Heparin modulates the expression of genes encoding pro and anti-apoptotic proteins in endothelial cells exposed to intestinal ischemia and reperfusion in rats

PURPOSE: To investigate if expression of genes encoding pro and anti-apoptotic proteins in the rat enteric endothelial cells stimulated by intestinal ischemia followed by reperfusion (IR) can be modified by treatment with heparin (HP).METHODS: Eighteen adult Wistar rats were divided in three groups: sham group submitted to laparotomy only (SG), ischemia followed by reperfusion group (IRG); ischemia followed by reperfusion plus pretreatment with HP 100 mg.kg-1 (IRG+HP). Ischemia was performed by clamping of the superior mesenteric artery. After 60 min of ischemia, metal clamps were removed for reperfusion for 120 min. Gene expression of encoding pro (Casp1, Casp6, Casp3, Cflar, Fas and Pgl) and anti-apoptotic (Bcl2, Bcl2l1 and Naip2) proteins in rat enteric endothelial cells was evaluated by PCR microarray method.RESULTS: Compared to rat endothelial cells of SG, the expression of pro-apoptotic genes was up-regulated in IRG while anti-apoptotic genes were down-regulated. In contrast, the expression of anti-apoptotic genes in IRG+HP was up-regulated while pro-apoptotic genes was down-regulated compared to SG.CONCLUSION: The attenuation by heparin of intestinal ischemia-reperfusion previously demonstrated in rodents could be related with ability of this drug to stimulate and reduce gene expression of encoding anti and pro-apoptotic proteins, respectively.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Federal University of São Paulo Department of SurgeryUNIFESP Pharmacology DepartmentUNIFESP Gynecology DepartmentUniversidade Federal de São Paulo (UNIFESP) Department of BiochemistryUniversidade Federal de São Paulo (UNIFESP) Department of SurgeryUNIFESP, Department of SurgeryUNIFESP, Pharmacology DepartmentUNIFESP, Gynecology DepartmentUNIFESP, Department of BiochemistryUNIFESP, Department of SurgerySciEL

Ischemic preconditioning and the gene expression of enteric endothelial cell biology of rats submitted to intestinal ischemia and reperfusion

PURPOSE: To investigate the effects of ischemic preconditioning (IPC) on the expression of pro and anti-apoptotic genes in rat endothelial cells undergoing enteric ischemia (I) and reperfusion (R). METHODS: Thirty rats underwent clamping of the superior mesenteric vessels. Sham group (GS) laparotomy only; Ischemia (GI): intestinal ischemia (60 min); Ischemia and Reperfusion (GIR): ischemia (60 min) and reperfusion (120 min); Ischemia and intestinal ischemic preconditioning (GI + IPC) : 5 minutes of ischemia followed by 10 min of reperfusion before sustained ischemia (60 min) ischemia and reperfusion and IPC (GIR + IPC): 5 min ischemia followed by 10 min of reperfusion before sustained ischemia (60min) and reperfusion (120 min). Rat Endothelial Cell Biology (PCR array) to determine the expression of genes related to endothelial cell biology. RESULTS: Gene expression of pro-apoptotic markers (Casp1, Casp6, Cflar, Fas, and Pgl) was down regulated in GI+IPC and in GIR + IPC. In contrast, the expression of anti-apoptotic genes (Bcl2 and Naip2), was up-regulated in GI + IPC and in GIR + IPC. CONCLUSION: Ischemic preconditioning may protect against cell death caused by ischemia and reperfusion.UNIFESPUNIFESPSciEL

The role of ischemic preconditioning and pentoxifylline in intestinal ischemia/reperfusion injury of rats

