Evaluation of insulin and ascorbic acid effects on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of STZ-induced diabetic rats

Abstract

Aims: Effects of insulin and ascorbic acid on expression of Bcl-2 family proteins and caspase-3 activity in hippocampus of diabetic rats were evaluated in this study.

Methods: Diabetes was induced in Wistar male rats by streptozotocin (STZ). Six weeks after verification of diabetes, the animals were treated for 2 weeks with insulin or/and ascorbic acid in separate groups. Hippocampi of rats were removed and evaluation of Bcl-2, Bcl-x(L), and Bax proteins expression in frozen hippocampi tissues were done by SDS-PAGE electrophoresis and blotting. The Bcl-2, Bcl-x(L), and Bax proteins bands were visualized after incubation with specific antibodies using enhanced chemiluminescences method. Caspase-3 activity was determined using the caspase-3/CPP32 Fluorometric Assay Kit.

Results: Diabetic rats showed increase in Bax protein expression and decrease in Bcl-2 and Bcl-x(L) proteins expression. The Bax/Bcl-2 and Bax/Bcl-x(L) ratios were found higher compared with non-diabetic control group. Treatments with insulin and/or ascorbic acid were resulted in decrease in Bax protein expression and increase in Bcl-2 and Bcl-x(L) proteins expression. The Bcl-2/Bax and Bcl-x(L)/Bax ratios were found higher in treated groups than untreated diabetic group. Caspase-3 activity level was found higher in diabetic group compared with non-diabetic group. Treatment with insulin and ascorbic acid did downregulated caspase-3 activity.

Conclusions: Our data provide supportive evidence to demonstrate the antiapoptotic effects of insulin and ascorbic acid on hippocampus of STZ-induced diabetic rats.

Fig. 1

Analysis of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi of rats groups C (Control) and D (Diabetic) by western blotting. (a) Bax gene expression, the level of expression of Bax in group D showed increase significantly compared with group C, (b) Bcl-2 gene expression, and (c) Bcl-x_L gene expression, the levels of Bcl-2 and Bcl-x_L were found lower in group D than group C

Fig. 2

Analysis of Bcl-2, Bcl-x_L, and Bax proteins expression in frozen hippocampi of rats groups C (Control), D (Diabetic), I (treated with insulin), AA (treated with ascorbic acid), and I + AA (treated with insulin + ascorbic acid) by western blotting. (a) Bax gene expression, the level of expression of Bax in groups I, AA, and I + AA showed decrease significantly compared with group D, (b) Bcl-2 gene expression, and (c) Bcl-x_L gene expression, the levels of Bcl-2 and Bcl-x_L were found higher in groups I, AA, and I + AA compared to the group D

Fig. 3

Bax/Bcl-x_L and Bax/Bcl-2 expression ratios in hippocampus tissue of control (C) and STZ-induced diabetic (D) groups of rat. (a) Bar graph indicates the mean ± SEM of Bax/Bcl-x_L ratio. P < 0.001, compared to the control group (n = 3 each group). (b) Bar graph indicates the mean ± SEM of Bax/Bcl-2 ratio. P < 0.01, compared to the control group (n = 3 each group)

Fig. 4

The effects of insulin and ascorbic acid on Bcl-x_L/Bax and Bcl-2/Bax expression ratios in hippocampal tissue of rats. Bar graph indicates the mean ± SEM. ^# P < 0.001, * P < 0.01 compared to the D group (n = 3 each group)

Fig. 5

Caspase-3 activity in hippocampal tissues of control (C) and STZ-induced diabetic (D) rat groups (n = 3 each group). Bar graph indicates the mean ± SEM P < 0.001, compared to the control group)

Fig. 6

Caspase-3 activity in hippocampal tissues rats groups C (Control), D (Diabetic), I (treated with insulin), AA (treated with ascorbic acid), and I + AA (treated with insulin + ascorbic acid) (n = 3 each group). Caspase-3 activity in groups I, AA, and I + AA showed decrease significantly compared with group D. Bar graph indicates the mean ± SEM, ^# P < 0.001, * P < 0.01 compared with group D