Abstract

The intestinal ischemia/reperfusion (I/R) injury is a common clinical event related with high mortality in patients undergoing surgery or trauma. Estrogen exerts salutary effect on intestinal I/R injury, but the receptor type is not totally understood. We aimed to identify whether the G protein–coupled estrogen receptor (GPER) could protect the intestine against I/R injury and explored the mechanism. Adult male C57BL/6 mice were subjected to intestinal I/R injury by clamping (45 min) of the superior mesenteric artery followed by 4 h of intestinal reperfusion. Our results revealed that the selective GPER blocker abolished the protective effect of estrogen on intestinal I/R injury. Selective GPER agonist G-1 significantly alleviated I/R-induced intestinal mucosal damage, neutrophil infiltration, up-regulation of TNF-α and cyclooxygenase-2 (Cox-2) expression, and restored impaired intestinal barrier function. G-1 could ameliorate the impaired crypt cell proliferation ability induced by I/R and restore the decrease in villus height and crypt depth. The up-regulation of inducible nitric oxide synthase (iNOS) expression after I/R treatment was attenuated by G-1 administration. Moreover, selective iNOS inhibitor had a similar effect with G-1 on promoting the proliferation of crypt cells in the intestinal I/R model. Both GPER and iNOS were expressed in leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) positive stem cells in crypt. Together, these findings demonstrate that GPER activation can prompt epithelial cell repair following intestinal injury, which occurred at least in part by inhibiting the iNOS expression in intestinal stem cells (ISCs). GPER may be a novel therapeutic target for intestinal I/R injury.

Introduction

Intestinal ischemia/reperfusion (I/R) injury is a common pathophysiological process, which has a high incidence in acute mesenteric ischemia, hemodynamic shock, severe infection, as well as in surgical operations, such as intestinal transplantation and aortic aneurysm repair [1–3]. After reperfusion multiple pathophysiological mechanisms, such as reactive oxygen species (ROS) attack [4], nitric oxide (NO) metabolism [4,5], inflammatory events [6,7], and so on, interact and ultimately lead to epithelial cell apoptosis or necrosis [8,9]. Intestinal epithelial barrier disruption may lead to systemic inflammatory response syndrome, multiple organ dysfunction syndrome or failure because of intestinal bacteria translocation, which is one of the important factors of death in patients undergoing surgery or trauma [10]. In this sense, treatment strategies to restore intestinal barrier function do help to protect patients suffering from intestinal I/R from various systemic complications and improve their survival rate [11].

Female rats were more resistant to intestinal pathological injury caused by hemorrhagic shock than male rats [12], suggesting a salutary effect of estrogen [13]. In fact the protective effect of estrogen on cardiac [14], cerebral [15], hepatic [16], renal [17], as well as intestinal I/R injury [18,19] has been confirmed. Various mechanisms were involved in the protective effects of estrogen on intestinal I/R injury, such as attenuation of inflammatory response, regulation of endothelial nitric oxide synthase (eNOS) or the inducible nitric oxide synthase (iNOS) expression, maintenance of mesenteric perfusion and intestinal integrity [18–21].

The intestinal salutary effect of estrogen was probably receptor mediated [22,23], which seemed critically dependent on the length of the treatment period [24,25]. Acute estrogen treatment has been shown to play a protective role in intestinal I/R injury [18,26]. It is well known that the nuclear estrogen receptors (ERs), ERα and ERβ mediate the conventional genomic actions of estrogen [27], while activation of membrane ER, like G protein–coupled ER (GPER, also known as GPR30) elicits rapid, non-genomic actions [28]. GPER has been proved to be involved in the regulation of colonic motility [29] and visceral pain [30], and played an important role in inflammatory bowel disease [23,31]. The location of GPER in the myenteric plexus of rat ileum [32] and human colonic mucosa [23] has been confirmed. Our group has found that in colon of mice GPER was expressed in submucous and intermuscular ganglia, as well as crypt [29]. Crypts are subunits that house intestinal stem cells (ISCs). The ISCs positive for leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5), located at the basement of crypt, possess the ability of indefinitely self-renewing while generating transit amplifying cells (TACs) and driving the quick renewal of intestinal epithelium [33]. ISCs may be an effective therapeutic target to promote intestinal recovery from injury since increased proliferation of undifferentiated progenitor contributes to regeneration of the intestinal barrier [34,35]. Previous research showed that there was GPER expression in hematopoietic stem cells [36], and GPER mediates estrogen-induced proliferation of primordial germ cells [37]. Notably, our preliminary studies showed that there was also GPER expression in crypt of jejunum in mice.

Based on these reports, in the present study we set out to identify whether GPER was involved in protecting the intestinal I/R injury, and further explore the underlying mechanism(s).

Materials and methods

Animals

Male C57BL/6 mice and lgr5-egfp-ires-CreERT2 (Lgr5-EGFP) mice weighing approximately 20 g were used for experiments. C57BL/6 mice were purchased from Animal Center of Shandong University, while Lgr5-EGFP mice were bought from Model Animal Research Center of Nanjing University, China. Animals were housed under a 12 h/12 h dark/light cycle in a temperature-controlled room with free access to food and water. All animal experiments were approved by Medical Ethics Committee for Experimental Animals, Shandong University, China.

