Ghrelin Inhibits Post-Operative Adhesions via Blockage of the TGF-β Signaling Pathway

Post-operative adhesions are a critical problem in pelvic and abdominal surgery despite a multitude of studies dedicated to finding modalities to prevent their occurrence. Ghrelin administration promotes an anti-fibrotic response in a surgical mouse model of adhesion-induction, but the mechanisms mediating this effect have not been established. In the current study, the molecular mechanisms that underlie the anti-adhesion effect of ghrelin were investigated. Post-surgical adhesions were experimentally created in C57BL/6 wild-type mice via a combination of ischemic peritoneal buttons and cecal multiple abrasions. Ghrelin or saline intraperitoneal injections were given twice daily from two days before surgery to selected time points post-surgically to assess the phenotypic and molecular effects of treatment (1 day (n = 20), 4 days (n = 20) and 20 days (n = 40) after surgery). Endpoints included the scoring of adhesions and gene and protein expression analysis of pro-fibrogenic factors conducted on peritoneal ischemic tissue by quantitative PCR and Western blot. Ghrelin administration significantly reduced post-surgical adhesions and down-regulated pro-inflammatory gene and protein expression, including Tgfb3 and Tgfbr2. The up-regulation of inhibitory proteins Smad6 and Smad7 confirmed the ghrelin-induced blockage of TGF-β signaling. Ghrelin is a candidate therapeutic drug for post-operative adhesion prevention, inhibiting inflammatory responses via blockage of the TGF-β signaling pathway at the onset of surgery before the occurrence of the granulation-remodeling phase.


Introduction
After pelvic or abdominal surgery, post-surgical adhesions are formed when abnormal fibrous connective tissue is produced by extracellular matrix secretion, fibrinolysis and neo-angiogenesis. Several pathways are involved in adhesiogenesis, many of which are also involved in normal wound healing [1]. Despite the diverse strategies developed to minimize and prevent post-operative adhesions in pelvic and abdominal surgery, adhesions remain a frequent complication [2].
The pathogenesis of post-operative adhesion formation is similar after almost all types of surgery. Surgical trauma evokes an inflammatory response, promoting pro-coagulatory and impacts wound healing and peritoneal adhesions by analyzing the expression of pro-fibrogenic factors at the mRNA and protein levels at various post-operative time-points. This study indicates that the inhibition of post-operative adhesions by intraperitoneal injections of ghrelin is mediated via blockage of TGF-β pathway at the onset of surgery before the occurrence of the granulation-remodeling phase.

Animals
Male C57BL/6 mice (n = 80) were purchased from Charles River Laboratories (Wilmington, MA). All mice, 50-55 days old, were housed under standard conditions (room temperature 25-28°C, humidity 30-70% and 12 hr dark-light cycle) in Brown University Animal Care Facility. The mice were fed a standard rodent chow (Purina Rodent Chow 5001, Farmer's Exchange, Framingham, MA) and ad libitum access to filtered tap water. The study was approved by the Brown University Institutional Animal Care and Use Committee and conducted in accordance with The Guide for the Care and Use of Laboratory Animals of the National Institute of Health. All surgery was performed under anesthesia, and all efforts were made to minimize suffering.

Experimental Design
Post-surgical intraperitoneal adhesions were induced as previously described [25]. In brief, mice underwent a laparotomy to create a combination adhesion model induced by ischemic peritoneal buttons and multiple cecal abrasions. Mice were randomly divided into two treatment groups: ghrelin group (0.16 mg/kg) and control group (saline). Mice received intraperitoneal injections of saline (0.1 ml) or 0.16 mg/kg of ghrelin diluted in 0.1 ml of saline, twice daily from 2 days pre-surgery to 1 day (n = 20), 4 days (n = 20) and 20 days (n = 40) post-surgery. Mice were euthanized by isoflurane overdose and cervical dislocation.

