Gastrointestinal disorders-induced pain

Gene Reports 18 (2020) 100580

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Gene Reports journal homepage: www.elsevier.com/locate/genrep

Gastrointestinal disorders-induced pain Megha Singhal a b c

a,1

, Vipin Arora

b,1

, Hee-Jeong Im

T

b,c,⁎

Department of Medicine, University of Illinois at Chicago, Chicago, IL, United States Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, United States Jesse Brown Veterans Affairs Medical Center (JBVAMC) at Chicago, IL, United States

A R T I C LE I N FO

A B S T R A C T

Keywords: Visceral pain Functional gastrointestinal disorders Irritable bowel syndrome Irritable bowel disorder NGF Microbiota Inflammasome VEGF

Functional gastrointestinal disorders (FGIDs), now better defined as disorders of gut-brain interaction (DGBI) are disorders of gut-brain interaction. Chronic episodic abdominal pain and discomfort constitute integral diagnostic criteria of these disorders. A common feature of these disorders is heightened pain sensitivity to experimental gut stimulation, termed visceral pain hypersensitivity (VPH). Visceral pain disorders are a tremendous health care burden; continues to pose a significantly unmet medical need, negatively affecting the lives of millions of individuals worldwide in all ethnic groups and all economic classes. The origin and maintenance of visceral pain involves peripheral and central mechanisms. In this review, we have highlighted the roles of the Microbiota, Nerve growth factor (NGF), NLRP3 inflammasome and vascular endothelial growth factor (VEGF) and tried to address the potential for manipulating these as a therapeutic target for FGIDs associated with visceral pain hypersensitivity which may warrant for drug development in the future.

1. Introduction Aristotle asked, “Why is it that an object held between crossed fingers appears to be two?” In 1942, Dr., Brown answered this and also the question: “Why is abdominal pain so often experienced at a distance from the organ (or tissue) in which it is produced?” (Brown, 1962). The starting point of his investigation is the well-known fact that visceral disorders are frequently accompanied by cutaneous tenderness, the pain occasioned by organic disturbance being “referred” by the patient to an area on the surface of the body (Mackenzie, 1893). In a series of papers published between 1893 and 1896, Dr. Henry Head has investigated the “Disturbances of Sensation with Especial Reference to the Pain of Visceral Disease” (Head, 1893). From the past one century to

the most recent Rome IV consensus document gave us a better understanding of the diagnostic criteria and management of functional gastrointestinal disorders (FGIDs) (Drossman and Hasler, 2016; Drossman, 2016). The most recent ROME IV criteria has replaced functional gastrointestinal disorders (FGID) with disorders of gut-brain interaction (DGBI) (Schmulson and Drossman, 2017). FGIDs or DGBI comprise 33 distinct clinical entities which can be grouped into one of six anatomical or organ domains: esophageal, gastroduodenal, gallbladder, bowel, anorectal along with the centrally mediated disorders of GI pain (Drossman and Hasler, 2016; Drossman, 2016). Chronic episodic abdominal pain and discomfort cause appreciable morbidity in FGIDs and are integral components of the diagnostic criteria in these disorders. It is estimated that in the United States a third of the general adult

Abbreviations: ACC, Anterior Cingulate Cortices; BDNF, Brain-Derived Neurotrophic Factor; BPS/IC, Bladder Pain Syndrome/Interstitial Cystitis; CAPS, CryopyrinAssociated Periodic fever Syndrome; CB2, Cannabinoid 2 receptor; CD, Crohn's Disease; CGRP, calcitonin gene-related peptide; CIPA, Congenital insensitivity to pain with anhidrosis; CNS, central nervous system; DAMPs, Damage Associated Molecular Pattern; DREADDs, Designer Receptor Exclusively Activated by Designer Drugs; DRG, Dorsal Root Ganglion; DSS, Dextran Sulfate Sodium; EC, Enterochromaffin cell; FGIDs, functional gastrointestinal disorders; fMRI, functional magnetic resonance imaging; GI, Gastrointestinal; IBD, Inflammatory Bowel Disease; IBS, irritable bowel syndrome; IL-18, interleukin-18; IL-1β, interleukin-1β; KO, Knock out; MEG, Magnetoencephalography; MOR1, mu-opioid receptor; mROS, Mitochondrial Reactive Oxygen Species; NRP1, Neuropilin 1; NRP2, Neuropilin 2; NGF, Nerve Growth Factor; NLR, Nod-Like Receptor; NLRP3, Nod-Like Receptor protein 3; HMA rats, Human Microbiota-Associated rats; NMS, Neonatal Maternal Separation; NOD2, Nucleotide-binding Oligomerization domain-containing protein 2; NTRK1, Neurotrophic Receptor Tyrosine Kinase 1; NTs, Neurotrophins; OA, Osteoarthritis; p75NTR, p75 Neurotrophin Receptor; PAMPs, Pathogen Associated Molecular Pattern; SPECT, Single-Photon Emission Computed Tomography; THAL, Thalamus; TLR, Toll like receptor; TNBS, Trinitrobenzene sulfonic acid; TrkA, Tropomyosin Receptor Kinase A; TRPV1, Transient Receptor Potential Cation Channel subfamily V member 1; UC, Ulcerative Colitis; VPH, Visceral Pain Hypersensitivity; VEGF, Vascular endothelial growth factor; DGBI, Disorders of gut-brain interaction; trK, Tropomyosin receptor kinase ⁎ Corresponding author at: Jesse Brown Veterans Affairs Medical Center (JBVAMC), 820 S. Damen Ave, Chicago, IL 60612, United States. E-mail addresses: [email protected], [email protected] (H.-J. Im). 1 The authors contributed equally to this work. https://doi.org/10.1016/j.genrep.2019.100580 Received 19 August 2019; Received in revised form 3 December 2019; Accepted 12 December 2019 Available online 16 December 2019 2452-0144/ © 2019 Published by Elsevier Inc.