Establishment of I/R injury model and study protocol

Mice were anesthetized with 3% isoflurane followed by maintenance at 1.5% isoflurane in oxygen. The intestinal I/R injury model was induced as previously described [38]. Briefly, the mice underwent a median laparotomy and the superior mesenteric artery was isolated and clamped with a microvessel clip. After 45 min of intestinal ischemia, the clip was removed to initiate blood reperfusion and followed by 2, 4, 24, and 72 h reperfusion. At the end of reperfusion time, the proximal jejunum from a point 10 cm distal to the ligament of Treiz was collected for Hematoxylin and Eosin staining (H&E staining), immunohistochemistry (IHC) analysis, or Western blot. The sham-operated mice underwent the same procedure without superior mesenteric artery occlusion.

We found that 45-min ischemia followed by 2 or 4 h reperfusion caused significant intestinal mucosal injury (Supplementary Figure S1). The intestinal mucosa damage scoring evaluated by Chiu’s score [39], increased significantly at 2 and 4 h of reperfusion and then markedly decreased at 24 and 72 h of reperfusion (Supplementary Figure S1). Acute 17β-estrogen (E2) administration alleviated the jejunal injury at 4 h of reperfusion, while it did not affect the intestinal mucosa damage scoring at 2 h of reperfusion (Supplementary Figure S2). Thus, we chose 45 min ischemia followed by 4 h reperfusion to establish I/R injury model in the following experiments.

The preliminary experiments showed that either acute E2 or G-1 (selective GPER agonist) administration had no effect on the jejunal histological features of sham-operated mice (data not shown). To test whether GPER mediated the effect of estrogen on intestinal I/R injury, the experiments were performed in five experimental groups: sham-operated vehicle (0.2 ml of 1% DMSO, i.p.) as control group, I/R vehicle (0.2 ml of 1% DMSO, i.p.), I/R treatment with E2 (3 μg per mouse prepared in 0.2 ml of 1% DMSO, i.p.), I/R treatment with E2 (3 μg per mouse prepared in 0.1 ml of 1% DMSO, i.p.) and ICI182,780 (selective estrogen nuclear blocker, 5 mg.kg−1 in 0.1 ml of 1% DMSO, i.p.) together, I/R treatment with E2 (3 μg per mouse prepared in 0.1 ml of 1% DMSO, i.p.) and G15 (selective GPER blocker, 300 μg.kg−1 in 0.1 ml of 1% DMSO, i.p.) together [40,41]. To observe the role of GPER or iNOS on intestinal I/R injury, mice were treated with G-1 (30 μg.kg−1 in 0.1 ml of 1% DMSO) [40] or a selective iNOS inhibitor, 1400W (3 mg.kg−1 in 0.1 ml of 1% DMSO) intraperitoneally [42]. All drugs were administrated at ischemia period (30 min after ischemia) except ICI182,780 and G15, which were given before ischemia.

H&E staining for histologic examination

The isolated jejunal segments were washed with ice-clod PBS and fixed in 4% paraformaldehyde for 24 h. Next, the tissues were dehydrated in a graded ethanol series and embedded in paraffin. The slices (4 μm) were subjected to H&E staining following the manufacturer’s instructions. The Chiu’s score was used to evaluate intestinal mucosal damage [39]. Higher Chiu score means more severe damage. The measurements were performed by a blinded observer and made in triplicate to get the mean value.

Myeloperoxidase activity assay

Tissue myeloperoxidase (MPO) activity, a reliable marker for neutrophil infiltration, was used to evaluate the degree of intestinal injury [43]. The jejunum segments were cut and washed with ice-cold PBS solution. MPO activity was measured in pieces of the jejunum with the MPO assay kit (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s instructions.

Measurement of in vivo intestinal permeability

In vivo intestinal permeability was determined by the concentration of FITC-dextran (molecular weight 4.0 kDa, Sigma–Aldrich) in blood to assess intestinal barrier function [44]. Briefly, mice were fasted overnight and administrated with FITC-dextran solution by gavage (400 mg/kg, dissolved in PBS at a concentration of 100 mg/ml) 4 h before being executed. Blood samples were drawn from eyeball and centrifuged to collect serum. The serum was taken and diluted ten-fold in PBS. Next, the fluorescence intensity of the diluted serum was measured by a microplate reader (Molecular Devices, U.S.A.) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The concentrations of plasma FITC-dextran were calculated from the standard curve of correlation between FITC fluorescence intensity (Y) and FITC-dextran concentration (X). The higher the concentration, the greater the intestinal permeability.

IHC and immunofluorescent assay

Paraffin sections (4 μm) of jejunum tissue were dewaxed with xylene and rehydrated with graded ethanol solutions. Then, sections were soaked with 10 mM citrate buffer (pH 6.0) and heated in a microwave oven. Endogenous peroxidase activity was inhibited by incubation with 3% H2O2 for 30 min. After rinsing with PBS, the sections were blocked with normal goat serum (ZSGB-BIO, Beijing, China) for 30 min.

