The ghrelin agonist, HM01 activates central vagal and enteric cholinergic neurons and reverses gastric inflammatory and ileus responses in rats

Electrical vagal stimulation alleviates abdominal surgery (AS)‐induced intestinal inflammation. Ghrelin receptors (GHS‐Rs) are expressed in the brain and peripheral tissues. We investigated the influence of HM01, an orally active ghrelin agonist crossing the blood–brain barrier, on AS‐induced gastric inflammation and emptying (GE) in rats.


| INTRODUC TI ON
In clinical setting, abdominal surgery (AS) is characterized by the suppression of propulsive motor function throughout the gastrointestinal tract including the delayed gastric emptying associated with nausea, vomiting, and the inability to tolerate food. 1 These functional alterations have a significant impact on the duration of hospitalization and associated with an economic burden. 2,3 Abdominal surgery consisting of laparotomy and manipulation of the gut performed in laboratory animals is well-established to investigate the mechanisms and treatments of postoperative gastrointestinal motor alterations. 4 In particular, experimental studies demonstrated that intestinal inflammation triggered by AS is an important underlying mechanism of postoperative ileus that has clinical relevance. 5,6 However, the gut inflammation induced by AS and its role in the treatment of the ileus have been studied so far primarily in the intestine [7][8][9] while it has been less investigated in the stomach. 10 Recently, we reported that AS activates M1 macrophages and increases proinflammatory cytokines expression and inflammation in the gastric submucosa plus muscle layers that are correlated with the delayed gastric emptying in rats. 11 The vagus nerve plays a key role in the cholinergic antiinflammatory reflex pathway 12 and electrical vagal activation dampened intestinal inflammation and postoperative ileus. 13,14 The stomach is richly innervated by vagus nerve fibers originating from neurons in the dorsal motor nucleus of the vagus (DMN). 15,16 Our previous studies also established that DMN activation induced by intracisternal injection of thyrotropin-releasing hormone (TRH) or the stable TRH agonist, RX-77368 dampened post-operative gastric ileus and abolished the expression of pro-inflammatory cytokines in the rat stomach 6 h after AS. 11,17 Ghrelin is a gut pleiotropic peptide influencing a wide range of biological processes including gastrointestinal motor and immune functions. [18][19][20] The ghrelin peptide interacts with the ghrelin receptor, a G-protein-coupled receptor also known as growth hormone secretagogue or GHS-R1a. 21 Nucleotide sequence analysis also revealed a truncated isoform GHS-R1b lacking transmembrane domains 6 and 7 apparently derived from the same gene, which has no affinity to ghrelin. 22 Transfected cell experiments showed that GHS-R1b oligomerized with GHS-R1a exerting a transcription-dependent modulatory role on GHS-R1a ability to signal. 22 Ghrelin receptors are localized in the rodent brainstem, vagus and gastrointestinal tract [23][24][25] and peripheral administration of ghrelin and ghrelin agonists have been reported to alleviate post-operative gastric ileus. [26][27][28][29] However, the central and/or peripheral sites through which ghrelin agonists prevent gastric stasis induced by AS need further investigations. 30 In particular, it is still unknown whether the ghrelinmediated beneficial effect on gastric post-operative ileus is associated with an anti-inflammatory effect in the stomach.
In the present study, we investigated the influence of the orally active and brain penetrant ghrelin agonist, HM01 30,31 on gastric ileus and the inflammatory response occurring 6 h after AS in rats. 11 To get insight on the central and/or peripheral sites of actions, we examined whether the peripheral administration of HM01 activates neurons in the DMN and/or gastric myenteric ganglia using Fos immunohistochemistry and double immunostaining with antibodies specifically against the peripheral isoform of acetylcholine transferase (pChAT) or common ChAT (cChAT) to mark enteric and central cholinergic neurons respectively. 32 Next, we determined whether the pretreatment with hexamethonium, to block nicotinic transmission, influenced the Fos induction by HM01 in neurons of the DMN and gastric myenteric plexus (MP). Lastly, we examined the mRNA expression levels of GHS-R1a and the truncated isoform GHS-R1b in the brain medulla and gastric corpus (mucosa vs submucosa plus muscle layers) to provide neuroanatomical support for direct neuronal activation at these sites by peripheral administration of HM01.

