Amitriptyline Ameliorates Arthritis by Downregulation of Inflammatory Mediators and Oxidative Stress; A Mechanistic Approach

Amitriptyline Ameliorates Arthritis by Downregulation of Inflammatory Mediators and Oxidative Stress; A Mechanistic Approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Amitriptyline Ameliorates Arthritis by Downregulation of Inflammatory Mediators and Oxidative Stress; A Mechanistic Approach Haseeb Ahsan, Rizwana Kauser, Hafiz Muhammad Irfan, Ihtisham Haider, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3828096/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study was designed to evaluate the antioxidant and anti-arthritic potential of amitriptyline. The albumin and formaldehyde were used to induce acute and chronic inflammation respectively. The adjuvant-induced arthritis model was developed in rats. In acute model, amitriptyline significantly (p < 0.001) decreased the thickness of paw at early as well as late stages. At high dose of test drug, the significant (p < 0.001) anti-arthritic effect was noted in formaldehyde-induced arthritic model. Likewise, Amitriptyline (40 mg/kg oral dose) produced a 4.7% decrease in swelling of paw prompted by CFA on day 14 that increased to17.09% on day 28. This effect significantly increased by higher dose of Amitriptyline 80 mg/kg (28.63%) on day 28th. The significant (p < 0.001) expression of antioxidant enzyme SOD (super oxide dismutase) was observed at maximum dose (80 mg/kg). Amitriptyline significantly reduced the levels of inflammatory cytokines like prostaglandin E2, TNF-α and NF-kB and the results were comparable with naproxen. Further, in-depth molecular docking and simulations studies of amitriptyline showed that it has good binding capacity with inflammatory cytokines showing the highest score with prostaglandin E2. In addition, ADMET studies also proved amitriptyline within the limits of Lipinski’s rule of five. These findings validate the anti-arthritic effect of amitriptyline, but it has limitations for clinical studies. Rheumatoid Arthritis Pro-inflammatory cytokines ROS Superoxide dismutase CFA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Autoimmune inflammatory disease, such as rheumatoid arthritis (RA), is an example of discrepancies inside the immune system usually arising from the effects of an accretion of environmental and expected insults over a specific period of time along with the genetic predispositions [ 1 ]. Prevalence of the rheumatoid arthritis is 2–4% internationally depending upon gender, age and geographical distribution [ 2 ]. The etiology of rheumatoid arthritis has not been clear yet. Immune-mediated diseases are generally initiated and determined by biomarkers of immune (T & B cells) as well as inflammatory cells (macrophages). In Rheumatoid arthritis, type II collagen and proteoglycan act as signal for activation of these immune cells to release pro-inflammatory molecules [ 3 ]. Some researchers have proposed that the enzymatic and non-enzymatic antioxidant systems are compromised in RA. Therefore, rheumatoid arthritis patients are at greater risk of being exposed to the free radicals. In different scenarios of oxidative stress, superoxide ions are formed by oxygen metabolism, which participate in the destruction of antioxidant system. Various anti-oxidants have been claimed to be very effective in the treatment of RA e.g. glutathione peroxidase, superoxide dismutase, glutathione reductase and catalase [ 4 ]. Synovial fluid obtained from the patients with RA were manifested to high level of antioxidant system damages. SOD (superoxide dismutase) is a necessary metallic enzyme containing copper and act as scavenger for free radicals formation [ 5 ]. The tissue injury takes place due to the prostaglandin’s synthesis and lipid peroxidation. Thus, the glutathione preventive effect might be due blockage of these reactions as recommended by Munthe et al. The available choices of therapeutic management for RA are disease-modifying anti rheumatic drugs, NSAIDs and novel biologics such as IL-1receptor blocker and TNF monoclonal antibodies. In recent studies, the drugs have been prescribed for symptomatic treatment and inhibit the chronic immunological events to lesser extent. Furthermore, precisely the undesired and hypersensitive reaction are limitations of the treatment [ 6 ]. RA is strongly associated with depression due to involvement of common biomarkers such as interleukin-6 (IL-6) and IL1β & tumor necrosis factor-α (TNF-α). As literature shown, roughly 30% of rheumatic have symptoms of depression [ 7 ] [ 8 ] [ 9 ]. The level of monoamines (norepinephrine, dopamine and Serotonin) is significantly influenced by these cytokines. Another cytokine-mediated depression mechanism is mitogen-activated protein kinase (MAPK) pathway [ 10 ]. These common targets of depression and RA are proposed by a study conducted of Zhang [ 7 ]. The current study was performed to explore the anti-arthritic and antioxidant potential of amitriptyline in Sprague Dawley rats against acute inflammation as well as chronic arthritis models and evaluation of its absorption, distribution, metabolism, excretion, toxicity (ADMET) and molecular docking profile. Material and Methods Drugs & reagent Amitriptyline was obtained from OBS®, Pakistan as a gift to conduct research. Complete Freund’s Adjuvant (CFA) and ELISA kits from Sigma-aldrich®, Germany Elab science® kits respectively. Experimental Animals Young healthy female and male Sprague-Dawley rats were used as experimental animals. The animals were kept in the controlled temperature (23-25°C) and for equal time interval (12 hr) in light and dark places at animal house, Faculty of Pharmacy, Sargodha University. Animals were fed with a standard-pellet diet and water ad-libitum. Experiments on animals were performed by using the guidelines of INH after the approval of Advance studies and research Board, University of Sargodha (SU/Acad/1723/2021). Inflammation flowed by egg albumin As procedure described by Ren et al [11], albumin was used for acute inflammatory model. After overnight fasting, animals were grouped into Control, Standard and Treated. Normal saline as vehicle for control, naproxen as standard and amitriptyline (20, 40 & 80 mg/kg) as treatment drugs.0.1 ml egg albumin was injected into the right hind paw of all animals. The swollen paw was observed for paw diameter evaluation using Vernier caliper at different time’s points (0, 1, 2, 3 and 4 hours). Percentage inhibition was calculated by using calculated paw volume [11]. Formaldehyde-induced arthritis This method was used to evaluate the anti-arthritic potential of amitriptyline against chronic arthritis induced by formaldehyde. Rats of either sex were categorized into 6 groups. Group I was administered distilled water. Group II was given control vehicle after, and arthritis was induced. Group III was given standard drug in dose of 10 mg/kg by oral route. Groups IV, V and VI were given amitriptyline as treated group. The same dosing as performed as in above experiment for test drugs. In this method, 30 minutes’ post administration of test drugs, 2% formaldehyde in the dose of 0.1 ml was administered to all groups except group I by sub planter route on day first, to induce chronic non immunological arthritis and was repeated on day 3. Vernier Caliper was used to asses’ arthritis by measuring the paw diameter after 10 days of treatment. The percentage inhibition of edema was measured in accordance with method given by Mahmood et al [13]. Complete Freund’s Adjuvant -induced arthritis Immunological arthritis model was developed using Complete Freund’s Adjuvant (CFA) according to our previously used methodwith slight modifications [14]. CFA was used to induce arthritis in left hind paws of animals and swelling of paw was assessed by using digital Vernier caliper at 0, 7-, 14-, 21- and 28-days’ post induction of phlogiston agent. Sprague-Dawley rats weighing from 150 to 200 g (either male or female) were randomized into six groups. Group I was considered arthritic control whereas Group II as normal control and both were given vehicle only. Group III was a standard group and was treated with reference drug naproxen (10 mg/kg). Group IV, V and VI were treated with amitriptyline in distilled water by oral rout, on day after induction of arthritis, at various doses (20, 40 & 80 mg/Kg). This study continued for 28 days starting from induction day (0-28). Determination of paw swelling Paw diameter was be observed on day 0, 7, 14, 21 & 28 th day. The body weight of animals was observed every week. After treatment, on 29 days animals was sacrificed and blood was collected. Measurement of weight variations: Weight of animals were observed on day 0, 7, 14, 21 and 28 th day. The weight of treated animals was increasing with increasing number of days i.e 7, 14, 21, 28 th day. But the weight of arthritic rats was increased on day 7 but decreased on 28 th day. Measurement of arthritic score: Severity of inflammation was observed with the help of arthritic score. Arthritic score was less in animals which were treated with amitriptyline dose 40 mg and was more less in rats treated with Amitriptyline 80 mg. Estimation of inflammatory cytokines by ELISA Blood samples were drawn by euthanizing the animals at the end of study. Mixed with EDTA and then centrifuged for 30 minutes to separate supernatant (serum). Serum levels of tumor necrosis factor, prostaglandin E-2 and nuclear factor kB were estimated by ELISA using kit methods (Elab science kits). Optical density was measured at 450 nm from which the concentrations were computed through standard curve method. Determination of antioxidant enzymes activities The animals were given anesthesia and liver sections were obtained. After that, the sections were homogenized according to formerly reported method. Protein content was estimated by Lowry’s method using bovine albumin as reference [15]. Estimation of superoxide dismutase activity (SOD) and Glutathione (GSH) Activity of superoxide dismutase enzyme and GSH was evaluated by SOD and GSH Kits protocol according to xanthine oxidase method in liver of sacrificed animals. The activity was presented in units/mg protein [16].[17]. Estimation of ADMET parameters The Pharmacokinetic parameters like absorption, distribution metabolism and excretion were noted by online data bank given by employing software (http://biosig.unimelb.edu.au/pkcsm/prediction) on May 25, 2022 [18]. Swiss ADMET software with given link: http://www.swissadme.ch/index.php was run to assess the polarity, permeability and other parameters of amitriptyline [19]. Molecular docking and Simulations Additionally, an in-silico studies were performed to examine the interaction, stability of amitriptyline with in the binding pocket of TNF-α, NF-κB, and Prostaglandin E2 (PGE2). Initial structure and system preparation In order to conduct the docking experiment, a 3D structure is required. Therefore, the structure of TNF-α (6X81 at 2.81 with 158 amino acids), NF-κB (1A3Q with 2.10 and 285 amino acids), and PGE2 (7CXR with 2.8 and 358 amino acids) were downloaded from the protein data bank (PDB) database for the current study ( Figure 1 ). A co-crystallized ligand of TNF-α [4-(isoquinolin-8-yl) phenyl] acetonitrile (PDB ID: UTJ), and PGE2 I.E., Prostaglandin E2 (P2E), required ions, and the protein chains (homodimers) were deleted from the crystal structure before they were used for further study. All hydrogen adds, non-polar hydrogen atom mergers, and Kollman charge were calculated using the AutoDock v4.2 program. Moreover, the ligand structure, amitriptyline, was also acquired from PubChem and stored in shell repository in the SDF files for further use. Open Babel was used to parse the SDF file to 3D PDB file format. By an improved force field and the steepest descent method, the ligand library was refined for 3000 steps. AutoDock was used to rotate all of the bonds that could be rotatable in order to induce torsion into the compounds. A PDB file with the minimize protein structure and amitriptyline was then saved in PDBQT for further docking studies. Molecular Docking Analysis Molecular docking was performed for amitriptyline and TNF-α, NF-κB, and PGE2. The prepared protein PDBQT files were loaded into AutoDock tool (receptor proteins), amitriptyline was used as docking compound. The receptor grid box with dimensions of x = 126, y = 126, and z = 126 was centered on the protein's ligand inside the active site. Around the existence co-crystalized ligand, the grid was centered on x = 90.977, y = 113.349, and z = 112.209 for PGE2, x = 9.071, y = 61.506, and z = 19.623 for NK-kb, x = -4.281, y = 28.68, and z = -13.491 for TNF-α respectively. Molecular Dynamic Simulation Studies In order to evaluate binding strength, stability, and flexibility, docked conformations complexes with high binding energies inside the binding site of proteins were subjected to molecular dynamics simulations using the GROMACS v2020 software [20]. Additionally, it keeps the systems' densities uniformly distributed. All of the bonds were constrained using the Linear Constraint Solver (LINCS) technique, and the long-range electrostatics were calculated using the Particle Mesh Ewald (PME) method with a cutoff value of 1.0 nm. After the system was brought to equilibrium, a 100 ns molecular dynamic simulation was run on a Linux computer to evaluate the trajectories and examine at interaction stability. Trajectory and structure analysis Moreover, VMD was utilized to extract coordinates for a certain time frame, show all trajectory movements over simulation time, and calculate trajectories. Chimera visualization tools were used to compare and visualize the structures. Gmx_rms was used to calculate the RMSF of the protein and ligand, whereas gmx_rmsf was used to calculate the RMSD. We investigated the protein-ligand hydrogen bond, gyration radius, and solvent accessibility surface analyses using the gmx_hbond, gmx_gyrate, and gmx_sasa functions. The trajectories' graphs were acquired using terminal-shell commands, and they were then drawn using the 2D charting program XMGRACE. Analysis by statistical tools The statistical analysis was performed by Graph-pad Prism version 8 software. ANOVA testing such as Two-way and One-was was applied with significant level of p< 0.005. Results Paw diameter in albumin induced inflammation Amitriptyline at three different doses (20, 40 & 80 mg/kg) were evaluated for its anti-inflammatory results in albumin induced inflammation. The test drug significantly ( p < 0.001 ) decreased the thickness of paw at early as well as late stage after the induction of inflammation in comparison to arthritic rats group shown in Fig. 2 . Similarly, the standard drug (naproxen 10 mg/kg) also revealed significant reduction in swollen paw. Paw diameter in formaldehyde induced arthritis. In this experiment. Amitriptyline was observed for its anti-arthritic effect. At high dose of test drug, the significant (p 0.005) effect up-to day 4 but shifted towards significant site at the end of study. The standard drug naproxen also displayed outstanding effect on inflamed paw throughout the experimental period as mentioned in Table 2 with maximum inhibition of 20.56%. Amitriptyline (80 mg/kg P.O) showed 21.74% inhibition on increase in paw diameter at the end of experiment (Day 10). Table 1 Measurement of arthritic score for evaluation of paw diameter Arthritic score Severity of inflammation 0 No inflammation 1 Mild inflammation 2 Moderate inflammation 3 Severe inflammation Table 2 Measurement of paw diameter on day 2nd, 4th, 6th, 8th & 10th day after induction of arthritis with formaldehyde. Days Paw diameter (mm) Arthritic Control Vehicle Control Naproxen 10 mg/kg Amitriptyline 20mg/kg Amitriptyline 40mg/kg Amitriptyline 80 mg/kg 2nd day 4.14 ± 0.068 3.94 ± 0.051 4.02 ± 0.066 n.s 3.92 ± 0.066 n.s 4.18 ± 0.086 n.s 3.88 ± 0.037 b 4th day 4.74 ± 0.068 3.94 ± 0.024 4.28 ± 0.058 a 4.60 ± 0.071 n.s 4.68 ± 0.058 n.s 4.18 ± 0.058 a 6th day 5.04 ± 0.051 3.88 ± 0.037 4.48 ± 0.058 a 4.74 ± 0.051 a 4.54 ± 0.060 a 4.18 ± 0.037 a 8th day 5.14 ± 0.051 3.84 ± 0.024 4.12 ± 0.037 a 4.84 ± 0.051 a 4.2 ± 0.032 a 4.02 ± 0.037 a 10th day 4.96 ± 0.051 3.94 ± 0.051 3.92 ± 0.037 a 4.80 ± 0.071 a 3.98 ± 0.037 a 3.88 ± 0.037 a Paw diameter in CFA experiment. In this Fig. 2 , there was significant inhibitory effect on paw diameter in CFA-induced arthritis. Amitriptyline (40 mg/kg oral dose) produced 4.7% decrease in swelling of paw prompted by CFA on day 14 that increased to 17.09% on day 28. This effect significantly increased by higher dose of Amitriptyline 80 mg/kg (28.63%) on day 28th. On the other side, standard drug naproxen showed 19.6% decrease in paw diameter on the same week. Weight variations after treatment with amitriptyline. Amitriptyline showed significant effect on weight variations at dose 80 mg/kg. There was a significant (p 0.005) at low dose of Amitriptyline (40 mg/kg orally) on the day 14 but became significant at the end of study as given in Fig. 3 . The weight of Amitriptyline treated rats (at 80 mg.kg dose) was 130 ± 5.62 g at day 7 but increased to 143 ± 1.78 g on day 28th .The weight of arthritic rats were 121 ± 1.87g on day 7 and decrease to 99.4 ± 0.81 g at day 28th . Effect of drug on arthritic score. Figure 3 displayed severity of inflammation in the form of arthritic index in arthritic animals throughout the experiment. This intensity of inflammation was reduced in animals treated with amitriptyline at high doses (40 & 80 mg/kg orally). There was no significant reduction in inflammatory signs with low dose of test drug (Amitriptyline 20 mg/kg). Maximum suppressant effect on inflammatory signs was observed on day 14–28 in treatment group of amitriptyline 80 m/kg orally. But the dose of 40 mg/kg P.O showed significant (p < 0.001) effect on inflammatory score on day 28. The standard drug naproxen also showed significant anti-inflammatory effect in comparison to arthritic animals. Effect of drug on inflammatory cytokines CFA significantly stimulated the enhanced release of pro-inflammatory cytokines in arthritic control animals in comparison to normal control group. Amitriptyline significantly reduced the levels of prostaglandin E2 in treatment groups at the doses of 20 as comparable to disease group animals. The serum concentration of TNF-α was also significantly lowered by amitriptyline at the doses of 40 (237.69 ± 5.21) and 80 mg/Kg (185.30 ± 2.71) when compared with arthritic control group (684.31 ± 30.35). The drug also declined the serum concentrations of NF-kB at all the doses of 20, 40 and 80 mg/Kg. The effects of the amitriptyline on prostaglandin E2, TNF-α and NF-kB were comparable with those of naproxen, used as reference drug as shown in Fig. 4 . Effect on SOD and GSH (antioxidant enzyme): The significant (p < 0.001) expression of antioxidant enzyme SOD (super oxide dismutase) was observed in animals treated with amitriptyline (80 mg/kg orally). Similarly, the effect on reducing agent GSH (glutathione) was less significant (p < 0.01) at the higher dose (80 mg/kg orally) of test drug amitriptyline. These results were comparable to arthritic group animals. There was significant suppression of these enzyme (SOD & GSH) in arthritic animals as depicted in Fig. 5 . ADMET profile of Amitriptyline: Before selecting drug for clinical trials, its absorption into central nervous system and gastrointestinal tract is very much important factor. The penetration of centrally acting drugs into blood brain barrier is necessary for its effects [ 21 , 22 ]. Amitriptyline has good absorption into human intestine with maximum values (98%) as given in Table 3 . It has blood brain barrier permeability of 0.96. Due to its maximum penetration into intestinal tract and minimum penetration into CNS contribute its minor CNS elated adverse effects. It is substrate of P-glycoprotein. It is substrate for CYP3A4 and has interactions with many drugs. The toxicity data showed its hepatotoxic behavior. Table 3 Evaluation of absorption, distribution, metabolism, excretion and toxicity parameters of amitriptyline Properties Parameters Amitriptyline Physicochemical Properties MW a (g/mol) 313.86 Rotatable bonds 3 HBA b 1 HBD c 0 Fraction Csp3 0.30 TPSA d 3.24A 2 Lipophilicity Log Po/w iLOGP 0.00 XLOGP3 5.84 MLOGP 4.54 Consensus 4.04 Absorption Human intestinal absorption 97.47% Skin Permeability -2.59 P-glycoprotein Substrate Yes Distribution Blood brain barrier Permeability 0.96 CNS permeability -1.196 Metabolism CYP3A4 substrate Yes CYP2D6 substrate No CYP2D6 inhibitor Yes Excretion Total clearance 1.198 Renal OCT2 substrate No Toxicity Oral rat acute toxicity (LD50) 2.802 Oral rate Chronic toxicity (LOAEL) 1.027 Hepatotoxicity Yes Molecular Docking Analysis Molecular docking was performed to better understand the reported pharmacological activity and binding mechanism of Amitriptyline against Nuclear factor-κB (NF-κB), Prostaglandin E2 (PGE2) and Tumor necrosis factor (TNF). The docking result shows the binding of Amitriptyline with NF-κB having significant binding energy of -7.2 kcal/mol and rmsd of 2.8 Å. It was observed that the Amitriptyline make a one hydrogen bonds with Asp186 of NF-kB with an energy of -12 kcal/mol and bond length of 2.99Å. One bond mediated with Ser188 of NF-kB with an energy of -0.5 kcal/mol and the bond length of 3.29Å. The Amitriptyline mediate also a strong hydrophobic interaction with Lys143, Ser222, Ser188, Asp219, Met185, Leu187, Tyr55, and His140 as shown in Fig. 6 A. It was also observed that the Amitriptyline having strong binding affinity toward TNF-α with a binding score of -6.17. It makes one hydrogen bond with Glu135 of TNF-α with 3.03 Å and energy of -1.9 kcal/mol. Whereas, mediate various hydrophobic interaction between TNF-α and Leu26, Gln25, Asn46, Pro139, Ilu136, Asn137, and Lys90 Fig. 6 B. Additionally, the docking result of Amitriptyline with PGE2 shows that ligand sets well in the pocket of receptor having a binding score of -8.49kcal/mol, highest among the rest of the proteins, and rmsd of 2.5. The ligand got a stable binding interaction within the pocket of PGE2. It mediated several electrostatic interactions comprised of ionic bond among Asp297 and N50 of the ligand having bond energy of -4.8kcal/mol and bond length of 2.9Å. Beside the ionic bond, it also makes various hydrophobic interactions between the ligand and Arg190, Val179, His191, Met286, Glu288, Asn287, Phe283, Arg292, Tyr285, Ser291, Ala284, Trp186, Phe280, Leu197, Ile189, Tyr181, Phe188, and Gln180 Fig. 6 C. Molecular Dynamic Simulation Studies The docked complex of PGE2 and ligand that had been chosen by docking studies was further examined for stability using MD simulation for up to 100 ns because of high affinity of Amitriptyline towards PGE2. The simulation findings are discussed with regard to RMSD, RMSF, hydrogen bond, radius of gyration, and SASA analysis. RMSD The protein-ligand complex's functioning and stability are evaluated using the RMSD. This helps us understand the structural variation of the protein-ligand complex throughout the duration of a 100 ns time trajectory. The amitriptyline appeared to be within the acceptable range of 0.5 nm, according to the RMSD, showing that it was favorably bound within the PGE2 binding cavity. The protein-compound complex system was shown to oscillate between 25 and 60 ns while equilibrating between 10 and 25 ns within the RMSD range of 0.35 to 0.5 nm initially. But after 60 ns, it was discovered that the amitriptyline had high stability for the entirety of the 100 ns simulation, as represented in the range of 0.35–0.55 nm, respectively. These persistent backbone alterations in PGE2 and ligand complex point to conformational changes during the whole simulation, as shown by the RMSD (Fig. 7 A). RMSF The RMSF trajectories provide significant details about the complex's protein residual variations. The broad range of plots indicates a higher degree of flexibility and fragility in the interactions. Lower values or less variance throughout the simulation parameter suggest complex regions with less distortion. But in the system examined for 100 ns of simulation, the PGE2 complex with chemicals had the same pattern of interactions, as seen in Fig. 7 B. The amitriptyline complexed with PGE2 demonstrated protein stability with minor high peaks shown as a continuous pattern. Considering this, the RMSF analysis verifies that the simulation had no impact on the protein-ligand structure. Radius of gyration (Rg) Additionally, to assess PGE2 compactness in the presence of amitriptyline, the radius of gyration was examined. Rg stands for the root mean square distance between the protein's atoms and its rotational axis. Plotting the Rg values of protein backbone atoms across time allowed researchers to examine changes in structural compactness. According to Fig. 7 C, the compound's simulated Rg ranges from 1.925 to 0.2.05 nm. The backbone Rg value is initially reduced by the binding of the molecule inside the binding cavity, while Rg was found to be constant with moderate fluctuation in the range of 2 nm. Additionally, the system was generally steady with a peak value of 2 nm after 60 ns. The Rg value demonstrated the protein's stability in the complex and showed that these chemicals bound steadily without undergoing any structural changes. Interaction Analysis At a simulation duration of 100 ns, the intensity of the interaction between ligands and PGE2 has been assessed. There was a variation of 2 hydrogen bonds found throughout the simulation. The amitriptyline mediated no hydrogen bonds for up to 60 ns of the simulation duration. In contrast, it mediates two persistent hydrogen bonds inside the PGE2 protein after 60 ns. In this way, numerous interactions of the same subtype with the ligand sustain the unique association Fig. 7 D. The substance defines the potential important in-silico inhibitory effect by demonstrating the contact is maintained and