For IHC assay, the sections were incubated with rabbit polyclonal anti-Ki67 antibody (dilution 1:300, CST, U.S.A.), mouse polyclonal anti-bromodeoxyuridine (BrdU) antibody (dilution 1:300, Proteintech, U.S.A.), or rabbit polyclonal anti-iNOS antibody (dilution 1:100, Abcam, U.K.) at 4°C overnight followed by biotin-labeled secondary antibodies (ZSGB-BIO, Beijing, China) at 37°C for 1 h. The slides were labeled with Streptomyces avidin peroxidase (ZSGB-BIO, Beijing, China) for 15 min at room temperature and visualized using a 3,3′-Diaminobenzidine tetrahydrochloride kit (ZSGB-BIO, Beijing, China) following the manufacturer’s protocol. Nuclei were counterstained with Hematoxylin.

For immunofluorescent (IF) assay, the sections were incubated with the primary antibody rabbit polyclonal anti-ZO-1 antibody (dilution 1:50, Proteintech, U.S.A.) or rabbit polyclonal anti-occludin antibody (dilution 1:25, Proteintech, U.S.A.) overnight at 4°C. After removal of the primary antibody by PBS washing, the sections were incubated with a secondary antibody mixture containing Rhodamine (TRITC)–conjugated goat anti-rabbit IgG (dilution 1:50, Proteintech, U.S.A.) in a humid box at 37°C for 60 min in the dark. To co-locate GPER or iNOS with Lgr5+ ISCs, the sections were incubated with the primary antibody mixture containing rabbit polyclonal anti-G-protein coupled receptor 30 antibody (dilution 1:50, GeneTex, U.S.A.) or rabbit polyclonal anti-iNOS antibody (dilution 1:50, Abcam, U.K.) and chicken polyclonal anti-GFP antibody (dilution 1:500, Aves, U.S.A.) followed by a secondary antibody mixture containing Rhodamine (TRITC)–conjugated goat anti-rabbit IgG (dilution 1:50, Proteintech, U.S.A.) and Alexa Fluor 488-labeled goat anti-chicken IgG (dilution 1:1000, Invitrogen, U.S.A.). The nuclei were counterstained with DAPI (Solarbio Life Sciences, China).

In the end, the section was mounted on a glass slide with a drop of 75% glycerol and then observed by fluorescene microscope (Nikon, Japan).

Protein isolation and Western blot analyses

Total protein for Western blotting was extracted from jejunum tissue and the protein concentration was quantitated using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, China). Proteins were separated by SDS/PAGE and transferred to PVDF membranes (Millipore, U.S.A.). Then, the membrane was blocked with 5% nonfat dry milk for 2 h at room temperature and membrane was incubated with rabbit polyclonal anti-G-protein coupled receptor 30 antibody (dilution 1:250, Abcam, U.K.), mouse monoclonal anti-TNF-α antibody (dilution 1:2000, Proteintech, U.S.A.), purified mouse anti-cyclooxygenase-2 (Cox-2) (dilution 1:1000, BD Biosciences), rabbit polyclonal anti-ZO-1 antibody (dilution 1:800, Proteintech, U.S.A.), rabbit polyclonal anti-occludin antibody (dilution 1:600, Proteintech, U.S.A.), or rabbit polyclonal anti-iNOS antibody (dilution 1:200, Abcam, U.K.) overnight at 4°C. To quantitate protein expression, the rabbit polyclonal anti-GAPDH antibody (dilution 1:5000, Proteintech, U.S.A.) or mouse polyclonal anti-β-actin antibody (dilution 1:5000, Proteintech, U.S.A.) incubation was performed overnight at 4°C. After rinsing with TBST the membrane was incubated with peroxidase-conjugated goat anti-rabbit IgG (dilution 1:5000, Proteintech, U.S.A.) or peroxidase–conjugated goat anti-mouse IgG (dilution 1:5000, Proteintech, U.S.A.) for 1 h at room temperature. The bands were visualized with BeyoECL Plus (Beyotime Institute of Biotechnology, China) and quantitated using ChemiDoc XRS system and Image Lab Software (Bio-Rad, U.S.A.).

Drugs and chemicals

E2 and ICI182,780 were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.), G-1 was purchased from ApexBio (Houston, U.S.A.) and G15 was purchased from Cayman Chemical (Ann Arbor, MI, U.S.A.). Isoflurane was obtained from RWD Life Science (Shenzhen, China).

Statistical analysis

All the data were present as mean ± S.E.M. unless otherwise stated. The difference between two groups was compared by unpaired t test, while one-way ANOVA followed by Student–Newman–Keuls method was used for the multi-group comparisons. The Kruskal–Wallis test followed by the Student–Newman–Keuls test was used for data with non-normal distribution. P-values of <0.05 were considered significant.

Results

Blockade of GPER abolished protective effect of estrogen on intestinal histopathological injury induced by I/R

The I/R group displayed destruction of the villi, severe villus edema, infiltration of inflammatory cells, lamina propria edema, hemorrhage, and increased gaps between epithelial cells (Figure 1B). By contrast, the sham-operated mice showed almost normal mucosal architecture (Figure 1A). Acute treatment with E2 dramatically attenuated the intestinal histological injury induced by I/R (Figure 1C,F). G15, a selective GPER blocker, abolished estrogen’s effect on jejunal I/R injury (Figure 1D,F). Although several estrogen and G15-treated I/R mice showed more severe damage than I/R mice vehicle, no statistical difference was found between the two groups. In contrary, nuclear ERs blocker ICI182,780 did not affect the effect of estrogen on intestinal injury following I/R (Figure 1E,F). The complete reversal of the effect of E2 by G15 indicated that the acute effect of E2 on intestinal I/R injury was mediated by GPER, not by nuclear receptor-dependent manner.

