Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells
Graphical abstract
Introduction
Disturbance of the intestinal epithelial barrier function is observed with the development of mucosal inflammation during acute and chronic enteropathies (Sharma and Tepas, 2010, Snoek et al., 2012, Pastorelli et al., 2013). Reactive oxygen species (ROS) are one of the major key players involved in the initiation and progression of inflammation (Mittal et al., 2014). Oxidative stress-induced epithelial cell damage leading to increased intestinal permeability and translocation of intraluminal endotoxins might trigger muscular inflammation during various conditions involving acute gastrointestinal (GI) inflammation such as postoperative ileus, septic ileus, necrotizing enterocolitis and acute intestinal ischemia/reperfusion (I/R) (Anup et al., 1999, de Winter et al., 2005, Baregamian et al., 2009, De Backer et al., 2009, Guan et al., 2009). Moreover, overproduction of ROS with extensive mucosal injury has also been observed for chronic GI inflammation in animal models of inflammatory bowel disease (IBD) (Ahn et al., 2001, Reifen et al., 2004, Cetinkaya et al., 2005) and in colonic samples of ulcerative colitis patients (Oshitani et al., 1993, Nishikawa et al., 2005). Antioxidant treatment appears to reduce oxidative stress and associated inflammation in animal models of colitis (Millar et al., 1996, Damiani et al., 2007, Vasina et al., 2010) and in patients with ulcerative colitis (Aghdassi et al., 2003, Barbosa et al., 2003, Seidner et al., 2005).
An array of pro-inflammatory cytokines is released within the intestinal mucosa during various GI inflammatory disorders (Kim et al., 2012) and, among others, tumor necrosis factor (TNF)-α is an early inflammatory mediator in inflamed intestine (Holtmann et al., 2002). Induction of intestinal epithelial cell (IEC) apoptosis and cell shedding by TNF-α is thought to play an important role in epithelial barrier dysfunction. We and others have reported that ROS play an important role in TNF-α-induced apoptotic cell death of IECs (Jin et al., 2008, Baregamian et al., 2009, Babu et al., 2012). In rat IEC-6 cells, the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family and the mitochondrial electron transport chain (ETC.) are the two major ROS-producing sources involved in TNF-α/cycloheximide (CHX)-induced cell death (Jin et al., 2008). The same accounts for murine MODE-K cells; particularly, in addition to NOX, complexes I and II of the mitochondrial ETC. were found to be the main sites of superoxide anion (O2−) production from the mitochondria, the primary ROS species originating from this organelle (Babu et al., 2015). These data indicate that the endogenous antioxidant defense system might not be sufficient to counteract TNF-α-induced ROS production, suggesting that mitigating excessive ROS production might be of therapeutic value to reduce intestinal barrier dysfunction during GI inflammation.
Among the endogenous intracellular antioxidant pathways, heme oxygenase-1 (HO-1) has a prominent role in the adaptation of tissues against oxidative stress since this redox-sensitive inducible enzyme generates biliverdin, a powerful antioxidant, and carbon monoxide (CO), which acts as a crucial signaling factor mediating a variety of important pharmacological effects (Ryter et al., 2006). CO-releasing molecules (CO-RMs), a class of organometallic compounds liberating CO in biological systems in a controllable manner, have been developed to mimic the antioxidant, anti-inflammatory and cytoprotective effects of CO (Motterlini et al., 2002, Sawle et al., 2005, Motterlini and Otterbein, 2010). The inhibitory effect of CO and CO-RMs on cytokine-induced changes in IECs might contribute to their beneficial effect in acute GI inflammation (Babu et al., 2014). The exact mechanism(s) of action of CO is still under scrutiny but emerging evidence indicates that the beneficial properties of CO may be linked to its ability to bind to heme-containing proteins such as NOX and mitochondrial complexes in different tissues (Taille et al., 2005, Bilban et al., 2008). The cytoprotective properties of CO/CO-RMs in IECs and their effect on cellular targets mediating ROS production in comparison to classical antioxidants have not been investigated so far. Resveratrol, a bioactive polyphenolic antioxidant present in red wine has been extensively studied with regard to cardiovascular and neuronal protection (Foti Cuzzola et al., 2011, Wang et al., 2012). Experimental data show that it also significantly ameliorates acute intestinal inflammation, such as induced by I/R (Ozkan et al., 2009) or by oral infection with Toxoplasma gondii (Bereswill et al., 2010). We previously showed that CORM-A1 as well as resveratrol reduced both TNF-α/CHX-induced ROS production and apoptosis in MODE-K IECs (Babu et al., 2012). The aim of the present study was therefore to investigate the influence of CORM-A1 and resveratrol on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells. We also examined the effects of CORM-A1 and resveratrol on mitochondrial function by assessing TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψm) and cellular oxygen consumption.