contributes to a stable binding with the desired protein over simulation duration. SASA Analysis The interaction of biomolecules with protein was presented in Fig. 7 E. However, the average SASA values (i.e., 155nm 2 ) for the amitriptyline-protein complex were slightly higher, especially in the first 30 ns. It was also discovered that the complex had larger solvent accessible surface area until 25 nanoseconds, but after 30 nanoseconds, the SASA value dropped. This suggests the MD simulation of a ligand-free structure can sample the binding pocket in its confined conformation as shown in Fig. 4 B. In such a dynamic manner, the ligand appears to be preferentially contained within the protein's pocket. While this suggests improved ligand-protein interactions due to the solvent-substitution process, the lower SASA trajectories (average 130 nm 2 ) until 80 ns into the simulation run support the opposite conclusion. After this point, the SASA tones increased progressively until they reached their maximum SASA values, after which they decreased until the MD simulation was completed. Amitriptyline may have migrated toward the solvent side of the protein pocket during simulated time frames 1–20 and 80–100 ns, where the protein pocket became strongly solvated and little compacted. Using the RMSD, RMSF, SASA, and Rg findings, we were able to display the dynamic differences between each of the studied ligands during the duration of the MD simulation. This is why it is important to study ligand-protein interactions throughout time as well as the varied trajectories of ligand-protein complexes so that the real stability of these complexes may be determined. Discussion RA is a chronic disease that usually develops in adults and elder people. The prevalence of RA is about 1% and more commonly occurs in women. Its prevalence is high in low-middle income countries [ 23 ]. The present investigations have been performed to evaluate the anti-arthritic potential of amitriptyline. Albumin-induced inflammatory model was used to evaluate its effect in acute phase. Injection of albumin stimulates the resident macrophages to release histamine and prostaglandins [ 24 ]. Amitriptyline confirmed its suppressor effect significantly on inflammation provoked by albumin as presented in Fig. 2 . This has been proved, that amitriptyline has inhibitory potential on inflammatory mediators. Arthritis developed in SD rats by formaldehyde is the most suitable method for ruling out the anti-arthritic behavior of test drug. This method showed a close similarity to the pattern of human arthritis [ 25 ]. The release of substance P is provoked by formaldehyde in neurogenic phase (early phase) whereas histamine, bradykinin and PGE2 are produced in inflammatory phase (late phase). These mediators are responsible for inflammatory changes such as alteration in caliber or permeability of vessels. This study results supported that amitriptyline restrained the arthritis by preventing the release of histamine, bradykinin and PGE2 with a dose of 20, 40 and 80 mg/kg orally as given in Table 2 . The mechanism behind CFA-mediated arthritis is the involvement of infiltrating cells. CFA induces response in three divisions less inflammation to joint swelling and then ultimately bone erosion due to involvement of PGE2 and autoantibodies [ 26 ]. The easiest way to evaluate the anti-arthritic activity is the measurement of paw edema. Exudate and inflammatory cells contribute to paw edema formation. The inhibitory effect on these factors results in decrease of paw diameter. As displayed in Fig. 2 , amitriptyline with high dose of 80 mg/kg caused a significant decrease in paw swelling throughout the experiment. This confirmed the anti-arthritic effect of amitriptyline as reported by Fig. 3 A. The loss of body weight in RA is due to decrease nutritional absorption [ 27 ]. Another study proposed that CFA enhances leptin level that contributes to weight loss. However, SD rats treated with amitriptyline (20, 40 and 80 mg/kg) significantly restored the loss in weight as depicted in Fig. 3 C. This might be due to suppression on level of leptin or increase abortion of nutrient. Other criteria for arthritic evaluation is arthritic score or index that is characterized by various symptoms such as redness or edema. Therefore, the treatment with amitriptyline reduced these inflammatory signs like swelling & redness at all doses (20, 40 and 80 mg/Kg) as shown in Fig. 3 B. Prostaglandin E-2, tumor necrosis factor and nuclear factor are the prospective targets of drugs which are used to restrain the progression of RA. PGE2, TNF-α and NF-kB are the causative agents which precipitate the certain vascular alterations such as dilation of blood vessels with increased permeation in the process of inflammation. These are habitually synthesized by macrophages which contribute the bone attrition mechanisms in RA. This study demonstrated a significant decline inculcated by amitriptyline at various doses (20, 40 and 80 mg/Kg) in the levels of PGE2, TNF-α and NF-kB estimated in serum samples of animals by ELISA. These outcomes offer a scientific comprehension to the mechanism of action of amitriptyline for management of arthritis induced by CFA ( Fig. 4 ). The oxidative stress in arthritic rats is due to CFA and result in elevation of SOD and CAT. Therefore, it could be imagined that increase in level of SOD and CAT by amitriptyline could be the future targets for inhibiting the gene expression of pro-inflammatory markers of arthritis as shown in Fig. 5 (a & b). Conclusion The findings of this study have validated the anti-arthritic behavior of amitriptyline by a reduction in paw edema, arthritic score, oxidative stress, and levels of pro-inflammatory cytokines including TNF-α, PGE2 & IL-1β. Therefore, it should be recommended for clinical investigations in future research. Declarations Funding: The authors did not receive any funding. Conflict of interest: The authors declare no conflict of interest. Author contributions: H.A designed the project, R.K performed the experimental work, Ihtisham Haider did ELISA work and paper writing. Z.K did graphs orientation. R.A in-vivo experimental work and data analysis, I.A . results interpretation and manuscript proof reading and editing, S.B and S.S.H did docking studies. Ethical approval: Experiments on animals were performed by using the guidelines of INH after the approval of Advance studies and research Board, University of Sargodha (SU/Acad/1723/2021). Data Availability Statement: Original data is preserved and will be submitted upon request. Acknowledgment: The authors thank the University of Sargodha, Pakistan for providing facilities to do work References Rosenthal, K.S.; Mikecz, K.; Steiner, H.L. 3rd; Glant, T.T.; Finnegan, A.; Carambula, R.E.; Zimmerman, D.H. Rheumatoid arthritis vaccine therapies: perspectives and lessons from therapeutic ligand epitope antigen presentation system vaccines for models of rheumatoid arthritis. Expert review of vaccines 2015 , 14 , 891–908, doi:10.1586/14760584.2015.1026330. Di, Y.M.; Zhou, Z.-W.; Guang Li, C.; Zhou, S.-F. Current and future therapeutic targets of rheumatoid arthritis. Anti-inflammatory & anti-allergy agents in medicinal chemistry 2011 , 10 , 92–120. Zimmerman, D.H.; Mikecz, K.; Markovics, A.; Carambula, R.E.; Ciemielewski, J.C.; Toth, D.M.; Glant, T.T.; Rosenthal, K.S. Vaccination by Two DerG LEAPS Conjugates Incorporating Distinct Proteoglycan (PG, Aggrecan) Epitopes Provides Therapy by Different Immune Mechanisms in a Mouse Model of Rheumatoid Arthritis. Vaccines 2021 , 9 , doi:10.3390/vaccines9050448. Aruoma, O.I.; Halliwell, B. Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase. The Biochemical journal 1987 , 248 , 973–976, doi:10.1042/bj2480973. Cimen, M.Y.; Cimen, O.B.; Kaçmaz, M.; Oztürk, H.S.; Yorgancioğlu, R.; Durak, I. Oxidant/antioxidant status of the erythrocytes from patients with rheumatoid arthritis. Clinical rheumatology 2000 , 19 , 275–277, doi:10.1007/pl00011172. Sukketsiri, W.; Chonpathompikunlert, P.; Tanasawet, S.; Choosri, N.; Wongtawatchai, T. Effects of Apium graveolens Extract on the Oxidative Stress in the Liver of Adjuvant-Induced Arthritic Rats. Preventive nutrition and food science 2016 , 21 , 79–84, doi:10.3746/pnf.2016.21.2.79. Zhang, C. Flare-up of cytokines in rheumatoid arthritis and their role in triggering depression: Shared common function and their possible applications in treatment (Review). Biomedical reports 2021 , 14 , 16, doi:10.3892/br.2020.1392. Hodes, G.E.; Ménard, C.; Russo, S.J. Integrating Interleukin-6 into depression diagnosis and treatment. Neurobiology of stress 2016 , 4 , 15–22, doi:10.1016/j.ynstr.2016.03.003. Abbott, R.; Whear, R.; Nikolaou, V.; Bethel, A.; Coon, J.T.; Stein, K.; Dickens, C. Tumour necrosis factor-α inhibitor therapy in chronic physical illness: A systematic review and meta-analysis of the effect on depression and anxiety. Journal of psychosomatic research 2015 , 79 , 175–184, doi:10.1016/j.jpsychores.2015.04.008. Dionisie, V.; Filip, G.A.; Manea, M.C.; Manea, M.; Riga, S. The anti-inflammatory role of SSRI and SNRI in the treatment of depression: a review of human and rodent research studies. Inflammopharmacology 2021 , 29 , 75–90, doi:10.1007/s10787-020-00777-5. Ren, M.; Tang, Q.; Chen, F.; Xing, X.; Huang, Y.; Tan, X. Mahuang Fuzi Xixin Decoction Attenuates Th1 and Th2 Responses in the Treatment of Ovalbumin-Induced Allergic Inflammation in a Rat Model of Allergic Rhinitis. Journal of immunology research 2017 , 2017 , 8254324, doi:10.1155/2017/8254324. Cicala, C.; Ianaro, A.; Fiorucci, S.; Calignano, A.; Bucci, M.; Gerli, R.; Santucci, L.; Wallace, J.L.; Cirino, G. NO-naproxen modulates inflammation, nociception and downregulates T cell response in rat Freund’s adjuvant arthritis. British journal of pharmacology 2000 , 130 , 1399–1405, doi:10.1038/sj.bjp.0703449. Mahmood, F.; Khan, J.A.; Mahnashi, M.H.; Jan, M.S.; Javed, M.A.; Rashid, U.; Sadiq, A.; Hassan, S.S.U.; Bungau, S. Anti-Inflammatory, Analgesic and Antioxidant Potential of New (2S,3S)-2-(4-isopropylbenzyl)-2-methyl-4-nitro-3-phenylbutanals and Their Corresponding Carboxylic Acids through In Vitro, In Silico and In Vivo Studies. Molecules (Basel, Switzerland) 2022 , 27 , doi:10.3390/molecules27134068. Ahsan, H.; Irfan, H.M.; Alamgeer; Shahzad, M.; Asim, M.H.; Akram, M.; Zafar, M.S. Anti-rheumatic activity of pseudoephedrine (a substituted phenethylamine) in complete Freund’s adjuvant-induced arthritic rats by down regulating IL-1β, IL-6 and TNF-α as well as upregulating IL-4 and IL-10. Inflammopharmacology 2021 , 29 , 673–682, doi:10.1007/s10787-021-00804-z. Akhtar, M.F.; Ashraf, M.; Anjum, A.A.; Javeed, A.; Sharif, A.; Saleem, A.; Akhtar, B. Textile industrial effluent induces mutagenicity and oxidative DNA damage and exploits oxidative stress biomarkers in rats. Environmental toxicology and pharmacology 2016 , 41 , 180–186, doi:10.1016/j.etap.2015.11.022. Sharif, A.; Ashraf, M.; Javeed, A.; Anjum, A.A.; Akhtar, M.F.; Akhtar, B.; Saleem, A. Oxidative stress responses in Wistar rats on subacute exposure to pharmaceutical wastewater. Environmental science and pollution research international 2016 , 23 , 24158–24165, doi:10.1007/s11356-016-7717-7. Adeyemi, W.J.; Olayaki, L.A. Diclofenac - induced hepatotoxicity: Low dose of omega-3 fatty acids have more protective effects. Toxicology reports 2018 , 5 , 90–95, doi:10.1016/j.toxrep.2017.12.002. Daina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports 2017 , 7 , 1–13, doi:10.1038/srep42717. Ul Hassan, S.S.; Muhammad, I.; Abbas, S.Q.; Hassan, M.; Majid, M.; Jin, H.Z.; Bungau, S. Stress driven discovery of natural products from actinobacteria with anti-oxidant and cytotoxic activities including docking and admet properties. International Journal of Molecular Sciences 2021 , 22 , 11432, doi:10.3390/ijms222111432. Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M.R.; Smith, J.C.; Kasson, P.M.; Van Der Spoel, D.; et al. GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit. Bioinformatics 2013 , 29 , 845–854, doi:10.1093/bioinformatics/btt055. Majid, M.; Farhan, A.; Asad, M.I.; Khan, M.R.; Hassan, S.S.U.; Haq, I.-U.; Bungau, S. An Extensive Pharmacological Evaluation of New Anti-Cancer Triterpenoid (Nummularic Acid) from Ipomoea batatas through In Vitro, In Silico, and In Vivo Studies. Molecules (Basel, Switzerland) 2022 , 27 , doi:10.3390/molecules27082474. Shams ul Hassan, S.; Abbas, S.Q.; Hassan, M.; Jin, H.