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hypersensitivity (Pusceddu and Gareau, 2018; SM et al., 2017; Moloney et al., 2016; O'Mahony et al., 2014; Hadizadeh et al., 2018). Accumulating evidence indicates that the gut microbiota communicates with the central nervous system (CNS) in a bidirectional manner thereby influencing brain function and behavior (Cryan and Dinan, 2012; Eisenstein, 2016; Liang et al., 2018; Mayer, 2011). The brain can influence microbiota indirectly, via changes in gastrointestinal motility and secretion, and intestinal permeability, or directly, via signaling molecules released into the gut lumen from cells in the lamina propria (enterochromaffin cells, neurons, immune cells) (Rhee et al., 2009). The roots of the concept seeded back in the late nineteenth century, when the physicians William James and Carl Lange first proposed that emotional response might be directly modulated by signals transmitted from the viscera to the brain (Eisenstein, 2016). Since then, there have been numerous observations that established the importance of the gut microbiome in the pathogenesis for numerous disease states, such as obesity cardiovascular disease, cancer and in intestinal conditions, such as inflammatory bowel disease. Thus, understanding microbiome activity is essential to the development of future personalized strategies of healthcare, as well as potentially providing new targets for drug development. In the present review we critically reinforce the concept that the gut microbiota is involved in the visceral pain hypersensitivity.

population fulfill the diagnostic criteria for a Rome IV FGID, with a prevalence rate as high as 35% (Aziz et al., 2018), hence presenting the impact is very widespread and carry heavy economic and social consequences. The most prevalent forms of visceral pain are categorized as FGID such as irritable bowel syndrome (IBS), which exceeds US$ 40 billion in medical costs (Quigley et al., 2006; Soubieres et al., 2015). Visceral pain disorders exert a tremendous burden on the health care system and are associated with psychological distress, sleep disorders and sexual dysfunction, negatively impacting overall patient quality of life (Hungin et al., 2003). It has been 35 years since Ritchie's landmark study which involves 67 patients with IBS and 16 control subjects, first demonstrated that inflation of a balloon to 60 ml in distal colon caused pain in 6% of the control subjects but caused pain in 65% of the IBS patients and this study instigate the other research investigators to further, demonstrated that a proportion of patients with FGIDs may display elevated pain sensitivity to experimental gut distension—VPH (Ritchie, 1973; Whitehead et al., 1990; Lemann et al., 1991) Many hypotheses have been proposed to explain the origin of symptoms in FGIDs, but no single factor has achieved primacy in the literature largely because of the heterogeneity of these disorders. However, a common feature of FGIDs is that patients often display a heightened sensitivity to experimental gut stimulation, termed visceral pain hypersensitivity (VPH) (Farmer and Aziz, 2009). This observation has spawned a large research effort in identifying the responsible molecular mechanisms. The germane hypothesis for the genesis and maintenance VPH in FGIDs involves a number of peripheral and central mechanisms. In the periphery, inflammatory mediators activate and sensitize nociceptive afferent nerves by reducing their transduction thresholds and by inducing the expression and recruitment of hitherto silent nociceptors culminating in an increase in pain sensitivity at the site of injury known as primary hyperalgesia. Centrally, secondary hyperalgesia, defined as an increase in pain sensitivity in anatomically distinct sites, occurs at the level of the spinal dorsal horn. Moreover, the stress responsive physiological systems, genetic and psychological factors may modulate VPH. We reviewed few of the important etiological factors for the management of VPH in FGIDs, namely the gastrointestinal microbiota, involvement of nerve growth factor (NGF), vascular endothelial growth factor (VEGF) and role of inflammasome as key targets causing abdominal pain. More importantly, we also tried to encapsulate the brain imaging studies, which also gave insights into different brain regions closely associated with the VPH in FGIDs. In the present review, we aim to provide a contemporaneous summary of mechanisms that may be identified as common coactivating factors or mediators to understand the entwined relationship between VPH and FGIDs.

2.2. Clinical and preclinical investigations probing the role of microbiota in visceral pain hypersensitivity & FGIDs Various animal models of visceral hypersensitivity have been exploited to determine the involvement of gut microbiota on visceral pain pathways (Larauche et al., 2012; Luczynski et al., 2016) and mainly mice raised in a sterile environment from birth, and as such, without gastrointestinal bacteria (germ-free), use of probiotics to restore the gastrointestinal bacteria and decrease in gastrointestinal bacteria using antibiotics. One of the studies done by Luczynski et al. (2016) used germ-free mice to study how the gut microbiota influences an animal's sensitivity to pain. These groups of investigators showed that, compared to mice with normal gut microbiota, the germ-free mice were more sensitive to pain from internal organs especially the gut. These mice also produced larger amounts of specific proteins involved in immune responses, which may have contributed to the animal's increased sensitivity to pain. The investigators also showed that the germfree mice had changes in the size of two areas of the brain involved in sensing pain: (i) the anterior cingulate cortex which became noticeably smaller and (ii) the periaqueductal gray region (Luczynski et al., 2016). The same group also showed that germ-free mice exhibited visceral hypersensitivity and an increase in cytokine and toll like receptor (TLR) expression in the spinal cord (Larauche et al., 2012). One of the studies, done by Verdu et al. (2006), showed the antibiotic induced increases the visceral sensitivity which was accompanied by increased substance P immunoreactivity. Moreover, disturbance of the gut microbiota in adult mice was shown to induce changes in local immune response in association with enhanced pain signaling (Verdu et al., 2006), one more interesting study showed that antibiotics administered early in life induces long-lasting effects on visceral pain responses coupled with alterations in receptors such as decreases in the transient receptor potential cation channel subfamily V member 1 (TRPV1), the alpha-2A adrenergic receptor, and cholecystokinin B receptor in the spinal cord (O'Mahony et al., 2014). Other investigation done by Distrutti and coworkers showed that the administration of mixture of 8 probiotics during the neonatal period prevents against the development of the visceral pain hypersensitivity in neonatal maternal rat separation rat model of IBS (Distrutti et al., 2013). Study done by Rousseaux et al. (2007) reported that orally administered the probiotic bacteria belonging to the Lactobacillus and Bifidobacterium for 15 consecutive days increased the m-opioid receptor (MOR1) and cannabinoid (CB2) expression in the Colon of rats and mice in comparison to that of untreated controls. They also found that oral administration of the