RNA and protein extraction
The two peritoneal ischemic buttons were harvested at 1 day (ghrelin-treated mice n = 10, saline-treated mice n = 10), 4 days (ghrelin-treated mice n = 10, saline-treated mice n = 10) and 20 days (ghrelin-treated mice n = 20, saline-treated mice n = 20) post-surgery. Both the ischemic buttons were frozen in liquid nitrogen. RNA was isolated from one peritoneal ischemic button using the RNeasy Micro Kit (Qiagen, Valencia, CA) with the additional recommended DNase step provided in the kit. The total RNA was reverse transcribed to cDNA using the RT 2 first strand kit (Qiagen, Valencia, CA). The RNA were assessed based on the RIN values generated from RNA nano kit and 2100 bioanalyzer system (Agilent technologies, Santa Clara, CA). Protein lysates were prepared by homogenizing the second peritoneal button with an electric homogenizer in radioimmunoprecipitation assay buffer (RIPA buffer: 50 mM Tris-HCl (pH 8), 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) with 1% phenylmethylsulfonyl fluoride (PMSF) and 1% protease and phosphatase inhibitor cocktail (Thermo Scientific, Rockford, IL). After the samples were centrifuged for 20 minutes at 12,000 rpm, supernatants were aspirated and placed in new tubes. The protein concentration was determined for each sample using Protein Assay kit (Bio-Rad, CA).

Real-time PCR array analysis
A custom SABiosciences PCR array (Qiagen, Valencia, CA) was created incorporating 40 genes generated from a pilot study using a Mouse Fibrosis PCR Array. Each PCR plate contained an internal positive control to detect synthetic DNA (PPC) and two negative controls to detect genomic DNA contamination, a genomic DNA control (GDC) and a no reverse transcriptase control (NRT). Five housekeeping genes were chosen (Actb, B2m, Gapdh, Gusb and Hsp90ab1). Samples were prepared according to manufacturer's instructions, and each group was divided among the plates ensuring that each plate contained control and treated samples. Samples were placed onto 384-well plates using an epMotion1 5075 automated pipetting system (Eppendorf, Hauppauge, NY) and run on a ViiA 7 RT-PCR System (Life Technologies, Grand Island, NY) using manufacturer recommended cycling conditions. Threshold cycle (Ct) values were calculated from the amplification plot. The target gene Ct values were normalized to the geometric mean of all stable housekeeping genes through the ViiA tm software AB (Applied Biosystems, Life Technologies) and analyzed using the comparative Ct method (ΔΔCt method).

Real-time qPCR Primer Assay
Real-Time PCR was performed using primers specific for Tgfb3, Tgfbr2, Il4, tPA, Mapk14, Icam-1 and Vcam-1 from SA Biosciences (Qiagen, Valencia, CA). RT-PCR reactions were performed according to manufacturer's instructions using RT2 SYBR Green (SA Biosciences). All assays including no template controls were done in triplicate. One stable housekeeping gene was used as internal control (B2m). Samples were placed onto 96-well plates using an epMo-tion1 5075 automated pipetting system (Eppendorf, Hauppauge, NY) and run on a ViiA 7 Real-Time PCR System (Life Technologies, Grand Island, NY) using manufacturer recommended cycling conditions. Data were analyzed using comparative Ct method (ΔΔCt method).

Western blot
The samples were denatured and reduced with a loading buffer containing 4% anionic denaturing detergent sodium dodecyl sulfate (SDS), 20% glycerol, 0.004% bromophenol blue, 10% βmercaptoethanol and 0.125 M Tris-HCl (pH 6.8) and boiled at 95°C for 10 minutes. Proteins were separated by SDS-PAGE and electrotransferred to ImmunoBlot PVDF membrane (Bio-Rad, Hercules, CA). PVDF membranes were blocked with 5% non-fat milk for 1 hour at room temperature. The following primary antibodies were used: anti-TGFβ3 . After washing the membranes with 0.01% TBS buffer, the membranes were incubated for 1 hour at room temperature with the following secondary antibodies: HRP-conjugated anti-rabbit (#7076S Cell Signaling Technology, Boston, MA), HRP-conjugated anti-mouse (#7074S Cell Signaling Technology, Boston, MA) and HRP-conjugated anti-goat (sc-2922 Santa Cruz Biotechnology, Dallas, TX). Band intensities were quantified by densitometric analysis using Image-J software and normalized for loading. The anti-γ-tubulin antibody was used as an internal control.

