This study investigates gastric mucosal injury associated with clopidogrel treatment in rats and gastric epithelial cells, highlighting its association with endoplasmic reticulum stress–related molecular changes, apoptosis, and systemic inflammation.
Research Article
Clopidogrel-Induced Gastric Mucosal Injury Associated with Endoplasmic Reticulum Stress in Rats
* These authors contributed equally
In This Article
Summary
Abstract
Clopidogrel is widely used as an antiplatelet agent for the prevention and treatment of arterial thrombotic diseases, but its clinical application may be limited by gastrointestinal adverse effects, including gastric mucosal injury. Because epithelial cell survival is essential for maintaining gastric mucosal integrity, we hypothesized that clopidogrel-induced gastric epithelial injury would be accompanied by alterations in ER stress- and apoptosis-related markers. To test this hypothesis, GES-1 human gastric epithelial cells were treated with different concentrations of clopidogrel, and cell viability, apoptosis, cell cycle distribution, and molecular changes were evaluated using the Cell Counting Kit-8 (CCK-8) assay, flow cytometry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. In parallel, Sprague-Dawley rats were administered clopidogrel by gavage to establish an in vivo gastric injury model. Gastric mucosal pathological changes were assessed by hematoxylin and eosin staining, and serum inflammatory cytokine levels were measured using enzyme-linked immunosorbent assay (ELISA). Clopidogrel reduced GES-1 cell viability, promoted apoptosis, and altered cell cycle progression. In rats, clopidogrel induced gastric mucosal erosion, edema, and inflammatory cell infiltration, accompanied by increased serum interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) levels. Clopidogrel also increased the expression of the ER stress markers C/EBP homologous protein (CHOP) and activating transcription factor 5 (ATF5), increased Bcl-2-associated X protein (Bax) expression, decreased B-cell lymphoma 2 (Bcl-2) expression, and elevated the Bax/Bcl-2 ratio in both gastric tissues and GES-1 cells. These findings suggest that clopidogrel-induced gastric mucosal injury is associated with increased ER stress-related marker expression and disruption of the Bax/Bcl-2 balance, although a direct causal ER stress–mitochondrial apoptosis pathway was not established in this study.
Introduction
The gastric mucosa’s innate and adaptive immune systems, comprising epithelial cells, immune cells, and signaling molecules, work together to maintain mucosal integrity and serve as a critical physiological barrier against harmful factors1,2. This protective function is essential for gastrointestinal homeostasis, and its disruption can contribute to the development of gastric diseases, including gastritis3. Clinically, drug-induced gastric mucosal injury is a common complication, with nonsteroidal anti-inflammatory drugs and antiplatelet agents among the most frequent causes4. Gastric mucosal integrity depends on a dynamic balance between cell proliferation and apoptosis5. Chronic exposure to environmental factors, including medications, can disrupt this balance, promote apoptosis, and result in mucosal injury6,7. The endoplasmic reticulum plays a central role in maintaining intracellular homeostasis. Under conditions of cellular stress, the accumulation of misfolded or unfolded proteins activates the unfolded protein response, including PERK/eIF2α/ATF4/CHOP signaling, which may promote apoptosis and alter the expression of B-cell lymphoma 2 (Bcl-2) family proteins8. ATF5 is also involved in mitochondrial stress responses and the crosstalk between mitochondrial dysfunction and endoplasmic reticulum stress9. Recent clinical evidence indicates that continued antiplatelet therapy, including clopidogrel, is associated with progressive gastric and small-intestinal mucosal injury10.
Clopidogrel is a widely prescribed antiplatelet agent that requires metabolic activation before irreversibly binding to platelet P2Y12 receptors, thereby inhibiting adenosine diphosphate-mediated platelet aggregation and producing antithrombotic effects11. This bioactivation occurs primarily through hepatic cytochrome P450 enzymes. Therefore, direct exposure of cultured gastric epithelial cells to the clopidogrel parent compound does not reproduce the complete metabolic and pharmacokinetic context of clopidogrel treatment in vivo. However, increasing evidence suggests that clopidogrel treatment may be associated with recurrent gastric ulcers and gastrointestinal bleeding, highlighting the importance of balancing antithrombotic efficacy with gastrointestinal safety12,13,14. Although these adverse effects have been reported, the mechanisms underlying clopidogrel-associated gastric mucosal injury remain incompletely understood, limiting the development of targeted preventive and therapeutic strategies. In the present study, we investigated the effects of clopidogrel on the gastric mucosa using Sprague–Dawley rats and GES-1 cells. Rats received clopidogrel by gavage, and complementary in vitro experiments were performed using GES-1 cells. Histopathological, molecular, and cellular analyses were conducted to investigate whether clopidogrel-induced gastric mucosal injury is associated with endoplasmic reticulum stress and apoptosis. Accordingly, the present study focused on characterizing the molecular changes associated with clopidogrel-induced gastric injury rather than establishing a causal signaling pathway. Our findings provide additional insight into the molecular changes associated with clopidogrel-induced gastric mucosal injury and may contribute to future studies aimed at preventing and managing these adverse effects.