2. Microbiota 2.1. Introduction Multicellular organisms exist as meta-organisms comprised of both the macroscopic host and its symbiotic commensal microbiota, the human body is home to far more than human cells: we harbor at least 100 trillion (1014) microbial cells (Clemente et al., 2012; Whitman et al., 1998; Belkaid and Hand, 2014). Hippocrates has been quoted as saying “death sits in the bowels” and “bad digestion is the root of all evil” in 400 BCE (Hawrelak and Myers, 2004; Sekirov et al., 2010), showed the importance of the gut microbiota in human health has been long recognized. Collectively, the microbial associates that reside in and on the human body constitute our microbiota, and the genes they encode is known as our microbiome, carrying approximately 150 times more genes than are found in the entire human genome (Ursell et al., 2014). This complex community of microbiota that interact with one another and with the host, greatly impact human health and physiology. Recent years have witnessed the rise of the gut microbiota as a major topic of research in the context of FGIDs associated visceral pain 2

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Fig. 1. Schematic representation of NGF and its receptors. TrKA: Tropomyosin receptor kinase A; Trk B: Tropomyosin receptor kinase B; TrkC:Tropomyosin receptor kinase C; p75: neurotrophin receptor p75; NGF: Nerve growth factor; NT3:neurotrophin-3; NT4: neurotrophin-4; BDNF: Brain-derived neurotrophic factor.

occurrence of Bacteroides vulgatus was low in Crohn's disease, ulcerative colitis and indeterminate colitis (Conte et al., 2006). In 2013 an interesting study conducted by Crouzet et al. (2013) in which they inoculated the germfree rats with the fecal microbiota from IBS patients characterized by hypersensitivity to colorectal distension (IBS HMA rats) or from non-hypersensitive healthy donors (Healthy HMA rats). The results demonstrated that the number of abdominal contractions in response to colorectal distensions was significantly higher in IBS HMA rats than in healthy HMA rats (Crouzet et al., 2013). This experiment showed that sensitivity to colonic distension of IBS patients can be transferred to the recipients by the fecal microbiota and proved that altered IBS microbiota may have important role in the hypersensitivity characterizing IBS patients. More recent study which summarizes the different clinical trials for the use probiotics for children with recurrent abdominal pain suggested that, children who were treated with probiotic preparations were more likely to experience improvement in pain (Newlove-Delgado et al., 2019). The evidences suggest that clinicians could consider probiotics as part of a holistic management strategy in recurrent abdominal pain.

probiotic decreased normal visceral perception (Rousseaux et al., 2007). More recent studies done by Guida et al. (2019) investigated that how the low Vitamin D dietary intake in mice leads the possible alteration in gut microbiota, pain sensitivity and endocannabinoid system signaling. They reported that vitamin D deficiency induced a lower microbial diversity. Concurrently, vitamin D deficient mice showed tactile allodynia associated with neuronal hyperexcitability and alterations of endocannabinoid system, not only at the spinal cord level but also in the duodenum and colon (Guida et al., 2019). These findings of reinforce the concept that the gut microbiota is involved in the visceral pain hypersensitivity. In clinical practice, evidence of microbiota communication with the central nervous system is clearly evident from the irritable bowel syndrome (IBS) and can be considered an example of the disruption of these complex relationships. As similar to the preclinical study, which uses the administration of Lactobacillus (probiotic), one of the clinical trials also evaluated the efficacy of Lactobacillus plantarum 299V (LP299V) in patients with IBS. It was found that patients treated with (LP299V) showed resolution of their abdominal pain as compared to 11 patients from a placebo group (Niedzielin et al., 2001). In one of the meta-analysis study done by Horvath et al. (2011), to evaluate the effect of Lactobacillus rhamnosus GG (LGG) for treating abdominal painrelated functional gastrointestinal disorders in children reported the use of Lactobacillus rhamnosus moderately increases treatment success in children with abdominal pain-related FGIDs with positive clinical outcomes. In a systematic review to evaluate the effectiveness of probiotics in IBS, with a total of 1793 patients demonstrated the beneficial effect of probiotics on abdominal pain, distension, bloating and flatulence, IBS diagnostic scores and IBS total symptoms with overall improved quality of life (Didari et al., 2015). Another clinical study which investigated the composition of the mucosa-associated intestinal microflora in colonoscopic biopsy specimens of the ileum, caecum and rectum obtained from 12 patients with Crohn's disease, 7 with ulcerative colitis, 6 with indeterminate colitis, 10 with lymphonodular hyperplasia of the distal ileum reported the higher number of mucosaassociated aerobic and facultative-anaerobic bacteria in biopsy specimens of children with inflammatory bowel disease (IBD) than in controls. An overall decrease in some bacterial species in particular,

3. Nerve growth factor 3.1. Introduction The discovery of nerve growth factor (NGF) is appropriately attributed to Rita Levi-Montalcini in the early 1950s and was the prototypical neurotrophic factor for many decades (Bradshaw et al., 2017). The cloning of brain-derived neurotrophic factor (BDNF) demonstrated that NGF is a member of a gene family dubbed neurotrophins (NTs), comprising in mammals also NT-3 and NT-4/5. All NTs have a common basic structure, along with variable domains that determine the specificity of biological actions resulting from the activation of the NT receptors 3, trks and p75NTR. Individual NTs activate different tropomyosin receptor kinase (trk) receptors (NGF acting at trkA, BDNF and NT-4/5 at TrkB, and NT-3 predominantly) (Thoenen and Sendtner, 2002) (Fig. 1). In 1995, Esteban and coworkers investigated the distribution of neurotrophin receptors (p75, trkA, trkB, and trkC-receptor proteins) 3