Statistical analysis
All data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism software. Student's t-test was used to determine statistical difference between control and ghrelin-treated groups. Values were considered to be significant at p-value < 0.05. The time course analysis between ghrelin and saline-treated mouse groups was conducted using a two-way analysis of variance (ANOVA) followed by individual comparisons using Fisher's LSD test. Differentially expressed genes (p <0.05) were uploaded into QIAGEN's Ingenuity Pathway Analysis software (IPA, QIAGEN Redwood City) to map genes onto pathways and identify upstream and downstream interactions.

Ghrelin administration increased mouse body weight after surgery
Ghrelin administered from preoperative day 2 to 20 days after surgery significantly (p<0.05) increased the body weight in mice compared to controls (2.742 ± 0.228 g versus 2.079 ± 0.229 g, p<0.05). The ghrelin treatment-related increase of body weight in mice at 20 days post-surgery was 25% compared to controls. At 1 and 4 days after surgery mice lost weight due to decreased food intake after surgery. No significant differences in body weight gain were detected in mice treated with ghrelin compared to the saline group at 1 day (-2.790 ± 0.197 g versus -2.300 ± 0.323 g, N.S.) or 4 days (-2.400 ± 0.362 g versus -2.940 ± 0.362 g, N.S.) after surgery ( Fig 1A).

Adhesion formation was altered by ghrelin administration
Adhesion formation was measured by a blinded surgeon using a scoring system described by Mazuji MK et al, 1964 [26]. Ghrelin administered intraperitoneally twice daily from 2 preoperative days to 20 days post-surgery, significantly (p<0.0001) decreased intra-abdominal adhesions by 42.11% compared to the controls [25]. At 20 days post-surgery in the control group the adhesions were fully formed, vastly different from the saline-treated group at 1 day postsurgery. No significant differences in adhesions were detected at 1 day post-surgery between the ghrelin and saline-treated group. The adhesion scores (mean ± SEM) at 1 day and 20 days after surgery were 1.278 ± 0.088 versus 1.050 ± 0.050 (N.S.) and 2.500 ± 0.213 versus 1.447 ± 0.126 (p<0.0001), respectively (Fig 1B).

Quantitative gene expression analysis showed altered mRNA levels in ghrelin-treated ischemic buttons
To evaluate the ghrelin-induced anti-fibrotic effect in our mouse surgical adhesion model, gene expression analysis was performed to analyze a panel of 40 genes that are involved in adhesiogenesis. Gene expression profiling was conducted on peritoneal ischemic tissue isolated from mice treated with ghrelin or saline at three different time-points: 1 day, 4 days, and 20 days post-surgery.

Western blot analysis showed altered protein expression in ghrelintreated ischemic buttons
Immunoblotting was used to validate the gene expression data analysis. Since phosphorylation is a post-translational modification that regulates the activity of MAPKs, we examined the effect of ghrelin on phospho-p38 MAPK. The ischemic buttons showed increased protein levels of phospho-p38 MAPK, but this increase was attenuated by treatment with ghrelin (phospho-p38 MAPK, p = 0.0526) (Fig 4B and 4C).
Phospho-SMAD2 and phospho-SMAD3 are the activated form of SMAD2 and SMAD3. The total SMAD2 and SMAD3 protein levels remained unchanged by ghrelin treatment. Phospho-SMAD3 (p = 0.0978) protein levels were decreased in ghrelin-treated samples compared to the control at 4 days post-surgery (S2 Fig). No significant differences were detected in IL-4 ( Fig 5A and 5B   Pathway and network analysis defined by IPA revealed inhibition of TGF-β signaling pathways in ischemic buttons after chronic ghrelin administration The p-value significant up-or down-regulated genes were subsequently subjected to Ingenuity Pathway Analysis (IPA) to obtain profiles of altered signaling pathways and to identify gene interaction networks. IPA revealed that ghrelin administration promotes inhibition of TGF-β/ Smads and p38-MAPK signaling pathways (Fig 6).

