Oral Phyto-thymol ameliorates the stress induced IBS symptoms

Physical stressors play a crucial role in the progression of irritable bowel syndrome (IBS). Here we report a heterogeneous physical stress induced IBS rat model which shows depression and subsequent modulation of IBS by oral treatment of thymol. Oral administration of Thymol reduces the stress induced IBS significantly altering the stress induced gastrointestinal hypermotility, prolonged the whole gut transit time, and increased abdominal withdrawal reflex suggesting gastrointestinal hypermotility and visceral discomfort caused the onset of depression. Immunohistochemical analysis in small intestine and colon of rats shows the decreased 5-HT3AR expression level while thymol treatment normalized the 5-HT3AR expression in the stressed rats. Molecular docking studies showed that thymol competes with endogenous serotonin and an antagonist, Tropisetron and all have similar binding energies to 5-HT3AR. Molecular dynamics simulations revealed that thymol and tropisetron might have similar effects on 5-HT3AR. Our study suggest that thymol improves IBS symptoms through 5-HT3AR, could be useful for the treatment of IBS.

Scientific RepoRtS | (2020) 10:13900 | https://doi.org/10.1038/s41598-020-70420-4 www.nature.com/scientificreports/ Asian herbs call Ajwaan, is used as traditional medicine and food component. It is typically used for bowel related complications and digestion problems 13 . Thymol derivatives have a pharmacologic use as a gastrointestinal modulator, but the biochemical mechanisms are largely unknown. Further studies are required to find out whether thymol could hold a putative potential for pain medication by alleviating the visceral pain associated with IBS and other diseases, modulating the opioid receptor. We used post-natal stress induced rat model of IBS 13 . Rats were given a mixture of heterotypic physical stressor (see "Experimental procedure" section) by alternating modality to mimic the humanized effects in social milieu for a period of 4-6 weeks. Cotton nest behavioral studies were performed preliminarily in order to see whether application of prolonged stress caused their mood fluctuation in terms of depression. We noticed that rat exposed to various stress factors showed visible signs of depression and mood alteration as evident from their participation to play with the cotton nest to reshuffle the cotton ( Fig. 1A-C). The tests were scored based on their zeal for participation and performance to play with cotton nest (Fig. 1D). At the end of 4 week of stress, we used gastrointestinal transit (GIT) as readout for both the consequence of stress and subsequent healing ability of thymol (for 2-week treatment starting from week 5 and continued in week 6) to ease out stress induced IBS. GIT is a surrogate marker for evaluating drug efficacy to inhibit bowel abnormalities in IBS, also clinically relevant for the assessment of "organic" disorder such as GI motility 8,14 . Indeed, drugs that target normalizing GIT such as Lubiprostone, Linaclotide turned out to be useful in relieving abdominal discomfort in Phase III trials 1,15 . We found that oral thymol treatment enhanced the GI transit (similar to untreated control animals) which was higher in stress induced animals possibly due to leaky gut ( Fig. 2A). This could be due to the stress driven change in local intestinal permeability resulting in modulation of mucosal inflammation and gut sensory visceromotor reflex. Since IBS is often associated with visceral hyperalgesia, fecal pellet output has also been used to study the response of stress and drugs in rodent models; this is in some instances homologous to and in some instances contrary to human GIT studies where the patients as well as healthy control subjects were evaluated in absence of acute stressor 16 . We noticed that fecal output was higher for stress induced rats (Fig. 2B). The thymol treatment significantly decreased the fecal count that was associated with stressed rats (Fig. 2B). This result corroborates other rat model studies that showed stress driven anxiety behavior and evaluation of corticotropin-releasing factor 1 (CRF1), an antagonist altering the accelerated fecal output in human IBS patients as well 16 . Besides leaky gut, stress can change the fecal microbiota that initiated many studies of fecal transplant to donor from IBS patients and subsequent analysis of recipient animal behavior such as visceral hypersensitivity 17 . This archetype is used for the study of anxiety associated psychiatry in mouse model by transferring other's behaviors. Next, abdominal withdrawal reflex (AWR) was used to measure the visceral hypersensitivity which is pre-clinically and clinically used to assess colorectal distension (CRD) 18 . This method is widely used in human and other rodent subjects as an index of visceral pain for the evaluation of analgesic compounds. We found that the animal under stress showed extreme abdominal discomfort which