A total of 112 male C57BL/6 (B6) mice (age, 6–8 weeks; weight, 22–25 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., Mice were maintained under specific pathogen-free conditions, all mice were provided free access to food and water, and conditions were a temperature of 23±2°C, humidity of 40–70% and a 12-h light/dark cycle. The procedures involving mice were carried out using protocols approved by the Ethics Committee of the First Affiliated Hospital of Soochow University (Suzhou, China).

The following primary antibodies were used: Anti-CD68 (Bio-Rad Laboratories, Inc., cat. no. MCA1957T) and anti-α7nAChR (Sigma-Aldrich; Merck KGaA, cat. no. M220). PNU282987 was purchased from Sigma-Aldrich (Merck KGaA, cat. no. P6499), methyllycaconitine citrate (MLA) was purchased from Sigma-Aldrich (Merck KGaA, cat. no. M168) and SHP099 was from Selleck Chemicals (cat. no. S8278). All drugs were dissolved in PBS, and PBS was used as vehicle control. DSS (molecular mass, 36,000-50,000, cat. no. 9011-18-1) was purchased from MP Biomedicals, LLC and dissolved in the drinking water at 3% (w/v) concentration.

Mice were provided 3% (wt/vol) DSS dissolved in the drinking water for 7 days. Weight loss was monitored daily. At the end of treatment, mice were euthanized by 100% CO₂ inhalation with 30% volume/minute flow rate; death was verified by cervical dislocation, colons were excised and colon length from the end of the cecum to the anus was recorded. In PNU282987-treated group, PNU282987 was dissolved in PBS and mice were intraperitoneal injection (i.p.) followed by DSS administration. For inhibiting a7nAChR, MLA was pre-administrated (i.p.) 1 day before PNU282987 treatment. PBS was used as vehicle control.

Colons from mice treated with or without 3% (wt/vol) DSS were excised and fixed in 4% paraformaldehyde at 4°C overnight, then colons were dehydrated with graduated ethanol (70,80,85,90,95 and 100%) and embedded in paraffin. Paraffin-embedded colons were sectioned into 5 µM slices and stained with hematoxylin and eosin, as described previously (25). Histological score was assessed (25) based on leukocyte infiltration: (0, no infiltration; 1: Basolateral; 2: Infiltration to muscularis mucosae; 3: Infiltration to submucosa), epithelial cell disruption (0: No epithelium disruption; 1: Crypt hyperplasia; 2: Mild crypt disruption; 3: Loss of crypt in large area). The histological score was conducted blind by two of the authors. Immunofluorescence analysis. Mouse colon specimens were fixed in 4% paraformaldehyde at 4°C overnight, dehydrated with 30 and 20% sucrose solution, then embedded with O.C.T. compound (Sakura Finetek USA, Inc.). Frozen sections (7 µM) were blocked with 10% normal goat serum (Beyotime Institute of Biotechnology) diluted with 1X PBS (1 ml normal goat serum supplied with 9 ml PBS) for 1 h at room temperature, then incubated with primary antibody to α7nAChR (1:200) or CD68 antibody (1:200 dilution) at 4°C overnight, and fluorophore-coupled secondary antibodies (Mouse IgG2a Cross-Adsorbed Secondary Antibody Alexa Fluor 488 (cat no. A-21131; Thermo Fisher Scientific, Inc., 1:500); Rabbit anti-Rat IgG (H + L), Texas Red^® (cat no. PA1-28571; Thermo Fisher Scientific, Inc., 1:500) for 1 h in the dark at room temperature. Sections were counterstained with 200 nM DAPI for 5 min and mounted in Fluorescent Mounting Medium. Fluorescent images were captured using a TCS SP8, laser-scanning confocal fluorescence microscope (Leica Microsystems GmbH).

Briefly, mice were treated with 3% (wt/vol) DSS for 7 days to induce colitis, and the large intestines were removed and carefully cleaned of their mesentery and fat tissue. The large intestine was then opened longitudinally, washed of fecal contents, cut into 0.5 cm sections, and subjected to two sequential 20-min incubations at 37°C in Hanks balanced salt solution (HBSS) plus 5% FBS (Gibco; Thermo Fisher Scientific) and 5 mM EDTA at 37°C with gentle agitation to remove epithelial cells. After each incubation step, media containing epithelial cells and tissue debris was discarded. The remaining tissue was minced with ophthalmic scissors and incubated for 30 min in HBSS plus 5% FBS, 1 mg/ml collagenase VIII (Sigma-Aldrich; Merck KGaA) and 0.1 mg/ml DNase I (Sigma-Aldrich; Merck KGaA) at 37°C with gentle agitation. Cell suspensions were collected and filtered through a 70-µm strainer and pelleted by centrifugation at 300 × g at room temperature for 10 min. Cells were incubated with CD45-Percific Blue (diluted in PBS with 1:1,000), CD11b-FITC (diluted in PBS with 1:1,000) and F4/80-PE (diluted in PBS with 1:1,000) antibody at room temperature in dark for 30 min, after washing twice with PBS, cells were resuspended in PBS with 1×10⁶ cells/ml. Colonic macrophages were sorted from CD45 positive cells, CD11b and F4/80 double positive gates by using flow cytometry (BD FACSAria™ III sorter). Purity of colonic macrophages was >85% analyzed with BD FACSCalibur™ with CD11b and F4/80 double positive gates. Purified colonic macrophages were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.).

