INTRODUCTION
With global warming, the morbidity and mortality of heatstroke have increased in recent years [
1,
2]. Clinically, heatstroke is characterized by extreme hyperthermia (core temperature ≥ 40 °C), central nervous system dysfunction, and multiple organ failure. The intestine is one of the primary target organs involved in the pathophysiological process of heatstroke. The intestine is considered to be the largest reservoir of bacteria and toxins in the body. Approximately 300–500 different types of bacteria are present in the human intestine [
3]. Endotoxins and pathogens can enter the circulatory system of the body through an impaired intestinal barrier, inducing endotoxemia and multiple organ dysfunction syndrome (MODS) [
4]. However, the underlying mechanisms of intestinal injury in heatstroke remain poorly understood.
The mechanical barrier of the intestine is composed of intestinal epithelial cells, tight junctions (TJs), and a mucus layer covering the epithelial cell surface [
5]. The intestinal mucosal epithelium provides a vital barrier against luminal pathogens. Endoplasmic reticulum (ER) is an important intracellular organelle that plays a pivotal role in maintaining cellular homeostasis. Recently, it has been reported that an increase in markers of ER stress was observed in the intestinal epithelia of patients with active inflammatory bowel disease (IBD), indicating that ER stress is relevant to the pathogenesis of IBD [
6]. However, little is known about the role of ER stress in mediating the intestinal epithelial barrier dysfunction caused by heatstroke.
Apoptosis is programmed cell death. The integrity of the intestinal structure depends on the balance of intestinal epithelial cell proliferation and apoptosis [
7]. Prior studies have found that excessive apoptosis can damage the intestinal epithelial barrier, and the apoptosis index is positively correlated with permeability [
8]. In recent years, ER stress has become a hot topic in the field of apoptosis. CCAAT/enhancer-binding protein homologous protein (CHOP), one of the most sensitive factors involved in the regulation of ER stress, is a pivotal marker of ER stress-mediated apoptosis [
9]. The upstream regulators of CHOP are three ER transmembrane proteins involving protein kinase R-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-required enzyme-1α (IRE1α)[
9]. Following the dissociation of glucose-regulated protein 78 (GRP78) from these three transmembrane proteins, three types of unfolded protein responses (UPRs) are triggered by ER stress. Among these pathways, the PERK/eukaryotic translation initiation factor 2α/activating transcription factor4 (PERK/eIF2ɑ/ATF4) signaling pathway is more important for CHOP protein expression compared with the other two pathways [
10,
11]. Thus, we hypothesized that CHOP may represent a key factor in regulating heatstroke-induced intestinal barrier dysfunction.
4-Phenylbutyrate (4-PBA), an inhibitor of ER stress, is currently approved by the Food and Drug Administration (FDA) for the treatment of patients with urea cycle disorders (UCDs). 4-PBA functions as a chemical chaperone that prevents misfolded protein aggregation and relieves ER stress [
12]. Recent studies have explored the potential therapeutic effects of 4-PBA in various experiments. For instance, in lipopolysaccharide (LPS)-induced acute lung injury (ALI) models, 4-PBA plays a protective role by inhibiting ER stress and autophagy [
13]. In a mouse model of alcoholic hepatitis, 4-PBA prevented CHOP upregulation and inflammasome activation in the proximal small intestine [
14]. The potential effect of 4-PBA on heatstroke and the underlying mechanisms require further study.
Therefore, our experiment was designed to investigate the role of CHOP in the pathogenesis of intestinal barrier dysfunction in the context of heatstroke and the therapeutic effect of the ER stress inhibitor 4-PBA. We applied two well-established models by performing heat stress (HS) on Caco-2 cells and mice to mimic heatstroke in vitro and in vivo.
MATERIALS AND METHODS
Cell Culture and Groups
In our study, tunicamycin (TM) was used as an ER stress inducer [
15]. Human gut–derived Caco-2 cells (Procell Life Science & Technology Co., Ltd., Wuhan, China) were used as a model. These cells have been approved as suitable for studying intestinal epithelial barrier function. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO
2. At approximately 80% confluence, the cells were divided into the following eight groups: control group, HS group, CHOP-siRNA + HS group, control-siRNA + HS group, CHOP-plasmid + HS group, control-plasmid + HS group, 4-PBA + HS group, and TM + HS group.
