapeutic reference group) (Figure 6). At the end of the experiment, the synovial tissue of the joint was evacuated and partially homogenized and centrifuged, yielding a supernatant that was stored at 20 °C for the examination.

Biochemical Assays
TNF-α, IL-1β, NF-B levels in serum were determined using an ELISA kit and the manufacturer's instructions. Rat NF kappaB p65 ELISA Kit (ab176648), was used and obtained from Abcam, United states. Rat TNF alpha ELISA kit (ab181421) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of TNF alpha protein (Abcam, united states), Rat IL-1 beta ELISA Kit (ab255730) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of IL-1 beta protein in serum, cell culture and plasma. The Lowry Method (1951) [66] was used to determine the protein levels in samples. For histopathological examination of synovial tissue, a portion of it was fixed in 4% paraformaldehyde buffer. In addition, immunohistochemistry was used to quantify inflammatory markers. Ohkawa et al. (1979) [67] described a method for measuring lipid peroxide (Malondialdehyde; MDA). Ellman (1959) [68] was used to measure the concentration of reduced glutathione (GSH). Glutathione reductase was measured using the method described by Hsiao et al. (2001) [69]. Fridovich (1989) [70] described a method for measuring Superoxide Dismutase (SOD) activity. The Sinha (1972) method was used to determine catalase activity [71].

Blood Sampling
Blood samples were drawn from the retro-orbital plexus and centrifuged at 3000 rpm for 15 min to determine uric acid levels in different groups.

Histopathological Examination
Tissue samples from various experimental groups were collected, fixed in 10% neutral buffered formalin, washed, decalcified with EDTA, dehydrated, cleared, and

Anti-Inflammatory and Antioxidant Properties of Malap electricus Skin Fish Methanolic Extract in Arthritic Rats: Therapeutic and Protective Effects
B levels in serum were determined using an ELISA kit and the manufacturer's instructions. Rat NF kappaB p65 ELISA Kit (ab176648), was used and obtained from Abcam, United states. Rat TNF alpha ELISA kit (ab181421) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of TNF alpha protein (Abcam, united states), Rat IL-1 beta ELISA Kit (ab255730) is a single-wash 90 min sandwich ELISA designed for the quantitative measurement of IL-1 beta protein in serum, cell culture and plasma. The Lowry Method (1951) [66] was used to determine the protein levels in samples. For histopathological examination of synovial tissue, a portion of it was fixed in 4% paraformaldehyde buffer. In addition, immunohistochemistry was used to quantify inflammatory markers. Ohkawa et al. (1979) [67] described a method for measuring lipid peroxide (Malondialdehyde; MDA). Ellman (1959) [68] was used to measure the concentration of reduced glutathione (GSH). Glutathione reductase was measured using the method described by Hsiao et al. (2001) [69]. Fridovich (1989) [70] described a method for measuring Superoxide Dismutase (SOD) activity. The Sinha (1972) method was used to determine catalase activity [71].

Blood Sampling
Blood samples were drawn from the retro-orbital plexus and centrifuged at 3000 rpm for 15 min to determine uric acid levels in different groups.

Histopathological Examination
Tissue samples from various experimental groups were collected, fixed in 10% neutral buffered formalin, washed, decalcified with EDTA, dehydrated, cleared, and embedded in paraffin. For histopathological examination, the paraffin-embedded blocks were sectioned at 5-micron thickness and stained with Hematoxylin and Eosin [72]. A light microscope (Olympus BX50, Ina, Japan) was used to examine stained sections).

Immunohistochemistry
The immunohistochemical analysis was performed in accordance to the methods described by Madkour et al., 2021 [74]. Tissue sections were deparaffinized in xylene and rehydrated in various alcohol grades. Antigen retrieval was accomplished by pretreating the sections for 20 min with citrate buffer at pH 6. In a humidified chamber, sections were incubated for two hours with rabbit polyclonal anti-iNOS antibody (ab15323; Abcam, Cambridge, UK). The sections were incubated with goat anti-rabbit IgG H&L (HRP) (ab205718; Abcam, Cambridge, UK), and the chromogen was 3,3 -diaminobenzidine tetrahydro-chloride (DAB, Sigma). The slides were then counterstained with hematoxylin and DPX mounted. PBS was used to replace the primary antibodies in the negative control slides.