Protocol
All animal experimental procedures were reviewed and approved by the Lab of Animal Experimental Ethical Inspection of Dr. Can Biotechnology (Zhejiang) Co., Ltd. The study was conducted in accordance with the institutional guidelines for the care and use of laboratory animals. The animal ethics approval number was DRK-20251015210.
Animals and Cell Lines
The ethical approval covered the use of 20 SPF-grade Sprague–Dawley rats. A total of 12 male SPF-grade Sprague–Dawley rats, aged 6–8 weeks and weighing 280–300 g at the beginning of the experiment, were included in the present study. The rats were obtained from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China; Production License No. SCXK [Zhejiang] 2024-0004) and randomly assigned to the control and clopidogrel-treated groups, with six rats per group. Rats were housed in a controlled environment at approximately 22°C under a 12 h light/dark cycle, with free access to standard chow and sterilized drinking water. All rats were allowed to acclimatize for one week before the experiment. Clopidogrel hydrogensulfate was suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na) at a concentration of 0.781 mg/mL. Rats in the clopidogrel-treated group received clopidogrel hydrogensulfate by oral gavage at 10 mL/kg body weight (equivalent to 1 mL/100 g body weight and a dose of 7.81 mg/kg) once daily for 3 consecutive days. Rats in the control group received an equal volume of 0.5% CMC-Na. The dose and 3-day treatment duration were selected based on a previously reported short-term rat model of oral clopidogrel administration. Following the final gavage on day 3, food was withheld for 12 h, whereas water was provided ad libitum. The rats were anesthetized in an induction chamber with 3% isoflurane in oxygen, and anesthesia was maintained with 1.5%–2.0% isoflurane delivered through a nose cone. Adequate anesthetic depth was confirmed by the absence of corneal and pedal withdrawal reflexes. Blood was collected by terminal cardiac puncture under deep anesthesia. While remaining deeply anesthetized, the rats were euthanized by exsanguination followed by bilateral thoracotomy. Death was confirmed by the absence of spontaneous respiration and cardiac activity for at least 5 min, together with the absence of corneal and pedal withdrawal reflexes. The stomach was then rapidly removed, opened along the greater curvature, and gently rinsed with ice-cold saline.
The human gastric mucosal epithelial cell line GES-1 was obtained from iCell Bioscience Inc. (Shanghai, China; Cat. No. iCell-h062) and was authenticated by short tandem repeat profiling by the supplier. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified incubator at 37°C with 5% CO₂. Early-passage cells between passages 3 and 10 were used for the experiments. When cell confluence reached approximately 80%–90%, the cells were passaged at a split ratio of 1:3. Cells in the logarithmic growth phase were used for subsequent experiments. For the CCK-8 assay, cells were seeded into 96-well plates at 6 × 103 cells/well. For apoptosis and cell cycle analyses, cells were seeded into 6-well plates at approximately 5 × 105 cells/well and allowed to adhere before clopidogrel treatment.
Chemicals and Solutions
Clopidogrel hydrogensulfate (Cat. No. C874834; purity ≥ 99%) was used for the animal and cell experiments. A clopidogrel stock solution was prepared by dissolving 100 mg of clopidogrel powder in dimethyl sulfoxide (DMSO) to obtain a 1 M stock solution. The stock solution was further diluted to the required working concentrations before use. The 1 M stock solution was serially diluted with DMSO to prepare 1000× dosing solutions at concentrations of 10, 100, 200, 400, 600, 800, and 1000 mM. Immediately before treatment, each 1000× dosing solution was added to complete culture medium at a ratio of 1:1000 (v/v) to obtain final clopidogrel concentrations of 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM, respectively. The vehicle control group received an equal volume of DMSO without clopidogrel. Thus, the final DMSO concentration was maintained at 0.1% (v/v) in all groups. Cultured GES-1 cells do not reproduce hepatic cytochrome P450-mediated bioactivation of clopidogrel. Therefore, direct clopidogrel treatment was used as an exploratory parent-compound exposure model. The concentration range was selected to characterize concentration-dependent cellular responses under the experimental conditions and should not be interpreted as equivalent to clinically achievable plasma or gastric tissue concentrations.
