2881

Regular paper

The endogenous ligand for guanylate cyclase-C activation reliefs intestinal inflammation in the DSS colitis model

Danfeng Lan1,2#, Yunling Wen2#, Xiangqian Dong2#, Qin Yang3#, Yan Liu2, Kunhua Wang4, Hongying Li2✉ and Yinglei Miao2✉

1Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan, 650500, China; 2Department of Gastroenterology, The First Affiliated Hospital of Kunming Medical University, Yunnan Institute of Digestive Disease, Kunming, Yunnan, P.R. China; 3Department of Critical Care Medicine, The First Peoples’ Hospital of Kunming, Kunming, Yunnan, P.R. China; 4Department of General Surgery, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, P.R. China

Ul cerative colitis (UC) is a major type of inflammatory bowel disease (IBD) and significantly impacts patient quality of life. Pr evious research revealed that the guanylate cyclase-C (GC-C) signaling pathway is associated with the severity of UC. We ai med to investigate the effect of the GC -C agonist, guanylin (Gn), on inflammatory injury in mice with colitis. An experimental UC model was established in Ba lb/c mice. Mesalamine served as a positive control. Th e Gn overexpression vector was administered once per day for 1 week. In testinal permeability of the mice was measured using fl uorescein isothiocyanate-dextran after the treatment. Hi s topathologic grading was estimated to assess the inflammatory injury of the colon. The expression level of crucial mediators of the GC-C signaling pathway (Gn, Ugn and GC-C) and tight junction proteins (occludin, claudin-1 and ZO-1) was measured in the colon. Additionally, the level of pro-inflammatory cytokines (IL-8 and TNF-α) in serum was measured. A fter injecting the UC mice with the Gn overexpression vector, the body weight increased, and the frequency of loose stools and bloody stools was decreased. Intestinal permeability and histopathologic score were significantly reduced (P<0.05). The expression level of GC-C, Gn, Ugn, claudin-1 and ZO-1 was significantly increased (P<0.05). The level of IL-8 and TNF-α in the serum was significantly decreased (P<0.01). Therefore, the application of Gn overexpression vector can ameliorate the intestinal inflammatory injury and repair the mucosal barrier in colitis mice, which further suggests the clinical therapeutic potential of GC-C agonists in IBD.

Key words: Gu anylate cyclase-C; Ulcerative colitis; Le ntiviral-mediated; Guanylin; Intestinal mucosal barrier

Received: 12 October, 2019; revised: 20 April, 2020; accepted:
05 May, 2020; available on-line: 07 September, 2020

✉e-mail: miaoyinglei@yeah.net (YM); licongcong6912@163.com (HL)

#Co-first authors: Danfeng Lan, Yunling Wen, Xiangqian Dong, Qin Yang

Acknowledgements of Financial Support: This work was supported by the National Natural Science Foundation of China (81860105, U1802282, 81670501); Applied Ba sic Research Projects of Yunnan Province (2019FE001); Yunnan Health Training Project of High Level Talents (H-2018041).

Abbreviations: cGMP, cyclic-guanosine-3’,5’-monophosphate; DSS, dextran sulfate sodium; IBD, inflammatory bowel disease; GC-C, guanylate cyclase-C; Gn, guanylin; STa, heat-stable enterotoxins; TJPs, Intestinal epithelial tight junction proteins; TNBS, trinitrobenzene sulfonic; UC, Ul cerative colitis; Ug, uroguanylin

Int roduction

Ulcerative colitis (UC) belongs to a group of chronic idiopathic inflammatory disorders that primarily affect the colon and is a major type of inflammatory bowel disease (IBD). IBD significantly impacts patients’ quality of life due to its repeated remissions and relapses. While the etiology of these diseases is still not well established, one of the contributing factors may be a poorly regulated immune response against the enteric microbiota in genetically predisposed individuals (Xa vier & Podolsky, 2007). The number of IBD patients has been increasing worldwide (Qua resma et al., 2019). Medical therapies for symptomatic relief of IBD include sulf asalazine, antibiotics, corticosteroids, azathioprine, biol ogic agents and so on. Although the treatment outcome improved with the use of biol ogic agents, a certain proportion of patients are resistant to the pharmacological therapy and suffer severe side effects (Ord ás et al., 2012; Engel et al., 2010). Therefore, there is a need to identify novel pharmacological targets that act locally at the site of inflammation to maximize the efficacy and minimize the systemic side effects of IBD treatment.

