Nutr Res Pract.  2014 Oct;8(5):494-500. 10.4162/nrp.2014.8.5.494.

Padina arborescens extract protects high glucose-induced apoptosis in pancreatic beta cells by reducing oxidative stress

• Park MH
• Han JS

• KMID: 2313769
• DOI: http://doi.org/10.4162/nrp.2014.8.5.494

Abstract

BACKGROUND/OBJECTIVES
This study investigated whether Padina arborescens extract (PAE) protects INS-1 pancreatic beta cells against glucotoxicity-induced apoptosis.
MATERIALS/METHODS
Assays, including cell viability, lipid peroxidation, generation of intracellular ROS, NO production, antioxidant enzyme activity and insulin secretion, were conducted. The expressions of Bax, Bcl-2, and caspase-3 proteins in INS-1 cells were evaluated by western blot analysis, and apoptosis/necrosis induced by high glucose was determined by analysis of FITC-Annexin V/PI staining.
RESULTS
Treatment with high concentrations of glucose induced INS-1 cell death, but PAE at concentrations of 25, 50 or 100 microg/ml significantly increased cell viability. The treatment with PAE dose dependently reduced the lipid peroxidation and increased the activities of antioxidant enzymes reduced by 30 mM glucose, while intracellular ROS levels increased under conditions of 30 mM glucose. PAE treatment improved the secretory responsiveness following stimulation with glucose. The results also demonstrated that glucotoxicity-induced apoptosis is associated with modulation of the Bax/Bcl-2 ratio. When INS-1 cells were stained with Annexin V/PI, we found that PAE reduced apoptosis by glucotoxicity.
CONCLUSIONS
In conclusion, the present study indicates that PAE protects against high glucose-induced apoptosis in pancreatic beta cells by reducing oxidative stress.

Keyword

Padina arborescens extract; high glucose; apoptosis; oxidative stress; INS-1 pancreatic beta cell

MeSH Terms

Apoptosis*
Blotting, Western
Caspase 3
Cell Death
Cell Survival
Glucose
Insulin
Insulin-Secreting Cells*
Lipid Peroxidation
Oxidative Stress*
Caspase 3
Glucose
Insulin

Figure

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  Fig. 1 Effect of PAE on the viability of high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with 5.5 mM or 30 mM glucose for 48 h, and subsequently incubated for 48 h in the presence or absence of 25, 50, or 100 µg/mL PAE. The use of 5.5 mM glucose was representative of normal glucose conditions and the 30 mM glucose treatments represent high glucose conditions. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

• 

  Fig. 2 Effect of PAE on intracellular ROS generation in the high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

• 

  Fig. 3 Effect of PAE on TBRAS generation in the high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

• 

  Fig. 4 Effect of PAE on NO level in high glucose treated INS-1 pancreatic β cells. Cells in wells of 96-well plates (2 × 104 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-cValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

• 

  Fig. 5 Effects of PAE on insulin secretion in high glucose-treated INS-1 pancreatic β cells. Cells in wells of 6-well plates (2 × 105 cells/well) were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. Each value is expressed as mean ± SD (n = 3). a-dValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

• 

  Fig. 6 Effects of PAE on the expression of Caspase-3, Bax and Bcl-2 (A) and Caspase-3, Bax and Bcl-2 ratio (B) in INS-1 pancreatic β cells. Relative expression was quantified by densitometry using the Multi Gauge V3.1 and calculated according to the reference bands of β-actin (mean, n = 3). a-cValues with different alphabets differ significantly at P < 0.05 as analyzed via Duncan's multiple range test.

• 

  Fig. 7 Identification of the type of cell death by Annexin V-FITC/PI staining. The status of cell death was determined by counting the cells stained with Annexin V-FITC/PI using glow cytometer. Cells were preincubated with glucose and incubated in the absence or presence of PAE as described in the legend to Fig. 1. A: 5.5 mM glucose; B: 30 mM glucose; C: 30 mM glucose + PAE 50 µg/mL; D: 30 mM glucose + PAE 100 µg/mL.

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