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American Journal of Physiology - Gastrointestinal and Liver Physiology logoLink to American Journal of Physiology - Gastrointestinal and Liver Physiology
. 2019 Sep 23;317(6):G862–G871. doi: 10.1152/ajpgi.00192.2019

NSAID-induced injury of gastric epithelial cells is reversible: roles of mitochondria, AMP kinase, NGF, and PGE2

Amrita Ahluwalia 1,*, Neil Hoa 1, Michael K Jones 1,2, Andrzej S Tarnawski 1,2,*,✉
PMCID: PMC6962499  PMID: 31545918

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Keywords: 5-amino-imidazole-4-carboxamide ribonucleotide, AMP-activated protein kinase, diclofenac, dm prostaglandin E2, indomethacin, mitochondria, nerve growth factor, non-steroidal anti-inflammatory drugs

Abstract

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN) and indomethacin (INDO) are extensively used worldwide. Their main side effects are injury of the gastrointestinal tract, including erosions, ulcers, and bleeding. Since gastric epithelial cells (GEPCs) are crucial for mucosal defense and are the major target of injury, we examined the extent to which DFN- and INDO-induced GEPC injury can be reversed by nerve growth factor (NGF), 16,16 dimethyl prostaglandin E2 (dmPGE2), and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), the pharmacological activator of the metabolic sensor AMP kinase (AMPK). Cultured normal rat gastric mucosal epithelial (RGM1) cells were treated with PBS (control), NGF, dmPGE2, AICAR, and/or NSAID (DFN or INDO) for 1–4 h. We examined cell injury by confocal microscopy, cell death/survival using calcein AM, mitochondrial membrane potential using MitoTracker, and phosphorylation of AMPK by Western blotting. DFN and INDO treatment of RGM1 cells for 2 h decreased mitochondrial membrane potential and cell viability. NGF posttreatment (initiated 1 or 2 h after DFN or INDO) reversed the dissipation of mitochondrial membrane potential and cell injury caused by DFN and INDO and increased cell viability versus cells treated for 4 h with NSAID alone. Pretreatment with dmPGE2 and AICAR significantly protected these cells from DFN- and INDO-induced injury, whereas dmPGE2 and AICAR posttreatment (initiated 1 h after NSAID treatment) reversed cell injury and significantly increased cell viability and rescued the cells from NSAID-induced mitochondrial membrane potential reduction. DFN and INDO induce extensive mitochondrial injury and GEPC death, which can be significantly reversed by NGF, dmPGE2, and AICAR.

NEW & NOTEWORTHY This study demonstrated that mitochondria are key targets of diclofenac- and indomethacin-induced injury of gastric epithelial cells and that diclofenac and indomethacin injury can be prevented and, importantly, also reversed by treatment with nerve growth factor, 16,16 dimethyl prostaglandin E2, and 5-aminoimidazole-4-carboxamide ribonucleotide.

INTRODUCTION

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DFN), indomethacin (INDO), and others are extensively used worldwide for the management of pain, arthritis, and inflammation. A recent report by the US Food and Drug Administration estimated that in 2017, over 89 million US households used over-the-counter NSAIDs, in addition to ~70 million prescriptions for NSAIDs (56a). However, NSAIDs have significant side effects, including gastrointestinal mucosal injury, erosions, ulcerations, and bleeding (11, 42, 48, 52, 53, 56). In vivo studies demonstrated that DFN and INDO treatment causes injury to the gastric mucosa in humans and rats (10, 14, 19). Studies in vitro in isolated cells under conditions independent of systemic factors such as blood flow and innervations have demonstrated that DFN and INDO induce direct cellular injury (5, 14, 18, 28). One of the main mechanisms of NSAID-induced cellular injury is inhibition of cyclooxygenases, COX1 and COX2, which causes reduced synthesis of prostaglandins (PGs) essential for maintenance of the mucosal integrity (13, 30, 33, 41, 43, 52). This is evidenced by the fact that supplementation with exogenous PGs significantly prevents NSAID-induced injury (47–49). Prostaglandins of E class (PGEs) and their synthetic analogs afford gastric mucosal protection and are clinically used to counteract the injurious side effects of NSAID-induced PG inhibition (2, 20, 24, 26, 27, 30). Recent studies have demonstrated that nerve growth factor (NGF) acts on various cell types besides neuronal cells, including gastric endothelial and epithelial cells (3, 17, 38). Our recent study demonstrated the protective effect of NGF on gastric epithelial cells against DFN-induced injury (4).