Section snippets
Chemicals and reagents
Reagents for cell culture, including Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, glutamax and fetal bovine serum were obtained from Gibco BRL (Grand Island, NY, USA). JC-10 was purchased from Enzo Life Sciences (Zandhoven, Belgium). Carboxylated analogue of 2′7′-dichlorodihydrofluorescein diacetate acetyl ester (carboxy-H2DCFDA), dihydrorhodamine 123 (DHR123), Hoechst blue 33342, MitoTracker Deep Red FM, MitoTracker Green FM, MitoSOX Red, Sytox Green, Sytox Red and
Effects of CORM-A1 and resveratrol on TNF-α/CHX-induced changes in intracellular total ROS production and cell death
To determine whether the modulation of ROS production by CORM-A1 and resveratrol could contribute to their corresponding cytoprotective activities, TNF-α/CHX-induced intracellular total ROS production and cell death were simultaneously measured by flow cytometry analysis. Treatment of MODE-K cells with CORM-A1 (100 μM) per se was without effect but CORM-A1 significantly reduced both TNF-α/CHX-induced ROS production (Fig. 1A and C) and dead cells (Fig. 1D). In contrast, resveratrol (75 μM) per se
Discussion
Treatment of MODE-K cells with TNF-α/CHX induces apoptosis, which is associated with increased production of ROS. We previously found that CORM-A1 and resveratrol reduce both these effects (Babu et al., 2012). We have also recently identified that NOX and mitochondrial ETC. complexes (I and II) are the major sources of ROS production during TNF-α/CHX-induced apoptosis (Babu et al., 2015). Here, we investigated the influence of CORM-A1 and resveratrol on these pathways. The principal finding of
Conclusion
In conclusion, the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. These data infer that interference of CORM-A1 and resveratrol with particular intracellular ROS production sites could have some therapeutic value and therefore should be further tested for the treatment of acute GI inflammatory diseases.
Abbreviations
- TNF-α
tumor necrosis factor (TNF)-α
- CHX
cycloheximide
- IEC
intestinal epithelial cell
- CO-RMs
carbon monoxide-releasing molecules
- CORM-A1
sodium boranocarbonate
- ROS
reactive oxygen species
- NADPH
nicotinamide adenine dinucleotide phosphate
- NOX
NADPH oxidase
- ETC.
electron transport chain
- OCR
oxygen consumption rate
- PKB
protein kinase B
- GSH
reduced glutathione
- SIRT
silent mating-type information regulation 2 homologue
- AMPK
adenine monophosphate-activated protein kinase
The following are the supplementary data related to this
Conflict of interest
The authors report no conflict of interest.
Authors' contributions
DB and RL conceived and designed the experiments. QR helped in designing some experiments during the initial stage of the project. DB and GL performed the experiments. VG assisted in selected experiments. GL, RM and PV contributed reagents or analytical tools. DB and RL wrote the manuscript. All authors read and approved the final manuscript.
Transparency Document
Acknowledgments
The study was supported by RL via a grant G.0021.09N from the Fund of Scientific Research Flanders (FWO) and COST Action BM1005 (European Network on Gasotransmitters). QR had a postdoc position from FWO. VG is paid by the Methusalem grant to PV. Research in Vandenabeele's unit is further supported by Belgian grants (Interuniversity Attraction Poles, IAP 6/18, IAP 7/32), Flemish grants (FWO G.0875.11, FWO G.0973.11N, FWO G.0A45.12N, FWO G.0172.12, FWO G.0787.13N, G0C3114N and FWO KAN 31528711)
References (83)
- et al.