Evaluation of iNOS Immunostaining
In each group, the quantitative immunoreactivity of iNOS was assessed in tissue sections [75], with five tissue sections examined. Immunoreactivity was assessed in 10 microscopical fields per section using a high-power microscope (×400). Color deconvolution image J 1.52 p software (Wayne Rasband, National Institutes of Health, Ina, Japan) was used to calculate the percentage of positively stained cells (%).

Preparation of Fatty Acids Methyl Esters
Methylation was carried out [69]. In a nutshell, 5 mg of fraction I was suspended in 1 mL of n-hexane. Next, in vials, a 2 mL aliquot of methanolic sulfuric acid (1%, v/v) was added and sealed. For 16 h, the sample was heated in a stopper tube at 50 • C. 2 mL aqueous sodium bicarbonate (2%, w/v) was added to finish the reaction. The products were then extracted using n-hexane (2.5 mL). Finally, samples were concentrated at room temperature for 48 h to remove acids.

GC-MS Analysis of Fatty Acids Methyl Esters
The recovered fatty acid methyl esters were chromatographically analyzed using GC-MS [76]. TRACE ® GC Ultra Gas Chromatograph (Thermo Scientific Corp., Berkeley, MO, USA) was used in conjunction with a Thermo MS detector (ISQ ® Single Quadrupole Mass Spectrometer, Thermo Fisher Scientific, Berkeley, MO, USA). The system included a TR-5 MS column (30 m × 0.32 mm i.d., 0.25 m film thickness).
The system was set up to analyze 1 L diluted samples (1:10 hexane, v/v), helium as the carrier gas, and the injector and detector at 210 • C. The flow rate was set to 1.0 mL/min with a split ratio of 1:10. The temperature program was 60 • C for 1 min, then rose at 4.0 • C/min to 240 • C for 1 min. Electron ionization (EI) at 70 eV yielded mass spectra with a spectral range of m/z 40-450. Finally, the obtained MS data were de-convoluted using AMDIS software (www.amdis.net, accessed on 20 October 2021) and identified by retention indices (relative to n-alkanes C8-C22), mass spectrum matching to authentic standards (when available), and Wiley spectral library collection and NIST library database.

Isolation and Purification of Compounds
Normal vacuum liquid chromatography (VLC) was used to fractionate the n-hexane fraction (4 g) using column 6 × 30 cm, 50 g n-hexane:EtOAC mixtures were used for gradient elution. The collected fractions (100 mL each) were concentrated and monitored by TLC using the n-hexane:EtOAC (8:2) system, and PAA was used to visualize them. Three sub-fractions were created by grouping and concentrating similar fractions (I 1 -I 3 ). Subfraction I 1 (1.50 g) was further fractionated by column chromatography on silica gel 60 (100 × 1 cm, 50 g), which was eluted as before to yield compounds 1 (20 mg) and 2 (10 mg), whereas subfractions I 2 and I 3 (1.00 g, each) yielded compounds 3 (50 mg) and 4 (30 mg).

Amino Acid Analysis
Fraction II (4 g) was also further using for amino acid analysis as follow:

Sample Preparation
300 mg of the sample was combined with 5 mL of hexane. For 24 h, the mixture was allowed to macerate. The mixture was then filtered through Whatman no. 1 filter paper, and the residue was transferred to a test tube and incubated in an oven with 10 mL 6N HCl for 24 h at 110 • C. Following incubation, the sample was filtered on Whatman no. 1 filter paper, evaporated on a rotary evaporator, and completely dissolved in 100 mL dilution buffer before diluting 1 mL in 3 mL vial, filtered using 0.22 m syringe filter, and 100 µL was injected.

Instrument Parameters
LCA K06/Na column, buffer a, buffer b, and the regeneration solution are all part of the mobile phase. Gradient elution mode 0.45 mL/min flow rate temperature: 57-74 • C gradient, wavelength: 440 and 570 nm, preparation of buffers and solutions (Table 9): Using the AutodockTools v.4.2 set, all torsions of the isolated compound structures were assigned and their Gasteiger charges were provided for all investigated atoms in structures [77]. Structures that had more than 32 torsions were eliminated.

