In accordance with the Committee on the Ethics of Animal Experiments of Sichuan University (Permit Number: 20240611004), a total of 60 Kunming mice (18–22 g, 6–8 week-old, 30 male and 30 female) were purchased from Da-Shuo Biological Technology Co., Ltd. (Chengdu, China) and housed under standard conditions (room temperature: 23 °C $\pm$ 1 °C; relative humidity: 55% $\pm$ 5%) with a 12-h day/night cycle (lights on at 8:00 am). After acclimation to the housing conditions for 5 days, all mice were randomly divided into six groups (10 mice per group, half male and half female): control group, ethanol group, esomeprazole injection (Chia Tai Tianqing Pharmaceutical Group Co., Ltd., lot no. 201230148, Jiangsu, China) high dose (EHI, 6.06 mg/kg, ip), low dose (ELI, 3.03 mg/kg, ip) and esomeprazole enteric capsule (Chia Tai Tianqing Pharmaceutical Group Co., Ltd., lot no. 19062501, Jiangsu, China) high dose (EHO, 6.06 mg/kg, orally), low dose (ELO, 3.03 mg/kg, orally) treatment groups. The esomeprazole enteric capsule was dissolved in 0.9% sodium chloride. The dose selection for esomeprazole was determined based on animal equivalent doses, in accordance with the recommended daily human doses [11, 12].

All treatment groups were pre-treated with the corresponding dose of esomeprazole, once a day for 5 days. The control group and the ethanol group were given the same volume of saline. Two hours after the last pre-treatment, 100% ethanol 0.2 mL was administered orally to induce acute gastric injury, according to our preliminary experiments [12]. The control group received saline 0.2 mL orally. After 1 h from induction, all mice were euthanized with pentobarbital sodium (20 mg/mL, 150 mg/kg, intraperitoneally).

The gastric tissues of all mice were removed and opened along the greater curvature. Gastric ulcerative lesions were assessed by two blinded observers and photographed. On the mucosal side of the stomach, according to the diameter of gastric ulcer, the ulcer index (UI) was scored as follows: 0: no lesion; 1: ulcer area with the longest diameter $\le$1 mm; 2: the longest diameter is $>$1 mm and $\le$2 mm; 3: the longest diameter is $>$2 mm and $\le$3 mm; 4: the longest diameter is $>$3 mm and $\le$4 mm; 5: the longest diameter $>$4 mm [13].

The gastric juice pH value was determined with pH test paper.

Then, the gastric tissues were cut into 2 halves. One was kept in 10% Neutral formalin solution for further histological studies, and one-half was kept at –80 °C.

After fixation in 10% Neutral formalin solution for 24 h, gastric tissues were embedded in paraffin and cut into 4 µm sections. These sections were stained with hematoxylin and eosin (HE) and Schiff’s periodic acid (PAS) for histological evaluation. The morphology of the gastric tissue was observed blindly using a light microscope (DM250, LEICA, Germany), and gastric injury was graded from 0 (normal) to 4 (severe). Ulcer depth, bleeding, and inflammation were scored, respectively. Finally, the gastric injury score was obtained by adding the individual scores for each type.

PAS stain was used to assess the mucus production in the gastric tissue.

For the IHC assay, the 4-µm sections were incubated with primary receptor-interacting protein kinase1 (RIPK1) antibodies (dilution 1:50, Proteintech, USA) overnight at 4 °C, washed three times with phosphate-buffered saline (PBS), and then stained with secondary antibody (goat anti-rabbit IgG labeled by horseradish peroxidase (HRP)) for 4 hours. 3,3’-diaminobenzidine (DAB) was used for IHC signal detection after washing three times with PBS, then hematoxylin counterstaining for 2 min. Observe the images using an optical microscope (DM250, LEICA, Germany) and perform semi-quantitative analysis blindly through ImageJ software.

Gastric tissues were homogenized with the extracting solution and centrifuged at 4 °C, 10,000 rpm for 10 min, and the protein concentrations in supernatant (Cpr) were measured by BCA at 562 nm. Pepsin activity from gastric tissues was determined by ultraviolet spectroscopy. The opticle density (OD) values were detected at 275 nm using a spectrophotometer. Pepsin activity (U•mg prot^-1) = 1.31 $\times$ (A_test – A_control)/Cpr.

Blood samples of mice were centrifuged at 3000 g for 15 min at 4 °C, then serum was collected, and the concentrations of IL-6 and TNF-$\alpha$ were measured using commercial ELISA kits (Boster, Wuhan, China) according to the manufacturer’s protocol. The absorbance was measured at 450 nm using an iMark microplate reader (BIO-RAD).