GPER involved in the protective effect of estrogen on I/R-induced intestinal injury in mice

Figure 1
GPER involved in the protective effect of estrogen on I/R-induced intestinal injury in mice

(AE) Representative images of intestinal histology (H&E staining, scale bar = 100 μm). Mice were subjected to 45 min of intestinal ischemia followed by 4 h of reperfusion or sham surgery. (A) Sham, (B) I/R vehicle, (C) I/R+E2, (D) I/R+E2+G15, (E) I/R+E2+ ICI182,780. (F) Intestinal mucosa damage scoring (Chiu’s score) at different groups. Data were expressed as median. Statistical analyses were performed using the Kruskal–Wallis test followed by the Student–Newman–Keuls test (n=9, ***P<0.001, **P<0.01).

Figure 1
GPER involved in the protective effect of estrogen on I/R-induced intestinal injury in mice

(AE) Representative images of intestinal histology (H&E staining, scale bar = 100 μm). Mice were subjected to 45 min of intestinal ischemia followed by 4 h of reperfusion or sham surgery. (A) Sham, (B) I/R vehicle, (C) I/R+E2, (D) I/R+E2+G15, (E) I/R+E2+ ICI182,780. (F) Intestinal mucosa damage scoring (Chiu’s score) at different groups. Data were expressed as median. Statistical analyses were performed using the Kruskal–Wallis test followed by the Student–Newman–Keuls test (n=9, ***P<0.001, **P<0.01).

GPER activation alleviated the intestinal mucosal damage and inflammation after intestinal I/R

Selective GPER agonist, G-1 administration improved histological images and decreased mucosal damage scoring compared with I/R group (Figure 2A,B). To detect whether GPER activation changed intestinal inflammation, we examined neutrophil infiltration (MPO activity) and the expression of inflammatory markers TNF-α and Cox-2 in intestinal tissue. Enhanced MPO activity in jejunum tissues was observed in I/R injury, which was down-regulated by G-1 treatment (Figure 2C), indicating GPER activation inhibited the neutrophil infiltration. Consistent with MPO activity, the expression of TNF-α and Cox-2 in I/R group was significantly higher than that in sham-operation group. Treatment with G-1 significantly alleviated the increase in TNF-α and Cox-2 expression induced by I/R (Figure 2D,E).

G-1 protected against I/R-induced intestinal injury in mice

Figure 2
G-1 protected against I/R-induced intestinal injury in mice

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Representative images of intestinal histology (H&E staining, scale bar = 100 μm). (B) Intestinal mucosa damage scoring (Chiu’s score) (n=9). (C) Effect of G-1 administration on MPO activity in jejunum tissue (n=5). (D) Effect of G-1 administration on TNF-α expression in jejunum tissue (n=4). (E) Effect of G-1 administration on Cox-2 expression in jejunum tissue (n=4). Data were expressed as median or mean ± S.E.M. Statistical analyses were performed using the Kruskal–Wallis test or one-way ANOVA followed by Student–Newman–Keuls method (***P<0.001, **P<0.01, *P<0.05).

Figure 2
G-1 protected against I/R-induced intestinal injury in mice

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Representative images of intestinal histology (H&E staining, scale bar = 100 μm). (B) Intestinal mucosa damage scoring (Chiu’s score) (n=9). (C) Effect of G-1 administration on MPO activity in jejunum tissue (n=5). (D) Effect of G-1 administration on TNF-α expression in jejunum tissue (n=4). (E) Effect of G-1 administration on Cox-2 expression in jejunum tissue (n=4). Data were expressed as median or mean ± S.E.M. Statistical analyses were performed using the Kruskal–Wallis test or one-way ANOVA followed by Student–Newman–Keuls method (***P<0.001, **P<0.01, *P<0.05).

GPER activation restored the I/R-induced disruption of intestinal mucosal barrier

In order to observe the intestinal mucosal barrier, we tested the expression of tight junction proteins, occludin and ZO-1. In sham group ZO-1 was expressed in apical side of the membrane in linear fashion along the jejunal villi. After I/R treatment the continuity and linear expression of ZO-1 in the intestinal epithelium was interrupted or absent (Figure 3A). Similar to ZO-1, occludin normally distributed at the periphery of the intestinal epithelial cell at areas of cell–cell contact from the crypts to the villi. There was loss of occludin staining or confused staining at I/R group in intestinal villi (Figure 3B). The abnormal staining pattern of ZO-1 and occludin induced by I/R was alleviated by G-1 administration (Figure 3A,B). Moreover, the expression of ZO-1 and occlusion in I/R group was lower than that in sham-operation group, and the difference was abolished after G-1 treatment (Figure 3C,D).