Cell Subculture
Complete medium was prepared by supplementing basal culture medium with FBS and penicillin–streptomycin. When cell confluence reached approximately 80%, the culture medium was removed, and the cells were washed three times with sterile phosphate-buffered saline (PBS). Cells were then digested with trypsin–ethylenediaminetetraacetic acid (EDTA) solution for approximately 1–2 min. After partial cell detachment, complete medium was added to terminate digestion. The cell suspension was centrifuged at 1,200 × g for 3 min. After removal of the supernatant, the cell pellet was resuspended in 1–2 mL of complete medium and seeded into new culture dishes according to the experimental requirements.
CCK-8 Cell Viability Assay
GES-1 cells in the logarithmic growth phase were seeded into 96-well plates at 6 × 103 cells/well and cultured overnight to allow cell attachment. After adherence, cells were treated with different concentrations of clopidogrel (0, 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mM) for 24 h. The control group received the same final concentration of vehicle. After treatment, CCK-8 solution was added to each well and incubated at 37°C according to the kit instructions. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as a percentage of the control group. Based on the concentration–response results, treatment with 0.1 mM clopidogrel for 24 h was selected for the subsequent apoptosis, cell cycle, qPCR, and Western blot analyses.
Flow Cytometry Analysis
GES-1 cells were seeded into 6-well plates and treated with 0.1 mM clopidogrel for 24 h after cell attachment. Following treatment, both floating and adherent cells were collected. The adherent cells were digested with trypsin without EDTA, washed twice with cold PBS, and resuspended in binding buffer. Cells were stained using an Annexin V-FITC/PI Apoptosis Kit according to the manufacturer’s instructions. After incubation at room temperature in the dark, apoptotic cells were detected using a RaiseCyte 2L6C flow cytometer. Data were acquired using the RaiseFlower acquisition software and analyzed using FlowJo software (version 10.8.1). Early apoptotic, late apoptotic, and total apoptotic cell populations were quantified using FlowJo software.
Flow Cytometric Analysis of Cell Cycle Distribution
GES-1 cells were seeded into 6-well plates and treated with 0.1 mM clopidogrel for 24 h. After treatment, cells were harvested, washed with PBS, and fixed in pre-cooled 70% ethanol at 4°C. The fixed cells were then washed to remove residual ethanol and incubated with RNase A and propidium iodide staining solution in the dark according to the manufacturer's instructions. Cell cycle distribution was analyzed using a flow cytometer. Data were acquired using the acquisition software and analyzed using FlowJo software. Cell cycle distributions were fitted using the Watson model, and the percentages of cells in the G0/G1, S, and G2/M phases were calculated.
Quantitative Real-Time PCR (qPCR)
Total RNA was extracted from GES-1 cells and rat gastric tissue samples using an RNA extraction kit according to the kit instructions. For tissue samples, gastric mucosal tissues were rapidly collected, homogenized under low-temperature conditions, and subjected to RNA extraction. RNA concentration and purity were determined by measuring the absorbance at 260 and 280 nm. Equal amounts of RNA were reverse transcribed into complementary DNA (cDNA) using a reverse transcription kit with genomic DNA removal. Quantitative real-time PCR was performed using a SYBR Green-based PCR master mix. Each reaction was performed in a total volume of 20 µL containing 10 µL of 2× master mix, 1 µL of cDNA, 0.4 µL of forward primer, 0.4 µL of reverse primer, and 8.2 µL of RNase-free ddH₂O. The thermal cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The expression levels of CHOP, ATF5, Bcl-2, and Bax were determined. GAPDH was used as the internal reference gene. Relative gene expression was calculated using the 2−ΔΔCt method. Primer sequences are listed in Supplementary Table 1.
Western Blot
Total protein was extracted from GES-1 cells and rat gastric tissue samples. For cell samples, treated GES-1 cells were washed with cold PBS and lysed using radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor. For tissue samples, gastric mucosal tissues were homogenized in RIPA buffer under low-temperature conditions and then centrifuged to collect the supernatant. Protein concentrations were determined using a BCA protein assay. A total of 20 µg of protein per lane was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with bovine serum albumin (BSA), the membranes were incubated overnight at 4°C with primary antibodies against CHOP (1:1000), ATF5 (1:1000), Bcl-2 (1:1000), Bax (1:1000), and β-actin (1:5000). After washing, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:10000). Protein bands were visualized using an enhanced chemiluminescence reagent and captured with a chemiluminescence imaging system. Band intensities were quantified using image analysis software and normalized to β-actin.