-Z. Computational Exploration of Anti-Cancer Potential of Guaiane Dimers from Xylopia vielana by Targeting B-Raf Kinase Using Chemo-Informatics, Molecular Docking and MD Simulation Studies. Anti-Cancer Agents in Medicinal Chemistry 2021 , 21 , 1–16, doi:10.2174/1871520621666211013115500. Tiwari, R.K.; Chanda, S.; M, U.; Singh, M.; Agarwal, S. Anti-Inflammatory and Anti-Arthritic Potential of Standardized Extract of Clerodendrum serratum (L.) Moon. Frontiers in pharmacology 2021 , 12 , 629607, doi:10.3389/fphar.2021.629607. Reanmongkol, W.; Noppapan, T.; Subhadhirasakul, S. Antinociceptive, antipyretic, and anti-inflammatory activities of Putranjiva roxburghii Wall. leaf extract in experimental animals. Journal of natural medicines 2009 , 63 , 290–296, doi:10.1007/s11418-009-0336-6. Thite, A.T.; Patil, R.R.; Naik, S.R. Anti-arthritic activity profile of methanolic extract of Ficus bengalensis: Comparison with some clinically effective drugs. Biomedicine & Aging Pathology 2014 , 4 , 207–217, doi:https://doi.org/10.1016/j.biomag.2014.03.005. Gautam, R.K.; Sharma, S.; Sharma, K.; Gupta, G. Evaluation of Antiarthritic Activity of Butanol Fraction of Punica granatum Linn. Rind Extract Against Freund’s Complete Adjuvant-Induced Arthritis in Rats. Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer 2018 , 37 , 53–62, doi:10.1615/JEnvironPatholToxicolOncol.2018025137. Jalalpure, S.S.; Mandavkar, Y.D.; Khalure, P.R.; Shinde, G.S.; Shelar, P.A.; Shah, A.S. Antiarthritic activity of various extracts of Mesua ferrea Linn. seed. Journal of ethnopharmacology 2011 , 138 , 700–704, doi:10.1016/j.jep.2011.09.042. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3828096","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267059184,"identity":"c6af4d09-cf91-4297-b917-ac9149cb59eb","order_by":0,"name":"Haseeb Ahsan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYBACAwYeEGUD4fGQoCWNdC2HSdBizn728IefO84nrp3dwPjgbRsRWix78tIke8/cTtx25wCz4VxitBgcyDFj4G0DarmRwCbNS5SW82+MP/5tOwfSwv6bOC03cgyAhh8A28JMpJY3ZtKybcnG224kNkvOOUeUw3KMP75ts5PddiP54Ic3ZURoQQKMDaSpHwWjYBSMglGAGwAAYY06gujTnoMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8060-1065","institution":"University of Minnesota Twin Cities Campus: University of Minnesota","correspondingAuthor":true,"prefix":"","firstName":"Haseeb","middleName":"","lastName":"Ahsan","suffix":""},{"id":267059185,"identity":"cd2c4c14-565c-4284-8606-6f370900377f","order_by":1,"name":"Rizwana Kauser","email":"","orcid":"","institution":"University of Sargodha","correspondingAuthor":false,"prefix":"","firstName":"Rizwana","middleName":"","lastName":"Kauser","suffix":""},{"id":267059186,"identity":"ecfdba63-f49f-4c44-afb1-92d688b2dd92","order_by":2,"name":"Hafiz Muhammad Irfan","email":"","orcid":"","institution":"University of Sargodha","correspondingAuthor":false,"prefix":"","firstName":"Hafiz","middleName":"Muhammad","lastName":"Irfan","suffix":""},{"id":267059187,"identity":"b7147f88-fa05-4602-82df-d9fe23c4f7dc","order_by":3,"name":"Ihtisham Haider","email":"","orcid":"","institution":"Mukabbir College","correspondingAuthor":false,"prefix":"","firstName":"Ihtisham","middleName":"","lastName":"Haider","suffix":""},{"id":267059188,"identity":"fee15a5b-5cf6-47e9-98c7-1597cb67ce17","order_by":4,"name":"Asma Ahsan","email":"","orcid":"","institution":"University of Minnesota Twin Cities: University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Asma","middleName":"","lastName":"Ahsan","suffix":""},{"id":267059189,"identity":"50bc6914-80e7-4795-aff5-04beb49ea56c","order_by":5,"name":"Syed Shams ul Hassan","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Syed","middleName":"Shams ul","lastName":"Hassan","suffix":""},{"id":267059190,"identity":"4739ff56-b7cb-46bc-b747-a579ed899a8e","order_by":6,"name":"Simona Bungau","email":"","orcid":"","institution":"University of Oradea: Universitatea din Oradea","correspondingAuthor":false,"prefix":"","firstName":"Simona","middleName":"","lastName":"Bungau","suffix":""},{"id":267059191,"identity":"5d54f400-8093-4f02-a01d-74d855e35683","order_by":7,"name":"irfan Anjum","email":"","orcid":"","institution":"Shifa Tameer-e-Millat University","correspondingAuthor":false,"prefix":"","firstName":"irfan","middleName":"","lastName":"Anjum","suffix":""}],"badges":[],"createdAt":"2024-01-01 21:30:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3828096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3828096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49690798,"identity":"2a9ea356-3461-498a-9a1a-8991c49f11f6","added_by":"auto","created_at":"2024-01-16 13:57:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":233188,"visible":true,"origin":"","legend":"\u003cp\u003e3D structure of TNF-α (A), PGE2 (B), NF-κB (C).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/ccc08e5ab1122bee534c5737.png"},{"id":49690460,"identity":"ae0b3d6d-646c-45db-91d9-36d18445de25","added_by":"auto","created_at":"2024-01-16 13:49:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34329,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of effect of amitriptyline (20, 40 \u0026amp; 80 mg/kg orally) on paw swelling in egg-albumin induced inflammation at different timepoints. Analysis was done by Two-Way ANOVA followed by Bonferroni test. ­­­\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/2001eddce8652ff44f7cd428.png"},{"id":49690462,"identity":"13465ca6-35e5-4cc7-ae8d-5e81e279c0f3","added_by":"auto","created_at":"2024-01-16 13:49:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":120780,"visible":true,"origin":"","legend":"\u003cp\u003ePresentation of effect of amitriptyline (20, 40 \u0026amp; 80 mg/kg orally) on (A) paw swelling, (B) Weight, and (C) arthritic score in CFA-induced arthritis at different days. Analysis was done by Two-Way ANOVA followed by Bonferroni,s test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/6086d977e10d90cbf59640aa.png"},{"id":49689679,"identity":"44c0aabd-6cf2-430f-b1bc-44c860125fdf","added_by":"auto","created_at":"2024-01-16 13:41:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":91934,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Amitriptyline at various doses of 20, 40 \u0026amp; 80 mg/kg orally on (A) PGE2, (B) TNF-α, and (C) NF-Kβ on day 28 after induction of arthritis with CFA. One-Way ANOVA followed by Dennett’s test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/a35d7d853de6ef9c75161e03.png"},{"id":49689683,"identity":"48ab2dc2-ab83-422e-aa67-b06d64b5b97d","added_by":"auto","created_at":"2024-01-16 13:41:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":78235,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of test drug (Amitriptyline 20, 40 \u0026amp; 80 mg/kg orally) on (A) SOD, and (B)GSH in CFA-induced arthritic animals on day 28. ONE- Way ANOVA was applied.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/534b34511507c7ba991d572c.png"},{"id":49689682,"identity":"7c8821d1-d4f2-430e-9971-b5d8a946a294","added_by":"auto","created_at":"2024-01-16 13:41:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":138052,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking analysis of (A) NF-κB, (B) TNF-α (C) PGE.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/e1136d06264a06cf2ccd700a.png"},{"id":49689684,"identity":"d682b334-82e7-4f82-94be-dcd6ecfa268c","added_by":"auto","created_at":"2024-01-16 13:41:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":62770,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;(A) The predicted stability and flexibility of amitriptyline and PGE2 for 100 ns simulation (B) RMSF plot for the fluctuation of PGE2 and ligand complex through the 100 ns demonstrating the same pattern of interacting of molecular docking (C) Radius of gyration of PGE2 in the presence of amitriptyline (D) The number of hydrogen bond observe within the binding cavity of PGE2 and amitriptyline for 100 ns simulation (E) The SASA analysis for the Protein and amitriptyline.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/c7233798ec1274211b4183de.png"},{"id":50806496,"identity":"ba36bebe-8ac6-434e-a204-3d5fed348742","added_by":"auto","created_at":"2024-02-07 15:47:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1273116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3828096/v1/83c12218-e1df-430a-8898-c345cce74c63.pdf"}],"financialInterests":"","formattedTitle":"Amitriptyline Ameliorates Arthritis by Downregulation of Inflammatory Mediators and Oxidative Stress; A\n\nMechanistic Approach","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAutoimmune inflammatory disease, such as rheumatoid arthritis (RA), is an example of discrepancies inside the immune system usually arising from the effects of an accretion of environmental and expected insults over a specific period of time along with the genetic predispositions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Prevalence of the rheumatoid arthritis is 2\u0026ndash;4% internationally depending upon gender, age and geographical distribution [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The etiology of rheumatoid arthritis has not been clear yet.\u003c/p\u003e \u003cp\u003eImmune-mediated diseases are generally initiated and determined by biomarkers of immune (T \u0026amp; B cells) as well as inflammatory cells (macrophages). In Rheumatoid arthritis, type II collagen and proteoglycan act as signal for activation of these immune cells to release pro-inflammatory molecules [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome researchers have proposed that the enzymatic and non-enzymatic antioxidant systems are compromised in RA. Therefore, rheumatoid arthritis patients are at greater risk of being exposed to the free radicals. In different scenarios of oxidative stress, superoxide ions are formed by oxygen metabolism, which participate in the destruction of antioxidant system. Various anti-oxidants have been claimed to be very effective in the treatment of RA e.g. glutathione peroxidase, superoxide dismutase, glutathione reductase and catalase [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Synovial fluid obtained from the patients with RA were manifested to high level of antioxidant system damages. SOD (superoxide dismutase) is a necessary metallic enzyme containing copper and act as scavenger for free radicals formation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The tissue injury takes place due to the prostaglandin\u0026rsquo;s synthesis and lipid peroxidation. Thus, the glutathione preventive effect might be due blockage of these reactions as recommended by Munthe et al.\u003c/p\u003e \u003cp\u003eThe available choices of therapeutic management for RA are disease-modifying anti rheumatic drugs, NSAIDs and novel biologics such as IL-1receptor blocker and TNF monoclonal antibodies. In recent studies, the drugs have been prescribed for symptomatic treatment and inhibit the chronic immunological events to lesser extent. Furthermore, precisely the undesired and hypersensitive reaction are limitations of the treatment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRA is strongly associated with depression due to involvement of common biomarkers such as interleukin-6 (IL-6) and IL1β \u0026amp; tumor necrosis factor-α (TNF-α). As literature shown, roughly 30% of rheumatic have symptoms of depression [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The level of monoamines (norepinephrine, dopamine and Serotonin) is significantly influenced by these cytokines. Another cytokine-mediated depression mechanism is mitogen-activated protein kinase (MAPK) pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. These common targets of depression and RA are proposed by a study conducted of Zhang [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe current study was performed to explore the anti-arthritic and antioxidant potential of amitriptyline in Sprague Dawley rats against acute inflammation as well as chronic arthritis models and evaluation of its absorption, distribution, metabolism, excretion, toxicity (ADMET) and molecular docking profile.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e\u003cstrong\u003eDrugs \u0026amp; reagent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmitriptyline was obtained from OBS\u0026reg;, Pakistan as a gift to conduct research. Complete Freund\u0026rsquo;s Adjuvant (CFA) and ELISA kits from Sigma-aldrich\u0026reg;, Germany Elab science\u0026reg; kits respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYoung healthy female and male Sprague-Dawley rats were used as experimental animals. The animals were kept in the controlled temperature (23-25\u0026deg;C) and for equal time interval (12 hr) in light and dark places at animal house, Faculty of Pharmacy, Sargodha University. Animals were fed with a standard-pellet diet and water ad-libitum. Experiments on animals were performed by using the guidelines of INH after the approval of Advance studies and research Board, University of Sargodha (SU/Acad/1723/2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;flowed by egg albumin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs procedure described by Ren et al\u0026nbsp;[11], albumin was used for acute inflammatory model. After\u0026nbsp;overnight fasting, animals were grouped into Control, Standard and Treated. Normal saline as vehicle for control, naproxen as standard and amitriptyline (20, 40 \u0026amp; 80 mg/kg) as treatment drugs.0.1 ml egg albumin was injected into the right hind paw of all animals. The swollen paw was observed for paw diameter evaluation using Vernier caliper at different time\u0026rsquo;s points (0, 1, 2, 3 and 4 hours). Percentage inhibition was calculated by using calculated paw volume\u0026nbsp;[11].