Total proteins were extracted using radioimmunoprecipitation assay (RIPA) Lysis Buffer (Beyotime Institute of Biotechnology, China), and the protein concentrations were measured with a BCA protein assay kit (Beyotime Institute of Biotechnology, China). Equal amounts of total protein (40 µg/10 µL) were loaded onto 8% SDS-PAGE gels at 120 v for 60 min, electro-transferred to polyvinylidene difluoride (PVDF) membranes, blocked in 5% non-fat milk at room temperature for 2 h, and incubated with the diluted primary antibodies, including Caspase8 (Cell Signalling Technology, 4790S, 1:1000), RIPK1 (Proteintech, 17519-1-AP, 1:1000), and NF-$\kappa$B (Cell Signalling Technology, 3033S, 1:1000) at 4 °C overnight in turn. After washing three times with Tween-20 (TBST), the membranes were probed with horseradish peroxidase-conjugated secondary antibody (Anti-rabbit IgG, Cell Signalling Technology, 7074P2, 1:2000 or Anti-mouse IgG, Cell Signalling Technology, 7076P2, 1:2000) at room temperature for 2 h. $\beta$-actin (Cell Signalling Technology, 3700S, 1:1000) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signalling Technology, 97166S, 1:1000) was the loading control. The protein was detected by chemiluminescence reagent (GE Healthcare), and the protein band density was quantified using Image J software (version 1.53, National Institutes of Health, Bethesda, MD, USA).

All values were expressed as mean $\pm$ SEM, and statistically analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction (GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA)). p 0.05 was considered statistically significant.

To investigate the gender difference of the gastroprotective effect of esomeprazole, the mouse acute ethanol (EtOH)-induced model of gastric injury was established by intragastric administration of 100% EtOH. First, the gastric mucosal injury was observed in gross tissues, and the ulcer index (UI) was used to evaluate the extent of gastric injury. As shown in Fig. 1A, no visible lesions developed in the control group, but 100% EtOH markedly resulted in gastric mucosa ulcer and bleeding in female and male mice, manifested by an increased UI compared with the control group (p 0.05, Fig. 1B). Interestingly, esomeprazole injection and enteric capsule demonstrated different degrees of gastroprotective effects. In female mice, pre-treatment with ELI at 3.03 mg/kg (clinical equivalent dose 20 mg) significantly attenuated gastric mucosal injury as shown by decreased UI compared with EtOH group (p 0.05, Fig. 1B), whereas in male mice, similar reduction in UI occurred in pre-treatment with ELO at 3.03 mg/kg (p 0.05, Fig. 1B).

Fig. 1.

Effects of esomeprazole on the EtOH-induced gastric injury in mice. Representative images of the mouse stomach from (A) control, EtOH, EtOH + esomeprazole 6.06 mg/kg i.p (EtOH + EHI), EtOH + esomeprazole 3.03 mg/kg i.p (EtOH + ELI), and EtOH + esomeprazole 6.06 mg/kg gavage (EtOH + EHO), EtOH + esomeprazole 3.03 mg/kg gavage (EtOH + ELO). (B) The ulcer index was compared between mice of different genders. Significant differences were observed after administration of esomeprazole at the dose of 3.03 mg/kg i.p in female mice and 3.03 mg/kg p.o in male mice. ^# p 0.05, as compared to control mice, ^& p 0.05, as compared to EtOH-group, * p 0.05, as compared to EtOH + ELO-group. Results are expressed as mean $\pm$ SEM for n = 5 mice per gender group.

Next, histopathological evaluation of the damage in the gastric mucosa was performed using HE. In the control group, the gastric mucosa of mice was intact, and there was no inflammatory cell infiltration and mucosal hemorrhage (Fig. 2A). 100% EtOH induced an extensive disruption of the gastric mucosal layer with a marked presence of ulcerative plaque, gland atrophy, and hemorrhage, inflammatory cell infiltration in the mucosal and submucosal layer, with an increased gastric injury score compared with the control group (Fig. 2B). As compared to the EtOH group, two dose forms of esomeprazole significantly reduced EtOH induced gastric mucosal injury with a significant inhibition in ulcer/hemorrhage score and total score, except for the ELI group (left and right panel, Fig. 2B). However, there was obviously inhibition in inflammatory cell infiltration only in female mice (middle panel, Fig. 2B). Therefore, as shown in the right panel of Fig. 2B, we found pre-treatment with ELO at 3.03 mg/kg (clinical equivalent dose 20 mg) significantly prevented the presence of ulcerative plaque, hemorrhage, and inflammatory cell infiltration, especially in female mice (p 0.05). Furthermore, we compared the different dose form of esomeprazole at the dose of 3.03 mg/kg (clinical equivalent dose 20 mg) in female mice and found that both ulcer/hemorrhage scores and inflammation scores were lower in ELO than those in ELI (p 0.05, Fig. 3), suggesting there was a good response to esomeprazole enteric capsule 3.03 mg/kg in female mice.