Intestinal epithelial tight junction proteins (TJPs) act in the intestinal epithelium to form a tight muco sal mechanical barrier. Maintenance of the intestinal epithelial cell barrier function is considered to be crucial for the host immune defense. Thus, a dysfunctional barrier and increased gut permeability might be the crucial pathophysiological factors underlying the etiology of IBD. Recent studies suggested that the guanylate cyclase-C (GC-C) signaling pathway is involved in the maintenance of a healthy intestinal barrier, anti-inflammatory effects, control of epithelial proliferation and tumorigenesis, and decrease of visceral pain sensation (Camilleri, 2015; Uranga et al., 2018). GC-C is a transmembrane enzyme expressed primarily on the intestinal epithelial cells (IECs) and serves as the receptor for the peptides guanylin (Gn) and uroguanylin (Ugn), as well as the receptor for heat-stable enterotoxins (STa) produced by enterotoxigenic Escherichia coli (Brierley, 2012). The endogenous ligands Gn, Ugn and the exogenous ligands STa of E. coli are structurally related peptides that activate the GC-C receptor, which is selectively expressed on the brush border membranes of enterocytes from the duodenum to the rectum (Forte, 1999). The biologically functional Gn, Ugn peptides are produced in situ in the goblet cells and enterochromaffin-like cells (Li et al., 1995; Perkins et al., 1997). Gn is most abundant in the large intestine and distal small intestine, while Ugn expression is higher in stomach and duodenum (Joo et al., 1998). Ligand-induced GC-C activation leads to intracellular elevation of cyclic-guanosine-3’,5’-monophosphate (cGMP). This physiological activation of GC-C regulates intestinal fluid and electrolyte homeostasis, preventing dehydration and intestinal obstruction (Basu, 2010).

Previ ous research showed that the guanylate cyclase-C signaling pathway was down-regulated in IBD and mice with 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis (Brenna et al., 2015). Consistent with this study, our study found that the expression level of GC-C, Gn and Ugn in the colonic mucosa of UC patients was significantly decreased compared to normal controls, and this decrease was more significant with increased disease activity, which suggests that the activity of GC-C signaling is reduced in UC patients and negatively correlates with the clinical severity of UC (Lan et al., 2016). In vitro studies revealed that the activity of GC-C signaling pathway can be restored by GC-C agonists. Activation of the GC-C signaling pathway ameliorated the impairment of barrier function and inflammatory injury of Caco-2 cells induced by interleukin-1β (Wang et al., 2016). Shailu bhai and others (Shailubhai et al., 2015) report ed that orally administered GC-C a gonists plecanatide and dolcanatide, the structural analogs of Ugn, could reduce intestinal inflammatory injury induced by chemical dextran sulfate sodium (DSS) or trinitrobenzene sulfonic (TNBS) in mice (Shailubhai et al., 2015). Chang and others (Chang et al., 2017) showed that orally administered plecanatide reduced the degree of inflammation-driven colonic dysplasia in mice (Chang et al., 2017). In accordance with these findings, Boulete and others (Boule te et al., 2018) indicated that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions (Boule te et al., 2018). Recent studies reported that GC-C agonists plecanatide and linaclotide can be used in the treatment of chronic idiopathic constipation (Lembo et al., 2011; Islam et al., 2018). As we know, diarrhea is the most common manifestation of UC. In this study, we focused on the lentiviral-mediated Gn expression and performed the experiment in DSS-in duced UC mouse model using the Gn overexpression vector, to determine the potential therapeutic value of GC-C agonist in UC. This in vivo study provided powerful experimental evidence for further clinical trials and exploring more efficacious treatments in IBD patients.