\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAVoAAABFCAYAAAALpi6MAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAABfaVRYdFNuaXBNZXRhZGF0YQAAAAAAeyJjbGlwUG9pbnRzIjpbeyJ4IjowLCJ5IjowfSx7IngiOjM0NiwieSI6MH0seyJ4IjozNDYsInkiOjY5fSx7IngiOjAsInkiOjY5fV1964rprwAACGdJREFUeF7t3clrFE0Dx/Hy/QPcTyIi6kHxIGhccLu5HkRBiCCIICgqeBBBUDwIKsSDBxE38CZEPbsf9BARRYUIHlVExFPc/gKf+Va68nb6GZ/JRCuZGb8fGCbV+3RP/6a6umYy4WdNkCRl87/iWZKUiUErSZkZtJKUmUErSZkZtJKUmUErSZkZtJKUmUErSZkZtJKUmUErSZkZtJKUmUErSZkZtJKUmUErSZkZtJKUmUErSZkZtBpXT548CRMmTAgbN26M5bNnz8byjh07Ynmk0nw8g+VRvnLlSixL48n/sKBxt3Tp0nDy5MmwefPm8OXLlxiS9+/fD9OmTSumaIz5pk+fHgYGBuJ8d+/eDbdv3w4XL14sppDGjzVajTuCduLEifHvgwcPhps3bzYVsmD6uXPnxmdC9/z58zFkqTEfOHCgmEoaHwatxt3s2bPD06dP42X/rl27wpw5c4Zqtlz+T506NU6XytVHMm/evBishDUh+/r167Bly5Zw6dKlYdNJY82g7TDlACJ0mpXaTH9nGaPx6NGj8OPHj9h8gBMnToQlS5YEWrZSey3NCZSrj7JTp04NhfWiRYvivH19ff+aThpTtTdgW6qdPJw58bFhw4ZiaGM9PT1xnv379xdDGhvNPOOF7WTfJAMDA3H/MLx2af3z3bt3xZhBvb29dfdf2r/lZeXCOrq6uorSoClTphR/jRzH6dixY0VpEK85N/YT664qv0frjWfb0nFRZ2vboAUnFSckYdIMTmoC5ldYbnd3d1Ea1Ggexl++fLko/fx5586dugFWb9l/UjUc2aa0PtZd/rBgv7H/quGLsQzaelL4sP7RbgOvj+PCc47XkfZRvSBlnzI87VveC+X3D+U0z68+7NQ52jpoCZDR1DJHE87NzMN0nDjV2tVYqIYj+yiVeS6f0Jzo9WpaSCFSXtZYYrtZ/+/sQ44Dxy2FbS58KFT3I+/L8nuT/Vj+8OC1lTFuvPa18mvrNtqHDx+GtWvXFqUQjh8/HtsV6drDjRBujnAjhXLC8NqbOnz69GlofLkdMrVNvn//vhhSf560zNSmyXCkbkYPHjwIZ86ciePSXe96y2Z6tptlMo4bPgzDSF5PI9+/fy/+Go5tuHr1atizZ08xpLXcuHEjtquePn26GNI8eiB8/fo1vHjxYkS9GNi/5WOUyunYNqP63pwxY0ao1W7jcrnxV/vAK8YMYh0MV2dq26AleL59+xaWLVsWy5yYq1atCrUaUOw/eevWrfD8+fMYkG/evInT4NmzZ/GkY/zbt2/j+PIbvHbJH4dxMyVJ8/DMPOvXr4/dh7B69eq4ToaB6QgI9Pf3x79TX856y965c2c8KV++fBn7gBKy586dG/HraWTbtm3hwoUL8W9eJzeYQPhz42gkAVRP9aZZvQcfGu2EY0sApmO0d+/eUKuVxuHNIlRnzpxZlMLQMf/8+XP48OHDsPcAKDNcnaltg5bQK4cWd5e5Y/3q1atYa6AmRIjwmDRpUpwGjx8/js/lmtKsWbOKv0IMsRSaCfNMnjw57Nu3L5YXL14cnxPWWa69EEK1S9Z417usumymo+ZLv1FeB9vKeJY30tfTyPbt22OtluDjzv7hw4eHasSsI9Wam60p8wHDh8h/Pegl0Eg5mHM/RiJtM9MTfH7hQX9C2wYt4VcNRBBchw4dKkqD5XXr1hWlwUu6NJ7aIzXJVCsGYVQOTTAPzQAJJyDBm7CO8jKoOdbbtuqy7927F7q6uobVblhXqnWi0etphGBO3aJSiLC8np6eeFVAyFOTvn79+rD1jJVqOOd8jBTH4HdRCaCpKeHDEjQh0G84lRPKDFdnatug5WSoBiLBgdQXkxpaudabmhvSeC7Fq0FHkC1YsKAo/X+ZaRrCmcv6rVu3xnKqvZaXQaBWa72oLpvaajmQOdkI/k2bNsVyo9czGteuXQvd3d2xts1VAZfHhDHr4HJ3pDqx6SBhH7Mv+KJD+u2EZnFcP378WJQGmwzSsVu5cuW/miMoM1ydqS2DlsAhMNPXNhOCg+BMuFTnJgOBxY+LML58E4LxhAzjWSbhAdrWuKQG83BZTcAShIQHJ0zqRE/tldpsWgcIVC7v0/Sot2xqrnxgpGUTgGwfl+Vo9HqaxTq4AUbzAfiCwGj9qaaDVsP+ZdsJxL6+vnD06NH4wdqsI0eOxHmT3bt3xzZxsO9YT1ouYU45HXd1oNqbqu2w2TxqgVcMGUSXoHJ3IPqyMl3qZtNoPH0e6Q7Ecvv7++Mw5qHrDsPTtOWuQmkZtYAcGs460vYxHvWWzfTMx7SMqy670fbWw/hfdRNieeW+nGwH28M6mYduUEnqgvQ3dTlKxyLtX/YNZR71usFxTNN4Hsxflvbhr+ZPy+dZna0tg1a/xolbLxyrQZqUPxTSBwD+xqCVcmnbNlo1h8tS+pNW0Zuh9j6IbYTVXhKtguYX2nxT80tqA05t2FKrM2g70Jo1a4YFUzPSTS6W0SroDUFbdWrDrNXCQ29vb8t+MEhVBm2HoXaaHqO5ucI8v7uMP23+/PnxpiXSh0e6GSm1A//DgtoCteyBgYH4Tbp27M2gv5s1WrWN9IPeCd2j6HpHCPu/wdTKDFq1BfoXpx/0Bn2C+b0GvuBBe+3ChQvjcKkV2XSgtkQnf74Ukn5/Qmpl1mjVtghavlVns4FanUGrtsTvAtB0sHz58rBixYpiqNSabDqQpMys0UpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGVm0EpSZgatJGUVwj8T3Rz9YZXNuQAAAABJRU5ErkJggg==\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormaldehyde-induced arthritis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis method was used to evaluate the anti-arthritic potential of amitriptyline against chronic arthritis induced by formaldehyde. Rats of either sex were categorized into 6 groups. Group I was administered distilled water. Group II was given control vehicle after, and arthritis was induced. Group III was given standard drug in dose of 10 mg/kg by oral route. Groups IV, V and VI were given amitriptyline as treated group. The same dosing as performed as in above experiment for test drugs. In this method, 30 minutes\u0026rsquo; post administration of test drugs, 2% formaldehyde in the dose of 0.1 ml was administered to all groups except group I by sub planter route on day first, to induce chronic non immunological arthritis and was repeated on day 3. Vernier Caliper was used to asses\u0026rsquo; arthritis by measuring the paw diameter after 10 days of treatment. The percentage inhibition of edema was measured in accordance with method given by Mahmood \u003cem\u003eet al\u0026nbsp;\u003c/em\u003e[13].\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComplete Freund\u0026rsquo;s Adjuvant\u003c/strong\u003e\u003cstrong\u003e-induced arthritis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunological arthritis model was developed using Complete Freund\u0026rsquo;s Adjuvant (CFA) according to our previously used methodwith slight modifications [14]. CFA was used to induce arthritis in left hind paws of animals and swelling of paw was assessed by using digital Vernier caliper at 0, 7-, 14-, 21- and 28-days\u0026rsquo; post induction of phlogiston agent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSprague-Dawley rats weighing from 150 to 200 g (either male or female) were randomized into six groups. Group I was considered arthritic control whereas Group II as normal control and both were given vehicle only. Group III was a standard group and was treated with reference drug naproxen (10 mg/kg). Group IV, V and VI were treated with amitriptyline in distilled water by oral rout, on day after induction of arthritis, at various doses (20, 40 \u0026amp; 80 mg/Kg). This study continued for 28 days starting from induction day (0-28).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of paw swelling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePaw diameter was be observed on day 0, 7, 14, 21 \u0026amp; 28\u003csup\u003eth\u003c/sup\u003e day. The body weight of animals was observed every week. After treatment, on 29 days animals was sacrificed and blood was collected.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of weight variations:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeight of animals were observed on day 0, 7, 14, 21 and 28\u003csup\u003eth\u0026nbsp;\u003c/sup\u003eday. The weight of treated animals was increasing with increasing number of days i.e 7, 14, 21, 28\u003csup\u003e\u0026nbsp;th\u003c/sup\u003e day. But the weight of arthritic rats was increased on day 7 but decreased on 28\u003csup\u003eth\u003c/sup\u003e day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of arthritic score:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeverity of inflammation was observed with the help of arthritic score. Arthritic score was less in animals which were treated with amitriptyline dose 40 mg and was more less in rats treated with Amitriptyline 80 mg.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstimation of inflammatory cytokines by ELISA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood samples were drawn by euthanizing the animals at the end of study. Mixed with EDTA and then centrifuged for 30 minutes to separate supernatant (serum). Serum levels of tumor necrosis factor, prostaglandin E-2 and nuclear factor kB were estimated by ELISA using kit methods (Elab science kits). Optical density was measured at 450 nm from which the concentrations were computed through standard curve method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of antioxidant enzymes activities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animals were given anesthesia and liver sections were obtained. After that, the sections were homogenized according to formerly reported method. \u0026nbsp;Protein content was estimated by Lowry\u0026rsquo;s method using bovine albumin as reference [15].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstimation of superoxide dismutase activity (SOD) and Glutathione (GSH)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivity of superoxide dismutase enzyme and GSH was evaluated by SOD and GSH Kits protocol according to xanthine oxidase method in liver of sacrificed animals. The activity was presented in units/mg protein\u0026nbsp;[16].[17].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstimation of ADMET parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Pharmacokinetic parameters like absorption, distribution metabolism and excretion were noted by online data bank given by employing software (http://biosig.unimelb.edu.au/pkcsm/prediction) on May 25, 2022 [18]. Swiss ADMET software with given link: http://www.swissadme.ch/index.php was run to assess the polarity, permeability and other parameters of amitriptyline [19].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking and Simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditionally, an in-silico studies were performed to examine the interaction, stability of amitriptyline with in the binding pocket of TNF-\u0026alpha;, NF-\u0026kappa;B, and Prostaglandin E2 (PGE2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eInitial structure and system preparation\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to conduct the docking experiment, a 3D structure is required. Therefore, the structure of TNF-\u0026alpha;\u0026nbsp;(6X81 at 2.81 with 158 amino acids), NF-\u0026kappa;B (1A3Q with 2.10 and 285 amino acids), and PGE2 (7CXR with 2.8 and 358 amino acids) were downloaded from the protein data bank (PDB) database for the current study (\u003cstrong\u003eFigure 1\u003c/strong\u003e). A co-crystallized ligand of TNF-\u0026alpha; [4-(isoquinolin-8-yl) phenyl] acetonitrile (PDB ID: UTJ), and PGE2 I.E., Prostaglandin E2 (P2E), required ions, and the protein chains (homodimers) were deleted from the crystal structure before they were used for further study. All hydrogen adds, non-polar hydrogen atom mergers, and Kollman charge were calculated using the AutoDock v4.2 program. Moreover, the ligand structure, amitriptyline, was also acquired from PubChem and stored in shell repository in the SDF files for further use. Open Babel was used to parse the SDF file to 3D PDB file format. By an improved force field and the steepest descent method, the ligand library was refined for 3000 steps. AutoDock was used to rotate all of the bonds that could be rotatable in order to induce torsion into the compounds. A PDB file with the minimize protein structure and amitriptyline was then saved\u0026nbsp;in PDBQT for further docking studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular Docking Analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking was performed for amitriptyline and TNF-\u0026alpha;, NF-\u0026kappa;B, and PGE2. The prepared protein PDBQT files were loaded into AutoDock tool (receptor proteins), amitriptyline was used as docking compound. The receptor grid box with dimensions of x = 126, y = 126, and z = 126 was centered on the protein\u0026apos;s ligand inside the active site.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAround the existence co-crystalized ligand, the grid was centered on x = 90.977, y = 113.349, and z = 112.209 for PGE2, x = 9.071, y = 61.506, and z = 19.623 for NK-kb, x = -4.281, y = 28.68, and z = -13.491 for TNF-\u0026alpha; respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMolecular Dynamic Simulation Studies\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to evaluate binding strength, stability, and flexibility, docked conformations complexes with high binding energies inside the binding site of proteins were subjected to molecular dynamics simulations using the GROMACS v2020 software\u0026nbsp;[20]. Additionally, it keeps the systems\u0026apos; densities uniformly distributed. All of the bonds were constrained using the Linear Constraint Solver (LINCS)\u0026nbsp;technique, and the long-range electrostatics were calculated using the Particle Mesh Ewald (PME)\u0026nbsp;method with a cutoff value of 1.0 nm. After the system was brought to equilibrium, a 100 ns molecular dynamic simulation was run on a Linux computer to evaluate the trajectories and examine at interaction stability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTrajectory and structure analysis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMoreover, VMD was utilized to extract coordinates for a certain time frame, show all trajectory movements over simulation time, and calculate trajectories. Chimera visualization tools were used to compare and visualize the structures. Gmx_rms was used to calculate the RMSF of the protein and ligand, whereas gmx_rmsf was used to calculate the RMSD. We investigated the protein-ligand hydrogen bond, gyration radius, and solvent accessibility surface analyses using the gmx_hbond, gmx_gyrate, and gmx_sasa functions. The trajectories\u0026apos; graphs were acquired using terminal-shell commands, and they were then drawn using the 2D charting program XMGRACE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis by statistical tools \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical analysis was performed by Graph-pad Prism version 8 software. ANOVA testing such as \u0026nbsp;Two-way and One-was was applied with significant level of p\u0026lt; 0.005.