Μελέτη της διαταραχής της ισορροπίας των οξειδωτικών / αντιοξειδωτικών μηχανισμών στον επαγόμενο από στρεπτοζοτοκίνη διαβήτη επίμυων και οι πιθανές της παρεμβολές στους μηχανισμούς ηπατικής αναγέννησης. Μελέτη της τροποποίησης της ανωτέρω ισορροπίας από τον αναστολέα του υποδοχέα της αγγειοτασίνης ΙΙ και του αναστολέα της φωσφοδιεστεράσης

Abstract

Background: Increased oxidative stress and alterations in enzymatic antioxidant defense system (EADS) seems to relate with some pathological events accompanying both clinical and experimental diabetes. Angiotensine II (Ang II) produced by local Ang II - generating system of vessel’s wall and islet’s β-cells, induces its pleiotropic effects via the AT₁ receptor through NADPH - and NOS - driven generation of ROS / RNS. Recent evidence supports that blockade of Ang II AT₁ receptor thus reducing the incidence of diabetes in “at risk” patients. Phosphodiesterase type IV (PDE4) specific inhibitor, Rolipram, increases intracellular cAMP levels and suppresses inflammatory cytokine production and ROS / RNS generation by lymphocytes and inflammatory cells. It has been recently shown that islet’s β-cells express PDE4, which can regulate their function through the cAMP - protein kinase A signaling pathways. Therefore, the aim of the present study was to assess whether diabetes induced changes in parameters of glucose metabolism, oxidative stress and the function of systemic enzymatic antioxidant defense system (EADS) are prevented by AT₁ receptor blocker, Losartan, and PDE4 specific inhibitor, Rolipram. As oxidative injury is a potential mechanism of hepatocyte apoptosis, oxidative stress may be a significant risk factor for liver regeneration after major hepatic resection and liver transplantation in patients suffering from diabetes. The PDE4 regulates intracellular cAMP level in rat hepatocytes and is sensitive to insulin. The cAMP PDEs inhibitors have been suggested to improve hepatic reperfusion injury but the effects of these agents on liver regeneration have not been thoroughly investigated. Thus, we assessed systemic and liver oxidative stress after partial hepatectomy in diabetic rats and explore the influence of Rolipram. Materials and methods: In this study male Wistar rats (205 - 250g) were used. Diabetes was induced by an intraperitoneal injection of Streptozotocin (STZ; 65mg/kg). The animals were diabetic if their blood glucose values were more than 200 mg/dl. Two series of experiments were performed, each of which lasted for eight days. In the first series of experiments rats were randomized into three groups: control rats, untreated diabetic rats and diabetic rats treated with Losartan (20 mg kg⁻¹ day⁻¹). In the second series of experiments rats were randomized into five groups: control rats, untreated diabetic rats and three groups of diabetic rats treated with 3,5, 7, and 10.5 mg kg⁻¹ day⁻¹ Rolipram, respectively. Rolipram was an offer of Shering Company (Berlin Germany). Blood glucose and body weight were determined as markers of metabolic state at 0, 3, 7 and 11 days after the administration of STZ. At the end of both experiments the rats were sacrificed by decapitation. Blood and pancreas were taken for analysis. Grading of islet destruction was performed using paraffin sections of pancreas stained with hematoxylin and eosin. Systemic lipid peroxidation was determined as a marker of oxidative stress by measuring plasma concentration of thiobarbituric acid - reactive substances (TBARS). Plasma activities of superoxide dismutase, (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were assessed to evaluate the state of EADS. To assess the effect of Rolipram on liver regeneration in STZ - induced diabetes, rats were divided into four groups: normal sham operated (N-SO), normal partially hepatectomized (N-PH), diabetic partially hepatectomized (STZ-PH) and diabetic partially hepatectomized Rolipram (10.5 mg kg⁻¹ day⁻¹) treated rats (STZ-Rol-PH). Experimental analyses were carried out 24 hours after hepatectomy by measuring plasma TBARS levels and the presence of 3 - nitrotyrosine in liver sections. Mitotic and PCNA indices were measured to evaluate liver regeneration. Results: STZ produced diabetes in rats 72 hours after its administration (blood glucose levels > 200mg/dl). Blood glucose levels enhanced and body weight reduced in a time-dependent manner during the eleven days of evolution of untreated diabetes which was in accordance with the degree of islets destruction (63% of islets were of 3+ grade) found at the end of experiment. TBARS levels were increased (~3 fold) in diabetic rats when compared to those in control group. The significant positive correlation between TBARS and glucose levels suggests the implication of hyperglycemia in systemic oxidative stress induction. At the end of the experimental period EADS activity was increased in STZ-treated rats and negatively correlated with lipid peroxidation products, indicating possible up regulation of the enzymes by these products. The comparison of the time course changes of metabolic and clinical signs of the disease in Losartan-treated diabetic rats with those of untreated diabetic animals showed a beneficial effect AT₁ inhibitor based on the above parameters. These results were further confirmed by the protective effect of Losartan on STZ-induced islets destruction. Losartan treatment significantly reduced TBARS levels in plasma of diabetic rats and improved changes in EADS function. Rolipram treatment improved clinical and biochemical signs of diabetes in dose - dependent manner. Administration of 10.5 mg kg⁻¹ day⁻¹ Rolipram to STZ-diabetic rats reduced the average grade of islets destruction from 2.64±0.10 to 1.71±0.13, p<0.001. Moreover, Rolipram treatment produced dose-dependent reduction in the systemic oxidative stress and restoration of EADS activity to almost its baseline level. Systemic oxidative stress was significantly higher in N-PH group in comparison to that in N-SO group, presumably due to non specific inflammatory response to the surgery. It was significantly increased in STZ-PH group compared to that in N-PH group and was reduced to nearly normal values in STZ-Rol-PH group. Hepatic regeneration was significantly decreased in STZ-PH group in comparison to that in N-PH group. The effect of diabetes on liver regeneration was improved by the administration of Rolipram. The absence of positive staining for 3-nitrotyrosine in regenerating livers of both N-PH and STZ-PH groups as well as in STZ-Rol-PH group might be an indication of high antioxidant capacity of this tissue, suggesting that oxidative stress does not form the reason of inhibited liver regeneration in diabetes. Conclusion: We have proved an increased systemic oxidative stress in STZ-treated diabetic rats, which leads to the increased expression of extracellular EADS. So, STZ-diabetic rats can mount an antioxidant defense against oxidant stress, at least at the onset of the disorder. The administration of both Losartan and Rolipram to STZ-treated diabetic rats, in addition to the antidiabetic effects, caused significant improvement of oxidative stress and the function of EADS. These findings could lead to implications for disease management and intervention strategies. Moreover, PDE4 specific inhibitors may constitute a novel therapeutic approach to prevent postoperative hepatic failure after major hepatic resection and liver transplantation. Potential mechanisms of beneficial effects of both AT₁ blocker and PDE4 inhibitor on diabetes - induced oxidative stress are discussed.