| Animals
Male Sprague-Dawley rats (Harlan Laboratory, San Diego, CA) weighing 280-310 g were housed under controlled conditions of temperature (22-24°C) and light (from 6:00 AM to 6:00 PM) for at least 1 week before the experiments. Animals had free access to

Key Points
• Oral administration of HM01 (6 mg/kg), an orally active and blood-brain barrier-crossing ghrelin agonist, normalized the delayed gastric emptying occurring 6 h after post abdominal surgery (AS) in rats. This is correlated with a gastric anti-inflammatory action shown by the rise of IL-10 and the suppression of AS-induced rise of pro-inflammatory cytokine expression in the rat stomach.
• Oral or intraperitoneal (ip) HM01 increased Fos expression in 52% and 55% of cholinergic neurons in the dorsal motor nucleus of the vagus (DMN) and gastric myenteric plexus (MP), respectively.
• HM01-induced Fos was not altered by ip nicotinic blockade in the DMN and reduced partially in the MP.
• GHS-R1a mRNA level was higher than that of the truncated GHS-R1b form in the brain medulla and in the gastric submucosa plus muscle layers than in the mucosa.
• These findings indicate that peripheral administration of a brain penetrant ghrelin agonist activates DMN and myenteric cholinergic pathways that may have translational application to improve inflammatory-related alterations of gastric emptying.

| Abdominal surgery
Abdominal surgery was performed as in previous studies 11,33 under isoflurane anesthesia. The abdomen was shaved and the area was wiped with 70% alcohol (Fisher Scientific, USA) followed by topical povidone-iodine antiseptic (Dynarex Mfg., Orangeburg, NY, Part # 36532301). After a median laparotomy (3-4 cm), the cecum was exteriorized, placed in saline-soaked gauze and gently manipulated between two fingers for 3 min. Then, the small intestine was exteriorized and ran throughout its entire length

| Gastric and brainstem preparations
The gastric corpus was dissected and rinsed with phosphate- Co.). Then, the gastric anterior wall was rinsed and dissected under a surgical microscope, scraping off the mucosa and removing the circular muscle to obtain the whole mount LMMP preparation as described previously. 32 To avoid regional differences, ∼0.5 cm 2 of preparation was dissected from the middle portion of the anterior part (∼0.4 mm from the boundary between the corpus and the antrum, 0.7 mm from the lesser curvature, and 1.0 mm from the greater curvature). The brain medulla was collected from the transverse sulcus to the occipital foramen, snapped frozen in dry ice and stored in −80°C until processing. Tissue samples were placed in the cryostat at −20°C for 1-h and then cut in 30μm-thick serial coronal sections using the following coordinates from the bregma (−14.08 to −13.68 mm) according to the Paxinos and Watson's rat atlas. 35

| Fos Immunohistochemistry
Fos Immunohistochemistry was performed as described previously. 32,36 Briefly, in experiment 2. Unilateral cell count was performed based on our previous studies in which we demonstrated no hemispheric differences. 37 The number of Fos + neurons in each nucleus or area was determined in a field of 100 μm × 100 μm using a 10× objective with a grid in the ocular of the microscope. The mean number from each animal was used to calculate the group mean.

| Gastric emptying of a non-nutrient meal
The gastric emptying of a non-nutrient viscous solution was determined by the phenol red/methyl cellulose method as used in our previous studies 11 and by others. 38  In the posterior wall of gastric corpus, the mucosa was removed and the submucosa plus muscle layers was snapped frozen on dry ice and stored at −80°C until processed for RT-qPCR as detailed in our previous studies. 11 Total RNA was extracted using RNA-Bee (TEL-TEST) according to manufacturer's recommended protocol and then followed by DNase-I treatment. cDNA was synthesized in a total volume of 20 μL reaction by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA).
RT-qPCR for rat IL-1 , TNF-, and IL10 was performed in duplicates using StepOnePlus Real-Time PCR System (Applied Biosystems) in a 25 μL reaction volume. The housekeeping gene, rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was also analyzed as an internal control. Selected forward and reverse primers are listed in Table S1. The specificity of the amplification reaction was determined by performing a melting curve analysis of the PCR fragments.
Each target gene was normalized with GAPDH and calculated using the comparative ∆∆Ct method. Results were calculated by the equation 2 −∆∆Ct and expressed as fold change in reference to the control group as previously described. 39

