Enhancement of reactive oxygen species and induction of apoptosis in streptozotocin-induced diabetic rats under hyperbaric oxygen exposure

Abstract

An important source of reactive oxygen species (ROS) production is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which on activation induces superoxide production via oxidation in the mitochondria, inflammation and stress; such ROS are implicated in the pathogenesis of diabetic complications, including neuropathy. Hyperbaric oxygen (HBO) treatments are applied various diseases including diabetic patients with unhealing foot ulcers, however, and also increases the formation of ROS. In a previous study, we showed that a clinically recommended HBO treatment significantly enhanced oxidative stress of pancreatic tissue in the diabetic rats. However, no study has been undertaken with regard to the effects of HBO on the activity and gene expression of the NADPH oxidase complex and on apoptosis in the pancreas of diabetic animals. The purpose of this study was to investigate the effect of HBO exposure on gene expression of the NADPH complex, and pancreatic expression of genes related to apoptosis via the mitochondria, using the NADPH oxidase inhibitor apocynin. The mRNA expression of genes related to NADPH oxidase complex and apoptosis increased significantly (P < 0.05) in the pancreas of diabetic rats under HBO exposure. Similarly, activities of NADPH oxidase and caspase-3 changed in parallel with mRNA levels. These results suggest that oxidative stress caused by HBO exposure in diabetic animals induces further ROS production and apoptosis, potentially through the up-regulation of NADPH oxidase complex. Thus, this study can contribute to development of a better understanding of the molecular mechanisms of apoptosis via the mitochondria in diabetes, under HBO exposure.

Keywords: Diabetes mellitus; apocynin; apoptosis; hyperbaric oxygen; nicotinamide adenine dinucleotide phosphate; reactive oxygen species.

Figure 1

Comparison of lipid peroxidation levels (TBARS) in the pancreas of non-diabetic rats in the non-HBO (control), non-diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. TBARS values are indicated as nmol/ mg protein in pancreatic tissue. Values are expressed as mean ± SEM (n = 6). * Represents significance at P < 0.05 compared with the control group untreated with apocynin. † Represents significance at P < 0.05 compared with the same group treated with or without apocynin.

Figure 2

NADPH oxidase activity (NADP⁺/NADPH ratios) in the pancreas of non-diabetic rats in the non-HBO (control), non-diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. NADPH oxidase activity is indicated as fold over control in pancreatic tissue. Values are expressed as mean ± SEM (n = 6). * Represents significance at P < 0.05 compared with the control group untreated with apocynin. † Represents significance at P < 0.05 compared with the same group treated with or without apocynin.

Figure 3

Apoptotic cells detected by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) technique (see Materials and Methods) in slides prepared from pancreatic tissue in the non-HBO (control), non-diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. Apoptotic nuclei in islets of Langerhans from the control, HBO, DM, and DM + HBO groups treated with or without apocynin (a). Scale Bar = 50 μm. Apoptotic index in pancreatic islets in the DM and DM + HBO groups treated with or without apocynin. Positive nuclei were counted in 100 islets per group and expressed as percentage of apoptotic nuclei with respect to the total nuclei in pancreatic islets (b). * Represents significance at P < 0.05 compared with the DM group untreated with apocynin (vs. DM + HBO group treated with or without apocynin). † Represents significance at P < 0.05 compared with the same group treated with or without apocynin.

Figure 4

Real-time PCR analysis of NADPH oxidase complex (a), apoptosis (b), and caspase-3 gene expression in the pancreas of non-diabetic rats in the non-HBO (control), non-diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. Values are expressed as mean ± SEM (n = 6). * Represents significance at P < 0.05 compared with the control group untreated with apocynin. † Represents significance at P < 0.05 compared with the same group treated with or without apocynin. gp91^phox, gp91^phox protein; p22^phox, p22^phox protein; p47^phox, p47^phox protein; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Figure 5

Caspase-3 activity in the pancreas of non-diabetic rats in the non-HBO (control), non-diabetic rats in the HBO (HBO), diabetic rats in the non-HBO (DM), and diabetic rats in the HBO (DM + HBO) groups. Caspase-3 activity is indicated as U/mg protein in pancreatic tissue. Values are expressed as mean ± SEM (n = 6). * Represents significance at P < 0.05 compared with the control group untreated with apocynin. † Represents significance at P < 0.05 compared with the same group treated with or without apocynin.