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human stressors). Neonatal maternal separation (NMS) in rodents, a well-documented animal model for early-life stress (Coutinho et al., 2002; Barreau et al., 2004a; Gareau et al., 2006), indeed induces various gastrointestinal dysfunctions, including hyperalgesia to colorectal distension, increased colonic mucosal permeability, and enhanced colonic motility (Coutinho et al., 2002; Barreau et al., 2004a; Gareau et al., 2006). One of the recent studies done by Wong et al. (2019) used the neonatal maternal separation model to investigate the function of NGF signaling in the regulation of intestinal homeostasis in response to early-life stress. The results showed that NMS leads to enterochromaffin hyperplasia and visceral hyperalgesia. This is reversed by inhibition of NGF-mediated trkA signaling. Barreau et al. (2004b) also reported that NGF is an important mediator to induced VPH and reported that NMS increases NGF and its mRNA level in peripheral tissue (colonic) at 14 days and later in life (12 weeks of age). These alterations of NGF mRNA expression are associated with an enhanced expression of TrkA and P75NTR receptor mRNA in colonic tissue in NMS pups but not in adult rats (Barreau et al., 2004b). Furthermore, NGF-mediated TrkA signaling has been implicated in the development of inflammation-associated visceral hyperalgesia (di Mola et al., 2000). Delafoy and coworkers showed that intraperitoneal injection of NGF lowered colonic distention thresholds in rats, which could be reversed by anti-NGF antibodies or a calcitonin gene-related peptide (CGRP) antagonist; injection of CGRP also induced similar hypersensitivity, while anti-NGF antibodies failed to reverse it, indicating that CGRP is a critical element downstream in NGF signaling (Delafoy et al., 2003; Delafoy et al., 2006). There are only a few articles reporting increased NGF expression in the colonic mucosa of IBS patients. One of the recent studies done by Xu et al. (2017) investigated the role of NGF, as well as mast cell- NGFnerve interaction in diarrhea-predominant IBS. It was reported that NGF gene expression, mast cell count and sensory nerve fibers were significantly increased in the IBS patients. Concurrently, the correlation analysis showed the NGF expression was positively correlated with the disease severity and visceral sensitivity thresholds were negatively associated with NGF expression (Xu et al., 2017). Another clinical study conducted by Dothel et al. (2015) examined the mucosal biopsy samples collected from 101 patients with IBS and 23 asymptomatic healthy individual. It was found that mucosa from patients with IBS had 89% increased staining density of NGF (NGF immunoreactivity was observed in lamina propria cells of patients with IBS compared with health controls). They also showed the 18% increase in the levels of NGF in tissues (mucosal biopsies from the proximal descending colon) from patients with IBS vs controls along 64% increase in the levels of neurotrophic receptor tyrosine kinase I (NTRK1) along with the expression of NTRK1 was markedly increased (193.8% increase) in mucosal biopsies obtained from IBS patients (Dothel et al., 2015). Similarly, analyses of the rectal biopsies from children with diarrhea-predominant IBS also showed the higher NGF content compared to control groups (Willot et al., 2012). Another group of investigators also demonstrated the increased levels of NGF and its high affinity receptor TrkA in patients of Crohn's disease (CD) and ulcerative colitis (UC). They found, in CD, NGF mRNA was increased in 60% (please make it consistent, % or percent) and TrkA mRNA in 54% compared to healthy control group. In UC, NGF mRNA expression was enhanced in 58% and TrkA mRNA expression in 50% as compared to their respective controls. One of the metanalyses which takes into consideration 295 cases bladder pain syndrome/interstitial cystitis (BPS/IC) showed an increased level of urinary NGF in BPS/IC patients in controlled participants (Chen et al., 2016). These results indicate that higher NGF levels act on the intestinal sensory nerve endings, promotes their growth and synapse formation, and thus mediates visceral hypersensitivity. Presently there is one ongoing clinical trial (NCT03675100) to evaluate the role of neurotrophic factors NGF to quantity tight junction proteins and cytokines in the colonic mucosa of IBS patients and also clarify sex differences in the pathophysiology of IBS. Despite its promising analgesic efficacy of anti-

(trK: Tropomyosin receptor kinase) by immunohistochemistry on sections of human gastrointestinal tract mucosa from esophagus through rectum. In all of the analyzed samples, there was specific immunoreactivity for trkB-receptor protein in EC in the duodenum and the sigmoid colon. EC displaying trkC-receptor protein immunoreactivity were also found in gastric fundus, duodenum, jejune, and colon; trkAreceptor protein labelling EC in the jejune, ileum and sigmoid colon. These results provide evidence for the occurrence of neurotrophin receptor proteins in non-neuronal tissues and suggest that neurotrophins, especially that binding trkB receptor proteins, can regulate a subpopulation of EC cells (Esteban et al., 1995). In one of the clinical investigations taking into consideration the 33 Inflammatory bowel disease (IBD) patients comprises primarily two disease states: Crohn's disease (CD) and ulcerative colitis (UC) studied the expression of NGF and trkA. Their findings showed that in CD, NGF mRNA was increased in 60% and trkA mRNA in 54% of samples. In UC, NGF mRNA expression was enhanced in 58% and trkA mRNA expression in 50% of samples. This showed that concomitant enhanced expression of NGF and its receptor suggested the activation of NGF-trkA pathway in chronic inflammation in CD and UC. In 1994, studies done McMahon & Katzenberg reported that 90% of visceral afferents expresses trkA. There are also other studies, which also showed the expression of high affinity NGF receptors (trkA) on the primary sensory neurons (Averill et al., 1995; Verge et al., 1989; McMahon et al., 1995; McMahon and Koltzenburg, 1994; McMahon et al., 1994). Evidence for a role of NGF both in supporting the development of sensory neurons, particularly those involved in sensing tissue damage and in changing pain thresholds has accumulated over five decades. Humans and mice harboring loss-of function mutation in the trkA gene lose sensory and sympathetic neurons and pain sensation, showing that NGF has an essential trophic role in supporting the survival of these neurons. The cell types that express trkA include not only peripheral sensory neurons but also small subgroups of CNS neurons (Holtzman et al., 1995). In the early 80's, study done by Otten and coworkers showed the correlation between substance P content in primary sensory neurons and pain sensitivity in rats exposed to antibodies to NGF (Otten et al., 1982). Further investigations also showed that hyperalgesic actions of NGF may be in parts is the consequence of an increase sensitivity of the peripheral terminals of high threshold nociceptors either as a result of direct action of NGF on trkA expressing sensory fibers or indirectly via the release of sensitizing mediators from trkA expressing inflammatory cells and postganglionic sympathetic neurons. NGF is also, however, retrogradely transported from peripheral tissues to sensory neurons in the dorsal root ganglion (DRG) where it alters transcription of a number of painrelated proteins and peptides (Woolf, 1996). Several clinical investigations were carried out by Miura and group to understand the congenital insensitivity to pain with anhidrosis (CIPA). CIPA is rare autosomal recessive disorder of the nervous system that prevents the feeling of pain. Their findings strongly suggest that defects in the NGFtrkA signaling pathway cause CIPA and that the NGF-trkA system has a crucial role in the development and function of the nociceptive reception. They further suggested trkA gene on chromosome 1q21-q22 encodes a receptor tyrosine kinase for NGF is responsible for CIPA (Miura et al., 2000a; Miura et al., 2000b; Mardy et al., 1999; Mardy et al., 2001; Indo, 2002; Indo et al., 1996). Since then, continuing studies revealed that inhibition of NGF effectively reduces various pain pathways, including inflammatory pain, neuropathic pain, osteoarthritis (OA) pain and diabetic neuropathy etc. 3.2. Clinical and preclinical investigations probing the Role of NGF in visceral pain hypersensitivity (VPH) & FGIDs Stress animal models present similar pathophysiologic abnormalities to that in IBS patients, such as VPH and intestinal barrier dysfunction. The main limitations of these models are that many have low construct validity (rodent stressors do not typically correspond to 4