Discussion
Bianchi E et al, 2015 established a new mouse surgical model for the induction of adhesions that is consistent and easily reproducible. This new surgical model showed that ghrelin administered intraperitoneally prevents post-operative adhesion formation in a GHSR-1a receptordependent manner [25]. In the present study, this surgical model was employed to define altered molecular events regulating post-operative adhesion formation after intraperitoneal administration of ghrelin. The genesis of adhesion formation is due to a trauma/injury that results in the initiation of an inflammatory response leading to the formation of a fibrin matrix [5]. During normal peritoneal healing, the fibrin bands are broken down by fibrinolysis into fibrin degradation products; alternatively, during abnormal healing, ischemia results in a reduction of fibrinolytic activity leading to adhesion formation [5]. Previous findings elucidated the importance of the peritoneal fibrinolytic system in the regulation of adhesion formation. Down-regulation of fibrinolytic activity results in increased adhesions, while up-regulation results in a reduction of adhesions [27][28][29]. Plat mRNA expression was significantly reduced in ghrelin-treated samples at 1 day, resulting in a down-regulation of the fibrinolytic activity. Thus, the mechanism by which ghrelin decreases adhesion formation is not associated with the activation of the fibrinolytic pathway. Additionally, significant upregulation of the matrix metalloproteinases mRNA levels in ghrelin-treated samples compared to the controls was not detected (S1 Fig). Since alterations in fibrinolysis did not explain the ghrelin-induced amelioration of adhesions, the role of inflammation was investigated by measuring the mRNA expression levels of several interleukins and cytokines that are implicated in the initial phase of the inflammatory process. The ability of ghrelin in alleviating post-operative adhesiogenesis may be associated with effects on these initiators of inflammation and a resulting down-regulation of Tgfb3 mRNA and protein levels (Fig 6). Expression of all three TGF-β isoforms increase after peritoneal injury at different times. TGF-β3 protein levels were minimally expressed after surgery increasing with the formation of adhesions. Exogenous addition of TGF-β3 at the time of surgery significantly increased adhesion formation in the mouse [13].
Our findings showed that TGFbr2 mRNA and protein levels were also decreased in ghrelintreated peritoneal ischemic tissue compared to the controls. This ghrelin-induced decreased expression of TGF-βR2 and TGF-β3 may attenuate TGF-βR1 pathway activation, resulting in the inhibition of TGF-β activity. Canonical and non-canonical TGF-β pathways are involved in the activation of myofibroblasts and extracellular matrix synthesis and deposition [30]. TGF-βR2 is required for intracellular signal transduction through the formation of a heterotetrameric complex with TGFβ-R1 (Fig 6). The TGFβ-R2 receptor is a constitutively active kinase whereas the TGF-βR1 needs to be activated by the TGFβ-R2. After ligand-induced formation of the heteromeric complex, TGFβ-R2 phosphorylates TGF-βR1 at glycine and serine/threonine residues. This phosphorylation event changes the conformation of the TGF-βR1 receptor, thereby activating its kinase. The activated TGF-βR1 receptor propagates the intracellular signal by phosphorylating specific proteins (SMADs) (Fig 6) [31].
Studies in both humans and animal models are supportive of these findings, in which the overproduction of TGF-β was correlated with pathological tissue fibrosis and adhesion formation [13]. Similarly, inhibition of TGF-β activity reduces adhesion formation [13,32]. The ghrelininduced blockage of TGF-β activity was also confirmed by up-regulation of Smad7 at 4 days post-surgery in our surgical mouse model. Smad7 is an inhibitor of TGF-β signaling by preventing Smad3 and/or Smad2 phosphorylation and recruitment of Smad2/Smad3 complex (Fig 6). Smad7 protects against TGF-β induced fibrosis in several organs [33][34][35]. Ghrelin-induced upregulation of Smad3 gene expression at 4 days post-surgery may be a compensatory effect generated by the inhibitory effect of Smad6 