NSAIDs have been shown to cause cell death and reduce mitochondrial membrane potential in cardiomyocytes, hepatocytes, and endothelial and epithelial cells (4, 5, 13, 14, 18, 32, 42). DFN has been shown to induce mitochondrial injury and inhibit adenosine triphosphate (ATP) synthesis (46) by preventing mitochondrial fusion in hepatocytes (28). INDO promotes mitochondrial hyperfission and dysfunction leading to apoptosis (32). Dysregulation of mitochondrial function can result in reduced bioenergetic capacity of cells and in cell death (58). Cell function critically depends on ATP. Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is the cells’ metabolic and energy sensor (22, 23, 35). Under conditions in which cellular AMP levels are very high (high AMP-to-ATP ratio), AMPK is activated and stimulates catabolic pathways to increase the cellular energy levels (22, 23). The AMP analog, 5-amino-imidazole-4-carboxamide ribonucleotide (AICAR) is a strong and highly specific pharmacological activator of AMPK (7, 22). Whether the metabolic sensor AMPK plays a role in NSAID-induced injury is not known.

We previously reported that DFN treatment of rat gastric mucosal epithelial (RGM1) cells for 4 h causes severe cell injury and that NGF pretreatment significantly prevents injury (4). That study was limited to one NSAID and did not examine the dependence of RGM1 cell injury on the duration of NSAID treatment nor whether this injury can be reversed.

This study was designed to determine whether DFN- and INDO-induced injury of gastric epithelial cells is reversible and to determine the time point following initiation of treatment with DFN or INDO at which injury could be reversed by subsequent treatment with NGF, 16,16 dimethyl prostaglandin E2 (dmPGE2), and AICAR. We also examined whether the mechanisms of protection, injury and reversal of injury involve mitochondria and the metabolic sensor AMPK.

This study demonstrated that DFN- and INDO-induced impairment of mitochondrial function precedes irreversible injury of gastric epithelial RGM1 cells. It also showed that cell injury is significantly reversible by NGF, dmPGE2, and AICAR posttreatment up to 2 h following initiation of treatment with DFN and INDO.

MATERIALS AND METHODS

This study was approved by the institutional review committee of the Veterans Affairs Long Beach Healthcare System (Long Beach, CA).

Cell culture and treatments.

The RGM1 cell line derived from normal rat gastric mucosa was maintained in Dulbecco’s modified Eagle’s medium/F12 medium supplemented with 2 mmol/L l-glutamine, antibiotics/antimycotics, and 20% fetal bovine serum at 37°C with 5% CO2 in a humidified incubator as described in our previous studies (4, 37).

Cell injury studies with DFN and INDO.

RGM1 cells were treated with DFN (0.5 mM; Sigma), INDO (0.25 mM; Sigma), or PBS (control) for 0, 1, or 2 h. Mitochondrial membrane potential and cell viability were then examined at the end of these treatments (time point 0, 1, or 2 h, respectively) as described below. The doses of DFN and INDO were based on our previous studies (4, 5, 14). The dose of DFN that induces significant injury of RGM1 cells was determined by treating these cells with either PBS (control) or DFN (0.25, 0.50, or 1.00 mM) for 4 h (4). The dose of INDO that induces significant injury of RGM1 cells was determined by treating these cells with either PBS (control) or INDO (0.01–0.5 mM) for 4 h (5, 14).

Pretreatment studies with NGF, dmPGE2, or AICAR.

Our previous study showed that DFN treatment of RGM1 cells for 4 h causes severe cell injury and that pretreatment with NGF protects these cells from DFN injury (4). In this study we examined the protective effect of pretreatment with NGF, dmPGE2, and AICAR against cell injury induced by two NSAIDs (DFN and INDO). RGM1 cells were pretreated with PBS, NGF, dmPGE2, or AICAR for either 1 or 2 h followed by addition of DFN or INDO to the culture medium and culture for an additional 4 h. Mitochondrial membrane potential and cell viability were then examined at the end of these treatments (time point 5 or 6 h, respectively) as described below.

Posttreatment studies with NGF, dmPGE2, or AICAR.