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Fig. 2. NLRP3 inflammasome complex and activation in disease states. Activation of NLRP3 (1) intracellular PAMPs and DAMPs; (2) ROS mode; (3) Pathogens; leads to the production of pro inflammatory cytokines IL-1β and IL-18 in a two-step process The first one being the priming step and second one is the activation signal. Activated caspase leads to cleavage of pro IL-1β and pro IL-18 leading to production of activated IL-1β and IL-18. Genetic susceptibility also affects the expression of NLRP3 inflammasome. The gut microbiota is separated from the intestinal cells by mucus layer and epithelial tight junctions. Under disease states, the epithelial barrier is compromised allowing the bacteria to attack the lamina propria. These bacteria interact with immune cells to via innate recognition receptors such as NLRs; the activation of which leads to the production of pro inflammatory cytokines. TLR: Toll like receptor, PAPMP: Pathogen associated molecular pattern, DAMP: Damage associated molecular pattern, ROS: Reactive Oxygen Species, ASC: Apoptosis-associated speck-like protein containing a caspase-recruitment domain.

proIL-1β, proIL-18 and inflammasome component such as; NLRP3 and NLRP1. (ii) The second step or the activating signal leads to inflammasome oligomerization, caspase-1 auto activation, cytokine cleavage and thus generating mature IL-1β and IL-18 and consecutive cellular release. These proinflammatory cytokines (IL-1β and IL-18) are well known to play critical roles in maintaining tissue homeostasis in intestinal epithelium and are key mediators of inflammatory response in cells (Chen et al., 2018; Rathinam and Fitzgerald, 2016). The activation of these cytokines by inflammasome leads to a wide range of biological effects associated with inflammation. Thus, inflammasomes play a vital role in coordinating the innate and adaptive immune responses in response to pathogenic microbes and cellular stress, and in the recruitment of neutrophils into damaged tissue. It is important to note that one pathogen can activate multiple inflammasomes, for instance; Listeria monocytogenes is able to activate NLRP3, AIM2 and NLRC4, while Candida albicans can activate NLRP3 and NLRC4 (Song et al., 2018; Wu et al., 2010).

NGF therapy on the gastrointestinal disorders, the mechanisms underlying the analgesic effects induced by the NGF-TrkA signaling is poorly understood as a knowledge gap, and yet to be discovered. 4. Inflammasome 4.1. Introduction The inflammasome is a large multiprotein complex that plays a key role in the production and activation of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18 (Sahoo et al., 2011). Many members of the nucleotide-binding oligomerization domain-like receptors, or NODlike receptors (NLRs), including NLRP1, NLRP2, NLRP3, NLRC4, NLRP6, NLRP7 and NLRP12 are able to form inflammasomes, which are cytoplasmic high-molecular protein complexes (Velloso et al., 2019). Among the NLRs, the multiprotein inflammasome complexes comprise of the sensor NLR protein, the adaptor-apoptosis-associated speck-like protein containing Caspase recruitment domain (ASC) and effector protein-caspase-1 (Belkaid and Hand, 2014; Bryan et al., 2009; Compan et al., 2015) (Fig. 2). The formation of inflammasome occurs in response to diverse stimuli, for example, pathogens, environmental irritants and structurally diverse, damage-associated molecular patterns (DAMPs) and pathogenassociated molecular patterns (PAMPs) and leads to the production of activated/mature IL-1β and IL-18 cytokines (Davis et al., 2011; Rock et al., 2010). This process involves 2 steps: (i) the first one being the priming step which involves the sensing of danger signals by TLRs to activate NF-κB transcription which in turn leads to the production of

4.2. Clinical and preclinical investigations probing the role of inflammasome in visceral pain hypersensitivity (VPH) & functional gastrointestinal disorders There are different line preclinical and clinical investigation that have highlighted the critical role of inflammasome in pathogenesis of IBD and the Pain (Soubieres et al., 2015; Guo et al., 2015; Perera et al., 2018). One of the inflammasomes which is best characterizes in various gastrointestinal disorders is NLRP3. Vast number of studies have reported polymorphism of NLRP3 gene, which increase the genetic 5