G-1 treatment restored the impaired barrier function in I/R-induced mice

Figure 3
G-1 treatment restored the impaired barrier function in I/R-induced mice

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Effects of G-1 on the expression of ZO-1 using immunofluorescence assays (scale bar = 50 μm). (B) Effects of G-1 on the expression of occludin using IF assays (upper trace: scale bar = 50 μm, lower trace was the high-power view of the white dotted-line box: scale bar = 20 μm). (C) Effects of G-1 on the expression of ZO-1 using Western blot assays (n=4). (D) Effects of G-1 on the expression of occludin using Western blot assays (n=4). (E) Intestinal epithelial paracellular permeability evaluated by FITC-dextran concentration in plasma. Higher the concentration, higher the permeability (n=6). Data were expressed as mean ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls method (**P<0.01, *P<0.05).

Figure 3
G-1 treatment restored the impaired barrier function in I/R-induced mice

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Effects of G-1 on the expression of ZO-1 using immunofluorescence assays (scale bar = 50 μm). (B) Effects of G-1 on the expression of occludin using IF assays (upper trace: scale bar = 50 μm, lower trace was the high-power view of the white dotted-line box: scale bar = 20 μm). (C) Effects of G-1 on the expression of ZO-1 using Western blot assays (n=4). (D) Effects of G-1 on the expression of occludin using Western blot assays (n=4). (E) Intestinal epithelial paracellular permeability evaluated by FITC-dextran concentration in plasma. Higher the concentration, higher the permeability (n=6). Data were expressed as mean ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls method (**P<0.01, *P<0.05).

Tight junction proteins have been shown to seal the gap between gut epithelial cells and play an important role in protecting the mucosal permeability [45]. Thus, we evaluated the intestinal mucosal permeability by the plasma FITC-dextran concentration. In-line with the change in tight junction proteins, we found that the intestinal mucosal permeability increased significantly following I/R. The increased permeability was restored by pretreatment with G-1 (Figure 3E).

Expression and location of GPER in small intestine

First, we used Western blot to detect the GPER expression in the small intestine. Using brain as a positive control, we confirmed that jejunum expressed GPER (Figure 4A). In order to identify the potential cell target that GPER activation protected I/R injury, we observed the GPER expression in the jejunum with immunolabeling experiments in Lgr5-EGFP mice. As shown in Figure 4B, GPER-positive cells were observed in jejunal epithelium, villus stroma, while predominantly located in jejunal crypt. By labeling Lgr5+ ISCs with GFP in Lgr5-EGFP mice we found that there was GPER expression in the Lgr5+ ISCs (Figure 4B).

Expression and localization of GPER in jejunum

Figure 4
Expression and localization of GPER in jejunum

(A) GPER protein expression in jejunum as determined by Western blot. (B) IF detection of GPER from the Lgr5-EGFP mouse. The lgr5+ ISCs were marked by GFP. DAPI was used as a nuclear stain. The upper and lower left quarter trace, scale bar = 50 μm. Lower right quarter trace was the high-power view of the white dotted-line box, scale bar = 10 μm.

Figure 4
Expression and localization of GPER in jejunum

(A) GPER protein expression in jejunum as determined by Western blot. (B) IF detection of GPER from the Lgr5-EGFP mouse. The lgr5+ ISCs were marked by GFP. DAPI was used as a nuclear stain. The upper and lower left quarter trace, scale bar = 50 μm. Lower right quarter trace was the high-power view of the white dotted-line box, scale bar = 10 μm.

GPER activation protected intestinal crypt cell proliferation at I/R injury

In consideration of GPER expression in Lgr5+ ISCs and the crucial role of stem cell proliferation on the epithelial repair following intestinal mucosal injury [34,35], we tested the effect of GPER activation on crypt cell proliferative ability. It showed that the jejunal villus height, crypt depth, and TACs number (Ki67+ positive cells) were significantly decreased following I/R injury. Administration of G-1 ameliorated decreasing of villus height, crypt depth, and TACs number induced by I/R (Figure 5A–C). Moreover, BrdU incorporation assay [46] verified that G-1 could reverse I/R-induced S-phase cell reduction (Figure 5D). Thus, these results suggested that GPER activation might protect I/R injury via improving proliferative ability of crypt cell.

G-1 treatment alleviated the injury of crypt cell proliferation induced by I/R

Figure 5
G-1 treatment alleviated the injury of crypt cell proliferation induced by I/R

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Representative images of intestinal morphology showing the villus height and crypt depth (H&E staining, scale bar = 100 μm). (B) Summarizes the villus height, crypt depth, and villus height-crypt depth ratio in three groups (n=9). (C) Representative figures and statistical chart of Ki67+ (a marker of proliferation) cells in jejunum crypt. The numbers of Ki67-positive cells were counted in each crypt within the different groups (n=9). (D) Representative figures and statistical chart of BrdU incorporation in crypt in jejunum. The numbers of BrdU positive cells were counted in each crypt within the different groups (n=9). Data were expressed as mean ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls method (***P<0.001, **P<0.01, *P<0.05).

