Clopidogrel-Induced Gastric Mucosal Injury Associated with Endoplasmic Reticulum Stress in Rats

Summary

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.

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 factors^1,2. 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 injury^6,7. 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 safety^12,13,14. 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 w...

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 epitheli...

Materials

List of materials used in this article

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