Mice were euthanized and their femurs and tibias were isolated and rinsed in 70% (vol/vol) ethanol. Bone marrow cells were flushed out with cold PBS and passed through 40 µM filters, then centrifuged at 2,500 × g for 5 min at room temperature. Pellets were suspended in 1 ml ACK red blood cell lysis buffer (Sangon Biotech Co., Ltd.) at room temperature for 1 min, then neutralized with 20 ml PBS. After centrifugation at 2,500 × g for 10 min at room temperature, the isolated bone marrow cells were resuspended in complete DMEM (supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin) containing 10 ng/ml macrophage colony stimulating factor (PeproTech, Inc.) and cultured at 37°C in a 5% CO₂ incubator to allow for differentiation for 7 days.

RNA was isolated from 30 mg of colon tissue. Biopsies were weighed and homogenized using liquid nitrogen and TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA following the manufacturer's instructions. cDNA was reverse transcribed from 1 µg total RNA with the ReverTra Ace qPCR RT kit (Toyobo Life Science). qPCR was performed with SYBR^® Green Realtime PCR Master Mix (Toyobo Life Science) and an ABI 7500 instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.), using the following primers were: α7nAChR, forward 5′-TCCTCATGTCAGCCCCAAAC-3′, reverse 5′-AGACCGACAGCTACAGGGAT-3′; IL-1β, forward 5′-AAGGGGACATTAGGCAGCAC-3′, reverse 5′-ATGAAAGACCTCAGTGCGGG-3′; IL-6, forward 5′-TGCCTTCTTGGGACTGATGC-3′, reverse 5′-TGAAGTCTCCTCTCCGGACT-3′; TNF-α, forward 5′-GCACCACCATCAAGGACTCA-3′, reverse 5′-TGTGAGGAAGGCTGTGCATT-3′; Il-12 p35, forward 5′-ATGATGACCCTGTGCCTTGG-3′, reverse 5′-CACCCTGTTGATGGTCACGA-3′; IL-23 p19, forward 5′-TGGAGCAACTTCACACCTCC-3′, reverse 5′-GGCAGCTATGGCCAAAAAGG-3′; IL-10, forward 5′-CCTCGTTTGTACCTCTCTCCG-3′, reverse 5′-CAAGTGTGGCCAGCCTTAGA-3′; GAPDH, forward 5′-CCTTCCGTGTTCCTACC-3′, reverse 5′-CAACCTGGTCCTCAGTGTA-3′. Gene transcripts were normalized against the loading control GAPDH, and relative expression levels were calculated using the 2^−ΔΔCq method (26).

ELISA: Colon (0.5 cm) was excised from mice with sterile scissors, washed three times in cold PBS plus 100 µg/ml gentamycin, 100 U/ml penicillin and 100 µg/ml streptomycin. The colons slices were cultured with RPMI-1640 medium supplemented with 10% FBS and 100 µg/ml gentamycin at 37°C, 5% CO₂ for 24 h. Supernatant was harvested for cytokine measurement. Cytokine production from colons was analyzed with Quantikine ELISA kits (R&D Systems; Cat. no. MLB00C, M6000B, MTA00B, M1270 and M1000B respectively), following the manufacturer's instructions. Cytokine production was normalized according to total colon tissue weight. Each treatment contained 3–4 mice.

Colon proteins were extracted as described previously (25). Briefly, RIPA buffer supplemented with protease inhibitors (Sangon Biotech Co., Ltd.) was used as lysis buffer. Colon proteins were separated by 10% SDS-PAGE, and the gels were then electro-transferred onto nitrocellulose filter membranes (Whatman; Cytiva). The membranes were incubated with antibodies against α7nAChR (Sigma-Aldrich; Merck KGaA, cat. no. M220, 1:1,000), phosphorylated (p-)SHP2 (Abcam; cat. no. ab62322, 1:500), SHP2 (Abcam, cat. no. ab131541, 1:500), p-STAT3 (Cell Signaling Technology, Inc.; cat. no. 9145, 1:1,000); STAT3 (Santa Cruz Biotechnology, Inc., cat. no. sc-482, 1:100), p-Jak2 (Abcam, cat. no. ab32101, 1:1,000); Jak2 (cat. no. ab108596, Abcam, 1:1,000) or GAPDH (Abcam, cat. no. ab181602, 1:2,000) overnight at 4°C. The membranes were then incubated with an IRDye 800CW-conjugated secondary antibody (Rockland Immunochemicals Inc. 1:20,000) for 1 h at room temperature. Images were acquired using an Odyssey infrared imaging system (Odyssey^® CLx Imaging System, LI-COR Biosciences) and ImageJ software v1.52 (NIH) was used for analysis.