Purpose: To investigate the role of ischemic preconditioning (IPC) and pentoxifylline (PTX) in intestinal mucosa ischemia/reperfusion injury (IR). Methods: Thirty rats were assigned to 5 groups (N= 6): (CG): no clamping of the superior mesenteric artery (90 min.)(IR-SS): saline + ischemia (30 min.) + reperfusion (60 min.)(IRPTX): PTX + ischemia (30min.) + reperfusion (60 min.)(IPC-IR-SS): 5 min. of ischemia + 5 minutes of reperfusion (IPC) + saline + ischemia (30 min.) + reperfusion (60 min.)(IPC-IR-PTX): 5 min. of ischemia + 5 min. of reperfusion (IPC) + PTX + 30 min. of I + 60 minutes of R. Results: The IR-PTX, IPC-IR-SS and IPC-IR-PTX groups had significantly lower scores of mucosa damage than the IR-SS group. IR-PTX group showed higher scores than the IPC-IR-PTX group, in accordance with the hypothesis of a favorable effect of IPC alone or in association with PTX. Additionally, IPC-IR-SS had a higher damage score than the IPC-IR-PTX. The villi height and crypt depth were similar in all groups. The villi height in the IR-SS was significantly lower. Conclusion: Ischemic preconditioning or pentoxifylline alone protect the intestinal mucosa from ischemia/reperfusion injury. However, they do not have a synergistic effect when applied together.UFGD, Ministerio da Educacao do BrasilUFGD, Sch Hlth Sci, Dourados, MS, BrazilUniv Sao Paulo UNIFESP, Dept Morfol & Genet, Sao Paulo, SP, BrazilUniv Estadual Campinas UNICAMP, Sch Med, Dept Publ Hlth, Publ Hlth, Campinas, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Surg, Div Surg Tech & Expt Surg, Sao Paulo, SP, BrazilUniv Sao Paulo UNIFESP, Dept Morfol & Genet, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Dept Surg, Div Surg Tech & Expt Surg, Sao Paulo, SP, BrazilUFGD, Ministerio da Educacao do BrasilWeb of Scienc

Low Concentrations of Hydrogen Peroxide or Nitrite Induced of Paracoccidioides brasiliensis Cell Proliferation in a Ras-Dependent Manner

Paracoccidioides brasiliensis, a causative agent of paracoccidioidomycosis (PCM), should be able to adapt to dramatic environmental changes inside the infected host after inhalation of air-borne conidia and transition to pathogenic yeasts. Proteins with antioxidant functions may protect fungal cells against reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocytic cells, thus acting as potential virulence factors. Ras GTPases are involved in stress responses, cell morphology, and differentiation in a range of organisms. Ras, in its activated form, interacts with effector proteins and can initiate a kinase cascade. in lower eukaryotes, Byr2 kinase represents a Ras target. the present study investigated the role of Ras in P. brasiliensis after in vitro stimulus with ROS or RNS. We have demonstrated that low concentrations of H2O2 (0.1 mM) or NO2 (0.1-0.25 mu M) stimulated P. brasiliensis yeast cell proliferation and that was not observed when yeast cells were pre-incubated with farnesyltransferase inhibitor. We constructed an expression plasmid containing the Byr2 Ras-binding domain (RBD) fused with GST (RBD-Byr2-GST) to detect the Ras active form. After stimulation with low concentrations of H2O2 or NO2, the Ras active form was observed in fungal extracts. Besides, NO2 induced a rapid increase in S-nitrosylated Ras levels. This alternative posttranslational modification of Ras, probably in residue Cys123, would lead to an exchange of GDP for GTP and consequent GTPase activation in P. brasiliensis. in conclusion, low concentrations of H2O2 or NO2 stimulated P. brasiliensis proliferation through Ras activation.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenacao Aperfeicoamento de Pessoal de Nivel SuperiorUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol CTCMol, Dept Bioquim Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol CTCMol, Dept Bioquim Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, BrazilFAPESP: 2011/14392-2CNPq: 477764/2009-6Web of Scienc

Study of Heparin in Intestinal Ischemia and Reperfusion in Rats: Morphologic and Functional Evaluation

To study whether treatment with heparin (HEP) attenuates intestinal dysfunction caused by ischemia (I) and reperfusion (R), rats were treated with HEP (100 U/kg intravenously) or saline solution (SS) before I (60 min), which was produced by occlusion of the superior mesenteric artery, and R (120 min). After I or I/R, we mounted 2-cm jejunal segment in an organ bath to study neurogenic contractions stimulated by electrical pulses or KCl, using a digital recording system. Thin jejunal slices were stained with hematoxylin and eosin for optical microscopy. Compared with the sham group, jejunal contractions were similar in the I + HEP and the I/R + HEP groups, but reduced in the I + SS and the I/R + SS groups. the jejunal enteric nerves were damaged in the I + SS and the I/R + SS, but not in the I + HEP and the I/R + HEP cohorts. These results suggested that HEP attenuated intestinal dysfunction caused by I and I/R.Universidade Federal de São Paulo, Escola Paulista Med, Dept Surg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morphol, BR-04023900 São Paulo, BrazilFed Univ Great Dourados, Sch Med, Dourados, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Surg, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pharmacol, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Morphol, BR-04023900 São Paulo, BrazilWeb of Scienc