Article Figures & Data
Figures
- Fig. 1.
GPER activation prevented DSS-induced colitis in mice. (A) Body weight change during the disease process within the different experimental groups (n = 8, **P < 0.01, ***P < 0.001, vs. DSS; $$P < 0.01, $$$P < 0.001, vs. DSS + G-1 + G15; #P < 0.05, ###P < 0.001, VS.DSS vs. DSS + G-1; &P < 0.05, &&P < 0.01, &&&P < 0.001, VS.DSS + G-1 vs. DSS + G-1+G15). (B) DAI change during the disease process within the different experimental groups (n = 8, ***P < 0.001, vs. DSS; $$P < 0.01, $$$P < 0.001, vs. DSS + G-1 + G15; ##P < 0.01, ###P < 0.001, VS.DSS vs. DSS + G-1; &&&P < 0.001, VS.DSS + G-1 vs. DSS + G-1 + G15). (C) Representative photograph of the colon from each group. (D) The statistical chart of colon length within the different experimental groups (n = 6, ***P < 0.001). (E) Representative images of colonic histology (H&E staining, scale bar = 100 μm). The black arrow indicates mucosal ulceration and necrosis. The red arrow indicates destruction, necrosis of crypt, and inflammatory cells infiltrated into the submucosa. (F) The statistical chart for histologic score within the different experimental groups (n = 8, ***P < 0.001). The mice were divided into four groups: control, DSS group, DSS + G-1 treatment, and DSS + G-1 and G15 treatment. Data are expressed as means ± S.E.M. or median. Statistical analyses were performed by one-way ANOVA or the Kruskal-Wallis test followed by the Student-Newman-Keuls method.
- Fig. 2.
Expression of GPER in colon epithelium and change of GPER expression in colitis. (A) Immunofluorescence detection of GPER in crypt from the Lgr5-EGFP mouse. The Lgr5+ intestinal stem cells were marked by GFP. DAPI was used as a nuclear stain. The green arrow indicates the GPER-positive Lgr5+ stem cell, and the yellow arrow indicates other GPER-positive cells in crypt (scale bar: lower right quarter trace was 10 μm; others were 20 μm). (B) GPER expression in acute colitis model with or without G-1 treatment (n = 4, **P < 0.01). Data are expressed as means ± S.E.M. Statistical analyses were performed by one-way ANOVA followed by the Student-Newman-Keuls method.
- Fig. 3.
GPER activation protected colonic mucosal barrier in DSS-induced colitis. (A) Immunohistochemical staining for JAM-1 in distal colon for each group (scale bars: 50 μm). (B) Immunohistochemical staining for occludin in distal colon for each group (scale bars: 50 μm). (C) Representative photographs of colon sections stained for mucous layers with PAS staining for each group (scale bars: 50 μm). (D) Representative images for immunohistochemical staining of Muc-2 in distal colon for each group (scale bars: 50 μm). (E) Statistical chart of (D) (n = 6 mice per group, five crypts were randomly calculated in each section in a blinded fashion, and the average value was obtained; **P < 0.01, ***P < 0.001). (F) Effect of G-1 treatment on colonic mucosal permeability in colitis mice. The permeability was evaluated by FITC-dextran concentration in serum. The mice received an oral gavage of FITC-dextran (400 mg/kg) 4 hours before collecting blood, and serum FITC-dextran concentrations were determined to reflect the colonic mucosal permeability (n = 8, **P < 0.01). The mice were divided into three groups: control, DSS treatment group, and DSS + G-1 treatment. Data are expressed as means ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by the Student-Newman-Keuls method.
- Fig. 4.
GPER activation inhibited the ERS and UPR in DSS-induced colitis. (A) Effect of G-1 treatment on GRP78 expression in colitis (the figure above was the original one, and the figure below was the statistical one; n = 4, **P < 0.01). (B) Effect of G-1 treatment on CHOP expression in colitis (the figure above was the original one, and the figure below was the statistical one; n = 4, ***P < 0.001, **P < 0.01). (C) Effect of G-1 treatment on PERK activity in colitis (the figure above was the original one, and the figure below was the statistical one; n = 4, *P < 0.05). (D) Effect of G-1 treatment on IRE1α activity in colitis (the figure above was the original one, and the figure below was the statistical one; n = 4, **P < 0.01, ***P < 0.001). (E) Effect of G-1 treatment on ATF6 expression in colitis (the figure above was the original one, and the figure below was the statistical one; n = 4, **P < 0.01, ***P < 0.001). The mice were divided into three groups: control, DSS treatment group, and DSS + G-1 treatment. Data are expressed as means ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by the Student-Newman-Keuls method.
- Fig. 5.