Cell Treatment
Caco-2 cells were divided into the 8 groups as mentioned above. In the control group, the cells were grown in an incubator at 37 °C. In the HS group, the cells were grown in an incubator at 43 °C for 2 h. In addition, the culture medium was refreshed, and the cells were further incubated at 37 °C for an additional 6 h. A previous study showed that the peak of cell injury and apoptosis was achieved 6 h after HS [
16]. In the CHOP-siRNA + HS group, the cells were transfected with CHOP-siRNA 48 h before HS. In the control-siRNA + HS group, the cells were transfected with a control-siRNA 48 h before HS. In the CHOP-plasmid + HS group, the cells were transfected with a CHOP overexpression plasmid 48 h before HS. In the control-plasmid + HS group, the cells were transfected with a control plasmid 48 h before HS. In the 4-PBA + HS group, the cells were pretreated with 5 mmol/L 4-PBA 1 h before exposure to HS. Finally, in the TM + HS group, the cells were pretreated with 5 µg/ml TM 1 h before exposure to HS.
Cell Transfection
Caco-2 cells were transfected with CHOP-siRNA (GenePharma, Shanghai, China) or the CHOP overexpression plasmid (Tsingke, Beijing, China) according to the manufacturer’s instructions. The cells were seeded in 6-well plates 24 h before transfection in antibiotic-free medium. The cell confluence rate before transfection was approximately 70%. Then, 5 μl of CHOP-siRNA, 5 μl of control siRNA, 5 μl of the CHOP overexpression plasmid, or 5 μl of control plasmid was diluted in 250 μl of Opti-MEM (Gibco, Carlsbad, CA, USA). Five microliters of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was also diluted in 250 μl of Opti-MEM for each reaction. The siRNA or plasmid solution was mixed gently with Lipofectamine 2000 and incubated for 20 min at room temperature. Then, the transfection complexes were added to 6-well plate cells at 500 µl per well. The culture medium was refreshed after culture for 6 h at 37 °C in a 5% CO2 incubator.
The sense sequences (5′ to 3′) were as follows: CHOP sense, GCU GAG UCA UUG CCU UUC UTT and CHOP antisense, AGA AAG GCA AUG ACU CAG CTT; negative control sense, UUC UCC GAA CGU GUC ACG UTT; and negative control antisense, ACG UGA CAC GUU CGG AGA ATT.
Cell Viability Assays
The cell survival rate of each group was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (Beyotime Co, Shanghai, China) according to the manufacturer’s instructions.
Lactate Dehydrogenase Analysis
Lactate dehydrogenase (LDH) is released when the cell membrane is damaged. Thus, LDH release was used to assess cell damage. LDH enzymatic activity was detected using an LDH kit following the manufacturer’s instructions (JianChen Co, Nanjing, China).
Hoechst 33258 Staining
Hoechst 33258 staining was performed to observe the morphological changes of cellular nuclei. The cells of each group were washed with PBS, fixed with 4% polyformaldehyde for 10 min, and then stained with Hoechst 33258 (Beyotime, Shanghai, China) solution for 10 min at room temperature in the dark. Morphological changes in the nuclei were then examined under a fluorescence microscope (Eclipse Ti-SR, Nikon Corporation, Tokyo, Japan). Under fluorescence microscopy, the living cellular nuclei stained by Hoechst dye present diffuse and uniform fluorescence, whereas the nuclei of dead cells exhibit blue dense staining or fragmentary dense staining.
Flow Cytometry Analysis
Caco-2 cell apoptosis in each group was measured by flow cytometry based on Annexin V FITC-propidine iodide (PI) double staining (Invitrogen, Carlsbad, CA, USA). According to the manufacturer’s instructions, the cells were digested with trypsin without ethylenediaminetetraacetic acid (EDTA) and centrifuged to harvest. The harvested cell number was approximately 1 × 105 cells. Next, Annexin V-FITC/PI staining was performed according to the protocol provided with the kit (Invitrogen, Carlsbad, CA, USA). After staining, the proportion of apoptotic cells was analyzed by flow cytometry (BD, New Jersey, USA). The apoptotic rate was calculated as the percentage of early apoptotic cells in the lower right quadrant.