| Reverse transcription polymerase chain reaction (RT-PCR) of ghrelin receptors in the gastric corpus and medullar oblonga
The gastric corpus and medullar oblonga (from the transverse sulcus to occipital foramen) were collected from 3 naïve (nonfasted) rats that were euthanized by decapitation. Gastric samples separated into mucosa and submucosa plus muscle layers and brainstem specimen were immediately frozen and stored at −80°C until processing. The RNA extraction and the assessment of quality, amount of RNA samples and total RNA reversed transcription were performed as detailed previously. 40,41 The primers specific for rat full length GHS-R1a and 1b coding sequences (Table S1) were designed to amplify GHS-R1a and 1b transcripts. PCR for acidic ribosomal protein (ARP), a housekeeping gene, served as an internal control to assess RNA quality and cDNA normalization.
All PCR products were separated by 1% agarose gel electrophoresis, visualized with ethidium bromide and extracted with QIAquick gel extraction kit (Qiagen) and sequenced to confirm their identities. Gel images acquired by Kodak EDAS 290 system were processed for densitometric analysis with NIH image software (Scion, Frederick, MD).

| Transcripts of ghrelin receptors in the brainstem and gastric corpus
GHS-R1a and GHS-R1b transcripts detected using the full-length primers (Table S1)

| DISCUSS ION
The present study showed that the increased expression of gastric inflammatory cytokines and gastric ileus occurring 6-h after abdominal surgery in rats was blocked by the oral administration of the of the ghrelin agonist with ghrelin receptors on DMN neurons is supported by our initial report that HM01 given orally has high brain penetrance shown by the brain/plasma ratio of 0.73 and 0.80 at 2 and 4 h post administration respectively in rats 31 that was further confirmed. 30 We also showed that HM01 displays high binding affinity to the active form of human GHS-R1a (Ki 1.42 ± 0.36 nM). 31 Rat GHS-R1a consists of 364 amino acid residues and displays 96.1% similarity with human GHS-R1a homologue. 47 We demonstrated that GHS-R1a mRNA level was 5.4-fold higher than the truncated GHS-R1b splice variant in the rat brainstem using the full length premiers coding for the receptors. In situ hybridization histochemistry study with a cRNA probe specific for GHS-R1a showed its ex- It is to note that our study pointing to the activation of DMN vagal gastric cholinergic anti-inflammatory pathways 12,13 in peripheral HM01-induced prevention of AS-induced gastric ileus, is mainly correlative and additional mechanisms contributing to HM01 anti-inflammatory effect need to be further investigated. Namely the role of the sympathetic pathway in the antiinflammatory response cannot be ruled out. 52,53 There is evidence of ghrelin receptor expression on autonomic preganglionic neurons of the intermediolateral cell columns 54 and the activation of sympathetic pathways has anti-inflammatory effect on the intestine in model of colitis. 55,56 Additionally, a direct anti-inflammatory effect on gut monocytes/macrophages cannot be ruled out.
However, in previous studies, ghrelin-induced downregulation of proinflammatory cytokines in sepsis was reported to be mediated through vagal pathways. 57 This was established based on the lack of ghrelin direct action on lipopolysaccharide-stimulated Kupffer cells or peritoneal macrophages while ghrelin anti-inflammatory action was blocked by vagotomy 57 which can be attributed to the involvements of the vagal efferents, but also to the lesion of the vagal afferents.
Growing evidence in transfected cell lines showed that the dimerization of GHS-R1b with GHS-R1a did not change the affinity of GHS-R1a for ghrelin, but completely blocked both ghrelin dependent and constitutive GHS-R1a intracellular signaling. 58 We found transcripts of GHS-R1b less prominently than those of GHS-R1a in the brainstem and gastric submucosa plus muscle layers. Whether such dual differential mRNA expression has functional relevance cannot be ascertained from the present study.
In summary, we demonstrated that peripheral administration of Other reports indicate that the oral administration of HM01 improved the delayed gastric emptying in the early phase of abdominal surgery 30 and by the standard L-dopa/carbidopa Parkinson's related medication in rats. 31 HM01 also suppressed emesis evoked by motion in musk shrew. 59,60 Collectively these data suggest that HM01, an orally active and brain penetrant ghrelin agonist, may have trans- We are grateful to Dr. Claudio Pietra (Helsinn Healthcare, Lugano, Switzerland) for the generous supply of HM01 and thank Ms.
Honghui Liang for her excellent technical assistance.

CO N FLI C T O F I NTE R E S T
All the authors have no competing interest.