[Frontiers in Bioscience E3, 201-211, January 1, 2011]

EPO attenuates inflammatory cytokines by Muller cells in diabetic retinopathy

Xia Lei1,2,3, Jingfa Zhang2, Jianfeng Shen1,3, Liu-Mei Hu4, Yalan Wu1, Lisha Mou1,3, Guoxu Xu5, Weiye Li2,4,6, Guo-Tong Xu1,2

1Institute of Health Sciences (IHS), Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) and Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai, China, 2 Tongji Eye Institute and Department of Regenerative Medicine, Tongji University School of Medicine, Shanghai, China, 3 Graduate School of Chinese Academy of Sciences, Beijing, China, 4Department of Ophthalmology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, China, 5Department of Ophthalmology, Second Affiliated Hospital of Soochow University, Suzhou, China, 6 Department of Ophthalmology, Drexel University College of Medicine, Philadelphia, PA, USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Muller cell culture
3.2. Experimental animals and intravitreal EPO treatment
3.3. Morphological examination and MTT assay
3.4. In situ detection of cell death by TUNEL assay
3.5. RNA purification and real-time PCR
3.6. ELISA
3.7. Luciferase assay for AP-1 and NF-kappaB activity
3.8. Immunostaining
3.9. Statistical analysis
4. Results
4.1. Effects of glyoxal on rMC-1 cells
4.2. Suppression by EPO on mRNA levels of TNF-alpha and IL-1beta but not IL-6 and VEGF
4.3. Attenuation by EPO on secretion of TNF-alpha and IL-1beta but not IL-6 and VEGF
4.4. Involvement of EPO/EPOR system in the inflammatory response
4.5. Modulation of AP-1 but not NF-kappaB activity by glyoxal and EPO
4.6. Attenuation of TNF-alpha and IL-1beta production by EPO in diabetic rats
5. Discussion
6. Acknowledgement
7. References

1. ABSTRACT

Diabetic retinopathy (DR) is a chronic, low-grade inflammatory disease. We aimed to investigate the regulatory effects of erythropoietin (EPO) on the inflammatory cytokine production by Muller cells under the condition of DR. The expression levels of TNF-alpha, IL-1beta, IL-6 and VEGF in cultured rat Muller cells were enhanced by 1 mM glyoxal. The elevated TNF-alpha and IL-1beta, but not IL-6 and VEGF, were decreased by 2 U/ml EPO as detected by real-time PCR and ELISA. Moreover, the activity of AP-1 but not NF-kappaB was modulated by glyoxal and EPO. Intravitreal injection of EPO performed 24 h prior to sacrifice significantly reduced TNF-alpha and IL-1beta production while moderately attenuating IL-6 and VEGF in the retinas of streptozotocin-induced diabetic rats. Furthermore, Muller cells were identified as the main source of IL-1beta production as indicated by co-localization of IL-1beta and CRALBP in situ. These findings implicate therapeutic potential of EPO in the amelioration of inflammation in diabetic retinas.