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epithelium (Matikainen et al., 2016). Taken together, NLRP3 inflammasome activation likely plays an adverse role in intestine injury and inflammation. There are several studies have looked at the role of inflammasomes other than NLRP3 in GI pathology. Previously published reports investigating the role of NLRC4 in the intestinal inflammation and experimental colitis showed conflicting results. Allen et al. (2010) demonstrated that the deletion of the NLRP4 inflammasome had no effect on the severity of DSS-colitis; albeit another study stated that NLRC4 KO mice had more severe colitis with 2% DSS; however, this effect was lost with higher dose of DSS (Allen et al., 2010; Carvalho et al., 2012). These mice were seen to have a lower colonic IL-18 secretion suggestive of the protective role of this cytokine against DSS colitis. On the other hand, a study in humans showed that neonatal-onset enterocolitis was caused by gain of function mutation in NLRC4 encoding a substitution in the HD1 domain of the protein (Romberg et al., 2014). The mutation led to constitutive IL1FC production and macrophage cell death (Romberg et al., 2014). These findings are interesting; albeit need further investigations on the role of NLRC4 in intestinal inflammation. Furthermore, following oral administration of C. rodentium infection, NLRC4 KO mice displayed significant weight loss, increased bacterial load, enhanced leukocyte infiltration as compared to wild type mice (Nordlander et al., 2014). Similarly, NLRC4 KO mice on BALB/c background infected with Salmonella exhibited exacerbated while on C57BL/6 background, they showed an increased mortality due to uncontrolled bacterial replication and systemic dissemination (Franchi et al., 2012). With reference to NLRP6, its role is mainly in connection with microbes in the gut, which could be related to its location in the intestine. The mice deficient in NLRP6 show decrease IL-18 secretion but do not have disturbed microflora (Lemire et al., 2017). Another study showed that the microbiota in NLRP6-deficient mice was described as “colitogenic” because it was linked to mucosal hyperplasia, infiltration of inflammatory cells and exacerbated DSS-colitis; and when this microbiota was transferred to normal mice it caused a similar colitis (Roy et al., 2017). In line with these studies, NLRP12 KO mice also showed more severe DSS-induced colitis as compared to wild type mice (Chen et al., 2018). These mice also had altered gut microbiota which comprised of increased abundance of colitogenic strains that induce colitis and decrease in the concentration of commensal strains that protect from colitis, suggesting the role of gut microbiota in more severe DSS-induced colitis in NLRP12 KO mice (Lai et al., 2017). A recent study by Grace et al. (2016) evaluated the inflammasome activation in pain. The authors showed that morphine, an opioid, which is used as pain reliever when given to male rats after 10 days of injury led to development of chronic neuropathic pain that lasted for months even after terminating the drug. It was shown that persistence of pain was due to activation of NLRP3 inflammasome in microglial cells in dorsal horn of spinal cord. The activation of NLRP3 inflammasome was associated with the concomitant release of IL-1β. The authors further confirmed this signaling in spinal cord by the selectively inhibiting spinal microglial cells in vivo after transfection with a novel Designer Receptor Exclusively Activated by Designer Drugs (DREADD) (Grace et al., 2018). Noteworthy, treatment with the DREADD-specific ligand clozapine-N-oxide not only prevented but also reversed morphine-induced constant sensitization for weeks to months after finishing clozapine-N-oxide (Manvich et al., 2018). These data emphasize the importance of microglial cells and their role in pain cessation. These findings highlight the vital role of NLRP3 inflammasome signaling in the development of acute to chronic pain sensitivity, and potentially NLRP3-induced activation of spinal microglial cells is involved in the pain cessation (Aziz et al., 2018). Although there is no clear mechanistic study showing the role of inflammasome in GI pain yet, it is likely that the similar role of inflammasome in GI-associated pain sensation. Further studies on defining the association of inflammasome and GIrelated pain may warrant for drug development in the future. Many compounds that block the inflammasome-associated signaling

Table 1 Polymorphisms of NLRP3 gene associated with susceptibility of CD & UC. Polymorphisms of NLRP3 gene that determine genetic susceptibility Crohn's disease

Ulcerative colitis

- SNPs in NLRP3 rs10733113 - CARD15/NOD2 - C10X allele in CARD8 and Q705K allele in NLRP3 - IL-18 (rs1946518 A > C, rs360718 A > C, and rs187238 G > C)

- SNPs in NLRP3 (rs10925019, rs10754558 and rs10754558)

susceptibility to CD and UC (Deng et al., 2015). Villani et al. (2009) reported that SNPs rs10733113 in the NLRP3 gene region contributed to CD susceptibility, although no such association was seen in other study conducted by Lewis et al., 2011 using a large cohort. Consequently, a study published in 2009 in American Journal of Gastroenterology reported that Swedish men carrying both the C10X allele in CARD8, Q705K allele in NLRP3, and wild-type alleles of NOD2 showed disease susceptibility to CD (Schoultz et al., 2009). Furthermore, polymorphisms of the NLRP3 effector IL-18 (rs1946518 A > C, rs360718 A > C, and rs187238 G > C) gene were linked to an increased susceptibility to CD (Gao et al., 2015). Similar line of investigation done by another group of investigators also showed that loss of function mutation in CARD8 gene could activate NLRP3 inflammasome and contributes to the development of CD (Gao et al., 2015; Mao et al., 2018). In addition, studies have shown that polymorphism in NLRP3 genes (rs10925019, rs10754558 and rs10754558) were liked to an increased susceptibility to UC (Zhang et al., 2014). These studies suggest that polymorphisms of NLRP3 gene may affect the expression of the NLRP3 inflammasome and hence the genetic susceptibility to IBD (Table 1). Besides, published reports suggest that increased expression and secretion of proinflammatory cytokines IL-1β from colonic tissues and macrophages of patients with IBD was positively correlated with the disease's severity. In preclinical studies done by Bauer et al. (2012) determined the role of NLRP3 in intestinal inflammation and found that NLRP3 deficiency in mice (NLRP3−/−) decreases the susceptibility to both dextran sulfate sodium (DSS)-induced colitis and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis animal models compared to control groups. Another hypothesis of the protective effect of NLRP3-deficiency could be the gut microbiome: the NLRP3 deficient mice that were cohoused with wild type mice for 2 weeks lost the protection from colitis conferred to them by the deficiency of NLRP3 (i.e. these mice were as susceptible as wild-type mice), indicating that gut microbiota exchange between the two mouse strains might have played a role in increased susceptibility of NLRP3−/− mice to DSS-induced colitis. More recent study by Perera et al. (2018) showed the efficacy of a compound MCC950 in the treatment of murine Ulcerative Colitis. MCC950 is a highly specific small molecule inhibitor of canonical and non-canonical activation of NLRP3 inflammasome. Furthermore, Umiker et al. (2019) demonstrated that NLRP3 inflammasome could be the major driving factor for intestinal inflammation in Nod2 KO mice. Conversely, studies by other researchers suggest that NLRP3 inflammasome plays a protective role in colitis and also pointed to its role in maintaining gut homeostasis (Itani et al., 2016). Studies by Zaki et al. (2010) illustrated that NLRP3 deficiency in mice exhibits more severe DSS-induced colitis as compared to wild type mice. Considering the fact that NLRP3−/− mice shows extensive leukocyte infiltration, increased systemic spreading of organisms and the lamina propria, it is possible that NLRP3−/− mice may have lost epithelial barrier function. These findings point towards the protective function of NLRP3 inflammasome in colitis. Alternatively, the production the production of IL-1β and IL18 is decreased in NLRP3 deficient mice, which may further impede the repair mechanisms partly by increasing the permeability of intestinal 6

