Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells

https://doi.org/10.1016/j.taap.2015.07.007Get rights and content

Highlights

  • In MODE-K IECs, TNF-α/CHX induces correlating ROS, mitochondrial O2radical dot and cell death.

  • CORM-A1 does not influence basal intracellular ROS and mitochondrial O2radical dot levels.

  • Resveratrol increases basal intracellular ROS but decreases mitochondrial O2radical dot levels.

  • CORM-A1 acts solely on NOX-derived ROS to protect from cell death by TNF-α/CHX.

  • Cytoprotection by resveratrol is predominantly due to reduction of mitochondrial O2radical dot.

Abstract

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H2O2-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrial superoxide anion (O2radical dot) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψm) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H2O2-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O2radical dot production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O2radical dot levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that 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. This might explain the more pronounced cytoprotective effect of resveratrol.

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 (O2radical dot) 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

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)

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