cytochrome-c-t and Intestinal-Diseases

The objectives of this study were to determine whether neutrophil depletion with anti-neutrophil serum (ANS) or preconditioning with the hydrogen sulfide (H(2)S) donor NaHS (NaHS-PC) 24 h prior to ischemia-reperfusion (I/R) would prevent postischemic mitochondrial dysfunction in rat intestinal mucosa and, if so, whether calcium-activated, large conductance potassium (BK(Ca)) channels were involved in this protective effect. I/R was induced by 45-min occlusion of the superior mesenteric artery followed by 60-min reperfusion in rats preconditioned with NaHS (NaHS-PC) or a BK(Ca) channel activator (NS-1619-PC) 24 h earlier or treated with ANS. Mitochondrial function was assessed by measuring mitochondrial membrane potential, mitochondrial dehydrogenase function, and cytochrome c release. Mucosal myeloperoxidase (MPO) and TNF-α levels were also determined, as measures of postischemic inflammation. BK(Ca) expression in intestinal mucosa was detected by immunohistochemistry and Western blotting. I/R induced mitochondrial dysfunction and increased tissue MPO and TNF-α levels. Although mitochondrial dysfunction was attenuated by NaHS-PC or NS-1619-PC, the postischemic increases in mucosal MPO and TNF-α levels were not. The protective effect of NaHS-PC or NS-1619-PC on postischemic mitochondrial function was abolished by coincident treatment with BK(Ca) channel inhibitors. ANS prevented the I/R-induced increase in tissue MPO levels and reversed mitochondrial dysfunction. These data indicate that neutrophils play an essential role in I/R-induced mucosal mitochondrial dysfunction. In addition, NaHS-PC prevents postischemic mitochondrial dysfunction (but not inflammation) by a BK(Ca) channel-dependent mechanism.

Topics: Animals; Benzimidazoles; Cytochromes c; Hydrogen Sulfide; Intestinal Diseases; Intestine, Small; Ischemic Preconditioning; Leukocyte Reduction Procedures; Male; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Diseases; Neutrophils; Peroxidase; Potassium Channels, Calcium-Activated; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sulfides; Tumor Necrosis Factor-alpha

Though recent studies have reported the importance of several endogenous cytoprotective factors including heat shock protein 70 (HSP70) that protect intestinal epithelial cells (IECs) from the effects of stress and injury, the exact mechanism of HSP70 underlying cytoprotection against hypoxia/reoxygenation induced IEC injury remains unclear. The present study was designed to investigate the possible mechanisms by which HSP70 protected IECs against hypoxia/reoxygenation injury and focused on the effects of HSP70 on IEC apoptosis induced by hypoxia/reoxygenation injury. Recombinant adenoviruses (Ad-HSP70) were transfected into the intestinal epithelial cell line in vitro and then suffered from 90 min of hypoxia followed by 60 min of reoxygenation. The LDH leaking, apoptosis, and mitochondrial membrane potential (Psi(m)) were evaluated after hypoxia/reoxygenation. The expression of HSP70, cytochrome c and Bcl-2 protein was determined by Western blot or immunofluorescence analysis. The results show that HSP70 protein was highly expressed in the IECs at 48h following Ad-HSP70 transfection. HSP70 overexpression could reduce LDH leakage and cell apoptosis in IECs following hypoxia/reoxygenation injury. Furthermore, the overexpression of HSP70 significantly reversed the decrease of mitochondrial membrane potential and the release of mitochondrial cytochrome c in IECs during hypoxia/reoxygenation. HSP70 overexpression was also associated with the increasing expression of Bcl-2 protein in IECs during hypoxia/reoxygenation. We conclude that HSP70 protects IECs against hypoxia/reoxygenation induced apoptosis through increasing Bcl-2 expression, which in turn could inhibit the mitochondria-related apoptotic pathway that involves the disruption of the Psi(m) and release of cytochrome c from mitochondria.

Topics: Animals; Apoptosis; Blotting, Western; Cell Hypoxia; Cell Line; Cytochromes c; HSP70 Heat-Shock Proteins; Humans; Intestinal Diseases; Intestinal Mucosa; Lactate Dehydrogenases; Membrane Potential, Mitochondrial; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Rats; Reperfusion Injury; Transfection; Up-Regulation