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Also, VEGF increases adhesion of leukocytes to the endothelium and chemotaxis of monocytes. VEGF activates NF-κB and causes production of many proinflammatory cytokines and chemokines (Chen et al., 2018).

pathways have been tested as therapeutic agents for IBD and experimental colitis in animal models. For instance, several published studies in THP-1 cells in a DSS colitis mouse model tested some compounds like Wogonoside, MI-2, mepazine, Alpinetin and Fraxinellone (Liu et al., 2016a). These NLRP3 inflammasome inhibitors use a common mechanism, for example, by blocking the NF-κB activation to alleviate the severity of experimental colitis and also suppress mucosal activation. Other therapeutic agents like Dimethyl fumarate, MitoQ and Asiatic acid act partly by decreasing mitochondrial reactive oxygen species (mROS) (Liu et al., 2016b). Another inhibitor Fc11a-2 considered to targeting the NLRP3 inflammasome awns evaluated for the treatment of DSS-induced experimental colitis in mice. Treatment with Fc11a-2 dose-dependently attenuated the loss of body weight and shortening of colon length induced by DSS. In addition, the disease activity index, histopathologic scores and myeloperoxidase activity were also significantly reduced by Fc11a-2 treatment (Liu et al., 2013). Other plantbased compounds of unknown mechanism which act like Fumigaclavine C, Magnesium lithospermate B, Apigenin and Astragalus polysaccharide have also been tested as NLRP3 inflammasome inhibitors (Jiang et al., 2016; Tian et al., 2017; Guo et al., 2015; Li et al., 2016). A cannabinoid receptor-2 agonist, HU 308 has also been tested in DSS-colitis mouse model (Ke et al., 2016). The compound works by promoting autophagy and blocking the activation of NLRP3 inflammasome. Interestingly, a recent study highlights the vital role of circadian rhythm disruption involving NLRP3 in pathophysiological development of IBD (Wang et al., 2018). The group showed evidence that the circadian rhythm genes is dysregulated in mice with DSS-induced colitis; targeted knock down of Rev-erbα gene, a key circadian rhythm regulator, aggravates the colitis in the Rev-erbaα−/− mice as compared to wild type mice. Administration of a synthetic drug SR009, an activator of Rev-erbα was able to activate Rev-erbα gene in mice with DSS-colitis and alleviate the severity of the disease. The authors in this detailed mechanistic study also described that Rev-erbα acts by repressing NLRP3/NF-κB axis since NLRP3 and Rev-erbα deficient mice do not show the protective effect conferred by the drug SR009. Rev-erbα was directly able to bind to NLRP3 promoter region thus serving as an attracting therapeutic target for treatment of colitis.

5.2. Clinical and preclinical investigations probing the role of VEGF in visceral pain hypersensitivity & FGIDs Several studies have highlighted the important role of VEGF in IBD and intestinal inflammation. In fact, studies showed that VEGF fosters inflammation by attracting leukocytes and cellular adhesion molecules (CAM) to the site of infection (Leick et al., 2014). One of the first studies by Schurer-Maly et al. in 1997 found that levels of VEGF were elevated in serum of IBD patients (Schurer-Maly et al., 1997). This was followed by another study from Germany in 1998 which reported significantly increased VEGF serum levels in patients with active Crohn's disease and Ulcerative colitis (Griga et al., 1998). The authors also mentioned that Peripheral Blood Mononuclear Cells (PBMCs) (Griga et al., 1999) and inflamed intestinal tissue was the source of VEGF in active IBD patients, which highlights the importance of VEGF in Crohn's disease and ulcerative colitis. It is known that VEGF and its receptor VEGFR2 play a critical role in pathogenesis of Ulcerative colitis by increasing vascular permeability and promoting the infiltration of inflammatory cells (Waldner et al., 2010). Various signaling cascades (e.g. Erk1/2, Akt, Src) carry out the effect of VEGF on endothelial cells (Fearnley et al., 2016). The study by Tolstanova et al. (2011) in a rat model of UC using 6% iodoacetamide showed the activation of Erk1/2 and Src kinase, while expression of total proteins Erk1/2 and Src was unchanged. In IL-10 KO mice model, which are known to develop spontaneous chronic colitis, the investigators showed an increase in total and activated Src proteins in IL10 KO mice compared to wild type, suggesting the VEGF/VEGFR2 axis plays a role in pathogenesis of ulcerative collitis through its signaling cascades, Akt and Src (Tolstanova et al., 2011). Another study by the same group also demonstrated that neutralizing anti-VEGF antibody markedly improved the clinical and morphologic features of ulcerative colitis in Sprague Dawley rats (Ulcerative Colitis induced with 6% iodoacetamide) (Tolstanova et al., 2009). There is no dearth of literature suggesting the role of VEGF in different gastrointestinal disorders, however, very few handful of investigations have explored the role of VEGF in visceral pain. One of the study done by Malykhina and coworkers (2012), studied the effects of VEGF on bladder function and found that upon VEGF instillation into the mouse bladder promotes a significant increase in peripheral nerve density together with alterations in bladder function and visceral sensitivity. Another study done by Lai et al. (2017) showed that Systemic blockade of VEGF signaling with anti-VEGF-neutralizing antibodies was effective in reducing pelvic/ bladder pain in the CYP cystitis model of bladder pain and warranted the further investigation of the use of anti-VEGF antibodies to manage bladder pain or visceral pain. Given the pathological role of VEGF in progression to IBD various investigators have evaluated the efficacy of anti-VEGF drugs/antibodies as therapeutic target. For example, DC101, an anti-VEGFR2 monoclonal antibody (mAb-VEGFR2) was tested in an experimental colitis animal model with promising results in weight change profile. However, overall disease severity was not affected suggesting VEGF-independent signal transduction (Knod et al., 2013). Results from another study demonstrated that the activation of VEGF-C/VEGFR3 pathway is protective in experimental colitis model and it was suggested as a potential therapeutic target for IBD (D'Alessio et al., 2014). The effects of VEGF-C were mediated by macrophages in a STAT6-dependent manner. As an example, study by Cromer et al. demonstrated that overexpression of rVEGF164b, an anti-angiogenic form of VEGF-A, has anti-inflammatory activity in a TNBS UC model, and both angiogenesis and lymphangiogenesis were also reduced their model of UC (Cromer et al., 2013). Recently, Salem and Wadie investigated the effect of niacin on VEGF in

