Kanho Rai

The pathophysiology of non-steroidal anti-inflammatory drug (NSAID)-induced mucosal injuries in stomach and small intestine

Non-steroidal anti-inflammatory drugs are the most commonly prescribed drugs for arthritis, inflammation, and cardiovascular protection. However, they cause gastrointestinal complications. The pathophysiology of these complications has mostly been ascribed to non-steroidal anti-inflammatory drugs’ action on the cyclooxygenase inhibition and the subsequent prostaglandin deficiency. However, recent clinical demonstrated the prevalence of non-steroidal anti-inflammatory drugs-induced small intestinal mucosal injury is more often than previously expected. In this review, we discuss the defense mechanisms of stomach, and the pathophysiology of non-steroidal anti-inflammatory drugs-induced injury of stomach and small intestine, especially focused on non-steroidal anti-inflammatory drugs’ action on mitochondria.

Gastric acid induces mitochondrial superoxide production and lipid peroxidation in gastric epithelial cells

BackgroundGastric hydrochloric acid (HCl) has been regarded as an inciting factor in gastric mucosal injuries and has been reported to induce lipid peroxidation in vitro. However, because HCl is not an oxidant per se, the exact mechanism by which the acid induces lipid peroxidation is unknown. We hypothesized that gastric acid may disrupt mitochondrial transmembrane potential and induce the production of superoxide in mitochondria, which subsequently may induce lipid peroxidation and apoptosis in gastric mucosal cells.MethodsFirstly we treated gastric epithelial RGM1 cells with solutions containing various concentrations of HCl (i.e., of varying pH), and examined cellular injury, lipid peroxidation, and apoptosis with specific fluorescent dyes. Secondly, we performed electron paramagnetic resonance (EPR) spectroscopy of isolated, acid-exposed mitochondria from the cells, using a spin-trapping reagent for superoxide, 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). Finally, we established novel RGM1 cells that overexpressed manganese superoxide dismutase (MnSOD), which removes superoxide from mitochondria, and examined the effect of acid treatment on cellular membrane lipid peroxidation.ResultsThe results indicated that the exposure to acid indeed induced cellular injury, cellular lipid peroxidation, apoptosis, and the demonstration of the exact superoxide spectra on EPR spectroscopy in gastric epithelial cells, and that overexpression of MnSOD decreased superoxide production and prevented cellular lipid peroxidation.ConclusionThese results suggested that gastric acid, like nonsteroidal anti-inflammatory drugs (NSAIDs), induces mitochondrial superoxide production, which induces gastric cellular injury by triggering cellular lipid peroxidation and apoptosis.

Lansoprazole inhibits mitochondrial superoxide production and cellular lipid peroxidation induced by indomethacin in RGM1 cells

Lansoprazole is effective in healing non-steroidal anti-inflammatory drugs induced ulcers, and antioxidant properties have been thought to play a key role in healing ulcers. We hypothesize that lansoprazole exerts a cytoprotective effect by inhibiting reactive oxygen species leakage from mitochondria and lipid peroxidation. We pretreated gastric epithelial RGM1 cells with lansoprazole and then treated them with indomethacin in vitro. We found that the lansoprazole pretreatment significantly reduced cellular injury, maintained mitochondrial transmembrane potential, and decreased lipid peroxidation. Furthermore, the signal intensity of the electron spin resonance spectrum of the indomethacin-treated mitochondria which were pretreated with lansoprazole showed considerable reduction compared to those without the lansoprazole pretreatment. These results suggest that lansoprazole reduced superoxide production in the mitochondria of indomethacin treated cells, and subsequently inhibited lipid peroxide and cellular injury in gastric epithelial cells.