is evident from the tolerance to external pain stimuli applied by Figure 1. Exposure to chronic stress reveals altered behavioral response in SD rats. Shredding of a cotton nestlet was measured in rat exposed to (A) control, (B) Stress, (C) Stress with treatment of thymol 50 mg/kg b w, and (D) Summary data showing that thymol 50 mg/kg b w treated increased the cotton nestlet shredded in chronic stress induced rat. The amount shredded is shown as percentage of total nestlet area. Data shown as mean ± S.E.M (n = 8). *P < 0.05 (one-way ANOVA and TMCT compared with stress). www.nature.com/scientificreports/ catheterization of balloon in colorectal cavity ( Fig. 2C and Fig. S1). The threshold of barostat tolerance was higher for the thymol treated rat upon pain stimuli as evident from the AWRanalysis (Fig. 2C). Next, we wanted to confirm whether the serotonin receptor is the target involved here for the regulation of stress induced IBS and subsequent thymol treatment of rats by oral administration. Serotonin agonist and antagonist are already known to treat IBS related visceral pain 19 . We have chosen 5-HT 3 AR among other serotonin receptor homologues, because 5-HT 3 A antagonist compound is capable of managing stress driven IBS defecation. After the rats were sacrificed, the intestine and colon were used for immunohistochemistry by hematoxylin and eosin staining (IHC), and immunohistofluorescence (IHF) analysis of serotonin receptor. IHC analysis clearly showed that visible symptoms of local intestinal tissue atrophy induced by physical stress, while thymol treatment showed the recovery of the intestine tissue architecture possibly by tissue remodeling (Fig. 3). Similar tissue damage was observed for the colonic tissue (Fig. S2). It was noticed that stress caused reduction of serotonin receptor (5-HT 3 AR) density presence on the surface membrane of the intestine tissue which was enhanced upon thymol treatment (Fig. 4A,B). 5-HT 3 A receptor expression was also upregulated in the case of colonic tissue (Fig. S3). This study indicates that possibly thymol is antagonizing the serotonin receptor to quench the stress mediated IBS that usually results in symptoms of GI hypermotility in rat.  www.nature.com/scientificreports/ During IBS, opioid receptors are one of the important regulators of pain sensation among the vast array of other receptors involved in pain perception 20,21 . We observed that physical stressor increased the pain sensation as evident from the overexpression of the mu (µ) opioid receptor that jumped higher upon stress induction (Fig. S4). Subsequent thymol treatment of the rats partly adjusts the µ opioid receptor as part of IBS allied visceral pain. However further study is desired to understand how thymol alters the µ opioid receptor axis during pain regulation spurred by IBS. Multi moderators in pain perception are the reason for the complexity to precisely perturb the pain regulator as an IBS related drug target.
A previous study indicated that thymol has a tendency to bind to the transmembrane region of the receptor 22 . Here, we used molecular docking and MD simulation calculations in order to predict whether thymol also has the ability to compete with serotonin towards 5-HT 3 AR that associates with the nociceptive pain signal in IBS via binding to the extracellular domain (ECD). 5-HT 3 A receptor is a pentamer with five equivalent binding sites; the neurotransmitter site is at the subunit interfaces in the ECD 23 . Our molecular docking studies against the entire receptor as well as the different conformations of the ECD of the receptor, as explained in the Experimental Procedure section, clearly revealed that serotonin and thymol bind into the same pocket of the 5-HT 3 AR with similar binding energies (Fig. 5, Fig. S5, Fig. S6 and Table S1, Table S2). Thymol, in all best poses (most energetically favorable), only, bound to the ECD of the structures. All the other poses of thymol also bound to the same domain in all structures except in 6HIS. In that last case, the 7th and 8th poses bound in the intracellular domains in an extreme region close to the extracellular domain. However, the binding region was still different from the one proposed by Lansdell et al. 22 The very same pocket is also a binding site for tropisetron, an antagonist for 5-HT 3 receptor, as was shown in crystal structure 6HIS 23 . Further, residue interaction analysis revealed that these compounds interact with protein residues in a similar manner (Table S5, Fig. S7). Interestingly, in 6HIS conformation both tropisetron and thymol interacted with identical residues TRP156 (chain A), TYR207 (chain A), ILE44 (chain E), TRP63 (chain E), ARG65 (chain E); while serotonin differed. However, we also observed similar interacting residues when serotonin binds to all other three conformations (6HIQ, 6HIO, 6HIN). Therefore, the difference in the agonist and antagonist may not be linked to the difference in the compounds' interacting residues.
