Inhibition of ROS production in peripheral blood mononuclear cells from type 2 diabetic patients by autologous plasma depends on Akt/PKB signaling pathway

Abstract

Objective: To compare the role of Akt/PKB signaling pathway in the modulation of reactive oxygen species (ROS) production by autologous plasma in peripheral blood mononuclear cells (PBMNC) from type 2 diabetic patients and healthy subjects.

Materials and methods: This study was approved by Santa Casa Ethical Committee and has included patients diagnosed with diabetes type 2 (DM2) and control group (non-diabetic) (ND). PBMNC were purified utilizing Ficoll-hypaque gradient. ROS was quantified by luminol-dependent chemiluminescence. The Akt/PKB phosphorylation was measured using a CASE kit. Statistical analyses were made with t Student test and chi-square (chi(2)). p<0.05 was considered significant.

Results: 12, 13-Phorbol dibutyrate (PDB) stimulated the production of higher levels of ROS in PBMNC from type 2 diabetic patients than that from healthy subjects. Autologous plasma, however, inhibited induced or not ROS production in PBMNC in both groups. The inhibition of PBMNC-ROS derived by autologous plasma from healthy subjects was higher than that from type 2 diabetic patients. Plasma phosphorylated (activated) Akt/PKB. The percentage of phosphorylation induced by autologous plasma in PBMNC from patients and healthy control were 14% and 93%, respectively. Inhibition of ROS production in PBMNC from DM2 were similar for PBMNC+plasma; PBMNC+Akti; and PBMNC+plasma+Akti. However, in ND control, plasma showed a higher ROS inhibition than Akti or plasma plus Akti.

Conclusions: Our results suggest that the low antioxidant capacity observed in autologous plasma from DM2 patients in conjunction with the decreased activation of PKB may cause an imbalance in the oxidizing/reducing responses, possible inducing an oxidative stress state, which could be associated with tissular damage.