Histopathological Evaluation
Gastric tissue samples were collected from Sprague–Dawley rats after clopidogrel treatment and fixed in 10% neutral buffered formalin. The samples were dehydrated, cleared, embedded in paraffin, and sectioned at a thickness of 4 µm. Paraffin sections were stained with hematoxylin and eosin. Histopathological changes were evaluated qualitatively under a light microscope based on mucosal morphology, epithelial integrity, edema, and inflammatory cell infiltration. Representative images were captured. No histological scoring system was applied.
Immunohistochemistry
Paraffin-embedded gastric tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Heat-induced antigen retrieval was performed in citrate buffer. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min, and nonspecific binding was blocked with normal goat serum for 30 min at room temperature. The sections were then incubated overnight at 4°C with primary antibodies against CHOP, ATF5, Bcl-2, and Bax. After washing with PBS, the sections were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody. Immunoreactive signals were visualized using DAB chromogenic solution, followed by counterstaining with hematoxylin. The sections were dehydrated, cleared, mounted, and examined under a light microscope. Representative images were captured, and the positive staining area was quantified using image analysis software.
Immunofluorescence Staining
Paraffin-embedded gastric tissue sections were deparaffinized, rehydrated, and subjected to heat-induced antigen retrieval in citrate buffer. After blocking with normal goat serum for 30 min, the sections were incubated overnight at 4°C with primary antibodies against CHOP (1:50), ATF5 (1:100), Bcl-2 (1:50), and Bax (1:200). After washing with PBS, the sections were incubated with the corresponding fluorescent secondary antibodies (1:500) for 1 h at room temperature in the dark and then counterstained with DAPI. Images were captured using a fluorescence microscope at ×200 magnification. Three randomly selected, non-overlapping fields from each animal were analyzed using Image-Pro Plus 6.0, and fluorescence intensity was expressed as IOD/Area. Three animals per group were included in the analysis.
Enzyme-Linked Immunosorbent Assay (ELISA)
Blood samples were collected from Sprague–Dawley rats after treatment and allowed to clot at room temperature. Serum was separated by centrifugation and stored at −80°C until analysis. The serum levels of IL-1β, IL-6, and TNF-α were measured using ELISA kits according to the kit instructions. The absorbance was measured using a microplate reader, and cytokine concentrations were calculated according to the standard curves.
Statistical Analysis
Statistical analyses were performed using statistical analysis software. Data are presented as the mean ± SD. Normality and homogeneity of variance were assessed using the Shapiro–Wilk test and Levene’s test, respectively. Comparisons between two groups were performed using Student's t-test. Comparisons among multiple groups were performed using one-way analysis of variance followed by Dunnett’s post hoc test. A value of P < 0.05 was considered statistically significant. All cell experiments were performed using at least three independent biological replicates.
Results
Clopidogrel Reduces GES-1 Cell Viability and Increases Apoptosis In Vitro
We first investigated whether clopidogrel affects GES-1 cell viability and apoptosis. CCK-8 assays showed that clopidogrel treatment significantly reduced the viability of GES-1 cells (Figure 1A,B), indicating reduced cell viability. Based on the overall reduction in cell viability across the tested concentration range, 0.1 mM clopidogrel for 24 h was selected for all subsequent cell experiments. Flow cytometry was subsequently performed to analyze apoptosis and cell cycle distribution (Figure 1C–F). Flow cytometric analysis showed an increased proportion of cells in the S phase, accompanied by a reduced G0/G1 population and no evident increase in the G2/M population, indicating S-phase accumulation rather than G2/M-phase arrest. The results demonstrated that clopidogrel significantly increased apoptosis and altered cell cycle distribution in GES-1 cells compared with the control group. Collectively, these findings indicate that clopidogrel reduces GES-1 cell viability and is associated with increased apoptosis and changes in cell cycle progression. These findings demonstrate cytotoxic responses following direct exposure to the clopidogrel parent compound under the selected experimental conditions. They do not establish that comparable effects occur at clinically relevant exposure levels or that the observed responses are mediated by the active clopidogrel metabolite.
