&lt;i&gt;Artemesia annua&lt;/i&gt; extract prevents glyoxal-induced cell injury in retinal microvascular endothelial cells during glaucoma

Shun Jiang,Jiawen Ling,Qin Wang

doi:10.4314/tjpr.v17i2.8

Abstract

Purpose: To investigate the effect of Artemesia annua extract on glyoxal-induced injury in retinal microvascular endothelial cells (HRECs). Methods: HRECs were cultured in a medium containing 500 μM glyoxal or glyoxal plus 50μM Artemesia annua extract, or in the medium alone for 24 h. Apoptosis was analysed by flow cytometry using annexin V and propidium iodide staining. Changes in mitochondrial membrane potential were determined by JC-1 staining. Results: When HRECs were cultured in a medium of 500 μM glyoxal, a significant (p < 0.05) decrease in caspase-3 expression was observed. However, treatment of HRECs with Artemesia annua extract (50 μM) inhibited the glyoxal-mediated decrease in caspase-3 expression. The extract also inhibited caspase-3 proteolysis, as was evident from the reduction in the level of cleaved caspase-3. Upregulation of ROS production by glyoxal in HRECs was inhibited by treatment with the extract. The viability of HRECs was significantly decreased by glyoxal (p < 0.05), but the decrease in viability was significantly reversed by Artemesia annua extract (p < 0.05). The extract also reduced gyloxal-induced apoptosis in HRECs from 17.3 to 2.6 % (p < 0.001). Results from JC-1 staining showed significantly (p < 0.001) higher level of green fluorescence in HRECs cultured with glyoxal. However, the glyoxal-induced increase in green fluorescence level was significantly (p < 0.01) reduced on exposure to Artemesia annua extract. Conclusion: Artemesia annua extract prevents oxidative damage to HRECs via inhibition of ROS production, up-regulation of caspase-3 expression and suppression of caspase-3 proteolysis. Therefore, Artemesia annua can potentially be used for the development of a new drug for the prevention of retinal injury in glaucoma. Keywords: Artemesia annua , Retinal injury, Glaucoma, Green fluorescence, cleaved caspase-3, ROS production

Highlights

Diabetic retinopathy is a common cause of vision loss in people with diabetes mellitus throughout the world [1]
The HRECs were cultured in a medium containing 500 μM glyoxal or glyoxal (500 μM) glyoxal alone or glyoxal and Artemesia annua extract (50 μM) for 24h, and washed twice with phosphate-buffered saline (PBS)
Proteolysis of caspase-3 was inhibited by Artemesia annua extract, as was evident in reduction of the level of cleaved caspase-3

Summary

INTRODUCTION

Diabetic retinopathy is a common cause of vision loss in people with diabetes mellitus throughout the world [1]. High intraocular pressure is considered a major pre-disposing factor for glaucoma, and it leads to optic nerve shrinkage and retinal ganglion cell apoptosis [9,10,11]. Studies have been carried out to investigate the protective effects of various compounds against retinal injury during glaucoma in animal models. Glyoxal was added to HRECs cultures to induce oxidative stress, and the protective effect of Artemesia annua extract was investigated. The blots were washed twice with PBS-T (0.1 % Tween-20 in PBS), and incubated with goat horseradish peroxidaseconjugated anti-mouse IgG (dilution 1: 5, 000; Pierce Biotechnology, Rockford, IL, USA) secondary antibody at room temperature for 1h. The HRECs were cultured in a medium containing 500 μM glyoxal alone (control) or glyoxal and Artemesia annua extract (50 μM) for 24h, and washed twice with phosphate-buffered saline (PBS).

Evaluation of cell viability

RESULTS

DISCUSSION

CONCLUSION

Conflict of Interest