Intracellular oxidative stress was measured using dichlorodihydrofluorescein diacetate oxidation. Macrophages were incubated with 100 µl of 1X DCFH-DA/media solution (Sangon Biotech Co., Ltd.) for 1 h at 37°C. After washed with DPBS three times, the fluorescence was read using a fluorometric plate reader (FlexStation 3, Molecular Devices, LLC) at 480 nm/530 nm.

Data are presented as the mean ± SD. All experiments were repeated three times. One-way ANOVA followed by Dunnett's multiple comparison post hoc test was performed when comparing all experimental groups with the control group, whereas Tukey's post hoc test was used for comparing the means of all treatments to the mean of every other treatment, using GraphPad software version 6.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

As a nicotinic acetyl cholinergic receptor, α7nAChR is expressed widely in the central and peripheral nervous systems; previous studies have shown that activation of α7nAChR could alleviate inflammation (12,27,28). However, the effects and associated mechanisms of receptor signaling in IBD is not completely understood. Immunofluorescence analysis was used to investigate the expression of α7nAChR in colonic tissues of mice, and the results demonstrated that α7nAChR expression was highly induced in inflammatory sites within the colon but was only sparsely expressed in healthy colon (Fig. 1A). Macrophages serve a crucial role in intestinal homeostasis (19), and expression of their specific surface marker, CD68, was more abundant in the inflamed compared with in the healthy colon (Fig. 1A); the merged panel demonstrates that these two proteins shared a degree of co-localization, which suggested that α7nAChR on macrophages may serve a crucial role in colitis. To clarify α7nAChR expression pattern after colitis onset, western blotting was used to analyze the protein expression levels of α7nAChR at day 0, 3, 5, 7 after DSS administration in mice. α7nAChR expression was significantly increased after 5 days of DSS treatment (Fig. 1B). To evaluate whether colitis-induced α7nAChR protein expression was due to reduced degradation or increased receptor synthesis, mRNA expression levels of α7nAChR in mice were assessed using RT-qPCR. The results showed α7nAChR mRNA levels have a similar expression pattern as protein levels in the mouse colon after DSS treatment (Fig. 1C). These data demonstrated that α7nAChR is highly expressed in the inflamed colon and suggested that the receptor on macrophages may play a critical role in colitis.

To investigate the role of α7nAChR in colitis, the α7nAChR specific agonist PNU282987 (0.3 or 3 mg/kg) was administered to mice by i.p. injection daily, following DSS administration. PNU282987 was dissolved in PBS and PBS was used as vehicle control. Pathological changes were observed in each group; progressive weight loss occurred in colitic mice treated with DSS, but the extent of weight loss was reduced with a 3 mg/kg of PNU282987 treatment in colitic mice compared with DSS+PBS-treated group (Fig. 2A). Moreover, the protective effects of PNU282987 were decreased by pre-treatment with MLA for 1 day, the antagonist of α7nAChR (Fig. S1). Colon shortening was lessened in colitic mice treated with PNU282987 at 3 mg/kg (Fig. 2B). Histological analysis of colons from colitic mice treated with the PBS control showed severe inflammation with loss of crypts (Fig. 2C, red arrow) and infiltration of leukocytes (Fig. 2C, blue arrow), whereas this damage was alleviated by treatment with PNU282987 (Fig. 2C). Histological scores were obtained based on colonic epithelial cell disruption and leukocyte infiltration, and the scores in the colitic mice were significantly lower after activation of α7nAChR by PNU282987 at 3 mg/kg (Fig. 2D). These results demonstrated that PNU282987 may exert a protective effect in DSS-induced mouse colitis.

Cytokines serve a crucial role in the pathology of colitis (1,2); therefore, IL-1β, IL-6, TNF-α, IL-12, IL-23 and IL-10 levels in colonic tissue were analyzed by ELISA. Compared with untreated control colitic mice, colitic mice treated with 3 mg/kg PNU282987 exhibited a significant reduction in IL-1β, IL-6, TNF-α, IL-12 and IL-23 concentrations, whereas IL-10 levels were significantly increased after treatment with PNU282987 (Fig. 3A-F). In DSS alone-treated group, IL-10 level was also enhanced possibly due to the macrophage or a small amount of Treg cell activation. TGF-α and IL-22 production showed no significant differences between mice treated with PNU282987 or PBS; these two cytokines play a negative role in IBD development (data not shown).