Figure 1. Clopidogrel reduces GES-1 cell viability and increases apoptosis. (A) Cell viability of the human gastric epithelial cell line GES-1 was assessed using the CCK-8 assay following treatment with increasing concentrations of clopidogrel (0–1.0 mM) for 24 h. (B) Quantitative comparison of cell viability between the control group and the group treated with 0.1 mM clopidogrel for 24 h. (C) Representative flow cytometric cell-cycle profiles analyzed using the Watson model. (D) Quantification of the percentages of cells in the G0/G1, S, and G2/M phases. Clopidogrel treatment increased the S-phase population without an evident increase in the G2/M population. (E) Representative flow cytometry dot plots of apoptosis following Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. (F) Quantification of the total apoptotic cell population. All cell experiments were performed using three independent biological replicates (n = 3). Data are presented as the mean ± standard deviation (SD). Statistical significance was determined by comparison with the control group. ***P < 0.001. Please click here to view a larger version of this figure.
Clopidogrel-induced gastric injury is associated with systemic inflammation
Sprague–Dawley rats were randomly divided into two groups (n = 6 per group): a control group and a clopidogrel-treated group that received daily gavage. The clopidogrel dose and 3-day treatment regimen were selected based on a previously reported rat model of clopidogrel-induced gastric injury16. After 3 days, gastric tissues were collected for hematoxylin and eosin (H&E) staining. Histopathological examination revealed gastric mucosal erosions, edema, and inflammatory cell infiltration in the clopidogrel-treated group compared with the control group (Figure 2A). Serum inflammatory cytokine levels were measured by ELISA. The clopidogrel-treated group showed significantly higher serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) than the control group (Figure 2B–D). These findings indicate that clopidogrel treatment is associated with gastric mucosal injury and increased systemic inflammatory responses15,16.
Figure 2. Clopidogrel induces gastric mucosal injury and increases circulating inflammatory cytokine levels in rats. (A) Representative hematoxylin and eosin (H&E)-stained sections of gastric mucosa from control and clopidogrel-treated Sprague–Dawley rats. Histological examination shows preserved gastric mucosal architecture in the control group and morphological alterations in the clopidogrel-treated group. Yellow arrows indicate gastric mucosal epithelial cells, red arrows indicate focal congestion within the mucosal layer, and black arrows indicate mild inflammatory cell infiltration. Original magnification, ×200; scale bar = 100 µm. n = 6 animals per group. (B–D) Serum concentrations of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA). Data are presented as the mean ± SD; n = 6 animals per group. ***P < 0.001 compared with the control group. Please click here to view a larger version of this figure.
Clopidogrel alters ER stress- and apoptosis-related markers in rat gastric mucosa
To investigate molecular changes associated with clopidogrel-induced gastric injury, immunohistochemical staining was performed on gastric tissue sections. Representative images showed stronger CHOP, ATF5, and Bax staining in the clopidogrel-treated group than in the control group, whereas Bcl-2 staining appeared weaker (Figure 3A). Quantitative analysis of IOD/area demonstrated that CHOP, ATF5, and Bax staining was significantly increased following clopidogrel treatment (Figure 3B,C,E). Bcl-2 staining showed a decreasing trend, although the difference did not reach statistical significance (Figure 3D). These results indicate that clopidogrel treatment is associated with altered expression of stress- and apoptosis-related proteins in rat gastric mucosa. Immunofluorescence analysis showed stronger CHOP and ATF5 signals in gastric mucosal tissue from the clopidogrel-treated group. Quantitative analysis confirmed significant increases in CHOP (Figures 4A and 5A) and ATF5 (Figures 4B and 5B), whereas Bcl-2 (Figures 4C and 5C) and Bax (Figures 4D and 5D) showed decreasing and increasing trends, respectively, without reaching statistical significance. Together, these findings indicate that clopidogrel treatment is associated with altered expression of ER stress- and apoptosis-related markers in rat gastric mucosa.
Figure 3. Immunohistochemical detection of endoplasmic reticulum stress- and apoptosis-related proteins in gastric tissue. (A) Representative immunohistochemical staining images of C/EBP homologous protein (CHOP), activating transcription factor 5 (ATF5), B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) in gastric mucosal tissue from control and clopidogrel-treated Sprague–Dawley rats. Brown staining indicates positive immunoreactivity, and nuclei were counterstained with hematoxylin. The images shown are representative of all samples analyzed, with three animals included in each group. Original magnification, ×200; scale bar = 100 µm. (B–E) Quantitative analysis of integrated optical density per unit area (IOD/Area) for CHOP, ATF5, Bcl-2, and Bax, respectively. Data are presented as the mean ± SD. *P < 0.05 and **P < 0.01 compared with the control group. Please click here to view a larger version of this figure.
