TNF-α/Cycloheximide-Induced Oxidative Stress and Apoptosis in Murine Intestinal Epithelial MODE-K Cells

Abstract

In the mouse postoperative ileus model, we have shown an increase in oxidative stress after intestinal manipulation occurring earlier in the mucosa than in the muscular layer, which might contribute to epithelial barrier dysfunction. To address these findings in vitro, we assessed TNF-α/cycloheximide (CHX)-induced oxidative stress and apoptosis in a mouse intestinal epithelial cell line, MODE-K. The influence of heme oxygenase (HO)-1-related products and agents known to reduce reactive oxygen species (ROS) production on TNF-α/CHX-induced oxidative stress and apoptosis were investigated. MODE-K cells were exposed to different concentrations of TNF-α/CHX in the absence/presence of the test agents.Cell viability, caspase-3/7 activity, apoptosis, reduced glutathione level (GSH) and intracellular ROS production were measured. TNF-α/CHX decreased cell viability, increased caspase-3/7 activity, induced apoptosis, reduced the GSH level and increased ROS production in a concentration-dependent manner in MODE-K cells. All these effects of TNF-α/CHX were partially prevented by pretreatment with a carbon monoxide-releasing agent (CORM-A1) and nitrite. The antioxidant resveratrol abolished TNF-α/CHX-induced increase in ROS production and caspase-3/7 activity, but apoptosis was only partially prevented. MODE-K cells are sensitive to TNF-α-induced apoptosis in the presence of CHX, which is associated with increased intracellular ROS production and caspase-3/7 activation. The effects were partially mitigated by CORM-A1, nitrite and resveratrol. Thus, these agents could be of potential use in protecting the epithelial barrier against oxidative stress during intestinal ischemia/reperfusion injury.

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

“…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).…”

“…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.…”

“…concentration without an effect per se on cell viability of MODE-K cells when incubated for 12 h, based on quantitation of ATP (see (Babu et al, 2012;Babu et al, 2015)). …”

“…fluorescence measurement of carboxy-H 2 DCFDA with a microtiter plate reader, and flow cytometric analysis of DNA hypoploidy upon staining with propidium iodide (PI), respectively (Babu et al, 2012). In the actual study, ROS production and cell death were measured simultaneously by flow cytometry, allowing measurement of ROS production in gated viable cells.…”

“…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 A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 6 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).…”