To determine the reversibility of NSAID-induced injury, RGM1 cells were treated with DFN (0.5 mM; Sigma), INDO (0.25 mM; Sigma), or PBS (control) for 0, 1, or 2 h, washed, and then cultured in media with or without NGF (100 ng/mL; R&D Systems, Minneapolis, MN), dmPGE2 (1 μg/mL), or AICAR (1 mM, Sigma-Aldrich, St. Louis, MO) for 4 h. Mitochondrial membrane potential and cell viability were then examined as described below.

Measurements of mitochondrial membrane potential using MitoTracker Red.

Quantitative assessment of mitochondrial structure and mitochondrial membrane potential was performed using MitoTracker Red dye in RGM1 cells similar to our previous study (4). Active mitochondria generate a mitochondrial membrane potential across the inner mitochondrial membrane, which is crucial for ATP production. Therefore, mitochondrial membrane potential serves as an important index of mitochondrial function. Mitochondrial molecular probes such as MitoTracker dye are absorbed by living cells and concentrate in mitochondria in a mitochondrial membrane potential-dependent manner. These probes have been used in combination with quantitative fluorescence microscopy and computer-based image analysis to quantitatively determine changes in the mitochondrial membrane potential (29). RGM1 cells were incubated with MitoTracker (10 nM; Invitrogen, Carlsbad, CA) for 45 min at 37°C, and then cells were fixed in 4% paraformaldehyde. Cells were mounted on glass coverslips and photographed using the AxioImager2 fluorescence microscope imaging system. The fluorescence signal intensity and area of MitoTracker staining in each cell were quantified using the MetaMorph 7.0 imaging system (Molecular Devices, Downingtown, PA). A reduction in the MitoTracker staining indicates reduced mitochondrial membrane potential.

Assessments of cell injury and viability using Calcein AM live cell tracking dye.

Cells were incubated with Calcein AM (10 μM, Invitrogen) for 30 min at 37°C. Cell injury was examined quantitatively by confocal microscopy and cell death/survival was examined using Calcein AM live cell tracking dye similar to our previous studies (4, 5). The total cell count and number of Calcein AM-stained cells were determined in five randomly selected fields. Cell viability was expressed as percent of Calcein AM-stained cells.

Western blot analysis for AMPK.

Proteins were isolated from RGM1 cells using standard radioimmunoprecipitation assay buffer. Western blot analysis was performed within the linear range to examine the expression of phosphorylated AMPK (pAMPK), total AMPK, and β actin proteins using methods described in our previous studies (6, 51). Antibodies used were pAMPK (1:500; Cell Signaling Technology), AMPK (1:1,000; Cell Signaling Technology), or β actin (1:1,000; A5316, Sigma).

Statistical analysis.

Data are presented as means ± SD. Data acquisition was performed within the linear range for all assays. Statistical significance was analyzed by either Student’s t test to compare data between two groups, one-way analysis of variance with Tukey’s multiple comparison post hoc testing for evaluating differences between multiple groups, or Pearson’s correlation using Prism (GraphPad Software, La Jolla, CA). A P value of < 0.05 was considered statistically significant.

RESULTS

DFN and INDO induce mitochondrial and cellular injury in RGM1 cells.

We examined the injury induced by treating RGM1 cells with either of the two NSAIDs examined, DFN or INDO. The extent of NSAID-induced injury of RGM1 cells was dependent on the duration of NSAID treatment. Treatment with either of the two NSAIDs, DFN or INDO, caused significant impairment of RGM1 mitochondrial function as reflected by dissipation of mitochondrial membrane potential and also caused significant cell damage as reflected by reduced cell viability. NSAID treatment of RGM1 cells significantly reduced mitochondrial membrane potential at 1 and 2 h after treatment with DFN (Fig. 1A) and INDO (Fig. 2A). NSAID treatment for 1 h did not significantly reduce RGM1 cell viability, whereas treatment for 2 h with either NSAID, DFN (Fig. 1B) or INDO (Fig. 2B), caused a significant reduction in RGM1 cell viability (P < 0.001) (Fig. 1B).

Fig. 1.

Fig. 1.