Materi al and Methods

Animal studies. Sixty male Balb/c mice weighing 18–22 g were obtained from the Animal Center of Kunming Medical University. All experiments were performed with the approval of the Animal Experimentation Committee of Kunming Medical University and in accordance with the institutional regulations. Mice w ere divided into 5 groups and the number of mice in each experimental group was 12. Contro l group: after normal drinking water for one week, 0.2 mL 0.9% normal saline (NS) was administered by gavage (1 time/day) and 50 µL of contro l vector without Gn (1.776×107 copies/mL) was administered by caudal vein injection (1 time/day) for one week. DSS+NS group: after drinking water with 3% DSS for one week, 0.2 mL 0.9% NS was administered by gavage (1 time/day) for one week. DSS+Me sal a mine group: after drinking water with 3% DSS for one week, 0.2 mL 30 mg/kg mesalamine suspension was administered by gavage (1 time/day) for one week. DSS+Gn group: after drinking water with 3% DSS for one week, 50 µL soluti on of Gn overexpression vector (1.776×107 copies/mL) was administered by caudal vein injection (1 time/day) for one week. DSS+ M esalamine+Gn group: after drinking water with 3% DSS for one week, 0.2 mL 30 mg/kg mesalamine suspension was administered by gavage (1 time/day) for one week. Concurrently, 50 µL of Gn overexpression vector (1.776×107 copies/mL) was administered by caudal vein injection (1 time/day) for one week.

Induction of colitis using DSS. The UC mouse model was induced by feeding mice 3% DSS (Sigma, United States) dissolved in UV-sterilized tap water for seven days. On day seven, all animals were returned to plain water. The successful establishment of the colitis model was indicated by the presence of any of the following symptoms: (1) loose stool, (2) diarrhea, (3) fecal occult blood positive, or (4) rectal bleeding. After DSS administration, the indicators including eating , activity, body weight, stool consistency, and rectal bleeding, were recorded from the first day of the experiment.

Constr uction of the recombinant lentivirus plasmid for Gn overexpression. The l e ntiviral expression vectors for Gn protein overexpression and the control vectors without Gn were purchased from GeneCo poeia (Guangzhou, China). The plasmids were EX-Mm0 2903-Lv109 and EX-NEG-Lv109, and their concentrations were 0.939 µg/µL and 0.964 µg/µL, respectively. First, 293Ta cells were thawed and su bcultured until they reached a density of 90% and inoculated into a 10 cm cell culture plate. When the cells grew to a density of 80%, the plasmid was transfected into the 293Ta cells to package the lentivirus. Afterwards, the lentivirus concentration was measured and the ti ter was determined. The viral RNA was extracted first, and then DNAse I was used for processing. The qPCR method was used to detect the ti ter of the plasmid lentivirus after the reverse transcription reaction. The titer was 1.776×107 copies/mL.

Intest inal permeability assay. On the 14th experimental day, at the end of the treatment, the intestinal permeability of mice was measured to assess the barrier function using the fluore scein isothiocyanate-dextran (FITC-D) method. Firstl y, mice were anesthetized with ketamine at 8 mg/100 g before the experiment. Then the abdominal wall was incised, the colon was separated 4 cm away, and both ends of the intestinal canal were clipped with an artery clip covered with a rubber sleeve to prevent the release of the fluorescent compound. 1 mL PBS solution containing 25 mg FITC-D (MW 4000, FD-4, Sigma) was injected into the intestinal lumen. After 2 h, 1 mL b lood was collected from the po rtal vein of the mice, diluted in 50 mM Tris solution and centrifuged at 1000 rpm for 7 min. Then, the supernatant was discarded and the remainders were placed into a 96-well plate specially designed for use in a fluorescence spectrophotometer. The concentration of FD-4 in the samples was detected using a fluore scence spectrophotometer (22331, Millipore, USA); the fluorescence intensity was measured (excitation 480 nm; emission 520 nm), and FD-4 concentrations were determined using standard curves generated by serial dilution of FD-4 (Nagy et al., 1989). Permeability was calculated by the linear regression analysis of the sample fluorescence.