5. VEGF 5.1. Introduction The VEGF family of glycoproteins comprises of signal proteins produced by the cells that are involved in vasculogenesis and angiogenesis. In mammals, the VEGF family comprises of five members namely VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF) (Fig. 3) (Holmes and Zachary, 2005). They bind to three types of receptor tyrosine kinases (RTKs), VEGFR1 (Flt-1), VEGFR2 (KDR/Flk1), and VEGFR3 (Flt-4), that can mediate ligand/receptor-specific signal transduction. VEGF-A binds to VEGFR1 and VEGFR2. VEGF-B and PlGF bind to VEGFR1. VEGF-C and VEGF-D bind to VEGFR2 and VEGFR3 (Holmes and Zachary, 2005). VEGF-A is a member of the VEGF family and is typically referred to as VEGF. It is also the most studied among the VEGF family members in context of IBD pathogenesis. VEGF has many isoforms that result from alternative splicing. In humans VEGF isoforms are VEGF121, VEGF165, VEGF189, and VEGF206 while murine counterparts of human VEGF are VEGF120, VEGF164, VEGF188, and VEGF205 (Park et al., 1993). VEGF signaling can be inhibited by endogenously produced soluble VEGFR-1 (sVEGFR-1/sFlt-1), which lacks an RTK and binds to VEGF (Shibuya, 2013). VEGF specific isoforms also bind to neuropilin 1 (NRP-1) and neuropilin 2 (NRP-2), which act as co-receptors that can enhance VEGFR signaling (Chidlow Jr. et al., 2007; Deban et al., 2008). VEGF plays an important role in wound repair, chronic inflammation, tumor occurrence, growth, and metastasis. The growth factor increases vascular permeability and induces EC proliferation (Ribatti et al., 1999). 7

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Fig. 3. Schematic representation of VEGF family ligands and their receptors. VEGF: vascular endothelial growth factor.

with increased functional and structural connectivity in endogenous pain facilitation regions (Witt et al., 2019). Another recent study done by Bhatt et al. (2019) investigated the brain structure and functional connectivity and its relation to pain perception in patients with IBS using the MRI technique, they found that girls with IBS had lower gray matter volume in the thalamus, caudate nucleus, nucleus accumbens, anterior midcingulate, and dorsolateral prefrontal cortex. Concurrently, they also report the correlation between the IBS patients with higher pain thresholds and lower thalamic GMV (Bhatt et al., 2019). Another more interesting study which presents the evidence for an association of gut microbiota with brain functional connectivity and gastrointestinal sensorimotor function in patients with IBS. This reported found the observed differences between controls and patients with IBS in the Lachnospiraceae incertae sedis, Clostridium XIVa, and Coprococcus subnetworks and suggested that disruptions in the brain-gut-microbiome axis in IBS patients involving mainly subcortical but also cortical brain regions may contribute to visceral hypersensitivity and altered perception of pain in patients with IBS (Labus et al., 2019)

a rat model of experimental colitis (Salem and Wadie, 2017). Intrarectal administration of iodoacetamide led to increase in the colonic levels of TNF-α, VEGF and angiostatin that were reversed by niacin. Niacin could therefore be working by inhibiting pathologic angiogenesis and inflammatory markers as a potential therapeutic drug. 6. Brain Imaging studies investigating interrelationship between visceral pain hypersensitivity & FGIDs Modern brain-imaging studies such as positron emission tomography (PET), functional MRI, single-photon emission computed tomography (SPECT) and more recently magnetoencephalography (MEG) have not only opened new avenues for noninvasive exploration of brain mechanisms but also adding to our basic knowledge about the fundamental pathogenesis of augmented VPH and FGIDs, providing practical tools for assessing the effects of pharmacological interventions on this complex process (Chopra and Arora, 2014). A growing number of reports on brain imaging results obtained in patients with chronic abdominal pain have been published (Mayer et al., 2009; Tillisch and Labus, 2011; Mayer et al., 2015; Bhatt et al., 2019). Results from two recent meta analyses of functional magnetic resonance imaging (fMRI) studies in IBS patients and healthy control subjects (HCs) using controlled rectal balloon distension as a stimulus have reported consistent activation of insular (INS) and anterior cingulate cortices (ACC) and thalamus (THAL) in both IBS patients and healthy control (Mayer et al., 2015; Sheehan et al., 2011; Tillisch et al., 2011). One of the investigation done by Icenhour et al. (2017) to understand the brain functional connectivity is associated with visceral sensitivity in women with IBS and findings suggest that visceral sensitivity in IBS is related to changes in brain functional connectivity is associated with the sensory processing. Converging lines of evidence point towards alterations in gut epithelial permeability as an important mechanism in altered gut brain communication (Kelly et al., 2015), and increased gut permeability has previously been implicated in the development of chronic VPH as a characteristic of IBS (Al-Chaer et al., 2000; Gebhart, 2004; Kelly et al., 2015). One of the recent studies done by Will and coworkers the relationship between resting state brain function and in vitro measures of gut barrier function in healthy subjects and patients with moderate to severe IBS. IBS participants with lower epithelial permeability reported increased IBS symptoms, which was associated

7. Conclusion Firstly, the role microbiota with pain manifestation and perception are altered as a consequence of the microbiota-gut-brain axis, whereby microbiota may be mediating pain response and address the potential for manipulating gastrointestinal microbiota as a therapeutic target for visceral pain. Secondly, NGF/TrkA signaling, despite its promising analgesic efficacy of anti-NGF therapy on the gastrointestinal disorders, the mechanisms underlying the analgesic effects induced by the NGFTrkA signaling are poorly understood as a knowledge gap, and yet to be discovered. Inflammasome and VEGF studies showed some promising role on defining the association of inflammasome and GI-related pain and may warrant for drug development in the future. Accumulating evidence suggests the pathological involvement microbiota NGF/TrkA, inflammasome, and VEGF in GI pathology and associated pain signaling pathways. Modulation of these pathways may emerge as a promising approach for the treatment of GI-related pathology and associated pain. Declaration of competing interest All authors have no conflict of interest to declare. 8

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Acknowledgements

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