Fig. 2.

Gastroprotective effect of esomeprazole on the EtOH-induced gastric injury in mice by histopathological evaluation. (A) Representative images of histopathological evaluation of the damage in the gastric mucosa stained with hematoxylin and eosin (HE) stains ($\times$20) from control, EtOH, EtOH + esomeprazole 6.06 mg/kg i.p (EtOH + EHI), EtOH + esomeprazole 3.03 mg/kg i.p (EtOH + ELI), and EtOH + esomeprazole 6.06 mg/kg gavage (EtOH + EHO), EtOH + esomeprazole 3.03 mg/kg gavage (EtOH + ELO). Scale bar: 0.1 mm. The slender arrow indicates mucosal damage, with altered villus morphology and disordered, shrunken cellular arrangement in the villi; the triangle, short, thick arrow denotes inflammation and bleeding, along with red blood cells. (B) Gastric injury scores include ulcer depth, bleeding, and inflammation score, were compared. ^# p 0.05, as compared to control mice; ^& p 0.05, as compared to EtOH-group; * p 0.05, as compared to the male group. Results are expressed as mean $\pm$ SEM for n = 5 mice per gender group.

Fig. 3.

Gastroprotective and anti-inflammatory effects of esomeprazole in the EtOH-induced gastric injury in female mice. (A) Gastric injury scores include ulcer depth, bleeding, (B) inflammation score and (C) total score were compared from control, EtOH, EtOH + esomeprazole 6.06 mg/kg i.p (EtOH + EHI), EtOH + esomeprazole 3.03 mg/kg i.p (EtOH + ELI), and EtOH + esomeprazole 6.06 mg/kg gavage (EtOH + EHO), EtOH + esomeprazole 3.03 mg/kg gavage (EtOH + ELO). * p 0.05, significant differences were observed after administration of esomeprazole at the dose of 3.03 mg/kg i.p and 3.03 mg/kg p.o in female mice; ^# p 0.05, as compared to control mice; ^& p 0.05, as compared to EtOH-group. Results are expressed as mean $\pm$ SEM for n = 5 mice per group.

To examine the gender different effect of esomeprazole on acid secretion and pepsin activity, we measured the pH value with pH test paper first and found the pH values of the EtOH group in both female and male mice were little difference compared with that of the control group (p $>$ 0.05, Fig. 4A). Interestingly, pretreatment with esomeprazole slightly increased the pH value in female mice, but slightly decreased it in male mice (p $>$ 0.05, Fig. 4A). However, EtOH-enhanced pepsin activity was significantly inhibited by pretreatment with esomeprazole in female and male mice (p 0.05, Fig. 4B). In detail, in female mice low dose of esomeprazole injection demonstrated the best efficacy, and in male mice, two dose forms of esomeprazole showed similar efficacy, except for ELO (Fig. 4B). These data also indicated that there was a gender difference in the effect of esomeprazole on the inhibition of gastric secretion.

Fig. 4.

Effects of esomeprazole on gastric acid secretion in the EtOH-induced gastric injury in mice. (A) pH value with pH test paper from control, EtOH, EtOH + esomeprazole 6.06 mg/kg i.p (EtOH + EHI), EtOH + esomeprazole 3.03 mg/kg i.p (EtOH + ELI), and EtOH + esomeprazole 6.06 mg/kg gavage (EtOH + EHO), EtOH + esomeprazole 3.03 mg/kg gavage (EtOH + ELO). (B) Pepsin activity was compared. Significant differences were observed after administration of esomeprazole EtOH + EHI, EtOH + ELI, and EtOH + ELO group in female mice, EtOH + EHI, EtOH + ELI, and EtOH + EHO in male mice. ^# p 0.05, as compared to control mice; ^& p 0.05, as compared to EtOH-group. Results are expressed as mean $\pm$ SEM for n = 5 mice per gender group.