Colon histopathologic grading. On the 15th day of the experiment, the mi ce were sacrificed by cervical dislocation under anesthesia, the peripheral blood was collected by the eyeball extraction, and the colon tissue was collected. In bri ef, the co lon tissue was washed with PBS and soaked in 4% paraformaldehyde for 48 h, the fixed tissue was soaked in alcohol to dehydrate, and the dehydrated tissue was put in dimethylbenzene. The tissue was soaked and permeated in paraffin, and the paraffin-embedded tissue was sliced into about 5-µm-th ick slices. The tissue was baked at 60°C in an incubator for 30 min. Then the tissue was hydrated, stained with hematoxylin at 37°C for 5 min, and stained with eosin for 3 min. Then it was washed, dehydrated, sealed and taken a picture. The da mage of colon tissue was assessed using microscopy by two pathologists in a blinded manner. The inflammation was graded as follows: severity of the epithelial damage (0–2: none, erosion, ulceration), depth of the ulcer (0–3: n o ne, submucosal, muscular is, serosa), edema (0 –3: none, slight, moderate, severe), infiltra tion of the inflammatory cells (0–3: none, slight, moderate, severe), the extent of the infiltration (0–3: none, submucosal, muscularis, serosa).

Immunohi stochemistry. Firstly, the tissue section was prepared using a slicer, the thickness of each piece was 5 µm. The slices w ere placed in xylene I and II to dewax for 10 min each, and soaked in anhydrous ethanol I and II for 2 min each. Then, they were rinsed with distilled water and the firs t antibody was added. The glass slides were put in a wet box and placed in a 4°C refrigerator overnight. After rinsing with PBS buffer, the slices we re added with the second antibodies respectively, including rabbit polyclonal anti-Gn antibody (1:200, Ab cam, USA), goat polyclonal anti-Ugn antibody (1:200, Santa Cruz Biotechnology, USA), mouse monoclonal anti-GC-C antibody (1:250, Si gma, USA), rabbit mo noclonal anti-occludin antibody (1:100, Gene Tex, USA), rabbit monoclonal anti-claudin-1 antibody (1:200, G ene Tex, USA), and rabbit monoclonal anti-ZO-1 antibody (1:250, Millipore, USA). Then, the DAB chromogen solution was added to terminate the staining. Finally, the experimental results were observed under an optical microscope, and the brown or brown-yellow particles in the cell membrane or cytoplasm were considered as the protein expression. Four fields of each section were randomly selected under a microscope with a magnification of 200 times, and HPIAS-2000 image analysis software was used for grayscale analysis. The immunohistochemical (IHC) scores were assigned by the percentage of positive cells and the staining intensity in each section. The percentage of positive cells was graded as follows: negative (0 points), 1–10% (1 point), 11–50% (2 points), 51–80% (3 points), 81–100% (4 points). The staining intensity was graded as follows: negative (0 points), weak positive staining (1 point), moderate positive staining (2 points), strong positive staining (3 points). IHC scores were the product of the above two values.

Enzyme-linked immunosorbent assay (ELISA) of the murine serum. The peripheral blood serum of the mice was diluted with the assay diluent. The level s of cytokines (IL-8 and TNF-α) in serum were measured with commercially available ELISA kits (RayBiotech, Minneapolis, MN, USA) according to the manufacturers’ instructions.

Statistics. Experiments were performed at least three times. Data were expressed as the mean ± SD, and normality was assessed using the Kolmogorov-Smirnov test. Variables with a non-normal distribution were logarithmically transformed before statistical analysis. Independen t sample t-test was used to compare the means between two groups, while the data among multiple groups were analyzed using one-way analysis of variance (ANOVA) followed by “LSD post hoc” test. Statistical significance was defined as a P-value<0.05. All statistical analyses were performed using SPSS 17.0 software.

