Dinesh Babu

CO and CO‐releasing molecules (CO‐RMs) in acute gastrointestinal inflammation

Carbon monoxide (CO) is enzymatically generated in mammalian cells alongside the liberation of iron and the production of biliverdin and bilirubin. This occurs during the degradation of haem by haem oxygenase (HO) enzymes, a class of ubiquitous proteins consisting of constitutive and inducible isoforms. The constitutive HO2 is present in the gastrointestinal tract in neurons and interstitial cells of Cajal and CO released from these cells might contribute to intestinal inhibitory neurotransmission and/or to the control of intestinal smooth muscle cell membrane potential. On the other hand, increased expression of the inducible HO1 is now recognized as a beneficial response to oxidative stress and inflammation. Among the products of haem metabolism, CO appears to contribute primarily to the antioxidant and anti‐inflammatory effects of the HO1 pathway explaining the studies conducted to exploit CO as a possible therapeutic agent. This article reviews the effects and, as far as known today, the mechanism(s) of action of CO administered either as CO gas or via CO‐releasing molecules in acute gastrointestinal inflammation. We provide here a comprehensive overview on the effect of CO in experimental in vivo models of post‐operative ileus, intestinal injury during sepsis and necrotizing enterocolitis. In addition, we will analyse the in vitro data obtained so far on the effect of CO on intestinal epithelial cell lines exposed to cytokines, considering the important role of the intestinal mucosa in the pathology of gastrointestinal inflammation.

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

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

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 (O2(-)) 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 O2(-) production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O2(-) 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.

Mitochondria and NADPH oxidases are the major sources of TNF-α/cycloheximide-induced oxidative stress in murine intestinal epithelial MODE-K cells

TNF-α/cycloheximide (CHX)-induced apoptosis of the mouse intestinal epithelial cell line MODE-K corresponds with the production of reactive oxygen species (ROS). The aim of the study is to investigate the sources of ROS production contributing to apoptotic cell death during TNF-α/CHX-induced oxidative stress in MODE-K cells. Total ROS or mitochondrial superoxide anion production was measured simultaneously with cell death in the absence or presence of pharmacological inhibitors of various ROS-producing systems, and of ROS scavengers/antioxidants. The influence of TNF-α/CHX on mitochondrial membrane potential (Ψ(m)) and cellular oxygen consumption was also studied. TNF-α/CHX time-dependently increased intracellular total ROS and mitochondrial superoxide anion production in MODE-K cells, starting from 2h. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) by a pan-NOX inhibitor (VAS-2870) and a specific inhibitor of Rac1 (NSC23766) significantly reduced TNF-α/CHX-induced total ROS and cell death levels. The mitochondrial electron transport chain inhibitors, amytal (IQ site of complex I) and TTFA (Qp site of complex II) showed a pronounced decrease in TNF-α/CHX-induced total ROS, mitochondrial superoxide anion and cell death levels. TNF-α/CHX treatment caused an immediate decrease in mitochondrial respiration, and a loss of Ψ(m) and increase in mitochondrial dysfunction from 1 h on. The results suggest that mitochondria and NOX are the two major sources of ROS overproduction during TNF-α/CHX-induced cell death in MODE-K cells, with superoxide anions being the major ROS species. Particularly, the quinone-binding sites of mitochondrial complex I (site I(Q)) and complex II (site Qp) seem to be the major sites of mitochondrial ROS production.

Toxicological Evaluation of Emblica officinalis Fruit Extract and its Anti-inflammatory and Free Radical Scavenging Properties

