Immunomodulatory role of Emblica officinalis in arsenic induced oxidative damage and apoptosis in thymocytes of mice

Abstract

Background: Arsenic is widely distributed in the environment and has been found to be associated with the various health related problems including skin lesions, cancer, cardiovascular and immunological disorders. The fruit extract of Emblica officinalis (amla) has been shown to have anti-oxidative and immunomodulatory properties. In view of increasing health risk of arsenic, the present study has been carried out to investigate the protective effect of amla against arsenic induced oxidative stress and apoptosis in thymocytes of mice.

Methods: Mice were exposed to arsenic (sodium arsenite 3 mg/kg body weight p.o.) or amla (500 mg/kg body weight p.o.) or simultaneously with arsenic and amla for 28 days. The antioxidant enzyme assays were carried out using spectrophotometer and generation of ROS, apoptotic parameters, change in cell cycle were carried out using flow cytometer following the standard protocols.

Results: Arsenic exposure to mice caused a significant increase in the lipid peroxidation, ROS production and decreased cell viability, levels of reduced glutathione, the activity of superoxide dismutase, catalase, cytochrome c oxidase and mitochondrial membrane potential in the thymus as compared to controls. Increased activity of caspase-3 linked with apoptosis assessed by the cell cycle analysis and annexin V/PI binding was also observed in mice exposed to arsenic as compared to controls. Co-treatment with arsenic and amla decreased the levels of lipid peroxidation, ROS production, activity of caspase-3, apoptosis and increased cell viability, levels of antioxidant enzymes, cytochrome c oxidase and mitochondrial membrane potential as compared to mice treated with arsenic alone.

Conclusions: The results of the present study exhibits that arsenic induced oxidative stress and apoptosis significantly protected by co-treatment with amla that could be due to its strong antioxidant potential.

Figure 1

DPPH free radical scavenging activity of fruit extract of Emblica officinalis. The concentration of extract above 20 μg/ml showed a saturation in the plot indicating the radical scavenging activity of more than 90%.

Figure 2

Effect of arsenic, amia and their co-treatment on cell viability in thymus of mice. Values are mean ± SEM of five animals in each group a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 3

Effect of arsenic, amla and their co-treatment on the levels of lipid peroxidation in thymus of mice. Values are mean ± SEM of five animals in each group a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 4

Effect of arsenic, amla and their co-treatment on the levels of reduced glutathione levels in thymus of mice. Values are mean ± SEM of five animals in each group a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 5

Effect of arsenic, amla and their co-treatment on the activity of superoxide dismutase in thymus of mice. Values are mean ± SEM of five animals in each group a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 6

Effect of arsenic, amla and their co-treatment on the catalase activity in thymus of mice. Values are mean ± SEM of five animals in each group a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 7

Effect of arsenic, amla and their co-treatment on the catalase-3 activity in thymus of mice. Values are mean ± SEM of five animals in each group a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 8

Effect of arsenic, amla and their co-treatment on the cytochrome c oxidase activity in thymus of mice. Values are mean ± SEM of five animals in each group a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 9

Effect of arsenic, amla and their co-treatment on the generation of reactive oxygen species in thymocytes of mice. Cells were incubated with DCFH-DA and fluorescence was measured using a flow cytometer with FL-1 filter. Values are presented as mean ± SEM of three assays performed independently a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 10

Effect of arsenic, amla and their co-treatment on mitochondrial membrane potential of thymocytes of mice. Cells were incubated with Rh 123 and fluorescence was measured using flow cytometer with FL-1 filter. Values are presented as mean ± SEM of three assays performed independently a-compared to control group, b-compared to arsenic treated group *Significantly differs (p < 0.05).

Figure 11

Effect of arsenic, amla and their co-treatment on the cell cycle progression of thymocytes of mice. Control and amla treated cells showed no sub-G1 peak while arsenic showed high numbers of cells in sub-G1 peak and group co-treated with of arsenic and amla showed reduce number of cells in sub-G1 peaks as compared to arsenic. Cells were incubated with propidium iodide and flourescence was measured using flow cutometer with FL-2 filter. Values are presented in the historam as mean ± SEM of three assays performed independently representing sub-G1 population of cells.

Figure 12

Effect of arsenic, amla and their co-treatment on the annexin-V/PI dual staining of thymocytes of mice. Cells in the upper right (UR) portion are late apoptotic cells, upper left (UL) are necrotic cells whereas cells in the lower left (LL) and lower right (LR) portion are viable and early apoptotic cells respectively. Values are presented as mean ± SEM of three assays performed independently.