Results

Changes in the general state and colon morphology of the mice

The contro l gr oup mice had f requent activity, active foraging, and formed sto ols. Each mouse had gained weight by the end of the experiment. The mice i n the DSS+NS gro up had less activity, eating and more loose stools. These mice lost weight by the end of the experiment. The mice of each treatment group were characterized by a better state, eating, and activity; their stools returned to normal gradually following the 10th day of the experiment, and their weight was higher. The body weight change relative to the beginning of the experiment was calculated from day 1 to day 14. DSS caused a reduction in body weight gain, the DSS+NS group mice were relatively smaller at the beginning of the treatment due to DSS administration during the previous week, while mesalamine or Gn treatment significantly alleviated this body weight loss (Fig. 1). There was not significant diarrhea observed in mice receiving treatment with Gn overexpression vector with 2–5 formed stools per day, without any mucus or blood. The frequency of defecation was higher than in the mice from DSS+Mesalamine group but lower than in DSS+NS group.

The appearance, length and general morphology of the mouse colon were assessed by visual observation. It showed that the abdominal cavity of mice in the control group had no adh esion of peritoneum, intestinal tube or mesentery, the intestinal mucosa was smooth and had no ulcers, and there was no thickening in the intest inal wall. The intestines of the DSS+NS group mice were shortened, the bowels were enlarged and had adhesions. There were hyperemia and edema on the surface of the serosa, as well as erosion in the intestinal mucosa without obvious ulcerations; thickening of the intestinal wall and lumen stenosis could be s een. In the mi ce of each treatment group, the shortening of the colon caused by inflammation was alleviated by the treatment. There were no obvious adhesions nor enlargement of the intestinal tube, and slight hyperemia and edema were obser ved on the surface of the serosa. Erosions and ulcers could not be observed in the intestinal mucosa (Fig. 2 ). During the experiment, 2 mice died in the DSS+NS group and the percentage of dead mice was 17%; 1 mouse died in the DSS+ Mesalamine group, and 1 mouse died in the DSS+Me sala mine+Gn group, resulting in the percentage of dead mice of 8% in both groups.

Changes in Gn, Ugn and GC-C level

Immunohist ochemical analysis showed the expression of Gn, Ugn and GC-C in the intest inal epithelial cells of the contro l group. The level of all the proteins was significantly lower in the DSS+NS group than in the control group (P<0.01) but increased significantly after treatments (P<0.05). The increa se in Gn, Ugn and GC-C was the most significant in the DSS+Gn group compared t o the DSS+NS group (Fig. 3, Supplementary Figs. 1–3 at https://ojs.ptbioch.edu.pl/index.php/abp). There was no significant difference in the expression of Gn, Ugn and GC-C between the DSS+Gn group and DSS+Mesalamine+Gn group (P=0.15).

Changes in IL-8 and TNF-α

The level of IL-8 and T N F-α was measured in the peripheral blood using ELISA, and the re sults showed that the level of both proteins was significantly higher in the DSS+NS gro up than the control group (P<0.01) but decreased significantly after treatments (P<0.01). The decrea se in IL-8 and T NF-α level was the most significant in the DSS+Gn group compared to the DSS+NS group (Fig. 4). There was no significant difference in IL-8 and TNF-α level between the DSS+Gn group and DSS+Mesala mine+Gn group (P=0.18).

Changes in intestinal inflammation and histopathologic grading

The histop athologic grading of colon dama ge was scored by two pathologists in a blinded manner. The colonic structure observed under a microscope was clear in the control group, including the mucosa , submucosa, muscularis and serosa. The arrangement of epithelial cells was neat, goblet cells were rich, and the intestinal gland was regular; capillaries and a small number of scattered lymphocytes could be s een in the lamina propria. The mucosa of colon tissue was partially absent, and goblet cells were largely lost in the DSS+NS group. The branched glands and the reparative hyperplasia of the surrounding glands could be s een. Numerous inflammatory cells infiltrated the lamina propria, including neutrophils, lymphocytes, and eosinophils. The histop athologic scores were significantly higher in the DSS+NS group than in the control group (P<0.01). After the treatment, intestinal inflammation was relieved and epithelial repair was obvious. The irregular regenerative epithelial structure was observed, and inflammatory cells were reduced in the submucosa and muscularis (Fig. 5). The decline of the histopatho logic scores was the most significant in the DSS+Mesalamine+Gn group compared to the DSS+NS group (P<0.01). There was no significant difference in the histopathologic scores between the DSS+Mesalamine group and the DSS+Gn group (P=0.12) (Fig. 6 ).