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Diclofenac (DFN) induces mitochondrial damage and cell injury in rat gastric mucosal epithelial (RGM1) cells. A: mitochondrial membrane potential and mitochondrial structure in RGM1 cells were examined using the mitochondrial stain, MitoTracker (red), which is readily absorbed by living cells and concentrates in mitochondria in a mitochondrial membrane potential-dependent manner. In control (PBS-treated) RGM1 cells, the MitoTracker dye accumulated in mitochondria is visualized under a fluorescence microscope as bright red structures in the cytoplasm of these cells around the nucleus (arrows). DFN treatment of RGM1 cells induced dissipation of mitochondrial membrane potential in a time-dependent manner and, as a result, less MitoTracker stain is retained in the mitochondria, which therefore stain less intensely. Data are means ± SD (n = 6). *P < 0.001. B: cell viability was determined using Calcein AM live cell tracking dye (green), which is retained by living cells (3, 4). In control (PBS-treated) cells, Calcein AM accumulated in the cells and labeled them bright green. DFN treatment induced injury of RGM1 cells, which did not retain the Calcein AM dye and therefore stained only faintly or did not stain. DFN treatment reduced RGM1 cell viability in a time-dependent manner. Data are means ± SD (n = 6). *P < 0.001.

Fig. 2.

Fig. 2.

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Indomethacin (INDO) induces mitochondrial damage and cell injury in rat gastric mucosal epithelial (RGM1) cells. A: mitochondrial membrane potential and mitochondrial structure were examined using the mitochondrial stain, MitoTracker (red). INDO treatment of RGM1 cells induced dissipation of mitochondrial membrane potential in a time-dependent manner. Data are means ± SD (n = 6). *P < 0.001. B: cell viability was determined using Calcein AM live cell tracking dye (green). INDO treatment induced injury of RGM1 cells and reduced RGM1 cell viability in a time-dependent manner. Data are means ± SD (n = 6). *P < 0.001.

NGF treatment reverses DFN- and INDO-induced injury of RGM1 cells.

We next examined the extent to which DFN-induced injury was reversible and the time points following initiation of DFN treatment that injury could still be reversed. RGM1 cells were treated with DFN or PBS for 1 or 2 h, washed, and then treated with NGF or PBS for 4 h. Posttreatment with NGF initiated 1 or 2 h after DFN treatment significantly reversed DFN-induced dissipation of mitochondrial membrane potential (both P < 0.001), respectively (Fig. 3A), preserved cell viability, and inhibited further cell damage versus cells treated with DFN alone (Fig. 3B).

Fig. 3.

Fig. 3.

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Nerve growth factor (NGF) posttreatment reverses diclofenac (DFN)-induced dissipation of mitochondrial membrane potential, preserves cell viability, and inhibits DFN-induced cell injury. A: NGF posttreatment for 4 h initiated 1 or 2 h after DFN treatment significantly reversed DFN-induced dissipation of mitochondrial membrane potential. The increase in mitochondrial membrane potential is visualized as red fluorescence by accumulation and retention of the MitoTracker stain in mitochondria of live cells. Data are mean ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. DFN-treated cells. B: NGF treatment initiated 1 or 2 h following DFN treatment significantly preserved cell viability and inhibited DFN-induced cell injury. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. DFN-treated cells.

We then examined the effect of NGF pretreatment initiated 1 h before INDO injury on mitochondrial membrane potential and cell viability. We pretreated RGM1 cells with NGF for 1 h followed by addition of INDO to the culture medium containing NGF and examined mitochondrial membrane potential and cell viability 4 h after INDO treatment. In addition, we examined whether INDO injury could be reversed by NGF posttreatment. We treated RGM1 cells with INDO or PBS for 1 h, washed them, and then treated the cells with NGF for 4 h. Pretreatment with NGF for 1 h protected the RGM1 cells from INDO-induced dissipation of mitochondrial membrane potential, preserved cell viability, and inhibited cell damage versus cells treated with INDO alone (Fig. 4A). Posttreatment of RGM1 cells with NGF initiated 1 h after INDO treatment reversed INDO-induced dissipation of mitochondrial membrane potential and significantly preserved cell viability versus cells treated with INDO alone for 4 h (Fig. 4B).

Fig. 4.

Fig. 4.

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Nerve growth factor (NGF) pretreatment prevents and reverses indomethacin (INDO)-induced dissipation of mitochondrial membrane potential and preserves cell viability in rat gastric mucosal epithelial (RGM1) cells. A: pretreatment with NGF for 1 h protected the RGM1 cells from INDO-induced dissipation of mitochondrial membrane potential and cell damage versus cells treated with INDO alone for 4 h. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. INDO-treated cells. B: posttreatment of RGM1 cells with NGF initiated 1 h after INDO treatment reversed INDO-induced dissipation of mitochondrial membrane potential, preserved cell viability, and inhibited INDO-induced injury versus cells treated with INDO alone for 4 h. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. INDO-treated cells.