qRT–PCR Analysis
Total RNA was extracted from the Caco-2 cells of each group using Trizol (Invitrogen, Carlsbad, CA, USA). RNA was transcribed into cDNA using a PrimeScriptTM RT Reagent Kit (Takara, Shiga, Japan). The PCR mixture (20-µl final volume per reaction) was prepared. Amplification was performed by quantitative real-time PCR with the TB Green Premix Ex TaqTM II kit (Takara, Shiga, Japan). The primer sequences are reported in Table
1. Actin served as the endogenous reference gene to normalize the data.
Table 1
Primer Sequences (5′-3′)
GRP78 | Forward | GAATTCCTCCTGCTCCTCGT |
Reverse | CAGCATCATTAACCATCCTTTCG |
PERK | Forward | ACGATGAGACAGAGTTGCGAC |
Reverse | ATCCAAGGCAGCAATTCTCCC |
eIF2ɑ | Forward | AAGCATGCAGTCTCAGACCC |
Reverse | GTGGGGTCAAGCGCCTATTA |
ATF4 | Forward | ACAAGACAGCAGCCACTA |
Reverse | CTTACGGACCTCTTCTATCAG |
CHOP | Forward | GGAAACAGAGTGGTCATTCCC |
Reverse | CTGCTTGAGCCGTTCATTCTC |
Bcl-2 | Forward | GGTGGGGTCATGTGTGTGG |
Reverse | CGGTTCAGGTACTCAGTCATCC |
Bax | Forward | TCACTGAAGCGACTGATGTCCC |
Reverse | ACTCCCGCCACAAAGATGGTC |
Actin | Forward | ACCCTGAAGTACCCCATCGAG |
Reverse | AGCACAGCCTGGATAGCAAC |
Transepithelial Electrical Resistance Measurement
Caco-2 cells were plated on collagen-coated membrane Transwell inserts (3-µm pore size filters, Corning, USA). The transepithelial electrical resistance (TEER) of cells was measured with an electrical resistance system (EVOM, World Precision Instruments, Berlin, Germany). The TEER was calculated by normalizing to the initial values and was expressed as percentages of the initial resistance values.
Paracellular Tracer Flux Assay
In this assay, FITC-dextran (2.5 mg/mL, Sigma–Aldrich, USA) was added to the upper chamber 2 h before the experimental endpoint. After incubation, the fluorescence concentration of FITC-dextran in the medium of the lower chamber was measured by fluorescence at Ex 490 nm/Em 520 nm (TecanGENios reader, Tecan Group Ltd., CH).
Animals
CHOP
−/− mice on a C57BL/6 background were purchased from Model Organism (Shanghai, China). Considering that estrogen can increase survival during heatstroke by relieving inflammatory responses and cardiovascular dysfunction [
17], only male mice were used in the study. Male wild-type (WT) and CHOP
−/− mice (12 weeks old) were used. The animal experiments were approved by the Ethics Committee of Hunan Provincial People’s Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha, China. All animals were housed in a controlled environment at a constant temperature of 21 ± 2 °C with a 12-h light/dark cycle.
Heatstroke Procedures
The mice were randomly divided into six groups: the WT + sham group (
n = 6), CHOP
−/− + sham group (
n = 6), WT + PBA + sham group (
n = 6), WT + HS group (
n = 6), CHOP
−/− + HS group (
n = 6), and WT + PBA + HS group (
n = 6). We applied HS to mice to mimic heatstroke
in vivo. To generate the heatstroke model, mice were transferred to an artificial climate chamber with an environmental temperature of 35.5 ± 0.5 °C and humidity of 60 ± 5%. Rectal temperature (Tc) was continuously measured using a mercury thermometer every 15 min. The time point at which the Tc reached 42 °C was taken as the point of heatstroke onset [
18,
19]. After the Tc reached 42 °C, the animals were allowed to recover at room temperature (24 ± 0.5 °C). In the WT + PBA + HS group, the WT mice were pretreated with 4-PBA (100 mg/kg, ip) before HS. In the WT + sham or CHOP
−/− + sham group, the mice were maintained under sham-heated conditions at a temperature of 24 ± 0.5 °C and humidity of 35 ± 5%. The mice in the WT + PBA + sham group were pretreated with 4-PBA (100 mg/kg, ip) before being maintained under the same conditions. A previous study demonstrated that the average survival time of mice with heatstroke was approximately 6 h even when cooling treatment was applied [
20]. Therefore, this time point was used for our subsequent experiment. The mice were sacrificed at this time point under anesthesia, and serum and the ileum were isolated.