One of the functions of macrophages is maintenance of intestinal homeostasis through cytokine production. In DSS-induced colitic mice treated with PNU282987 or with PBS, colonic macrophages were sorted >85% purity by flow cytometry using CD11b and F4/80 antibody staining (Fig. S2). IL-1β, IL-6, TNF-α, IL-12 p35, IL-23 p19 and IL-10 mRNA expression levels in macrophages were measured. In colitic mice treated with PNU282987, mRNA expression levels of IL-1β, IL-6, TNF-α, IL-12 p35 and IL-23 p19 were significantly decreased compared with colitic mice treated with PBS, whereas IL-10 mRNA expression was significantly raised in colonic macrophages of colitic mice treated with PNU282987 (Fig. 3G-L). Also due to the activation of macrophages in the colitic colon, IL-10 mRNA level was enhanced in the DSS-treated group compared with the control. These results are consistent with previous reports that suggest acute DSS-induced colitis development depends on macrophage activation (29,30).

SHP2 has previously been identified as a detrimental factor in the colonic immune system (24). To study SHP2 activation in colonic macrophages and the effect of treatment with PNU282987, mice were treated with DSS and colonic macrophages were isolated. JAK2 and STAT3 phosphorylation levels were notably upregulated in the colitic model mice, whereas a markedly decreased level was seen in the PNU282987-treated group (Fig. 4A). Moreover, SHP2 phosphorylation levels were also enhanced in the colitic group and were unchanged in the colitic group treated with PNU282987, indicating that PNU282987 has no direct effect on SHP2 activation (Fig. 4A). As STAT3 is involved in both α7nAChR and SHP2 pathways, activating α7nAChR or inhibiting SHP2 leads to upregulating the level of p-STAT3, which is beneficial for recovery in colitis (16,24). To investigate the possible role of SHP2 in PNU282987 effects on macrophages, SHP2 expression in BMDMs was knockdown by three siRNA, and the third siRNA show the highest efficacy on SHP2 knockdown (knockdown efficacy was verified by western blot; Fig. 4B). Cells were incubated with PNU282987 followed with LPS challenge which is a classical regent to inducing inflammation in macrophages. In BMDMs which had been challenged with LPS and treated with PNU282987, TNF-α and IL-6 cytokine production was significantly reduced in cells depleted of SHP2 or PNU282987 treatment compared with LPS only administration group. In addition, a combination of siRNA-SHP2 and PNU282987 decreased TNF-α and IL-6 cytokine protein level (Fig. 4C and D). These results indicated that inhibiting SHP2 activation may have a combined effect with PNU282987 in suppression of inflammatory macrophages.

SHP2 is a ubiquitously expressed tyrosine phosphatase and previous studies reported that it has an inhibitory effect on the JAK/STAT signaling pathway (24). To investigate whether inhibiting SHP2 has a synergistic effect in combination with PNU282987, BMDMs were treated with SHP099, a selective pharmacological inhibitor of SHP2, and PNU282987 simultaneously. Co-treatment with SHP099 and PNU282987 resulted in a significant decrease in TNF-α and IL-6 expression compared with PNU282987 only treatment in LPS-induced macrophages, whereas no significant decrease was identified in TNF-α and only a slight decrease in IL-6 production with SHP099 treatment alone (Fig. 5A). Excessive accumulation of reactive oxygen species (ROS) can induce further damage in inflamed colonic tissue (31). Consistent with the cytokine production data, ROS levels induced by LPS in macrophages were significantly decreased by a combination of SHP099 and PNU282987 treatments, whereas there were no differences identified between the SHP099-alone treatment group (Fig. 5B).

To further investigate the effect of SHP099 and PNU282987 in the experimental animal model, DSS-induced colitic mice were treated with PNU282987 combined with SHP099. Administration of DSS lead to weight loss, colon shortening and severe colonic disruption. The aim of the present study was to find a combined drug that could lower the dose of PNU282987 and protect mice from DSS-induced colitis, so 0.3 mg/kg PNU282987 or 1 mg/kg SHP099 treatment alone were chosen, which had no significant effect on colitis. However, when 0.3 mg/kg PNU282987 was combined with SHP099 administration, this resulted in attenuated weight loss and colon shortening (Fig. 6A and B). Histopathologically, in the PNU282987 and SHP099 co-treatment groups, mice displayed markedly mitigated mucosal damage and less leukocyte infiltration compared with PNU282987 treatment alone (Fig. 6C). Statistical analysis of histological scores supported these conclusions (Fig. 6D).