Changes in the intestinal permeability and TJPs

Intestinal permeability of the mice was evaluated by measuring the rate of intestinal passage of FD-4 ma cromolecular compound. The results showed that the intestinal permeability was significantly higher in the DSS+NS group than in the control group (P<0.01) but decreased significantly after treatment (P<0.05). The decline in intestinal permeability was the most significant in the DSS+Gn group compared to the DSS+NS group (Fig. 7). There was no significant difference in intestinal permeability between the DSS+Gn group and DSS+Mesala mine+Gn group (P=0.23).

The TJPs (occludin, claudin-1 and ZO-1) were expressed in the intestinal epithelial cells of the control group, as determined by immunohistochemistry. The level of all the proteins was significantly lower in the DSS+NS group than in the control group (P<0.01). Expression of claudin-1 and ZO-1 increased significantly after treatments (P<0.05 for both proteins), and the increase was the most significant in the DSS+Mesalamine+Gn group compared to the DSS+NS group. There was no significant difference in the level of claudin-1 and ZO-1 between the DSS+Gn group and DSS+Mesalamine group (P>0.05 for both proteins) (Fig. 8, Supplementary Figs. 4–6 at https://ojs.ptbioch.edu.pl/index.php/abp/).

Discussion

Treatment of IBD faces great challenges due to the prolonged course and a high recurrence rate. Common therapeutic drugs for IBD include anti-TNFα and anti-integrin agents, glucocorticoids, immunosuppressants, 5-aminosalicylates, enteral nutrition, antibiotics, probiotics, which have limited efficacy and may cause considerable side effects or problems with adherence (Lee et al., 2015). Unresponsive patients may require colectomy, which exposes them to the risk of sequelae, such as pouchitis. With a series of basic studies, many new treatment strategies for IBD emerged, such as stem cell transplantation, fecal microbiota transplantation, and the use of some diets (CDED, CD-TREAT) (Shimizu et al., 2019; Moayyedi et al., 2015; Pigneur et al., 2019). However, fecal micr obiota transplantation has very poor efficacy in adults with IBD. Stem cell transplants are not perceived as a great opportunity either. In this study, after treatment of mice with colitis with the Gn ove rexpression vector, alone or in combina tion with mesalazine, intestinal permeability was decreased, the general condition of the mice improved and remission of colonic congestion, edema, and adhesion was observed. Histopathologic scores and pro-inflammatory cytokines (IL-8, TNF-α) were reduced. The protein level of crucial mediators of the GC-C signaling pathway (GC-C, Gn, Ugn) and TJPs (claudin-1, ZO-1) was increased. These results suggest that the application of the Gn ove rexpression vector alo ne or in combination with mesalazine recovered the activity of the GC-C signaling pathway and that intestinal inflammation and perturbed barrier function can be mitigated by GC-C agoni sts. Accordingly, Amarachintha et al. reported that GC-C may reduce the invasion of intestinal epithelial cells by bacterial pathogens (Amarachintha et al., 2018).

The endogenous peptides Gn and Ugn play t heir role mainly by activating GC-C expressed on the apical surface of intestinal epithelial cells (Forte, 2004). The binding of Gn/Ugn to GC-C can increase the level of intracellular cGMP, which leads to the secretion of liquid into the gut lu men through a series of signaling reactions. GC-C endog enous ligands function as “fluid sensors”, maintaining the balance of water and electrolytes in the intestinal tract and the optimal hydration of the intestinal mucosa (Lucas, 2000). Recent studies showed that orally administered GC-C agoni sts are rarely absorbed into the systemic circulation and act locally in the gut lumen (Shailubhai et al., 2013; Miner et al., 2013). Previously, we reported that the transcription and expression of GC-C, Gn a nd Ugn were downregulated in the colonic mucosa of UC patients and negatively correlated with UC disease activity (Lan et al., 20 16). Consistently with our studies, Janecke and others (Janecke et al., 2016) reported t hat a number of patients with GUCY2C (encoding intestinal receptor GC-C) and SLC9A3 (encoding Na/H antiporter 3, a downstream target of GC-C) mutations developed IBD. Primary Na/H antiporter 3 and GC-C malfunction is implicated as a predisposition for IBD in a subset of patients (Janecke et al., 2016). In this st udy, when mice with colitis were treated with a Gn overexpression vector through the caudal vein, the defecation of loose and bloody stools was relieved, and the level of the pro-inflammatory cytokines IL-8 and TNF-α was significantly reduced in the peripheral blood, indicating that the anti-inflammatory effects of GC-C receptor agonists may be related to their inhibition of the production of pro-inflammatory cytokines.