Figure 5
G-1 treatment alleviated the injury of crypt cell proliferation induced by I/R

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Representative images of intestinal morphology showing the villus height and crypt depth (H&E staining, scale bar = 100 μm). (B) Summarizes the villus height, crypt depth, and villus height-crypt depth ratio in three groups (n=9). (C) Representative figures and statistical chart of Ki67+ (a marker of proliferation) cells in jejunum crypt. The numbers of Ki67-positive cells were counted in each crypt within the different groups (n=9). (D) Representative figures and statistical chart of BrdU incorporation in crypt in jejunum. The numbers of BrdU positive cells were counted in each crypt within the different groups (n=9). Data were expressed as mean ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls method (***P<0.001, **P<0.01, *P<0.05).

GPER activation protected the proliferative ability of crypt cell via down-regulating iNOS expression

To further verify the underlying mechanism of GPER activation protected I/R, we tested the iNOS expression. Consistent with previous report [5], we found that the expression of iNOS in jejunal crypt increased significantly after I/R injury, while G-1 administration decreased the expression of iNOS in crypt (Figure 6A,B). The immunostaining in Lgr5-EGFP mice showed that iNOS was expressed in the intestinal crypt and positioned with Lgr5+ ISCs (Figure 6C), suggesting that Lgr5+ ISCs might be the target cells of GPER down-regulating iNOS expression.

G-1 treatment inhibited iNOS expression in I/R-injured mice

Figure 6
G-1 treatment inhibited iNOS expression in I/R-injured mice

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Effect of G-1 on the expression of iNOS proteins. iNOS expression was quantitated by Western blot. Data were expressed as mean ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls method (n=4, *P<0.05). (B) IHC staining of iNOS at three groups (scale bar = 20 μm). (C) IF detection of iNOS from the Lgr5-EGFP mouse. The lgr5+ ISCs were marked by GFP. DAPI was used as a nuclear stain (the upper and lower left quarter trace, scale bar = 20 μm. Lower right quarter trace was the high-power view of the white dotted-line box, scale bar = 10 μm).

Figure 6
G-1 treatment inhibited iNOS expression in I/R-injured mice

The mice were divided into three groups: sham, I/R vehicle, and I/R+G-1. (A) Effect of G-1 on the expression of iNOS proteins. iNOS expression was quantitated by Western blot. Data were expressed as mean ± S.E.M. Statistical analyses were performed using one-way ANOVA followed by Student–Newman–Keuls method (n=4, *P<0.05). (B) IHC staining of iNOS at three groups (scale bar = 20 μm). (C) IF detection of iNOS from the Lgr5-EGFP mouse. The lgr5+ ISCs were marked by GFP. DAPI was used as a nuclear stain (the upper and lower left quarter trace, scale bar = 20 μm. Lower right quarter trace was the high-power view of the white dotted-line box, scale bar = 10 μm).

To clarify the potential role of iNOS inhibition in I/R injury, we investigated the effect of selective iNOS inhibitor 1400W on I/R injury. It showed that after 1400W treatment, the mucosal injury score of I/R injury group was significantly lower (Figure 7A). Similar to the effect of G-1, 1400W also restored the decrease in TACs number and BrdU+ cells induced by I/R (Figure 7B,C).

Selective iNOS inhibitor 1400W treatment protected intestinal injury and enhanced the intestinal crypt cell proliferation in I/R injury model

Figure 7
Selective iNOS inhibitor 1400W treatment protected intestinal injury and enhanced the intestinal crypt cell proliferation in I/R injury model

(A) Representative images of intestinal histology (H&E staining, scare bar = 100 μm) and intestinal mucosa damage scoring (n=9). (B) Representative figures and statistical chart of Ki67+ cells in jejunum crypt. The numbers of Ki67 positive cells were counted in one crypt within the different groups (n=9). (C) Representative figures and statistical chart of BrdU incorporation in crypt in jejunum. The numbers of BrdU positive cells were counted in one crypt within the different groups (n=9). Data were expressed as median or mean ± S.E.M. Statistical analyses were performed using the Kruskal–Wallis test or one-way ANOVA followed by Student–Newman–Keuls method (***P<0.001, **P<0.01).

Figure 7
Selective iNOS inhibitor 1400W treatment protected intestinal injury and enhanced the intestinal crypt cell proliferation in I/R injury model

(A) Representative images of intestinal histology (H&E staining, scare bar = 100 μm) and intestinal mucosa damage scoring (n=9). (B) Representative figures and statistical chart of Ki67+ cells in jejunum crypt. The numbers of Ki67 positive cells were counted in one crypt within the different groups (n=9). (C) Representative figures and statistical chart of BrdU incorporation in crypt in jejunum. The numbers of BrdU positive cells were counted in one crypt within the different groups (n=9). Data were expressed as median or mean ± S.E.M. Statistical analyses were performed using the Kruskal–Wallis test or one-way ANOVA followed by Student–Newman–Keuls method (***P<0.001, **P<0.01).

Discussion

Intestinal I/R injury is an important clinical topic, which is a significant cause of morbidity and mortality in surgical patients. In the present study, we showed for the first time that the membrane ER, GPER was expressed in Lgr5+ ISCs in jejunum crypt of mice. In agreement with previous reports [18,19], we found that estrogen exerted an acute protective effect on intestinal I/R injury, while the GPER blocker G15 eliminated this effect, suggesting the key role of GPER. This notion was further supported by the observations that selective GPER agonist G-1 relieved morphological injury, neutrophil infiltration and intestinal mucosal barrier disruption in jejunum induced by I/R. Further studies confirmed that the salutary effect of GPER was related to protect the proliferation ability of crypt cell via inhibiting the up-regulation of iNOS expression.