Treatment with dmPGE2 reverses DFN- and INDO-induced mitochondrial and cell injury in RGM1 cells.

We next examined whether dmPGE2 could protect and/or reverse DFN- and/or INDO-induced mitochondrial and cellular injury. Pretreatment with dmPGE2 for 1 h protected the RGM1 cells from dissipation of mitochondrial membrane potential induced by DFN (Fig. 5A) or INDO (Fig. 6A), preserved cell viability, and inhibited cell damage induced by DFN and INDO versus cells treated with only DFN (Fig. 5A) or INDO (Fig. 6A) alone. Posttreatment of RGM1 cells with dmPGE2 initiated 1 h after NSAID treatment reversed the dissipation of mitochondrial membrane potential induced by DFN (Fig. 5B) or INDO (Fig. 6B), preserved cell viability, and inhibited further cell damage induced by DFN (Fig. 5B) or INDO (Fig. 6B) versus cells treated with either NSAID alone.

Fig. 5.

Fig. 5.

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16,16-Dimethyl prostaglandin E2 (dmPGE2) prevents and reverses diclofenac (DFN)-induced dissipation of mitochondrial membrane potential and preserves rat gastric mucosal epithelial (RGM1) cell viability. A: pretreatment with dmPGE2 for 1 h protected the RGM1 cells from DFN-induced dissipation of mitochondrial membrane potential and cell injury versus cells treated with DFN alone for 4 h. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. DFN-treated cells. B: posttreatment of RGM1 cells with dmPGE2 initiated 1 h after DFN treatment reversed DFN-induced dissipation of mitochondrial membrane potential, preserved cell viability, and inhibited DFN-induced injury versus cells treated with DFN alone. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. DFN-treated cells.

Fig. 6.

Fig. 6.

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16,16-Dimethyl prostaglandin E2 (dmPGE2) prevents and reverses indomethacin (INDO)-induced dissipation of mitochondrial membrane potential and preserves cell viability in rat gastric mucosal epithelial (RGM1) cells. A: pretreatment with dmPGE2 for 1 h protected the RGM1 cells from INDO-induced dissipation of mitochondrial membrane potential and cell damage versus cells treated with INDO alone for 4 h. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. INDO-treated cells). B: posttreatment of RGM1 cells with dmPGE2 initiated 1 h after INDO treatment reversed INDO-induced dissipation of mitochondrial membrane potential, preserved cell viability, and inhibited INDO-induced injury versus cells treated with INDO alone. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. INDO-treated cells.

Treatment with AICAR, the pharmacological activator of the metabolic sensor AMPK, reverses DFN- and INDO-induced mitochondrial and cellular injury in RGM1 cells.

We next examined whether activation of the metabolic sensor AMPK could protect and/or reverse NSAID-induced mitochondrial and cellular injury. Pretreatment of the RGM1 cells with AICAR for 1 h protected the cells from the dissipation of mitochondrial membrane potential induced by 4-h treatment with DFN (Fig. 7A) or INDO (Fig. 8A), preserved cell viability, and inhibited cell damage versus cells treated with either NSAID alone. Posttreatment of RGM1 cells with AICAR initiated 1 h after NSAID treatment reversed DFN- and INDO-induced dissipation of mitochondrial membrane potential (Figs. 7B and 8B), preserved cell viability, and inhibited further cell damage (Figs. 7B and 8B) versus cells treated with either NSAID alone.

Fig. 7.

Fig. 7.

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5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) prevents and reverses diclofenac (DFN)-induced dissipation of mitochondrial membrane potential and preserves rat gastric mucosal epithelial (RGM1) cell viability. A: pretreatment with AICAR for 1 h protected the RGM1 cells from DFN-induced dissipation of mitochondrial membrane potential and cell injury vs. cells treated with DFN alone for 4 h. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. DFN-treated cells. B: posttreatment of RGM1 cells with AICAR initiated 1 h after DFN treatment reversed DFN-induced dissipation of mitochondrial membrane potential, preserved cell viability, and inhibited DFN-induced injury versus cells treated with DFN alone. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. DFN-treated cells.

Fig. 8.

Fig. 8.