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePaw diameter in albumin induced inflammation\u003c/h2\u003e \u003cp\u003eAmitriptyline at three different doses (20, 40 \u0026amp; 80 mg/kg) were evaluated for its anti-inflammatory results in albumin induced inflammation. The test drug significantly (\u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.001\u003c/em\u003e) decreased the thickness of paw at early as well as late stage after the induction of inflammation in comparison to arthritic rats group shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Similarly, the standard drug (naproxen 10 mg/kg) also revealed significant reduction in swollen paw.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePaw diameter in formaldehyde induced arthritis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this experiment. Amitriptyline was observed for its anti-arthritic effect. At high dose of test drug, the significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) anti-arthritic effect was noted the whole study period of 10 days. But the low dose depicted non-significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.005) effect up-to day 4 but shifted towards significant site at the end of study. The standard drug naproxen also displayed outstanding effect on inflamed paw throughout the experimental period as mentioned in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e with maximum inhibition of 20.56%. Amitriptyline (80 mg/kg P.O) showed 21.74% inhibition on increase in paw diameter at the end of experiment (Day 10).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMeasurement of arthritic score for evaluation of paw diameter\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArthritic score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeverity of inflammation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo inflammation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMild inflammation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eModerate inflammation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSevere inflammation\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMeasurement of paw diameter on day 2nd, 4th, 6th, 8th \u0026amp; 10th day after induction of arthritis with formaldehyde.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDays\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003ePaw diameter (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArthritic Control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVehicle Control\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNaproxen 10 mg/kg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAmitriptyline 20mg/kg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAmitriptyline 40mg/kg\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAmitriptyline 80 mg/kg\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2nd day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.066\u003csup\u003en.s\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.066\u003csup\u003en.s\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.086\u003csup\u003en.s\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4th day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.058\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.071\u003csup\u003en.s\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.058\u003csup\u003en.s\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.058\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6th day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.058\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.060\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8th day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10th day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.071\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePaw diameter in CFA experiment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, there was significant inhibitory effect on paw diameter in CFA-induced arthritis. Amitriptyline (40 mg/kg oral dose) produced 4.7% decrease in swelling of paw prompted by CFA on day 14 that increased to 17.09% on day 28. This effect significantly increased by higher dose of Amitriptyline 80 mg/kg (28.63%) on day 28th. On the other side, standard drug naproxen showed 19.6% decrease in paw diameter on the same week.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWeight variations after treatment with amitriptyline.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAmitriptyline showed significant effect on weight variations at dose 80 mg/kg. There was a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) increase on the weight of treated (Amitriptyline 80 mg/kg orally) animals on day 14, 21 and 28 in comparison to arthritic rats. This effect was non-significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.005) at low dose of Amitriptyline (40 mg/kg orally) on the day 14 but became significant at the end of study as given in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The weight of Amitriptyline treated rats (at 80 mg.kg dose) was 130\u0026thinsp;\u0026plusmn;\u0026thinsp;5.62 g at day 7 but increased to 143\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78 g on day 28th .The weight of arthritic rats were 121\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87g on day 7 and decrease to 99.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 g at day 28th .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of drug on arthritic score.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displayed severity of inflammation in the form of arthritic index in arthritic animals throughout the experiment. This intensity of inflammation was reduced in animals treated with amitriptyline at high doses (40 \u0026amp; 80 mg/kg orally). There was no significant reduction in inflammatory signs with low dose of test drug (Amitriptyline 20 mg/kg). Maximum suppressant effect on inflammatory signs was observed on day 14\u0026ndash;28 in treatment group of amitriptyline 80 m/kg orally. But the dose of 40 mg/kg P.O showed significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) effect on inflammatory score on day 28. The standard drug naproxen also showed significant anti-inflammatory effect in comparison to arthritic animals.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEffect of drug on inflammatory cytokines\u003c/h2\u003e \u003cp\u003eCFA significantly stimulated the enhanced release of pro-inflammatory cytokines in arthritic control animals in comparison to normal control group. Amitriptyline significantly reduced the levels of prostaglandin E2 in treatment groups at the doses of 20 as comparable to disease group animals. The serum concentration of TNF-α was also significantly lowered by amitriptyline at the doses of 40 (237.69\u0026thinsp;\u0026plusmn;\u0026thinsp;5.21) and 80 mg/Kg (185.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.71) when compared with arthritic control group (684.31\u0026thinsp;\u0026plusmn;\u0026thinsp;30.35). The drug also declined the serum concentrations of NF-kB at all the doses of 20, 40 and 80 mg/Kg. The effects of the amitriptyline on prostaglandin E2, TNF-α and NF-kB were comparable with those of naproxen, used as reference drug as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eEffect on SOD and GSH (antioxidant enzyme):\u003c/h2\u003e \u003cp\u003eThe significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) expression of antioxidant enzyme SOD (super oxide dismutase) was observed in animals treated with amitriptyline (80 mg/kg orally). Similarly, the effect on reducing agent GSH (glutathione) was less significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) at the higher dose (80 mg/kg orally) of test drug amitriptyline. These results were comparable to arthritic group animals. There was significant suppression of these enzyme (SOD \u0026amp; GSH) in arthritic animals as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eADMET profile of Amitriptyline:\u003c/h2\u003e \u003cp\u003eBefore selecting drug for clinical trials, its absorption into central nervous system and gastrointestinal tract is very much important factor. The penetration of centrally acting drugs into blood brain barrier is necessary for its effects [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Amitriptyline has good absorption into human intestine with maximum values (98%) as given in Table \u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It has blood brain barrier permeability of 0.96. Due to its maximum penetration into intestinal tract and minimum penetration into CNS contribute its minor CNS elated adverse effects. It is substrate of P-glycoprotein. It is substrate for CYP3A4 and has interactions with many drugs. The toxicity data showed its hepatotoxic behavior.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEvaluation of absorption, distribution, metabolism, excretion and toxicity parameters of amitriptyline\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProperties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmitriptyline\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003ePhysicochemical Properties\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMW \u003csup\u003ea\u003c/sup\u003e (g/mol)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e313.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRotatable bonds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHBA \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHBD \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFraction Csp3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPSA \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.24A\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eLipophilicity Log Po/w\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eiLOGP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXLOGP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMLOGP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConsensus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAbsorption\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman intestinal absorption\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97.47%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSkin Permeability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-2.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP-glycoprotein Substrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDistribution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBlood brain barrier Permeability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCNS permeability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-1.196\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eMetabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCYP3A4 substrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCYP2D6 substrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCYP2D6 inhibitor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExcretion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal clearance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRenal OCT2 substrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eToxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOral rat acute toxicity (LD50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.802\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOral rate Chronic toxicity (LOAEL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.027\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHepatotoxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eMolecular Docking Analysis\u003c/h2\u003e \u003cp\u003eMolecular docking was performed to better understand the reported pharmacological activity and binding mechanism of Amitriptyline against Nuclear factor-κB (NF-κB), Prostaglandin E2 (PGE2) and Tumor necrosis factor (TNF).