Abstract
Purpose:
Zinc ion is an essential cofactor for normal cell function, the zinc transporters play a crucial role in zinc homeostasis. This study is to explore the regulation of erythropoietin (EPO) on zinc transporter 8 (ZnT8) in diabetic rats
Methods:
Diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley rats. Four days after intravitreal injection of EPO, the retinas were harvested for detection. The rat Muller cell line (rMC-1) was treated by cobalt chloride (CoCl2) with or without EPO. The changes of HIF-1α, VEGF, ZnT8, phosphor-ERK and ERK were detected with real-time PCR, Western blot and immunofluorescence.
Results:
In 4-week diabetic rat retinas, the expression of HIF-1α was up-regulated, while ZnT8 was down-regulated; intravitreal injection of EPO could down-regulate HIF-1α and up-regulated ZnT8 expressions. Retinal HIF-1α was increased at both transcriptional (increased by 23%) and translational (1.7-fold) levels, which were down-regulated by16% (mRNA) and 71% (protein), respectively, by EPO. For ZnT8, the mRNA level and total protein were decreased by 32% (n=5, p=0.002) and 30% (n=5, p=0.0014), separately, in diabetic rat retinas, which were increased by 23% (n=5, p=0.02) and 29% (n=6, p=0.025), separately, by EPO. Membrane protein of ZnT8 protein was significantly decreased by 62% in diabetic rat retinas and was increased by EPO (63%, n=3, p=0.025). The changes of HIF-1α and ZnT8, as well as EPO’s effect on them, were confirmed in CoCl2-treated rMC-1 cells. In CoCl2-treated rMC-1 cells, the ratio of pERK/ERK was decreased by about 40%, and EPO could increase this ratio to normal level. This effect was abolished with ERK inhibitor U0126. The immunofluorescence result showed that rMC-1 expressed ZnT8 both in cytoplasm and cell membrane.
Conclusions:
The present data indicated that intravitreal injection of EPO could increase ZnT8 expression in diabetic rat retina possible through its regulation of ERK and HIF-1α pathway.