Receptor Preparation
Docking screening was performed on human COX-1 and COX-2 structures with co-crystallized ligands CEL and arachidonic acid (PDB codes: 3kk6 and 3hs5, respectively). PDBfixer [78] was used to repair missing residues and atoms, as well as to remove co-crystalized H2O and hetero-atoms from the downloaded structure. Following that, AutodockTools v.4.2 was used to provide polar hydrogen and Gasteiger charge to all receptors [77].

Structural Docking
For the docking step, the PyRx platform's Auto-Dock Vina software was used [79,80]. The docking search binding sites were estimated based on the enzyme's co-formed ligands (i.e., CEL and arachidonic acid for COX-1 and COX-2, respectively). For the active sites of COX-1 and COX-2, the grid box co-ordinates were x = −31.94; y = 42.74; z = −4.39, and x = 20.95; y = 15.73; z = 66.82, respectively. The grid box size was set to 15 while the exhaustiveness was set to 24. Pymol software was used to analyze and visualize docking poses [80].

Molecular Dynamics Simulation
MDS experiments were carried out using the Desmond v. 2.2 software [81][82][83][84]. The OPLS-2005 force field is used in this software. Protein systems were created using the System Builder option, which checked the protein structure for any missing hydrogens, set the protonation states of the amino acid residues (pH = 7.4), and removed the co-crystalized water molecules. Following that, the entire structure was immersed in an orthorhombic box of TIP3P water containing 0.15 M Na + and Cl − ions in a solvent buffer of 20. Following that, the ready systems were energy minimized and equilibrated for 10 ns. The top-scoring poses for protein-ligand complexes were used as starting points for simulation. During the system building step, the Desmond software automatically parameterizes inputted ligands based on the OPLS force field. The absolute binding free energy (Gbinding) was determined using NAMD simulations [85], and the protein structures were built and optimized using the VMD software's QwikMD toolkit. The compounds' parameters and topologies were calculated using the VMD plugin Force Field Toolkit (ffTK). Following that, the generated parameters and topology files were loaded into VMD to easily read the protein-ligand complexes and then run the simulation steps.

Absolute Binding Free Energy Calculations
The free energy perturbation (FEP) method was used to calculate binding free energy (G). Kim and colleagues [86] recently published a detailed description of this method. The value of each G is estimated using NAMD software from a separate simulation. The online website Charmm-GUI (https://charmm-gui.org/?doc=input/afes.abinding, accessed on 18 May 2021) can be used to prepare all input files required for NAMD simulation). Following that, we can use these files in NAMD to generate the required simulations using the FEP calculation function. In the presence of the TIP3P water model, the equilibration (5 ns long) was achieved in the NPT ensemble at 300 K and 1 atm (1.01325 bar) with Langevin piston pressure (for "Complex" and "Ligand"). Next, for each compound, 10 ns FEP simulations were run, and the final 5 ns of free energy values were measured for the final free energy values [86].

Statistical Analyses
Statistical analysis is performed using the SPSS computer program (One Way Analysis of Variance, ANOVA) in conjunction with the co-state computer program, with different letters being significant at p ≤ 0.05% Change is calculated compared to control group as: (mean of treated − mean of negative/mean of negative control) × 100%. Reduction: (mean of positive control-mean of treated group/mean of positive control) × 100%.

Conclusions
Nutrition has a significant impact on a person's health. Omega-3 fatty acids and protein-rich FE have been shown to reduce inflammation. Not only because of the absence of harmful side effects and the positive health benefits of FE, but also because of the exceptional therapeutic impact demonstrated in this study in arthritis-induced rats. M. electricus FE effects could be attributed to inhibitory and modulatory actions on the production and release of nitric oxide and cytokines, both of which are involved in disease pathogenesis. According to molecular modelling and dynamics simulation, one of the major components of the crude extract (compound 4) has the potential to target and inhibit COX isoforms with a higher affinity for COX-2. For all of this, we aim that the FE will provide an impressive and encouraging therapy option for arthritis, contributing to the reduction and progression of this chronic-inflammatory disorder.