and Smad7 on TGF-β signaling. To examine the TGF-β downstream signaling molecules mediating the effect of ghrelin, western blot analysis was conducted to quantify the phospho-SMAD2 and phospho-SMAD3 protein levels on the total levels of SMAD2 and SMAD3 at 4 days post-surgery. In comparison to the control samples, ghrelintreated samples were suggestive of an attenuation of phospho-SMAD3 protein levels relative to the total SMAD3 proteins levels at 4 days post-surgery (S2 Fig). TGF-β may also work through an alternative non-Smad signaling pathway to convey the same invasive fibrinogenic signals [36]. Tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGF-β associated kinase I (TAK1) are crucial for the activation of MAPK signaling [37].
The current study revealed that ghrelin administration significantly decreases Tnf, Mapk14 (p38α) and Myc mRNA levels and increases Smad6 mRNA levels. These results suggest that the ghrelin anti-adhesion effect is also mediated by down-regulation of p38-MAPK signaling pathway (Fig 6). c-Myc is a downstream target of p38α and it is known to be involved in inflammation [38] and apoptosis [39]. p38-MAPK signaling is a compensatory pathway that amplifies the response to TGF-β via a non-canonical pathway in case of reduced expression of TGF-β receptors [40]. Jung S.M. et al. 2013, showed that Smad6 negatively regulates the activation of the noncanonical TGF-β pathway through recruitment of the de-ubiquitinase A20 to TRAF6 [41]. The inhibition of p38-MAPK results in a reduction of alpha-SMA expression which is a key factor involved in the differentiation of fibroblasts to a contractile myofibroblastic phenotype [42].
Il-4 mRNA expression was significantly decreased at 4 and 20 days post-surgery in ghrelintreated peritoneal ischemic buttons compared to controls. IL-4-activated M2 macrophages are involved in the peritoneal fibrotic process with peritoneal M2 macrophages acting as potential targets for the interventional therapy of peritoneal fibrosis. The IL-4 cytokine promotes alternative activation of macrophages into the M2 phenotype and inhibits classical activation of macrophages into the M1 phenotype. An increase in M2 macrophages, also known as repair macrophages, is coupled with the secretion of TGF-β. The release of arginase, proline, polyaminases and TGF-β by the activated M2 cells is associated with peritoneal fibrosis [43].
In addition, IL13RA2 mRNA was significantly decreased at 4 days post-surgery in ghrelintreated peritoneal ischemic buttons compared to controls, suggesting that the anti-fibrogenic response of ghrelin is potentiated by down-regulation of IL13RA2 signaling upstream. IL-13 induces monocytes and macrophages to produce TGF-β1, a potent regulator of extracellular matrix deposition and tissue remodeling [44]. Fichtner-Feigl S et al. 2006, suggested that Il13RA2 signaling is an important therapeutic target for the prevention of fibrosis [45].
Adhesion formation is associated with an increase of local inflammatory mediators, including ICAM-1 and VCAM-1 [46]. The present study shows that ghrelin significantly decreases Icam-1 mRNA levels in peritoneal ischemic tissue at 4 and 20 days post-surgery. The first step in adhesion formation is the initiation of a local inflammatory response at the site of injury via expression of ICAM-1 and VCAM-1 by mesothelial cells lining the peritoneal cavity to facilitate leukocyte migration through the wall of the blood vessel [5].
In conclusion, these experiments suggest that ghrelin inhibits the TGF-β/Smads and p-38 MAPK signaling pathways activated during the inflammatory response at the onset of injury before the granulation-remodeling phase occurs. We provide evidence that ghrelin reduces upstream collagen deposition and myofibroblastic differentiation via down-regulation of proinflammatory factors, TGFβ3, TGFβR2 and up-regulation of inhibitor proteins such as Smad7 and Smad6. Therefore, the attenuation of extracellular matrix component production and differentiation of fibroblasts into myofibroblasts via inhibition of the inflammatory process at the onset of injury rather than up-regulation of fibrinolytic activity may be a viable therapeutic strategy for the prevention of post-surgical adhesions.