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5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) prevents and reverses indomethacin (INDO)-induced dissipation of mitochondrial membrane potential and preserves cell viability in rat gastric mucosal epithelial (RGM1) cells. A. pretreatment with AICAR for 1 h protected the RGM1 cells from INDO-induced dissipation of mitochondrial membrane potential and cell damage versus cells treated with INDO alone for 4 h. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. INDO-treated cells. B: posttreatment of RGM1 cells with AICAR initiated 1 h after INDO treatment reversed INDO-induced dissipation of mitochondrial membrane potential, preserved cell viability, and inhibited INDO-induced injury versus cells treated with INDO alone for 4 h. Data are means ± SD (n = 6). *P < 0.001 vs. PBS-treated cells; †P < 0.001 vs. INDO-treated cells.

NGF, dmPGE2, and AICAR induce phosphorylation of AMPK in RGM1 cells.

We examined whether treatment with NGF, dmPGE2, or AICAR affects the cellular metabolic and energy sensor AMPK. We determined AMPK levels and phosphorylation/activation using Western blot analysis. Treatment of RGM1 cells with AICAR, NGF, and dmPGE2 significantly increased AMPK levels (all P < 0.001) and induced phosphorylation of AMPK (all P < 0.001) versus PBS-treated control cells (Fig. 9).

Fig. 9.

Fig. 9.

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Treatment of rat gastric mucosal epithelial (RGM1) cells with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), nerve growth factor (NGF), and 16,16 dimethyl prostaglandin E2 (dmPGE2) induces phosphorylation of AMP kinase (AMPK). Levels of AMPK and phosphorylated AMPK (pAMPK) were higher in RGM1 cells treated for 4 h with AICAR, NGF, or dmPGE2 versus vehicle-treated cells. Treatment of RGM1 cells with AICAR, NGF, and dmPGE2 increased the levels of AMPK and induced its phosphorylation. Data are means ± SD (n = 3). *P < 0.001 vs. control (PBS-treated cells).

DISCUSSION

This study demonstrated that gastric epithelial cell injury induced by the NSAIDs DFN and INDO can be prevented and, importantly, was also reversed by NGF, dmPGE2, and AICAR, the pharmacological activator of cellular metabolic and energy sensor AMPK. DFN and INDO induce dissipation of mitochondrial membrane potential in gastric epithelial cells and cause significant cell damage. Treatment of gastric epithelial cells with NGF, dmPGE2, and AICAR reversed the effect of DFN and NGF on dissipation of mitochondrial membrane potential in the cells that were injured but still viable, preserved cell viability, and inhibited further cell damage. One potential mechanism can be that activation of AMPK, metabolic sensor, by rescue treatment with NGF, dmPGE2, and AICAR restores uptake and retention of MitoTracker by mitochondria.

Our previous study demonstrated that NSAIDs such as indomethacin induce severe injury to isolated human gastric glands consisting of epithelial cells, and that dmPGE2 pretreatment significantly protects against this injury (48). The clinical relevance and significance of NSAID-induced injury are underscored by our previous study performed in 29 healthy human volunteers. That study showed that a single intragastric instillation of 600 mg of aspirin (suspended in isotonic saline) caused significant damage to ~21% of surface epithelial cells, microscopic erosions of the mucosal surface in 60% of subjects and a marked reduction in gastric potential difference within 15 min of aspirin instillation (50). Although injury after a single dose of NSAIDs is rarely clinically significant, in certain circumstances, e.g., aging, portal hypertension or healing ulcer, it may trigger a deep injury (8, 9, 54). In the latter condition, one can envision a healing gastric ulcer in which granulation tissue is covered with a single layer of epithelial cells. NSAIDs-induced damage of these epithelial cells can trigger acid and pepsin digestion of granulation tissue and ulcer reactivation. It should be pointed out that the cultured cells are more sensitive to a single dose of injurious agent than in vivo gastric mucosa, because they lack appropriate defense mechanisms such as gastric mucus, mucosal blood flow, and innervation (30).

In the present study, we examined the effect of NGF, dmPGE2, and AICAR on DFN- and INDO-induced cellular and mitochondrial injury in RGM1 cells. We assessed mitochondrial structure and mitochondrial membrane potential using the cell-permeable MitoTracker Red dye. The nonfluorescent, reduced form of this dye is taken up by live cells oxidized to its fluorescent form, which accumulates in mitochondria and is an index of the cellular and mitochondrial health. The oxidized MitoTracker Red is retained in mitochondria after fixation and can be used to detect changes in mitochondrial membrane potential in combination with quantitative fluorescence microscopy and computer-based image analysis (29).