Background: Emblica officinalis (Euphorbiaceae), popularly known as Indian gooseberry or “Amla” in India, is used in Ayurveda as “rejuvenating herb” since ancient times. Objective: This study was carried out to estimate toxicity, anti-inflammatory, and antioxidative activities of the methanolic extract of Emblica officinalis fruit (MEO) in an animal model. Materials and Methods: Antioxidative property of MEO was assessed by in vitro assays such as phosphomolybdenum assay (total antioxidant capacity), free radical scavenging assays 1,1-diphenyl-2-picrylhydrazyl and 2,2’-azino-bis and 3-ethylbenzthiazoline-6-sulphonic acid (DPPH and ABTS method) and lipid peroxidation assay (LPO). The anti-inflammatory property was evaluated by carrageenan-induced acute inflammation in rats by measuring rat paw volume at different time intervals and toxicological analysis using mice. Results and Discussion: High performance liquid chromatography studies revealed the presence of gallic acid (2.10%), mucic acid (4.90%), ellagic acid (2.10%), quercetin (28.00%), rutin (3.89%), and β-glucogallin (1.46%). MEO showed highest antioxidant activities by using DPPH (17.33–89.00%), ABTS (23.03–94.16%), nitric oxide scavenging activity (12.94–70.16%), LPO (56.54%), and phosphomolybdenum assay (142 ± 6.09 μg/ml). The LD50 was found to be approximately 1125 mg/kg (p.o). High dose of MEO showed significant reduction (72.71%) in the inflammation after 4 h of treatment, which was comparable to diclofenac (10 mg/kg) (61.57%) treated group. Significant reduction (P < 0.05) in the inflammatory cytokine (interleukin-1β and tumor necrosis factor-α) markers were also observed (57.25% and 35.41%, respectively) in serum of MEO treated animals as compared to control. Conclusion: Taken together, phenolic compounds of MEO may serve as a potential herbal drug for amelioration of acute inflammation due to their modulatory action on free radicals. SUMMARY The methanolic extract of Emblica officinalis fruit (MEO) has potent antioxidant activity as assessed by DPPH, ABTS and LPO assays MEO has potent anti-inflammatory activity in carrageenan induced paw edema model The phenolic compounds of MEO might be a potential herbal drug for amelioration of acute inflammation. Abbreviations used: ROS, reactive oxygen species; RNS, reactive nitrogen species, LPO, lipid peroxidation, NO, nitric oxide, IL, interleukin; TNF α tumor necrosis factor alpha; NSAIDs, nonsteroidal anti inflammatory drugs; AA, ascorbic acid; MEO, methanolic extract of Emblica officinalis fruit; ABTS+; 2,2’ azino bis 3 ethylbenzthiazoline 6 sulphonic acid; DPPH, 1,1 diphenyl 2 picrylhydrazyl; HPLC, high performance liquid chromatography; MDA, malondialdehyde; DMSO, dimethyl sulphoxide; ELISA, enzyme linked immunosorbent assay.

Differential effects of CORM-2 and CORM-401 in murine intestinal epithelial MODE-K cells under oxidative stress

Carbon monoxide (CO)-releasing molecules (CO-RMs) are intensively studied to provide cytoprotective and anti-inflammatory effects of CO in inflammatory conditions including intestinal inflammation. The water-soluble CORM-A1 reduced apoptosis and NADPH oxidase (NOX)-derived reactive oxygen species (ROS) induced by tumor necrosis factor (TNF)-α/cycloheximide (CHX) in mouse MODE-K intestinal epithelial cells (IECs), without influencing TNF-α/CHX-induced mitochondrial superoxide anion (O2•–). The aim of the present study in the same model was to comparatively investigate the influence of lipid-soluble CORM-2 and water-soluble CORM-401, shown in vitro to release more CO under oxidative conditions. CORM-2 abolished TNF-α/CHX-induced total cellular ROS whereas CORM-401 partially reduced it, both partially reducing TNF-α/CHX-induced cell death. Only CORM-2 increased mitochondrial O2•– production after 2 h of incubation. CORM-2 reduced TNF-α/CHX-, rotenone- and antimycin-A-induced mitochondrial O2•– production; CORM-401 only reduced the effect of antimycin-A. Co-treatment with CORM-401 during 1 h exposure to H2O2 reduced H2O2 (7.5 mM)-induced ROS production and cell death, whereas CORM-2 did not. The study illustrates the importance of the chemical characteristics of different CO-RMs. The lipid solubility of CORM-2 might contribute to its interference with TNF-α/CHX-induced mitochondrial ROS signaling, at least in mouse IECs. CORM-401 is more effective than other CO-RMs under H2O2-induced oxidative stress conditions.