Serum d-Lactate and Diamine Oxidase Levels
Serum samples were collected and measured using a corresponding enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Co. Ltd., Nanjing, China) to detect serum d-lactate (d-LA) and diamine oxidase (DAO) following the manufacturer’s instructions.
Histopathology
Ileum specimens from each group were fixed in 10% neutral-buffered formalin. The specimens were then embedded in paraffin blocks, sectioned at 5–7 µm, and stained with hematoxylin and eosin (H&E). The histopathological changes in ileal tissue were assessed under light microscopy.
Ultrastructural Observation by Transmission Electron Microscopy
The ultrastructural changes in Caco-2 cells or ileal tissues were observed by transmission electron microscopy. Caco-2 cell or ileum tissue specimens were fixed with 2.5% glutaraldehyde and then postfixed with 1% osmium tetroxide. Then, the specimens were dehydrated using a graded ethanol series (concentrations of 50%, 70%, 80%, 90%, and 100%) into pure acetone. After dehydration, the specimens were embedded with graded mixtures of acetone and SPI-PON812 resin and then polymerized for 12 h at 45 °C and 48 h at 60 °C. Finally, ultrathin Sects. (70 nm) were stained and viewed under transmission electron microscopy (Hitachi, Tokyo, Japan).
Western Blotting
Proteins were extracted from Caco-2 cells or ileal tissues using RIPA buffer (Beyotime, Shanghai, China) to obtain total protein. The protein concentration was detected using a BCA protein assay kit (Applygen Technologies, Inc., Beijing, China). Equal amounts of protein (30 μg) were separated on SDS–PAGE gels and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were blocked in a 5% skim milk-TBS solution at room temperature for 1 h. Then, the membranes of cell proteins were incubated with diluted primary antibodies against the following proteins: GRP78, PERK, eIF2α, p-eIF2α, and CHOP (Cell Signaling Technology, Danvers, Massachusetts, USA); ATF4, B-cell lymphoma-2 (Bcl-2), and Bcl-2 Associated X Protein (Bax) (Proteintech Group, Inc. Rosemont, Illinois, USA); and zonula occluden-1 (ZO-1) and occludin (Abcam, Massachusetts, United States). The membranes of tissue proteins were incubated with diluted primary antibodies against ZO-1 and occludin. Afterward, the membranes were washed thrice in PBS/Tween-20 for 5 min with shaking. The membranes were then incubated with the secondary antibody (Zhongshan Inc., China) for 2 h at room temperature. The membranes were developed using a GE ImageQuant LAS 500 (GE Healthcare, USA). Quantification of the digitized images of the Western blot bands was performed using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
Statistical Analysis
All the data were presented as the mean ± standard deviation, and differences were analyzed with one-way analysis of variance (ANOVA) among groups using SPSS statistical software (SPSS for Windows, version 19.0, Chicago, IL). A value of P < 0.05 was considered statistically significant.
DISCUSSION
Intestinal barrier dysfunction plays a critical role in heatstroke progression, but the underlying mechanisms remain poorly understood. To our knowledge, the current study identifies a novel mechanism underlying heatstroke-induced intestinal injury. Our results indicate that CHOP mediates intestinal epithelial apoptosis and barrier dysfunction induced by heatstroke. 4-PBA prevents apoptosis and improves the integrity of the intestinal barrier, underscoring its utility as a potential treatment for heatstroke.
As one of the key transcription factors in ER stress, CHOP is widely expressed in a variety of mammalian body cells. Under normal physiological conditions, CHOP expression is extremely low. However, CHOP expression is significantly increased in response to ER stress, thereby activating a series of downstream apoptotic molecules to induce apoptosis and participate in the occurrence and development of various pathological processes [
21]. Apoptosis is a type of programmed cell death that is implicated in intestinal epithelial cell death [
22]. The dynamic balance of intestinal epithelial cell proliferation and apoptosis maintains the homeostasis of the intestinal barrier. Under pathological conditions, repair and proliferation of the intestinal mucosa are difficult to achieve, and excessive apoptosis of intestinal mucosal cells will inevitably cause damage to the intestinal barrier [
23]. In the pathophysiology of MODS secondary to heatstroke, the gastrointestinal tract is one of the first onset organs. Severe heatstroke can lead to increased intestinal epithelial apoptosis and impaired intestinal mucosal barrier followed by an endotoxemia-induced inflammation cascade and systemic inflammatory response syndrome (SIRS), which ultimately leads to MODS or even death [
24]. In our study, we first established a cellular model of heatstroke using Caco-2 cells and observed the role of CHOP in apoptosis-mediated intestinal epithelial injury. We observed that CHOP was activated in Caco-2 cells after HS. Reductions in CHOP expression via transfection with siRNA increased cell viability and decreased apoptosis, whereas CHOP overexpression significantly decreased cell survival and increased apoptosis. These results revealed that CHOP was essential in HS-induced Caco-2 cell apoptosis.