Bisphosphonate-induced gastrointestinal mucosal injury is mediated by mitochondrial superoxide production and lipid peroxidation

Bisphosphonates such as alendronate and risedronate are commonly used for the treatment of postmenopausal osteoporosis. They have the gastrointestinal adverse effects such as erosions and ulcers in stomach and small intestine. However, the detailed biological mechanism remains to be elucidated. Since alendronate is suggested to increase the risk of non-steroidal anti-inflammatory drug-related gastropathy, we hypothesized that bisphosphonates and non-steroidal anti-inflammatory drugs have the same pathophysiological mechanisms in gastrointestinal mucosa: Bisphosphonates may induce cellular lipid peroxidation by inducing the production of mitochondrial superoxide. We also hypothesized that geranylgeranylacetone, an antiulcer drug, may prevent lipid peroxidation by reducing superoxide production. We treated gastric RGM1 cells and small intestinal IEC6 cells with alendronate or risedronate, and examined cellular injury, lipid peroxidation and superoxide production with specific fluorescent dyes, and underwent electron paramagnetic resonance spectroscopy to detect the production of superoxide in vitro. The results indicated that bisphosphonates indeed induced cellular injury, cellular lipid peroxidation, and superoxide production. We also demonstrated that the pretreatment of geranylgeranylacetone decreased superoxide production and prevented cellular lipid peroxidation. These results suggested that bisphosphonates, like non-steroidal anti-inflammatory drugs, induce lipid peroxidation by producing mitochondrial superoxide, which was prevented by geranylgeranylacetone.

Rebamipide Attenuates Nonsteroidal Anti-Inflammatory Drug (NSAID)-Induced Superoxide Production and Lipid Peroxidation by Increasing the Expression of Manganese Superoxide Dismutase (MnSOD) Protein in Gastric and Small Intestinal Epithelial Cells

Early mucosal restitution occurs as a consequence of intestinal epithelial cell (IEC) migration to reseal superficial wounds, a process independent of cell proliferation. Canonical transient receptor potential-1 (TRPC1) is highly expressed in IECs and functions as a Ca2+ permeable channel mediating store-operated Ca2+ entry (SOCE) and plays an important role in GI mucosal restitution. However, the exact mechanism by which TRPCs are activated after wounding remains elusive. Caveolin-1 (Cav1) is a major component associated with caveolar lipid rafts in the plasma membrane and was recently identified as a regulator of SOCE. This study tests hypothesis that Cav1 plays a role in the regulation of GI epithelial restitution by activating TRPC1 channel activity.Methods: Studies In Vitrowere conducted in differentiated IEC-Cdx2L1 cells and stable TRPC1-transfected cells (IEC-TRPC1). The level of Cav1 was decreased by siRNA targeting Cav1 (siCav1) but it was increased by ectopic Cav1 overexpression. Function of Cav1 and its interaction with TRPC1 In Vivo were measured by using Cav1 knock-out mice. [Ca]cyt was measured by fluorescence digital imaging analysis, and SOCE was elicited by passive store depletion using cyclopiazonic acid (CPA). Restitution In Vitro was measured in a model that mimics early cell division-independent stages of epithelial restitution, whereas it was examined In Vivo by using hypertonic NaClinduced mucosal injury model. Results: Cav1 was highly expressed in the GI mucosa of the mouse and in cultured IECs. Cav1 protein interacted with TRPC1 as measured by immunoprecipitation assays. Cav1 silencing by transfection with siCav1 reduced store depletion-induced SOCE in stable IEC-TRPC1 cells, although it did not reduce resting [Ca]cyt. The levels of CPA-induced Ca2+ influx was inhibited by ~70% in Cav1-silenced IEC-TRPC1 cells. Inhibition of Cav1 expression and subsequent decrease in Ca2+ influx by siCav1 repressed restitution as indicated by a decrease in cell migration (by ~50%) after wounding. In contrast, stable ectopic Cav1 overexpression (IEC-Cav1) increased SOCE (by ~55%) and enhanced epithelial restitution after wounding. The numbers of cells migrating over the wounded edge in stable IEC-Cav1 cells were increased by almost ~45% at 6 h after wounding compared to cells transfected with the empty vector lacking Cav1 cDNA. Results from In Vivo studies further revealed that interactions of Cav1 with TRPC1 decreased in Cav1 knockout mice, which was associated with delayed intestinal mucosal healing compared to wildtype animals. Conclusions: These results indicate that 1) Cav1 physically interacts with and activates TRPC1 channel activity and 2) induced [Cav1/TRPC1] complexes after injury stimulate gut epithelial restitution as a result of increase in TRPC1-mediated Ca2+ signaling.