Figure 4. Immunofluorescence detection of endoplasmic reticulum stress- and apoptosis-related proteins in gastric tissue. Representative immunofluorescence images showing the localization of C/EBP homologous protein (CHOP), activating transcription factor 5 (ATF5), B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) in gastric mucosal tissue from control and clopidogrel-treated Sprague–Dawley rats. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue), and positive immunofluorescence staining is shown in red. Merged images are shown in the right column. (A) CHOP. (B) ATF5. (C) Bcl-2. (D) Bax. Original magnification, ×200; scale bar = 100 µm. The images shown are representative of all samples analyzed, with n = 3 animals per group. Please click here to view a larger version of this figure.
Figure 5. Quantitative analysis of immunofluorescence for endoplasmic reticulum stress- and apoptosis-related proteins in gastric tissue. (A–D) Quantification of integrated optical density per unit area (IOD/Area) for C/EBP homologous protein (CHOP), activating transcription factor 5 (ATF5), B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) in gastric mucosal tissue from control and clopidogrel-treated Sprague–Dawley rats. For each animal, three randomly selected non-overlapping microscopic fields per tissue section were analyzed using image analysis software, and the mean value was used for statistical analysis. Data are presented as the mean ± SD; n = 3 animals per group. *P < 0.05 and **P < 0.01 compared with the control group. Please click here to view a larger version of this figure.
Clopidogrel alters ER stress- and apoptosis-related gene and protein expression in GES-1 cells and rat gastric tissue
Previous studies have indicated that ER stress plays a key role in cellular stress responses and apoptosis17,18. To evaluate changes in ER stress-related markers following clopidogrel treatment, the expression of CHOP and ATF5 was examined in GES-1 cells. qRT-PCR demonstrated increased mRNA expression of CHOP and ATF5 following clopidogrel treatment. Western blot analysis further confirmed increased CHOP and ATF5 protein expression (Figure 6A–E). The expression of apoptosis-related proteins was subsequently evaluated in GES-1 cells and rat gastric tissue. Compared with the control group, clopidogrel treatment increased Bax expression and decreased Bcl-2 expression, resulting in an increased Bax/Bcl-2 ratio (Figure 6A–J). These expression patterns were consistently observed at both the mRNA and protein levels in GES-1 cells and rat gastric tissue. Together, these findings demonstrate that clopidogrel treatment is associated with altered expression of ER stress- and apoptosis-related markers in both experimental models.
Figure 6. Expression of endoplasmic reticulum stress- and apoptosis-related markers in GES-1 cells and rat gastric tissue following clopidogrel treatment. (A–D) Relative messenger RNA (mRNA) expression of C/EBP homologous protein (CHOP), activating transcription factor 5 (ATF5), B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) in the human gastric epithelial cell line GES-1 determined by quantitative real-time polymerase chain reaction (qRT-PCR). (E) Representative Western blot images with corresponding densitometric quantification of CHOP, ATF5, Bcl-2, and Bax protein expression in GES-1 cells. Protein expression levels were normalized to β-actin. Cell experiments were performed using three independent biological replicates (n = 3). (F–I) Relative mRNA expression of CHOP, ATF5, Bcl-2, and Bax in rat gastric tissue determined by qRT-PCR. (J) Representative Western blot images with corresponding densitometric quantification of CHOP, ATF5, Bcl-2, and Bax protein expression in rat gastric tissue. Protein expression levels were normalized to β-actin. Tissue experiments included n = 3 animals per group. Data are presented as the mean ± SD. Statistical significance was determined by comparison with the control group. *P < 0.05, **P < 0.01, and ***P < 0.001. Please click here to view a larger version of this figure.
Collectively, the results demonstrate that clopidogrel treatment reduced GES-1 cell viability, increased apoptosis, altered the expression of ER stress- and apoptosis-related markers, and was associated with gastric mucosal injury accompanied by increased circulating inflammatory cytokine levels in rats. These findings support the study hypothesis that clopidogrel treatment is associated with gastric mucosal injury and concurrent changes in ER stress- and apoptosis-related markers.
Data Availability:
All data generated or analyzed during this study are included in this published article and its supplementary materials. The primer sequences used for RT-PCR are provided in Supplementary Table 1. The raw data supporting all figures and individual figure panels, including original experimental datasets and source files, are provided in the accompanying Raw Data supplementary archive (zipped file).
Supplementary Table 1. Primer sequences used for RT-PCR analysis. Forward (F) and reverse (R) primer sequences used for RT-PCR analysis of GAPDH, CHOP, ATF5, Bcl-2, and Bax in this study are listed in the 5′–3′ orientation.Please click here to download this file.