This study demonstrated that treatment with DFN or INDO for 1 h decreased mitochondrial membrane potential without significantly affecting cell viability. Treatment with DFN or INDO for a longer duration (2–4 h) caused extensive, time-dependent cell injury, mitochondrial disintegration, and mitochondrial membrane potential dissipation. Importantly, it also demonstrated that NGF, even when administered up to 2 h after initiation of DFN treatment, significantly reverses DFN-induced mitochondrial and cellular injury.

Mitochondria play a central role in cellular viability, cell signaling, and apoptotic pathways (34), and alterations to mitochondrial membrane potential and mitochondrial function are detrimental to cell survival (39). Mitochondrial membrane potential is an important index of mitochondrial function, as it is crucial for ATP production, which provides energy for all cellular processes. The lipophilic and charged structure of DFN facilitates cellular and mitochondrial injury by uncoupling oxidative phosphorylation, increasing reactive oxygen species, and reducing ATP synthesis (12, 44, 45). A recent study demonstrated that DFN induces cellular and mitochondrial damage in a human small intestinal cell monolayer culture system as evidenced by increased cellular toxicity and reduced mitochondrial integrity and mitochondrial membrane potential (12). The present study also demonstrated that DFN- and INDO-induced cellular and mitochondrial damage of RGM1 cells can be reversed by treatment with NGF, dm PGE2, and the AMPK activator AICAR within 1–2 h following initiation of NSAID treatment. DFN and INDO inhibit both COX1 and COX2, and generation of COX-induced PGs critical for cell and mucosal integrity, accordingly cell and mucosal injury, is significantly prevented by PG supplementation (13, 30, 33, 41, 43, 52). Mitochondria express the prostaglandin receptors EP2, EP3, and EP4 (15). The EP4 PG receptor is a critical regulator of mitochondrial biogenesis, and EP4 knockout mice have increased mitochondrial biogenesis, pAMPK, and oxidative capacity in white adipose tissue (59). Since COX-mediated PG generation is inhibited by DFN and INDO, the protection afforded by NGF and/or AICAR against DFN-induced mitochondrial and cellular injury likely involves mechanisms that are independent of PGs. Therefore, protection and reversal of DFN-induced injury involves both PG-dependent and PG-independent mechanisms.

AMPK plays a critical role in mitochondrial homeostasis by regulating mitochondrial fission, fusion, and biogenesis (16, 21, 25, 31, 55, 60, 61). The phosphorylation of mitochondrial fission factor by AMPK results in mitochondrial fission and degradation of damaged mitochondria by autophagy (60) and mitophagy (25). Under metabolic stress conditions, AMPK phosphorylates the peroxisome proliferator-activated receptor gamma coactivator 1α protein to promote mitochondrial biogenesis (16, 25). In hepatocytes, DFN has been demonstrated to uncouple oxidative phosphorylation, reduce mitochondrial membrane potential, deplete ATP, and promote apoptosis (36, 40, 57). DFN-mediated dysregulation of mitochondrial function in hepatocytes includes reduction in mitochondrial fusion protein Mfn1 levels, which is reversed by AICAR treatment (28). INDO promotes mitochondrial hyperfission and dysfunction leading to apoptosis (32). This study demonstrated that treatment with NGF, dmPGE2, and AICAR activates AMPK in RGM1 cells and that activation of AMPK is associated with increased mitochondrial membrane potential. Taken together, these studies suggest that activation of AMPK by AICAR and/or NGF or dmPGE2 may protect and/or reverse NSAID-induced mitochondrial and cellular injury by regulating mitochondrial function. These findings have important basic science and mechanistic implications for epithelial cell physiology and gastrointestinal injury and have important clinical implications.

GRANTS

This work was supported by Merit Review Award no. I01 BX000626-05A2 from the US Department of Veterans Affairs Biomedical Laboratory Research and Development Service (to A. S. Tarnawski).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.A. and A.S.T. conceived and designed research; A.A., N.H., and A.S.T. performed experiments; A.A., N.H., and A.S.T. analyzed data; A.A., N.H., and A.S.T. interpreted results of experiments; A.A., N.H., and A.S.T. prepared figures; A.A. and A.S.T. drafted manuscript; A.A., N.H., M.K.J., and A.S.T. edited and revised manuscript; A.A., N.H., M.K.J., and A.S.T. approved final version of manuscript.

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