To further analyze protein-ligand complexes, MD simulations were performed for the ECD, and root mean square deviation (RMSD), radius of gyration (Rg), root mean square fluctuations (RMSF) and hydrogen bond formation were calculated. RMSD, Rg and hydrogen bond results are presented in Fig. S8. According to RMSD results, all the complexes were stable, and no ligand dissociation was observed. The maximum observed ligand RMSD was ~ 1.25 Å for all ligands except tropisetron in 6HIS which increased up to ~ 2 Å. We also observed that tropisetron moves from its original position that was presented in the crystal structure towards the inner part of the binding pocket (Data not shown). Rg also did not show any significant variation. Concerning hydrogen bonding, serotonin formed a higher number of hydrogen bonds than thymol had in all cases (and also more than tropisetron had in 6HIS). Interestingly, in both 6HIO and 6HIQ, Thymol seemed to rearrange and adopt a more stable pose shown here with its more consistent hydrogen bonding toward the end of the simulation. www.nature.com/scientificreports/ Thymol in 6HIS presented a special case in which the compound does not make any hydrogen bond, but it is mainly stabilized by hydrophobic contacts. Even though, serotonin, thymol and tropisetron are binding to the same binding pocket of the receptor and showing similar residue interaction pattern, RMSF results revealed that the protein is behaving differently in the presence of serotonin and thymol (Fig. 6). In general, residues showed more flexibility when serotonin was bound to protein, contrary to both thymol and tropisetron (6HIS). In the antagonist (tropisetron) bound conformation of the receptor (6HIS), protein residues presented similar behavior in the presence of thymol or tropisetron (Fig. 6). These results might be an indication of antagonist behavior of thymol. 3 AR antagonists have been used for IBS treatment modulating the visceral pain situation 19,24 . Thymol has been also used as an essential oil component for various biological benefits 25 . In our study, we find that thymol can manage the intestinal hypermotility and ameliorates the visceral sensitivity that is associated with physical stressor mediated IBS (Fig. 7). Thus, oral thymol administration could be a potential option to handle the leaky gut with subsequent soothing of the IBS symptoms possibly by regulating the serotonin receptor (5-HT 3 AR). Besides animal model, molecular docking simulation study confirms that thymol competes with native ligand towards the same binding site of 5-HT 3 AR. Whether thymol can intervene in the nociceptive pain triggered by noxious stimuli mediating through pain receptor such as opioid and cannabinoid, could certainly lead to a new direction in developing pain medication related to an array of multiple disorders encompassing viscera and innervated spinal cord to GI tract. Thymol mediated recovery of intestinal tissue architecture that was locally damaged by stress induction also requires further evaluation as to whether thymol has the ability of tissue remodeling during IBS mediated tissue loss. Similarly, we could not find conclusive changes from the CaCO-2 cellular competition experiment to obtain more molecular insight for the thymol mediated 5-HT 3 AR antagonism (data not shown). However, 5-HT 3 R genetic polymorphism (greater C/C genotype) is associated with the severity of IBS patient symptoms, with enhanced anxiety and amygdala 26 . Further investigation is required to understand the precise molecular mechanism of thymol driven 5-HT 3 AR antagonism, even though the 5-HT 3 R antagonists are viable therapeutic modules to treat IBS and anxiolytic effects.. None the less the non-invasive (oral) validation of thymol as therapeutic target for taming preclinical anxiety associated IBS model in rats warrants further investigation to translate this phytochemical for the application of anxiety associated IBS, other hepatobiliary disease and psycho somatosensory disorders.

5-HT
Comparing ligand structures, thymol does not present the indole ring present in both serotonin and tropisetron. This moiety is very common among bioactive compounds on the 5-HT 3 A receptor (CHEMBL4972). Indeed, except for its aromatic ring which is common among to 5-HT 3 A receptor antagonists, thymol lacks the carbonyl and the basic amine which are considered as key pharmacophoric point for 5-HT 3 A receptor antagonists. However as previously shown, from histological analysis of 5-HT 3 A receptors, and MD simulation analysis of binding similarity as serotonin and tropisetron, thymol interact with the 5-HT 3 A receptor during stress mediated IBS. These interactions are mainly driven by hydrophobic contacts on its aromatic ring and its substituents as evident from the MD simulation. Hence thymol may present a different scaffold for a new class of 5-HT 3 A modulator and may be worthy of additional structure-activity relation (SAR) study.  Induction of stress protocol. The stressor was as follows: (a) no water for 12 h, (b) no food for 12 h, (c) wet/dirty bed for 10 min, (d) press tail for 2 min, (e) day and night reverse, (f) shake the cage for 10 min, (g) tight the legs for 3 min. The animals were exposed to one of the seven various restraint stressors for different time manner. The animals exposed to stressor were returned to the animal house after completion following stressor exposure to minimize the disturbance of the control group, group 1. The sequences of the stressors were exchanged alternatively every week. Rats were kept under rest remaining days of the week. After four weeks of stressors the animals were randomly divided into two groups, groups 2 and 3. Each group had eight to ten animals (n = 8-10). Group 3 was treated with 50 mg thymol/kg body weight (Sigma Aldrich, Catalog No: G8802B). Thymol was dissolved in 0.9% NaCl along with tween 80 (1%) and then orally fed on the fifth and sixth consecutive weeks to the animals of group-3 as indicated. The experiments were conducted for six weeks.