Discussion
Clopidogrel is widely used for the prevention and treatment of cardiovascular disease; however, its clinical use is associated with bleeding risks and gastrointestinal complications, including peptic ulcers, bleeding, and perforation19. Therefore, understanding the molecular changes associated with clopidogrel-induced gastric injury is important for improving the prevention and management of these adverse effects. In the present study, we investigated the effects of clopidogrel on gastric epithelial cells and rat gastric tissue and observed changes in cell viability, apoptosis, inflammatory responses, and the expression of ER stress- and apoptosis-related markers. These findings suggest that clopidogrel-induced gastric mucosal injury is associated with alterations in ER stress- and apoptosis-related marker expression. However, because no pharmacological rescue or genetic loss-of-function experiments were performed, these findings do not establish a causal relationship between ER stress and apoptosis.
Previous studies have indicated that clopidogrel may impair gastric mucosal repair and delay gastric ulcer healing20. In the present study, CCK-8 assays showed that clopidogrel reduced the viability of GES-1 cells, and flow cytometry demonstrated increased apoptosis and altered cell cycle progression (Figure 1). These findings are consistent with previous reports of clopidogrel-induced apoptosis in gastric epithelial cells21. These observations suggest that clopidogrel treatment is associated with impaired gastric epithelial cell viability and increased apoptosis, which may contribute to gastric mucosal injury11. In the rat model, H&E staining demonstrated gastric mucosal injury following clopidogrel treatment (Figure 2A). Serum levels of IL-1β, IL-6, and TNF-α were also significantly increased (Figure 2B–D), indicating an inflammatory response associated with gastric mucosal injury22. In addition, immunohistochemical and immunofluorescence analyses demonstrated altered expression of ER stress- and apoptosis-related markers in gastric tissue from clopidogrel-treated rats compared with the control group (Figure 3, Figure 4, Figure 5).
CHOP, ATF5, Bcl-2, and Bax are important regulators of cellular stress responses and apoptosis23,24,25,26. In this study, clopidogrel treatment was associated with increased expression of CHOP and ATF5 and altered expression of Bax and Bcl-2 in both GES-1 cells and rat gastric tissue (Figure 6). These findings indicate concurrent alterations in stress- and apoptosis-related markers. However, the present study evaluated expression changes only and did not determine whether these molecules directly mediate clopidogrel-induced gastric mucosal injury. Likewise, although an increased Bax/Bcl-2 ratio was observed, the study did not directly demonstrate activation of the mitochondrial apoptosis pathway. Therefore, these findings should be interpreted as associations rather than evidence of a causal signaling mechanism.
The present study combines an in vivo rat model with exploratory direct-exposure experiments in GES-1 cells to evaluate the morphological and molecular changes associated with clopidogrel treatment. The in vivo model provides the primary evidence of gastric mucosal injury, whereas the GES-1 experiments provide supportive evidence regarding cellular responses following direct exposure to the parent compound. The combination of histopathological analysis, inflammatory cytokine measurement, immunohistochemistry, immunofluorescence, qRT-PCR, and Western blotting enables the comprehensive assessment of morphological and molecular changes associated with gastric injury. This experimental approach may be useful for investigating the gastrointestinal effects of antiplatelet agents and for evaluating potential protective interventions in future studies.
Several limitations should be acknowledged. Clopidogrel is a prodrug that requires hepatic metabolic activation, whereas direct exposure of GES-1 cells to the parent compound does not reproduce this process or fully reflect clinical pharmacokinetics. In addition, the relatively high concentrations used in vitro may have caused nonspecific cytotoxicity. Therefore, the GES-1 experiments should be regarded as exploratory evidence of cellular responses under direct exposure conditions. The CCK-8 assay did not include a positive control for reduced cell viability, limiting independent verification of assay performance. The upstream mechanisms responsible for the observed ER stress-related changes were not investigated, and pharmacological inhibition, gene knockdown, or gene knockout experiments were not performed to determine whether ER stress directly regulates the Bax/Bcl-2 balance and apoptosis. Future studies should compare the parent compound with its active metabolite, incorporate an appropriate metabolic activation system, and perform functional intervention experiments to clarify these relationships. In addition, a well-established apoptosis-inducing positive control was not included in the Annexin V-FITC/PI assay. Therefore, although clopidogrel treatment was associated with an increased Annexin V-positive cell population compared with the vehicle control, the technical performance of the apoptosis assay was not independently verified using a positive-control condition. Future studies should include an apoptosis inducer, such as staurosporine, in the same experimental batch.