Both nuclear ERs [47,48] and GPER [49,50] have been reported to be involved in the protective effect of estrogen on I/R injury, whereas the receptor-mediating effect of estrogen on intestinal I/R injury remains not to be elucidated. We found that GPER blocker G15 completely abolished the acute effect of estrogen on intestinal I/R, suggesting that GPER activation was involved in the protection of intestinal I/R injury. On the contrary, ICI182,780 did not affect the acute effect of estrogen on intestinal I/R injury. ICI182,780 binds to nuclear ERs and accelerates the degradation of ERs, and fully blocks the effects of exogenous estrogen on ER-α and ER-β non-selectively [51]. ER-α and ER-β have divergent functions in the body and even play opposite effects on some processes [52]. Therefore, these results suggested that the acute salutary effect of estrogen did not depend on nuclear ERs, but the exact roles of ER-α and ER-β in I/R injury were still unclear and needed to be clarified in future studies. On the other hand, ICI182,780 is known to bind GPER with a high affinity [53] and exert estrogenic actions via activation of GPER [41]. This also might be related with the inability of ICI182,780 to block the role of estrogen.

Inflammation and intestinal epithelial barrier disruption are key pathophysiological changes of intestinal I/R injury [6,7,11]. Therefore, we selected relevant parameters to characterize the effects of GPER activation on intestinal I/R injury. First, we confirmed that GPER activation alleviated the histological injury induced by I/R. In-line with histological results, G-1 treatment attenuated neutrophil infiltration following intestinal I/R treatment, represented by a marked decrease in MPO activity [43]. Reperfusion of the gut leads to production of a lot of inflammatory factors, such as TNF-α, IL-1β, IL-6, and Cox-2, which contribute to increase in vasopermeability, local intestine, and distant organ injury in I/R [54–56]. We found that the inflammatory markers TNF-α and Cox-2 in intestinal tissue in G-1 treated I/R group were significantly lower than those in I/R alone, suggesting that GPER activation attenuated the heightened inflammatory response. Loss of intestinal barrier function is a particularly dangerous complication of intestinal I/R injury, since the increased mucosa permeability induced by intestinal barrier disruption not only hinders the absorption of nutrients in the intestinal mucosa, but also is associated with life-threatening bacterial translocation from the gut [10,57]. So, next we observed the effect of GPER on intestinal barrier function. Transmembrane protein occludin [58] and intracellular protein ZO-1 [59] are key tight junction proteins in maintaining intestinal structure, which are essential for the integrity of the intestinal barrier by building the most apical structure and regulating paracellular permeability and polarity of the cell [45]. Besides alterations of expression level of tight junction proteins, change in their intracellular localization has been involved in disruption of intestinal barrier function [58–60]. Consistent with previous report [60], we found the alteration of the expression and distribution of tight junction protein in mice following I/R treatment. G-1 treatment significantly improved confused distribution of occludin and ZO-1, increased the reduced tight junction protein expression in I/R group, and then restored the permeability of mucosa. This finding agrees with those reported by Lu et al. [61], who found that GPER activation ameliorated the blood–brain barrier permeability in global cerebral ischemia via reducing tight junction disruption. Estrogen could reduce post-ischemic glomerular endothelial hyperpermeability at least in part through GPER activation [50]. Overall, our current study documented that GPER activation at ischemia period resulted in improvement of intestinal injury as observed by the normalized morphological appearance, the decreased neutrophil infiltration and expression of inflammatory markers, restoration of impaired barrier function.

GPER activation has been shown to promote the proliferation of various tumor cells [46,62,63], as well as bovine satellite cells [64] and primordial germ cells [37] with stem cell characteristics. After G-1 stimulation, the expression of CyclinD1 was up-regulated and the S-phase cells increased [46]. In our research we found that activation of GPER significantly improved the decrease in TACs number, as well as the cells in S-phase in I/R injury. In-line with this, H&E staining revealed that G-1 treatment improved the decrease in villus height and crypt depth in jejunum induced by I/R. The IF assay showed that GPER was predominantly expressed in jejunum crypt, where it was expressed in Lgr5+ ISCs. The stemness of Lgr5+ ISCs is a critical factor to maintain the intestinal homeostasis [33]. Increased loss of stem cell proliferation ability occurs in response to a variety of insults, and enhancing crypt cell proliferation contributes to restore intestinal epithelial barrier function [11,35,65]. Cultured Lgr5+ cells generated crypt–villus structures in vitro [66] and were expected to be transplanted for stem cell therapy to repair damaged epithelium [67]. Hogan et al. showed that the crypt size and the proliferative activity of epithelial cells in colon fluctuated slightly during the estrous cycle, suggesting a possible role of estrogen on epithelial cell proliferation [68]. Our present findings provided data first to confirm that GPER activation enhances crypt cell proliferation and protects the intestine from I/R injury by enhancing epithelial cell repair after I/R injury. The Lgr5+ ISCs might be the target cell of GPER acting on intestinal I/R injury. Nevertheless, the role of GPER in cell proliferation is still controversial. In some cells, GPER activation has been shown to inhibit cell proliferation [69,70]. This paradox may be related to cell type, animal and experimental method, suggesting cell or tissue-specific effect of GPER on proliferation.