\u003c/p\u003e \u003cp\u003eThe docking result shows the binding of Amitriptyline with NF-κB having significant binding energy of -7.2 kcal/mol and rmsd of 2.8 \u0026Aring;. It was observed that the Amitriptyline make a one hydrogen bonds with Asp186 of NF-kB with an energy of -12 kcal/mol and bond length of 2.99\u0026Aring;. One bond mediated with Ser188 of NF-kB with an energy of -0.5 kcal/mol and the bond length of 3.29\u0026Aring;. The Amitriptyline mediate also a strong hydrophobic interaction with Lys143, Ser222, Ser188, Asp219, Met185, Leu187, Tyr55, and His140 as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt was also observed that the Amitriptyline having strong binding affinity toward TNF-α with a binding score of -6.17. It makes one hydrogen bond with Glu135 of TNF-α with 3.03 \u0026Aring; and energy of -1.9 kcal/mol. Whereas, mediate various hydrophobic interaction between TNF-α and Leu26, Gln25, Asn46, Pro139, Ilu136, Asn137, and Lys90 Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eAdditionally, the docking result of Amitriptyline with PGE2 shows that ligand sets well in the pocket of receptor having a binding score of -8.49kcal/mol, highest among the rest of the proteins, and rmsd of 2.5. The ligand got a stable binding interaction within the pocket of PGE2. It mediated several electrostatic interactions comprised of ionic bond among Asp297 and N50 of the ligand having bond energy of -4.8kcal/mol and bond length of 2.9\u0026Aring;. Beside the ionic bond, it also makes various hydrophobic interactions between the ligand and Arg190, Val179, His191, Met286, Glu288, Asn287, Phe283, Arg292, Tyr285, Ser291, Ala284, Trp186, Phe280, Leu197, Ile189, Tyr181, Phe188, and Gln180 Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eMolecular Dynamic Simulation Studies\u003c/h2\u003e \u003cp\u003eThe docked complex of PGE2 and ligand that had been chosen by docking studies was further examined for stability using MD simulation for up to 100 ns because of high affinity of Amitriptyline towards PGE2. The simulation findings are discussed with regard to RMSD, RMSF, hydrogen bond, radius of gyration, and SASA analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eRMSD\u003c/h2\u003e \u003cp\u003eThe protein-ligand complex's functioning and stability are evaluated using the RMSD. This helps us understand the structural variation of the protein-ligand complex throughout the duration of a 100 ns time trajectory. The amitriptyline appeared to be within the acceptable range of 0.5 nm, according to the RMSD, showing that it was favorably bound within the PGE2 binding cavity. The protein-compound complex system was shown to oscillate between 25 and 60 ns while equilibrating between 10 and 25 ns within the RMSD range of 0.35 to 0.5 nm initially. But after 60 ns, it was discovered that the amitriptyline had high stability for the entirety of the 100 ns simulation, as represented in the range of 0.35\u0026ndash;0.55 nm, respectively. These persistent backbone alterations in PGE2 and ligand complex point to conformational changes during the whole simulation, as shown by the RMSD (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eRMSF\u003c/h2\u003e \u003cp\u003eThe RMSF trajectories provide significant details about the complex's protein residual variations. The broad range of plots indicates a higher degree of flexibility and fragility in the interactions. Lower values or less variance throughout the simulation parameter suggest complex regions with less distortion. But in the system examined for 100 ns of simulation, the PGE2 complex with chemicals had the same pattern of interactions, as seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. The amitriptyline complexed with PGE2 demonstrated protein stability with minor high peaks shown as a continuous pattern. Considering this, the RMSF analysis verifies that the simulation had no impact on the protein-ligand structure.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRadius of gyration (Rg)\u003c/h3\u003e\n\u003cp\u003eAdditionally, to assess PGE2 compactness in the presence of amitriptyline, the radius of gyration was examined. Rg stands for the root mean square distance between the protein's atoms and its rotational axis. Plotting the Rg values of protein backbone atoms across time allowed researchers to examine changes in structural compactness. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, the compound's simulated Rg ranges from 1.925 to 0.2.05 nm. The backbone Rg value is initially reduced by the binding of the molecule inside the binding cavity, while Rg was found to be constant with moderate fluctuation in the range of 2 nm. Additionally, the system was generally steady with a peak value of 2 nm after 60 ns. The Rg value demonstrated the protein's stability in the complex and showed that these chemicals bound steadily without undergoing any structural changes.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eInteraction Analysis\u003c/h2\u003e \u003cp\u003eAt a simulation duration of 100 ns, the intensity of the interaction between ligands and PGE2 has been assessed. There was a variation of 2 hydrogen bonds found throughout the simulation. The amitriptyline mediated no hydrogen bonds for up to 60 ns of the simulation duration. In contrast, it mediates two persistent hydrogen bonds inside the PGE2 protein after 60 ns. In this way, numerous interactions of the same subtype with the ligand sustain the unique association Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD. The substance defines the potential important in-silico inhibitory effect by demonstrating the contact is maintained and contributes to a stable binding with the desired protein over simulation duration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eSASA Analysis\u003c/h2\u003e \u003cp\u003eThe interaction of biomolecules with protein was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE. However, the average SASA values (i.e., 155nm\u003csup\u003e2\u003c/sup\u003e) for the amitriptyline-protein complex were slightly higher, especially in the first 30 ns. It was also discovered that the complex had larger solvent accessible surface area until 25 nanoseconds, but after 30 nanoseconds, the SASA value dropped. This suggests the MD simulation of a ligand-free structure can sample the binding pocket in its confined conformation as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. In such a dynamic manner, the ligand appears to be preferentially contained within the protein's pocket. While this suggests improved ligand-protein interactions due to the solvent-substitution process, the lower SASA trajectories (average 130 nm\u003csup\u003e2\u003c/sup\u003e) until 80 ns into the simulation run support the opposite conclusion. After this point, the SASA tones increased progressively until they reached their maximum SASA values, after which they decreased until the MD simulation was completed. Amitriptyline may have migrated toward the solvent side of the protein pocket during simulated time frames 1\u0026ndash;20 and 80\u0026ndash;100 ns, where the protein pocket became strongly solvated and little compacted.\u003c/p\u003e \u003cp\u003eUsing the RMSD, RMSF, SASA, and Rg findings, we were able to display the dynamic differences between each of the studied ligands during the duration of the MD simulation. This is why it is important to study ligand-protein interactions throughout time as well as the varied trajectories of ligand-protein complexes so that the real stability of these complexes may be determined.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRA is a chronic disease that usually develops in adults and elder people. The prevalence of RA is about 1% and more commonly occurs in women. Its prevalence is high in low-middle income countries [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present investigations have been performed to evaluate the anti-arthritic potential of amitriptyline. Albumin-induced inflammatory model was used to evaluate its effect in acute phase. Injection of albumin stimulates the resident macrophages to release histamine and prostaglandins [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Amitriptyline confirmed its suppressor effect significantly on inflammation provoked by albumin as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This has been proved, that amitriptyline has inhibitory potential on inflammatory mediators.\u003c/p\u003e \u003cp\u003eArthritis developed in SD rats by formaldehyde is the most suitable method for ruling out the anti-arthritic behavior of test drug. This method showed a close similarity to the pattern of human arthritis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The release of substance P is provoked by formaldehyde in neurogenic phase (early phase) whereas histamine, bradykinin and PGE2 are produced in inflammatory phase (late phase). These mediators are responsible for inflammatory changes such as alteration in caliber or permeability of vessels. This study results supported that amitriptyline restrained the arthritis by preventing the release of histamine, bradykinin and PGE2 with a dose of 20, 40 and 80 mg/kg orally as given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe mechanism behind CFA-mediated arthritis is the involvement of infiltrating cells. CFA induces response in three divisions less inflammation to joint swelling and then ultimately bone erosion due to involvement of PGE2 and autoantibodies [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe easiest way to evaluate the anti-arthritic activity is the measurement of paw edema. Exudate and inflammatory cells contribute to paw edema formation. The inhibitory effect on these factors results in decrease of paw diameter. As displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, amitriptyline with high dose of 80 mg/kg caused a significant decrease in paw swelling throughout the experiment. This confirmed the anti-arthritic effect of amitriptyline as reported by Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003eThe loss of body weight in RA is due to decrease nutritional absorption [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Another study proposed that CFA enhances leptin level that contributes to weight loss. However, SD rats treated with amitriptyline (20, 40 and 80 mg/kg) significantly restored the loss in weight as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC. This might be due to suppression on level of leptin or increase abortion of nutrient.\u003c/p\u003e \u003cp\u003eOther criteria for arthritic evaluation is arthritic score or index that is characterized by various symptoms such as redness or edema. Therefore, the treatment with amitriptyline reduced these inflammatory signs like swelling \u0026amp; redness at all doses (20, 40 and 80 mg/Kg) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eProstaglandin E-2, tumor necrosis factor and nuclear factor are the prospective targets of drugs which are used to restrain the progression of RA. PGE2, TNF-α and NF-kB are the causative agents which precipitate the certain vascular alterations such as dilation of blood vessels with increased permeation in the process of inflammation. These are habitually synthesized by macrophages which contribute the bone attrition mechanisms in RA. This study demonstrated a significant decline inculcated by amitriptyline at various doses (20, 40 and 80 mg/Kg) in the levels of PGE2, TNF-α and NF-kB estimated in serum samples of animals by ELISA. These outcomes offer a scientific comprehension to the mechanism of action of amitriptyline for management of arthritis induced by CFA \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe oxidative stress in arthritic rats is due to CFA and result in elevation of SOD and CAT. Therefore, it could be imagined that increase in level of SOD and CAT by amitriptyline could be the future targets for inhibiting the gene expression of pro-inflammatory markers of arthritis as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e \u003cb\u003e(a \u0026amp; b).\u003c/b\u003e\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings of this study have validated the anti-arthritic behavior of amitriptyline by a reduction in paw edema, arthritic score, oxidative stress, and levels of pro-inflammatory cytokines including TNF-α, PGE2 \u0026amp; IL-1β. Therefore, it should be recommended for clinical investigations in future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors did not receive any funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: H.A\u003c/strong\u003e designed the project, \u003cstrong\u003eR.K\u003c/strong\u003e performed the experimental work, Ihtisham Haider did ELISA work and paper writing. Z.K did graphs orientation. \u003cstrong\u003eR.A\u003c/strong\u003e in-vivo experimental work and data analysis, \u003cstrong\u003eI.A\u003c/strong\u003e. results interpretation and manuscript proof reading and editing, \u003cstrong\u003eS.B\u003c/strong\u003e and \u003cstrong\u003eS.S.H\u003c/strong\u003e did docking studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e Experiments on animals were performed by using the guidelines of INH after the approval of Advance studies and research Board, University of Sargodha (SU/Acad/1723/2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e Original data is preserved and will be submitted upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment:\u003c/strong\u003e The authors thank the University of Sargodha, Pakistan for providing facilities to do work\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRosenthal, K.S.; Mikecz, K.; Steiner, H.L. 3rd; Glant, T.T.; Finnegan, A.; Carambula, R.E.; Zimmerman, D.H. Rheumatoid arthritis vaccine therapies: perspectives and lessons from therapeutic ligand epitope antigen presentation system vaccines for models of rheumatoid arthritis. \u003cem\u003eExpert review of vaccines\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e, \u003cem\u003e14\u003c/em\u003e, 891\u0026ndash;908, doi:10.1586/14760584.2015.1026330.\u003c/li\u003e\n\u003cli\u003eDi, Y.M.; Zhou, Z.