Antioxidant vitamin supplementation in Crohn's disease decreases oxidative stress. A randomized controlled trial
Am. J. Gastroenterol.
(2003) - et al.
Surgical stress and the small intestine: role of oxygen free radicals
Surgery
(1999) - et al.
Mitochondria and NADPH oxidases are the major sources of TNF-alpha/cycloheximide-induced oxidative stress in murine intestinal epithelial MODE-K cells
Cell. Signal.
(2015) - et al.
Decreased oxidative stress in patients with ulcerative colitis supplemented with fish oil omega-3 fatty acids
Nutrition
(2003) - et al.
Carbon monoxide orchestrates a protective response through PPARgamma
Immunity
(2006) - et al.
Production of reactive oxygen species by mitochondria: central role of complex III
J. Biol. Chem.
(2003) - et al.
RIP kinase-dependent necrosis drives lethal systemic inflammatory response syndrome
Immunity
(2011) - et al.
Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death
Free Radic. Biol. Med.
(2010) - et al.
A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species
Free Radic. Biol. Med.
(2011) - et al.
Compartmentalized oxidative stress in dopaminergic cell death induced by pesticides and complex I inhibitors: distinct roles of superoxide anion and superoxide dismutases
Free Radic. Biol. Med.
(2013)
Resveratrol protects primary rat hepatocytes against oxidative stress damage: activation of the Nrf2 transcription factor and augmented activities of antioxidant enzymes
Eur. J. Pharmacol.
NADPH oxidases 1 and 4 mediate cellular senescence induced by resveratrol in human endothelial cells
Free Radic. Biol. Med.
An oral supplement enriched with fish oil, soluble fiber, and antioxidants for corticosteroid sparing in ulcerative colitis: a randomized, controlled trial
Clin. Gastroenterol. Hepatol.
CO from enhanced HO activity or from CORM-2 inhibits both O-2(−) and NO production and downregulates HO-1 expression in LPS-stimulated macrophages
Biochem. Pharmacol.
Topology of superoxide production from different sites in the mitochondrial electron transport chain
J. Biol. Chem.
Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle
J. Biol. Chem.
Immortalization of mouse intestinal epithelial cells by the SV40-large T gene. Phenotypic and immune characterization of the MODE-K cell line
J. Immunol. Methods
Carbon monoxide protects against hyperoxia-induced endothelial cell apoptosis by inhibiting reactive oxygen species formation
J. Biol. Chem.
Targeting mitochondria-derived reactive oxygen species to reduce epithelial barrier dysfunction and colitis
Am. J. Pathol.
Pro-oxidant activity of low doses of resveratrol inhibits hydrogen peroxide-induced apoptosis
Ann. N. Y. Acad. Sci.
Efficacy of use of colonoscopy in dextran sulfate sodium induced ulcerative colitis in rats: the evaluation of the effects of antioxidant by colonoscopy
Int. J. Colorectal Dis.
Carbon monoxide and mitochondria-modulation of cell metabolism, redox response and cell death
Front. Physiol.
TNF-alpha/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells
Curr. Pharm. Des.
CO and CO-releasing molecules (CO-RMs) in acute gastrointestinal inflammation
Br. J. Pharmacol.
Tumor necrosis factor-alpha and apoptosis signal-regulating kinase 1 control reactive oxygen species release, mitochondrial autophagy, and c-Jun N-terminal kinase/p38 phosphorylation during necrotizing enterocolitis
Oxid. Med. Cell. Longev.
Uncoupling protein 2 negatively regulates mitochondrial reactive oxygen species generation and induces phosphatase-mediated anti-inflammatory response in experimental visceral leishmaniasis
J. Immunol.
Nox4 NADPH oxidase mediates oxidative stress and apoptosis caused by TNF-alpha in cerebral vascular endothelial cells
Am. J. Physiol. Cell Physiol.
Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-alpha-induced apoptosis
Am. J. Physiol. Cell Physiol.
Pivotal role of NOX-2-containing NADPH oxidase in early ischemic preconditioning
FASEB J.
Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation
PLoS ONE
Heme oxygenase and carbon monoxide initiate homeostatic signaling
J. Mol. Med.
NADPH oxidase promotes NF-kappaB activation and proliferation in human airway smooth muscle
Am. J. Physiol. Lung Cell. Mol. Physiol.
Beneficial effects of N-acetylcysteine on acetic acid-induced colitis in rats
Tohoku J. Exp. Med.
Hypoxia-inducible factor 1alpha stabilization by carbon monoxide results in cytoprotective preconditioning
Proc. Natl. Acad. Sci. U. S. A.
NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon
Mol. Cell. Biol.
Age-associated proinflammatory secretory phenotype in vascular smooth muscle cells from the non-human primate Macaca mulatta: reversal by resveratrol treatment
J. Gerontol. A Biol. Sci. Med. Sci.
Oxidative stress and metabolism in animal model of colitis induced by dextran sulfate sodium
J. Gastroenterol. Hepatol.
Inhibition of cellular respiration by endogenously produced carbon monoxide
J. Cell Sci.
Water-soluble CO-releasing molecules reduce the development of postoperative ileus via modulation of MAPK/HO-1 signalling and reduction of oxidative stress
Gut
Resveratrol as an antioxidant and pro-oxidant agent: mechanisms and clinical implications
Biochem. Soc. Trans.
Role of oxidative stress in the pathogenesis of septic ileus in mice
Neurogastroenterol. Motil.
Cited by (36)
Colon delivery of resveratrol for the treatment of inflammatory bowel disease
2024, Journal of Drug Delivery Science and TechnologyThe effects of carbon monoxide releasing molecules on paraquat-induced pulmonary interstitial inflammation and fibrosis
2021, ToxicologyCitation Excerpt :Of particular therapeutic interest are the water-soluble molecules carbon monoxide releasing molecule 3 (CORM-3) and carbon monoxide releasing molecule A1 (CORM-A1). Treatment of lipopolysaccharide-stimulated murine macrophages with CORM-3 caused a marked increase in HO-1 expression and heme oxygenase activity, reduced nitrite generation, and significantly suppressed the inflammatory response with no evident cytotoxicity (Sawle et al., 2005; Babu et al., 2015). Another study in cultured rat PA-SMCs revealed that CORM-3 inhibits pulmonary vascular remodeling viap21 and reverses pulmonary hypertension and distal pulmonary artery muscularization in mice exposed to chronic hypoxia (Abid et al., 2014; Lawendy et al., 2014).
Heme is required for carbon monoxide activation of mitochondrial BK<inf>Ca</inf> channel
2020, European Journal of PharmacologyCitation Excerpt :While CORM-A1 has been shown to inhibit amiloride-sensitive sodium currents in human bronchiolar epithelial cells (Althaus et al., 2009), little is known about the action of CORM-A1 as a potassium channel modulator. Moreover, CORM-A1 has been studied, as have CORM-2 and CORM-401, in a model of oxidative stress in endothelial cells (Babu et al., 2015, 2017). The application of all tested CORMs resulted in antioxidant and cytoprotective effects.
Resveratrol protects against PM2.5-induced heart defects in zebrafish embryos as an antioxidant rather than as an AHR antagonist
2020, Toxicology and Applied PharmacologyInvolvement of Nrf2 and Keap1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent peptides from soft-shelled turtle
2020, Process BiochemistryCitation Excerpt :The Arg 415 was bond to the peptide amino acid residues “Glu”, and the Arg 380 was interactive with “Gly”, these two amino acid residues was the “head” and “tail” of the peptide EDYGA, so it can get a very stable posture in the active pocket of Keap1 Kelch domain (Fig. 4). It is believed that overproduction of reactive oxygen species generates oxidative stress in cells [41,42]. Oxidative stress results in various pathophysiological conditions, especially cancers and neurodegenerative diseases.