To further explore the pathogenesis that GPER activation prompted stem cell proliferation, we tested the effect of GPER activation on iNOS expression in I/R injury. Since expression of iNOS increased during the intestinal I/R injury and inhibition of iNOS exerted salutary effect on intestinal I/R injury [5,71]. Furthermore, iNOS inhibition was related to the beneficial effect of GPER [72]. Our present research revealed that iNOS expression increased significantly in I/R injury model, which was inhibited by G-1 treatment. The up-regulation of iNOS could induce cell apoptosis or inhibit cell proliferation, oxidative stress, and apoptosis mechanism might be involved in this process [73]. Overexcessive NO induced by iNOS up-regulation reacts with superoxide anion and produces peroxy nitroso, which would damage structural macromolecules of cells like DNA, proteins and lipids, ultimately leading to reperfusion injury [3]. Our location studies showed that iNOS was located in Lgr5+ ISCs of crypt. Therefore, we suspected the decrease in crypt cell proliferation ability might be related with the up-regulation of iNOS expression during I/R injury and GPER might improve proliferative ability of crypt cell by inhibiting iNOS expression. Next research confirmed the speculation. We found that selective iNOS inhibitor 1400W up-regulated the number of Ki67+ and BrdU+ cells in the crypt following I/R injury and improved the intestinal mucosal injury score.

We should note that our study still has limitations. First, the present study confirmed that GPER activation might protect intestine from I/R injury by acting on Lgr5+ ISCs. However, immunohistochemical staining showed that Lgr5+ ISCs were not the only cell type expressing GPER in crypt, and GPER was also expressed in intestinal villus besides crypt. Therefore, we can not rule out that the effect of GPER may involve other potential mechanisms that need to be elucidated in the future. Second, we did not further explore the mechanism that GPER activation down-regulated iNOS expression. Third, we identified that acute GPER activation could protect the intestine from I/R injury, while the chronic effects of GPER have not been examined in the present study.

In summary, the present study demonstrated that GPER is located in the lgr5+ ISCs in crypt and its activation protects mice from intestinal I/R injury through prompting the crypt cell proliferation in vivo, which occurred at least in part by inhibiting the iNOS expression in stem cell. Stem cell proliferation is essential for the epithelial cell repair following intestinal injury. Therefore, GPER may become a new target for the treatment of intestinal I/R injury by triggering the healing process of intestinal I/R injury.

Clinical perspectives

  • Intestinal I/R injury is a clinical event with a high morbidity and mortality, but treatment remains non-specific. The destruction of intestinal mucosal barrier is one of the key risk factors for serious complications and even death induced by intestinal I/R injury.

  • This research for the first time demonstrates that GPER can alleviate the intestinal mucosal injury, inhibit the inflammatory response and restore the impaired intestinal mucosal barrier via protecting the crypt cell proliferation. The mechanism was at least partially related to inhibiting the up-regulation of iNOS in stem cell.

  • Since GPER activation lacks the feminizing effects associated with agonists of the classical nuclear ERs, selective activation of GPER may be a promising strategy for treatment of intestinal I/R injury by triggering the healing process of intestinal epithelium.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) [grant number 31771278]; the Natural Science Foundation of Shandong Province [grant numbers ZR2016HM51, ZR2012HM015]; and the Key Research and Development Program Foundation of Shandong Province [grant number 2017GSF218011].

Author contribution

S.Chai performed the experiments, collected and analyzed the data. K.L., W.F., T.L., Q.W., R.Z., S.Chen, G.C., and T.M. performed some experiments and collected the data. L.W. provided technical and material support. J.Z. revised the paper and provided financial support. C.L. revised the paper. B.X. designed this research, wrote the paper, and provided financial support. All authors reviewed and approved the manuscript.

Abbreviations

     
  • BrdU

    bromodeoxyuridine

  •  
  • Cox-2

    cyclooxygenase-2

  •  
  • CST

    cell signaling technology

  •  
  • ER

    estrogen receptor

  •  
  • E2

    17β-estrogen

  •  
  • GPER

    G protein–coupled ER

  •  
  • H&E staining

    Hematoxylin and Eosin staining

  •  
  • IF

    immunofluorescent

  •  
  • IHC

    immunohistochemistry

  •  
  • IL

    interleukin

  •  
  • iNOS

    inducible nitric oxide synthase

  •  
  • i.p.

    intraperitoneal injection

  •  
  • ISC

    intestinal stem cell

  •  
  • I/R

    ischemia/reperfusion

  •  
  • Lgr5

    leucine-rich repeat containing G-protein coupled receptor 5

  •  
  • Lgr5-EGFP

    lgr5-egfp-ires-CreERT2

  •  
  • MPO

    myeloperoxidase

  •  
  • TAC

    transit amplifying cell

  •  
  • TNF

    tumor necrosis factor

  •  
  • ZO-1

    Zonula-Occludens-1

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Supplementary data