Considering the gender difference in esomeprazole efficacy, we chose female mice as the animal model in the subsequent experiment. PAS staining was used to evaluate the content of gastric mucosal glycoprotein. As we expected, the PAS staining of the gastric mucus was weaker in the EtOH group than that in the control group, which was significantly increased in all esomeprazole pretreatment groups (Fig. 5A).

Fig. 5.

Effects of esomeprazole on serum inflammatory factors and gastric mucosal glycoprotein expression in the EtOH-induced gastric injury in female mice. (A) Representative PAS staining images of the mouse stomach from control, EtOH, EtOH + esomeprazole 6.06 mg/kg i.p (EtOH + EHI), EtOH + esomeprazole 3.03 mg/kg i.p (EtOH + ELI), and EtOH + esomeprazole 6.06 mg/kg gavage (EtOH + EHO), EtOH + esomeprazole 3.03 mg/kg gavage (EtOH + ELO). PAS staining of the gastric mucus was weaker in the EtOH group than in the control group; mucosal glycoprotein expression increased in all esomeprazole groups. Scale bar: 0.1 mm. (B) Serum concentrations of inflammatory factors IL-6 and TNF-$\alpha$ in mice were quantified with an enzyme-linked immunosorbent assay (ELISA) kit from control, EtOH, EtOH + esomeprazole 6.06 mg/kg i.p (EtOH + EHI), EtOH + esomeprazole 3.03 mg/kg i.p (EtOH + ELI), and EtOH + esomeprazole 6.06 mg/kg gavage (EtOH + EHO), EtOH + esomeprazole 3.03 mg/kg gavage (EtOH + ELO). ^# p 0.05, as compared to control mice; ^#⁢# p 0.05, as compared to control mice; * p 0.05, as compared to EtOH-group; *** p 0.05, as EtOH-ELO compared to EtOH-EHI group. n = 5 mice per female group.

Our studies demonstrated that the score of the EtOH-induced gastric injury tissues in female mice was significantly attenuated following esomeprazole pretreatment (p 0.05, Fig. 3). The histological analysis results showed that the gastric injury score, including ulcer depth, bleeding, and inflammation score were significantly increased in the EtOH group compared to the normal control group (p 0.05, Fig. 3). After pretreatment with esomeprazole, these scores decreased to varying degrees in both the injection and gavage groups, as well as the high-dose and low-dose groups. Interestingly, we found that compared to the low-dose injection group, the low-dose gavage group was more effective in improving gastric inflammation damage (p 0.05, Fig. 3). Plasma IL-6 and TNF-$\alpha$ concentrations, two effective biomarkers for inflammation, were measured by ELISA. As shown in Fig. 5B, compared to the control group, the EtOH group significantly increased plasma IL-6 concentrations in mice (p 0.05), and TNF-$\alpha$ showed no obvious change; and ELISA results also showed that esomeprazole significantly decreased IL-6 concentrations (p 0.05). Next, as expected, WB analysis showed that the important factors, including RIPK1 and NF-$\kappa$B levels in the inflammatory transmission pathway, were significantly increased in EtOH-treated mice, whereas notably ameliorated by 6.06, 3.03 mg/kg esomeprazole pretreatment (Fig. 6A). Our data also showed that EtOH markedly decreased caspase-8 expression in the gastric tissue, while EtOH +esomeprazole could increase anti-inflammation protein caspase level in contrast to EtOH mice (Fig. 6A). In addition, we found esomeprazole pretreatment (injection groups) suppressed RIPK1 expression by IHC assay, that consistent with previous studies (Fig. 6B).

Fig. 6.

Anti-inflammatory effects of esomeprazole expression via receptor-interacting protein kinase1 (RIPK1)/NF-$\kappa$B pathway in the EtOH-induced gastric injury in mice. (A) Representative Western blotting images of CASP-8, RIPK1, and NF-$\kappa$B in the mouse stomach. GAPDH was used as an internal standard (left panel); quantitative analysis from different groups by Image J software (2.1.0/1.53c) (right panel). (B) Representative IHC images of RIPK1 staining in the EtOH-induced gastric injury mice (n = 5) from control, EtOH, EtOH + esomeprazole 6.06 mg/kg i.p (EtOH + EHI), EtOH + esomeprazole 3.03 mg/kg i.p (EtOH + ELI), and EtOH + esomeprazole 6.06 mg/kg gavage (EtOH + EHO), EtOH + esomeprazole 3.03 mg/kg gavage (EtOH + ELO). Scale bar: 0.1 mm. ^# p 0.05, as compared to control mice; * p 0.05, as compared to EtOH group; ** p 0.05, as compared to EtOH group. n = 5 mice per group.