In conclusion, oral clopidogrel treatment caused gastric mucosal injury in rats and was accompanied by changes in ER stress- and apoptosis-related markers. Direct exposure of GES-1 cells to the clopidogrel parent compound also reduced cell viability and altered related molecular markers under the experimental conditions. However, because the cell model does not reproduce hepatic bioactivation and relatively high concentrations were used, the in vitro findings cannot be directly extrapolated to clinical exposure, and nonspecific cytotoxicity cannot be excluded. Overall, the findings indicate that clopidogrel-induced gastric mucosal injury is associated with alterations in ER stress- and apoptosis-related marker expression, although further mechanistic studies are required to establish causal relationships.
Disclosures
Conflict of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study.
Acknowledgements
This work was supported by the project “Fundamental Research on the CHOP/ATF5/Bcl-2 Signaling Pathway in Clopidogrel-Induced Gastric Mucosal Epithelial Cell Apoptosis” (Project No. 2023BWKY009).
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Annexin V-FITC/PI Apoptosis Detection Kit | Elabscience | E-CK-A211 | Apoptosis detection kit |
| ATF5 Antibody | Proteintech | 67066-1-AP | Primary antibody |
| BCA Protein Assay Kit | Solarbio | PC0020 | Protein concentration assay |
| Bax Antibody | Proteintech | 50599-1-AP | Primary antibody |
| Bcl-2 Antibody | Proteintech | 26593-1-AP | Primary antibody |
| β-actin Antibody | Proteintech | 66009-1-Ig | Primary antibody |
| Blocking Buffer | Beyotime | P0252 | Western blot blocking solution |
| CCK-8 Cell Counting Kit | KeyGen Biotech | KGA317 | Cell viability assay |
| cDNA Synthesis Kit | TransGen Biotech | AU341 | First-strand cDNA synthesis kit with genomic DNA removal |
| Cell Cycle Detection Kit | Lianke Biotechnology | CCS012 | Cell cycle analysis kit |
| CHOP Antibody | Proteintech | 15204-1-AP | Primary antibody |
| Clopidogrel Hydrogensulfate (Purity ≥99%) | Macklin | C874834 | Experimental drug |
| DAB Chromogen Kit | Beyotime | P0201M | Immunohistochemistry substrate |
| Dimethyl Sulfoxide (DMSO) | MedChemExpress | HY-Y0320C | Solvent |
| Enhanced Chemiluminescence (ECL) Reagent | Meilunbio | MA0186 | Chemiluminescent substrate |
| Fetal Bovine Serum (FBS) | Gibco | 10099-141 | Cell culture supplement |
| GES-1 Human Gastric Epithelial Cell Line | iCell Bioscience | iCell-h062 | Human gastric epithelial cell line |
| Hematoxylin and Eosin (H&E) Staining Kit | Beyotime | C0105S | Histological staining |
| Horseradish Peroxidase (HRP)-Conjugated Secondary Antibody | Proteintech | RGAM001/RGAR001 | Secondary antibody |
| Hydrogen Peroxide Solution | Absin | ABS9333-100mL | Endogenous peroxidase blocking reagent |
| IL-1β ELISA Kit | MEIMIAN | MM-0047R2 | Serum cytokine assay |
| IL-6 ELISA Kit | MEIMIAN | MM-0190R2 | Serum cytokine assay |
| Microplate Reader | Beijing Liuyi Biotechnology | WD-2102B | Absorbance measurement |
| Penicillin-Streptomycin Solution | Solarbio | P1400 | Antibiotic solution |
| Phosphate-Buffered Saline (PBS) | Servicebio | G4202-500ML | 1× PBS |
| Polyvinylidene Difluoride (PVDF) Membrane | Millipore | IPVH00010 | Western blot membrane |
| Primary Antibody Diluent | Beyotime | P0023A | Antibody diluent |
| Protein Molecular Weight Marker | Yeasen | WJ103 | Prestained protein ladder |
| Quantitative Real-Time PCR Master Mix (SYBR Green) | CWBio | CW3008 | qPCR reagent |
| Radioimmunoprecipitation Assay (RIPA) Lysis Buffer | Solarbio | R0010 | Protein extraction buffer |
| RNase A | Lianke Biotechnology | CCS012 | Cell cycle analysis reagent |
| RPMI-1640 Medium | Servicebio | G4535-500ML | Basal cell culture medium |
| Sprague-Dawley Rats | Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. | N/A | SPF grade |
| TNF-α ELISA Kit | MEIMIAN | MM-0180R2 | Serum cytokine assay |
| Total RNA Extraction Kit | TransGen Biotech | ER501-01 | RNA extraction kit |
| Trypsin-EDTA Solution | Solarbio | T1300 | Cell dissociation reagent |
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