G-1 treatment reduced the epithelial cell apoptosis in DSS-induced colitis model. (A) Representative immunohistochemical staining of cleaved caspase-3 (brown) in colonic mucosa for each group (scale bar: 20 μm). (B) Bar graphs to analyze the cleaved caspase-3–positive cells per crypt within the different experimental groups. For each mouse, five colonic crypts were randomly calculated in each section in a blinded fashion, and the average value was obtained (n = 5, ***P < 0.001). (C) Representative images for TUNEL staining in colonic mucosa for each group (scale bar: 50 μm). (D) Bar graphs to analyze the TUNEL-positive cells per 200× field within the different experimental groups. Percentage of TUNEL-positive cells was counted for each slice under two random fields (×200) in a blinded fashion, and the average was calculated (n = 5 mice per group, ***P < 0.001). The mice were divided into three groups: control, DSS group, and DSS + G-1 treatment. Data are expressed as means ± S.E.M. or medians. Statistical analyses were performed using one-way ANOVA or the Kruskal-Wallis test followed by the Student-Newman-Keuls method.
- Fig. 6.
G-1 treatment protected the crypt cell proliferation in DSS-induced colitis. (A) Representative figures for Ki67 staining (scale bar: 50 μm) and statistical chart of Ki67-positive cells per crypt. Five colonic crypts were randomly calculated in each section in a blinded fashion, and the average value was obtained (n = 6, ***P < 0.001). (B) Representative figures for BrdU incorporation in crypt in colon (scale bar: 50 μm) and statistical chart of BrdU-positive cells per crypt. Five colonic crypts were randomly calculated in each section in a blinded fashion, and the average value was obtained (n = 6, **P < 0.01, ***P < 0.001). (C) Representative Western blot photographs for cyclin D1 and cyclin B1. (D) Densitometry analysis of cyclin D1 in colonic tissue within the different experimental groups (n = 4, **P < 0.01, ***P < 0.001). (E) Densitometry analysis of cyclin B1 in colonic tissue within the different experimental groups (n = 4, *P < 0.05, **P < 0.01). (F) Statistical chart of Ki67-positive cells per crypt after G-1 treatment in control mice. Five colonic crypts were randomly calculated in each section in a blinded fashion, and the average value was obtained. In addition to (F), mice were divided into three groups: control group, DSS group, and DSS group. In (F) mice were divided into control and control + G-1 group. Data are expressed as means ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by the Student-Newman-Keuls method or unpaired t test.
- Fig. 7.
G-1 treatment inhibited the ERS in cultured CCD841 cells. (A) Representative Western blot photographs for GRP78 and CHOP expression in cultured CCD841 cells. (B) The statistical chart of GRP78 expression within different experimental groups (n = 3, *P < 0.05, ***P < 0.001). (C) The statistical chart of CHOP expression within different experimental groups (n = 3, **P < 0.01). CCD841 cells were treated with TG or TG + G-1. The control group was treated with 0.05% DMSO only. Cells were stimulated with TG for 6 hours to induce ERS, and G-1 (10−7 M) was administrated for 12 hours. Data are expressed as means ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by the Student-Newman-Keuls method.
- Fig. 8.
GPER activation inhibited the cell injury induced by ERS in vitro. (A) Fluorescence image of in vitro cultured CCD841 cells treated with EdU for 3 hours to show EdU-positive cells (red) in different groups (scale bar: 50 μm). The EdU incorporation test was performed 48 hours after G-1 treatment. (B) Quantitative results of EdU incorporation test. For each sample, three 200× fields were randomly selected under fluorescence microscope, the proportions of EdU staining–positive cells to Hoechst staining–positive ones was calculated by a blind observer, and the mean value was taken (n = 4, **P < 0.01, ***P < 0.001). (C) Effect of G-1 on the number viable cells proliferation under ERS. The cell was evaluated by CCK-8 test (n = 3, *P < 0.05, ***P < 0.001, vs. TG; #P < 0.05, ##P < 0.01, vs. TG + G-1; &&& P < 0.001, vs. control). (D) Representative Western blots showed specific bands for cyclin D1. The cells were collected for Western blot 12 hours after G-1 treatment. (E) The statistical chart for (D) within different experimental groups (n = 3, *P < 0.05). CCD841 cells were treated with TG or TG + G-1. The control group was treated with 0.05% DMSO only. Cells were stimulated with TG for 6 hours to induce ERS, and the treatment time of G-1 (10−7 M) was selected according to the experimental needs. Data are expressed as means ± SEM. Statistical analyses were performed using one-way ANOVA followed by the Student-Newman-Keuls method.
Tables
Epithelium Score Normal morphology 0 Loss of goblet cells 1 Loss of goblet cells in large areas 2 Loss of crypts 3 Loss of crypts in large areas 4 Infiltration No infiltrates 0 Infiltrate around crypt basis 1 Infiltrate reaching to lamina muscularis mucosae 2 Extensive infiltration reaching the lamina muscularis mucosae and thickening of mucosa with severe edema 3 Infiltration of the lamina submucosa 4 - TABLE 2
Body weight of each group before and 7 days after DSS administration
Colitis was induced by adding DSS (2.5%) in the animals’ drinking water for 7 days. Data are expressed as means ± S.E.M. (n = 8).
Group Body weight (g) 0 day 7th day Control 21.99 ± 0.54 23.36 ± 0.48 DSS 21.45 ± 0.26 17.36 ± 0.28*** DSS + G-1 21.71 ± 0.29 19.11 ± 0.52# DSS + G-1 + G15 22.04 ± 0.10 17.48 ± 0.21&&
In this issue
GPER Activation Prevented the Development of Acute Colitis