In addition, our study revealed that GC-C, Gn and Ugn were downregulated in colitis mice, intestinal permeability was decreased, and the expression of claudin-1 and ZO-1 increased significantly after treatment with the Gn overexpression vector, which suggests that the anti-inflammatory effect of the GC-C signaling pathway may be related to the maintenance of the intestinal barrier function. TJPs regulate the barrier function of intestinal epithelial cells (IECs), whi ch control the penetration of pathogens and allergens to the submucosa and beyond (Turner, 2009; Matter & Balda, 2003). Among many members of TJPs, occludin, claudins and ZO-1 are membrane proteins that connect the adjacent IECs and comprise the epithelial barrier (Mitic & Anderson, 1998; Ebnet et al., 2004). The TJP occludin behaves differently from claudin and ZO-1. Occludin is important for blocking the gap between adjacent epithelial cells and endothelial cells to maintain the barrier function of tight junctions. The integrity of the intestinal mucosa is maintained by the prolif eration, migration, differentiation and apoptosis of IECs (Eastwood, 1992). Previous studies found that GC-C can r egulate the proliferation, apoptosis, migration and regeneration of IECs (Lin et al., 2010; Shailubhai, 2002), and aberrant GC-C signaling can lead to the dysfunction of the intestinal epithelial barrier (Mann et al., 2013). The occurrence and severity of colitis in mice were reduced through drug intervention to protect the intestinal mucosal barrier (Arrieta et al., 2009; Madsen et al., 1999). Similar results were obtained in previous studies, showing that microorganisms in the intestinal lumen infiltrate the intestinal submucosa and below if the intestinal barrier function is impaired, the accumulation of a large number of white blood cells and macrophages disrupts the intestinal homeostasis, leading to the rapid increase of intestine pro-inflammatory factors and promoting the inflammatory reactions in the intestine (Neuman, 2007; Heller et al., 2005). Additionally, it was found that the intestinal permeability of GC-C-/- an d Ugn-/- mice was higher than that of wild-type mice, and the expression of TJPs in GC-C-/- mice was decreased in intestinal mucosa. The GC-C signaling pathway plays an important role in maintaining the balance of proliferation of intestinal epithelial cells and the integrity of intestinal mucosa (Neuman et al., 2007; Steinbrecher et al., 2011).

The limita tion of the current study was the failure to detect the level of Gn, Ugn and GC-C in the serum and other tissues, and lack of the group of animals receiving normal drinking water + Gn overexpression vector solution. The wet an d dry weight of feces produced at the end of the study was not recorded. These data will be included in the future study to test the efficacy of GC-C agonists for IBD therapy.

Conclusions

Taken together, the present study indicated that the application of a Gn overexpression vector, alone or in combination with mesalazine, can alleviate the inflammatory injury in the colon and restore the barrier function of intestinal mucosa in UC mice. In accordance with our previous findings, these observations further support the notion that the GC-C signaling pathway plays a protective role in intestinal inflammatory injury and epithelial barrier function, and GC-C agonists are a potential treatment approach for UC patients. Nevertheless, excessive activation of GC-C signa ling might lead to excessive secretion of water into the gut lumen and cause diarrhea. Future studies will be necessary to demonstrate whether the oral GC-C agonists have therapeutic potential in IBD.

Conflict of interest

The authors declare no conflict of interest.

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