-W.; Guang Li, C.; Zhou, S.-F. Current and future therapeutic targets of rheumatoid arthritis. \u003cem\u003eAnti-inflammatory \u0026amp; anti-allergy agents in medicinal chemistry\u003c/em\u003e \u003cstrong\u003e2011\u003c/strong\u003e, \u003cem\u003e10\u003c/em\u003e, 92\u0026ndash;120.\u003c/li\u003e\n\u003cli\u003eZimmerman, D.H.; Mikecz, K.; Markovics, A.; Carambula, R.E.; Ciemielewski, J.C.; Toth, D.M.; Glant, T.T.; Rosenthal, K.S. Vaccination by Two DerG LEAPS Conjugates Incorporating Distinct Proteoglycan (PG, Aggrecan) Epitopes Provides Therapy by Different Immune Mechanisms in a Mouse Model of Rheumatoid Arthritis. \u003cem\u003eVaccines\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e9\u003c/em\u003e, doi:10.3390/vaccines9050448.\u003c/li\u003e\n\u003cli\u003eAruoma, O.I.; Halliwell, B. Action of hypochlorous acid on the antioxidant protective enzymes superoxide dismutase, catalase and glutathione peroxidase. \u003cem\u003eThe Biochemical journal\u003c/em\u003e \u003cstrong\u003e1987\u003c/strong\u003e, \u003cem\u003e248\u003c/em\u003e, 973\u0026ndash;976, doi:10.1042/bj2480973.\u003c/li\u003e\n\u003cli\u003eCimen, M.Y.; Cimen, O.B.; Ka\u0026ccedil;maz, M.; Ozt\u0026uuml;rk, H.S.; Yorgancioğlu, R.; Durak, I. Oxidant/antioxidant status of the erythrocytes from patients with rheumatoid arthritis. \u003cem\u003eClinical rheumatology\u003c/em\u003e \u003cstrong\u003e2000\u003c/strong\u003e, \u003cem\u003e19\u003c/em\u003e, 275\u0026ndash;277, doi:10.1007/pl00011172.\u003c/li\u003e\n\u003cli\u003eSukketsiri, W.; Chonpathompikunlert, P.; Tanasawet, S.; Choosri, N.; Wongtawatchai, T. Effects of Apium graveolens Extract on the Oxidative Stress in the Liver of Adjuvant-Induced Arthritic Rats. \u003cem\u003ePreventive nutrition and food science\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e21\u003c/em\u003e, 79\u0026ndash;84, doi:10.3746/pnf.2016.21.2.79.\u003c/li\u003e\n\u003cli\u003eZhang, C. Flare-up of cytokines in rheumatoid arthritis and their role in triggering depression: Shared common function and their possible applications in treatment (Review). \u003cem\u003eBiomedical reports\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e14\u003c/em\u003e, 16, doi:10.3892/br.2020.1392.\u003c/li\u003e\n\u003cli\u003eHodes, G.E.; M\u0026eacute;nard, C.; Russo, S.J. Integrating Interleukin-6 into depression diagnosis and treatment. \u003cem\u003eNeurobiology of stress\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e, 15\u0026ndash;22, doi:10.1016/j.ynstr.2016.03.003.\u003c/li\u003e\n\u003cli\u003eAbbott, R.; Whear, R.; Nikolaou, V.; Bethel, A.; Coon, J.T.; Stein, K.; Dickens, C. Tumour necrosis factor-\u0026alpha; inhibitor therapy in chronic physical illness: A systematic review and meta-analysis of the effect on depression and anxiety. \u003cem\u003eJournal of psychosomatic research\u003c/em\u003e \u003cstrong\u003e2015\u003c/strong\u003e, \u003cem\u003e79\u003c/em\u003e, 175\u0026ndash;184, doi:10.1016/j.jpsychores.2015.04.008.\u003c/li\u003e\n\u003cli\u003eDionisie, V.; Filip, G.A.; Manea, M.C.; Manea, M.; Riga, S. The anti-inflammatory role of SSRI and SNRI in the treatment of depression: a review of human and rodent research studies. \u003cem\u003eInflammopharmacology\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e29\u003c/em\u003e, 75\u0026ndash;90, doi:10.1007/s10787-020-00777-5.\u003c/li\u003e\n\u003cli\u003eRen, M.; Tang, Q.; Chen, F.; Xing, X.; Huang, Y.; Tan, X. Mahuang Fuzi Xixin Decoction Attenuates Th1 and Th2 Responses in the Treatment of Ovalbumin-Induced Allergic Inflammation in a Rat Model of Allergic Rhinitis. \u003cem\u003eJournal of immunology research\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e2017\u003c/em\u003e, 8254324, doi:10.1155/2017/8254324.\u003c/li\u003e\n\u003cli\u003eCicala, C.; Ianaro, A.; Fiorucci, S.; Calignano, A.; Bucci, M.; Gerli, R.; Santucci, L.; Wallace, J.L.; Cirino, G. NO-naproxen modulates inflammation, nociception and downregulates T cell response in rat Freund\u0026rsquo;s adjuvant arthritis. \u003cem\u003eBritish journal of pharmacology\u003c/em\u003e \u003cstrong\u003e2000\u003c/strong\u003e, \u003cem\u003e130\u003c/em\u003e, 1399\u0026ndash;1405, doi:10.1038/sj.bjp.0703449.\u003c/li\u003e\n\u003cli\u003eMahmood, F.; Khan, J.A.; Mahnashi, M.H.; Jan, M.S.; Javed, M.A.; Rashid, U.; Sadiq, A.; Hassan, S.S.U.; Bungau, S. Anti-Inflammatory, Analgesic and Antioxidant Potential of New (2S,3S)-2-(4-isopropylbenzyl)-2-methyl-4-nitro-3-phenylbutanals and Their Corresponding Carboxylic Acids through In Vitro, In Silico and In Vivo Studies. \u003cem\u003eMolecules (Basel, Switzerland)\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e27\u003c/em\u003e, doi:10.3390/molecules27134068.\u003c/li\u003e\n\u003cli\u003eAhsan, H.; Irfan, H.M.; Alamgeer; Shahzad, M.; Asim, M.H.; Akram, M.; Zafar, M.S. Anti-rheumatic activity of pseudoephedrine (a substituted phenethylamine) in complete Freund\u0026rsquo;s adjuvant-induced arthritic rats by down regulating IL-1\u0026beta;, IL-6 and TNF-\u0026alpha; as well as upregulating IL-4 and IL-10. \u003cem\u003eInflammopharmacology\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e29\u003c/em\u003e, 673\u0026ndash;682, doi:10.1007/s10787-021-00804-z.\u003c/li\u003e\n\u003cli\u003eAkhtar, M.F.; Ashraf, M.; Anjum, A.A.; Javeed, A.; Sharif, A.; Saleem, A.; Akhtar, B. Textile industrial effluent induces mutagenicity and oxidative DNA damage and exploits oxidative stress biomarkers in rats. \u003cem\u003eEnvironmental toxicology and pharmacology\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e41\u003c/em\u003e, 180\u0026ndash;186, doi:10.1016/j.etap.2015.11.022.\u003c/li\u003e\n\u003cli\u003eSharif, A.; Ashraf, M.; Javeed, A.; Anjum, A.A.; Akhtar, M.F.; Akhtar, B.; Saleem, A. Oxidative stress responses in Wistar rats on subacute exposure to pharmaceutical wastewater. \u003cem\u003eEnvironmental science and pollution research international\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, \u003cem\u003e23\u003c/em\u003e, 24158\u0026ndash;24165, doi:10.1007/s11356-016-7717-7.\u003c/li\u003e\n\u003cli\u003eAdeyemi, W.J.; Olayaki, L.A. Diclofenac - induced hepatotoxicity: Low dose of omega-3 fatty acids have more protective effects. \u003cem\u003eToxicology reports\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e5\u003c/em\u003e, 90\u0026ndash;95, doi:10.1016/j.toxrep.2017.12.002.\u003c/li\u003e\n\u003cli\u003eDaina, A.; Michielin, O.; Zoete, V. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. \u003cem\u003eScientific Reports\u003c/em\u003e \u003cstrong\u003e2017\u003c/strong\u003e, \u003cem\u003e7\u003c/em\u003e, 1\u0026ndash;13, doi:10.1038/srep42717.\u003c/li\u003e\n\u003cli\u003eUl Hassan, S.S.; Muhammad, I.; Abbas, S.Q.; Hassan, M.; Majid, M.; Jin, H.Z.; Bungau, S. Stress driven discovery of natural products from actinobacteria with anti-oxidant and cytotoxic activities including docking and admet properties. \u003cem\u003eInternational Journal of Molecular Sciences\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e22\u003c/em\u003e, 11432, doi:10.3390/ijms222111432.\u003c/li\u003e\n\u003cli\u003ePronk, S.; P\u0026aacute;ll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.; Shirts, M.R.; Smith, J.C.; Kasson, P.M.; Van Der Spoel, D.; et al. GROMACS 4.5: A high-throughput and highly parallel open source molecular simulation toolkit. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cstrong\u003e2013\u003c/strong\u003e, \u003cem\u003e29\u003c/em\u003e, 845\u0026ndash;854, doi:10.1093/bioinformatics/btt055.\u003c/li\u003e\n\u003cli\u003eMajid, M.; Farhan, A.; Asad, M.I.; Khan, M.R.; Hassan, S.S.U.; Haq, I.-U.; Bungau, S. An Extensive Pharmacological Evaluation of New Anti-Cancer Triterpenoid (Nummularic Acid) from Ipomoea batatas through In Vitro, In Silico, and In Vivo Studies. \u003cem\u003eMolecules (Basel, Switzerland)\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, \u003cem\u003e27\u003c/em\u003e, doi:10.3390/molecules27082474.\u003c/li\u003e\n\u003cli\u003eShams ul Hassan, S.; Abbas, S.Q.; Hassan, M.; Jin, H.-Z. Computational Exploration of Anti-Cancer Potential of Guaiane Dimers from Xylopia vielana by Targeting B-Raf Kinase Using Chemo-Informatics, Molecular Docking and MD Simulation Studies. \u003cem\u003eAnti-Cancer Agents in Medicinal Chemistry\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e21\u003c/em\u003e, 1\u0026ndash;16, doi:10.2174/1871520621666211013115500.\u003c/li\u003e\n\u003cli\u003eTiwari, R.K.; Chanda, S.; M, U.; Singh, M.; Agarwal, S. Anti-Inflammatory and Anti-Arthritic Potential of Standardized Extract of Clerodendrum serratum (L.) Moon. \u003cem\u003eFrontiers in pharmacology\u003c/em\u003e \u003cstrong\u003e2021\u003c/strong\u003e, \u003cem\u003e12\u003c/em\u003e, 629607, doi:10.3389/fphar.2021.629607.\u003c/li\u003e\n\u003cli\u003eReanmongkol, W.; Noppapan, T.; Subhadhirasakul, S. Antinociceptive, antipyretic, and anti-inflammatory activities of Putranjiva roxburghii Wall. leaf extract in experimental animals. \u003cem\u003eJournal of natural medicines\u003c/em\u003e \u003cstrong\u003e2009\u003c/strong\u003e, \u003cem\u003e63\u003c/em\u003e, 290\u0026ndash;296, doi:10.1007/s11418-009-0336-6.\u003c/li\u003e\n\u003cli\u003eThite, A.T.; Patil, R.R.; Naik, S.R. Anti-arthritic activity profile of methanolic extract of Ficus bengalensis: Comparison with some clinically effective drugs. \u003cem\u003eBiomedicine \u0026amp; Aging Pathology\u003c/em\u003e \u003cstrong\u003e2014\u003c/strong\u003e, \u003cem\u003e4\u003c/em\u003e, 207\u0026ndash;217, doi:https://doi.org/10.1016/j.biomag.2014.03.005.\u003c/li\u003e\n\u003cli\u003eGautam, R.K.; Sharma, S.; Sharma, K.; Gupta, G. Evaluation of Antiarthritic Activity of Butanol Fraction of Punica granatum Linn. Rind Extract Against Freund\u0026rsquo;s Complete Adjuvant-Induced Arthritis in Rats. \u003cem\u003eJournal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer\u003c/em\u003e \u003cstrong\u003e2018\u003c/strong\u003e, \u003cem\u003e37\u003c/em\u003e, 53\u0026ndash;62, doi:10.1615/JEnvironPatholToxicolOncol.2018025137.\u003c/li\u003e\n\u003cli\u003eJalalpure, S.S.; Mandavkar, Y.D.; Khalure, P.R.; Shinde, G.S.; Shelar, P.A.; Shah, A.S. Antiarthritic activity of various extracts of Mesua ferrea Linn. seed. \u003cem\u003eJournal of ethnopharmacology\u003c/em\u003e \u003cstrong\u003e2011\u003c/strong\u003e, \u003cem\u003e138\u003c/em\u003e, 700\u0026ndash;704, doi:10.1016/j.jep.2011.09.042.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Rheumatoid Arthritis, Pro-inflammatory cytokines, ROS, Superoxide dismutase, CFA","lastPublishedDoi":"10.21203/rs.3.rs-3828096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3828096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study was designed to evaluate the antioxidant and anti-arthritic potential of amitriptyline. The albumin and formaldehyde were used to induce acute and chronic inflammation respectively. The adjuvant-induced arthritis model was developed in rats. In acute model, amitriptyline significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) decreased the thickness of paw at early as well as late stages. At high dose of test drug, the significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) anti-arthritic effect was noted in formaldehyde-induced arthritic model. Likewise, Amitriptyline (40 mg/kg oral dose) produced a 4.7% decrease in swelling of paw prompted by CFA on day 14 that increased to17.09% on day 28. This effect significantly increased by higher dose of Amitriptyline 80 mg/kg (28.63%) on day 28th. The significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) expression of antioxidant enzyme SOD (super oxide dismutase) was observed at maximum dose (80 mg/kg). Amitriptyline significantly reduced the levels of inflammatory cytokines like prostaglandin E2, TNF-α and NF-kB and the results were comparable with naproxen. Further, in-depth molecular docking and simulations studies of amitriptyline showed that it has good binding capacity with inflammatory cytokines showing the highest score with prostaglandin E2. In addition, ADMET studies also proved amitriptyline within the limits of Lipinski\u0026rsquo;s rule of five. These findings validate the anti-arthritic effect of amitriptyline, but it has limitations for clinical studies.\u003c/p\u003e","manuscriptTitle":"Amitriptyline Ameliorates Arthritis by Downregulation of Inflammatory Mediators and Oxidative Stress; A\nMechanistic Approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-16 13:41:08","doi":"10.21203/rs.3.rs-3828096/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5aa94209-548a-4bbb-8cd3-01032c5963ba","owner":[],"postedDate":"January 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-07T15:39:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-16 13:41:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3828096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3828096","identity":"rs-3828096","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}