CHOP is regulated by PERK, ATF6, and IRE1, but the PERK/eIF2ɑ/ATF4 signaling pathway plays a more important role in CHOP activation compared with the other two UPR pathways [
25]. In this pathway, PERK stimulates eIF2α phosphorylation to enhance the translation of ATF4, thereby increasing CHOP transcription. Bcl-2 and Bax are downstream target genes of the PERK-CHOP apoptosis signaling pathway [
26]. Our findings showed that HS increased the mRNA and protein expression of factors involved in the PERK-CHOP pathway in Caco-2 cells. By silencing CHOP before HS, Bax mRNA and protein expression levels were reduced, whereas Bcl-2 mRNA and protein expression levels were increased. However, CHOP overexpression upregulated Bax expression and downregulated Bcl-2 expression. Our data strongly support our hypothesis that CHOP plays a pivotal role in HS-induced apoptosis by regulating Bcl-2 and Bax.
Interestingly, after CHOP silencing, the expression of the ER stress chaperone proteins GRP78, PERK, eIF2ɑ, and ATF4 was decreased, albeit without statistical significance. Similarly, in a study on the mechanism of rifampicin-associated liver damage, GRP78, PERK, and ATF4 expression was also downregulated after CHOP silencing [
27]. Regarding the effect of silencing CHOP on GRP78 and the upstream factors of CHOP, such as PERK, eIF2ɑ, and ATF4, previous studies have reported the presence of a feedback loop in the ER stress pathway [
28]. Thus, we hypothesize that these upstream and downstream kinases may affect each other through a feedback loop in the same signaling pathway.
In addition, the results also indicated that pretreating Caco-2 cells with the ER stress inhibitor 4-PBA can significantly attenuate HS-induced changes in cell morphology and apoptosis by inhibiting PERK-CHOP pathways, subsequently downregulating the proapoptotic protein Bax and upregulating the antiapoptotic protein Bcl-2. In contrast, the ER stress-inducer TM aggravated Caco-2 cell apoptosis. Our results explore the potential therapeutic effects of 4-PBA in heatstroke.
Next, we explored the role of CHOP in regulating intestinal barrier function
in vitro and
in vivo. Maintenance of intestinal barrier integrity relies on a variety of mucosal structural components, such as tight junctions, which form zonula occludentes at the apical surface between cells [
29]. It has been reported that extreme heat induces intestinal permeability and TJ protein disruption [
30]. A previous study demonstrated that CHOP
−/− mice are protected from bile duct ligation (BDL)-induced disruption of intestinal barrier function [
31]. Our results showed that HS induced morphological injuries and ultrastructural damage (especially to TJs) of the intestinal mucosa, accompanied by an increase in intestinal permeability and disruption of epithelial integrity
in vitro and
in vivo. CHOP silencing significantly attenuated the HS-induced decrease in TEER and increase in FITC-dextran release in Caco-2 cells by ameliorating the changes in tight junction structure and regulating TJ protein expression.
In vivo studies with CHOP
−/− mice further demonstrated that CHOP deficiency obviously alleviated HS-induced intestinal tissue damage and barrier dysfunction. In addition, our data showed that 4-PBA repaired dysfunction of the intestinal barrier following HS both
in vitro and
in vivo, indicating that ER plays a central role in the maintenance of intestinal mucosal homeostasis.
In conclusion, we have demonstrated for the first time that CHOP deficiency attenuates heatstroke-induced intestinal mucosal damage and barrier dysfunction. Here, we have provided strong evidence that targeting ER stress represents a novel therapeutic strategy for heatstroke.
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