Avaliação do estresse oxidativo em ilhotas pancreáticas humanas e em cultura de células INS-1E / Evaluation of oxidative stress in human pancreatic islets and INS-1E cells culture

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Human pancreatic islet transplantation is considered a promising strategy to cure the cure Diabetes Mellitus type I. However, transplantation efficiency is dramatically affected by sub-optimum islet recovery in the isolation/purification procedure and islet viability after transplantation. Isolated pancreatic islets are obtained through collagenase perfusion and cell purification in a Ficoll gradient. Reactive oxygen species (ROS) play an important role during human pancreatic islet isolation and may contribute to the decrease in cell viability. The aim of this study was evaluated the response of human pancreatic islets during its isolation/purification and culture time. Activities of superoxide dismutase (SOD) and catalase as well as protein oxidation levels increased in most of analyzed samples, mainly during the Ficoll gradient islet purification step and further culture. Ficoll seems to be the critical step for ROS generation. Nevertheless, it was observed that donors characteristics (aging, cause of death, habits, etc.) and organ preservation conditions (ischemic time, preservation solution, etc.) may be related to our results. To minimize these variations, a physiological cellular model based on INS-1E cells was chosen. The antioxidant enzymes SOD, catalase, glutathione peroxidase (GPx) and glutathione reductase (GR) activities as well the oxidative damage to proteins and lipids, reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, cellular viability and the protein levels of some enzymes responsible for apoptotic signaling like p38, JNK-1, ERK 1-2 and PI3-K upon exposure to polysucrose (1100 mg/mL), a similar of Ficoll, were determined. The SOD, catalase and GPx in samples exposed to polysucrose displayed hight activities. In all cultures, the activity of mitochondrial isoform of SOD (Mn-SOD) corresponds to 50% of total SOD activity. In the presence of polysucrose, the activity of Mn-SOD increased up to 80%. Lipids and protein oxidation levels were also increased and the GSH levels with the GR activity decreased. These results indicated that the exposure of INS-1E cells to polysucrose is associated with oxidative stress. However, the polysucrose exposure was not responsible for cell death although JNK-1, ERK1-2 and PI3-K levels showed hight levels but not p38, upon polysucrose exposure. The expression and activities of antioxidants enzymes are known to be very low in pancreatic islets. N-acetylcysteine (NAC) was added to the INS-1E cultures to prevent oxidative stress. Under these conditions, NAC was able to protect INS- 1E cells from induced oxidative damage by increasing intracellular GSH levels. Taken together, these results suggest that the exposure to polysucrose is related to the oxidative stress in INS-1E cells and NAC seems to be able to maintain cell viability.

ASSUNTO(S)

transplante de ilhotas pancreáticas pancreatic islet transplantation antioxidants enzymes diabetes mellitus enzimas antioxidantes diabetes mellitus oxidative stress estresse oxidativo radicais livres free radicals

ACESSO AO ARTIGO

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