Cotton nestle shredding test. Rats were individually placed in a clean cage with a cotton material comprising 5 cm 2 squares of compressed cotton allowed in the cage at 12 h; the percentage of the area of cotton shredded was measured by NIH software (ImageJ). www.nature.com/scientificreports/ Whole gut transit time (GIT) test. In the morning at 8 a.m. of each experimental animal were transferred into individual empty polyethylene cage and were left to acclimatize to the cage for 1 h. 5% Evan blue and 5% gum Arabic dissolved in 0.9% saline was feed orally to those rats. Rats were returned to their individual cage. The time from the end of the experiment to the notice of the first blue fecal pellet was measured in minutes and constituted the whole gut transit time. During the whole gut transit time the number of fecal pellets in each cage was counted for gastro intestinal hypermotility.

Abdominal withdraw reflex (AWR) test. Rats were anaesthetized by ethyl ether and catheter with
syringe was inserted (about 8 cm after lubrication with paraffin oil) through anus. When the rats woke up, they were put in a special transparent plastic cage; after the rat adapted to the environment, air was gradually injected into the catheter to expand the intestinal tract, recording the volume of injected air when rats reach to three points. This experiment was repeated three times, with an interval of five minutes.
Tissue processing for histology and immunofluorescence. Intestine and colon for histology. At the end of the behavioral experimental, animals were autopsied after anesthesia with ether. 2 cm of intestine and colon were removed, placed in 4% paraformaldehyde overnight, and sent for histologic processing. The small intestine and colon were fixed in formalin, dehydrated in grade of alcohols and xylene, embedded in paraffin wax, cut into 5 µm cross section by microtome. A cross section of intestine and colon were attached in pre albumin coated slide to be stained with hematoxylin and eosin. Intestine and colon section slides were imaged using a NIKON Eclipse e100. Statistical analysis. All data were analyzed using Prism 6 software (GraphPad software). Experiments were analyzed using one-way ANOVA and Tukey corrections for multiple testing between categories. Data is presented with means ± standard error of the mean (SEM) shown as line and sticker. (r012) 27 . The docking validation was, first, done by re-docking serotonin to the 5-HT 3 receptor (6HIQ) using blind docking approach via QuickVina-W 28 . The RMSD value between crystalized and docked serotonin was RMSD 2.2 Å (Fig. S9). Blind docking experiments were, then, repeated against full length protein (4PIR) as well as against the ECD domain in four different conformations. Thymol, serotonin, tropisetron and NAG (N-acetlyd-Glucosamine, the co-crystal ligand in 4PIR) were docked in the 5 five structures. The complete docking parameters are in Table S3 and Table S4. Root mean square deviation (RMSD) values were computed using GROMACS (Version 5.1.2) 29 without least-squares fitting of the structures.

Molecular dynamics simulations and analysis.
Nine MD simulations were done using the ECD of the receptor extracted from different conformations. TMPPAA was not included in the simulations. Systems were simulated in a dodecahedron box. The distance between the solute and the box was set to 1.0 nm. TIP3P water model was used with a concentration of 0.15 M Na + (sodium) and Cl− (chloride) ions. Energy minimization was done via steepest descent method with a maximum force set at < 1,000.0 kJ/mol/nm and a maximum number of steps to 50,000. This was followed by equilibration at 300 K and 1 atm with 50 ps of MD simulation in the isothermal-isobaric ensemble and subsequently in the canonical one. A cutoff of 10 Å was used for the Lennard-Jones and short-range electrostatic interactions. The smooth particle mesh Ewald method and a fourth-order interpolation scheme were used for the long-range electrostatic interactions. The leap-frog algorithm was used for integration. Ligand topology files were generated via ACPYPE 30 using a total charge of zero for all ligands. Simulations were conducted on a remote machine at Center for High Performance Computing (CHPC) with GROMACS version 2016 using the Amber ff99SB-ILDN 31 force field. The generated trajectories were analyzed with GROMACS modules using RMSD, radius of gyration (Rg) and root mean square fluctuations (RMSF) to