Objective To investigate the effect of pigment epitheliumderived factor (PEDF）on the expression of glutamine synthetase in retinal Muuml;ller cells of diabetic rats.Methods Diabetic rats were induced with streptozotocin injection.Before and after injection of 10 mu;l (0.1 mu;g/mu;l) PEDF (experimental group) or 10 mu;l PBS (control group) into the vitreous cavities of diabetic rats respectively for 48 hours,the expressions of GS and IL-1beta; in retina were analyzed by immunohistochemistry and real time RTPCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions,the expressions of GS and IL-1beta; in cultured Muuml;ller cells were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V fluorescein isothiocyanate/Propidium idoium (Annexin V-FITC/PI) staining.Results By immunohistochemistry (the protein level) and real time RT-PCR (the mRNA level),it was found that the expression of GS decreased and the expression of IL-1beta; increased obviously (real time RT-PCR：GS:t=4.23,P＜0.01；IL-1beta;:t=16.73,P＜0.01；immunohistochemistry：GS: t=5.13,P＜0.01；IL-1beta;:t=9.32,P＜0.01) in diabetic rats. After injection of 10 mu;l (0.1 mu;g/mu;l) PEDF into the vitreous cavities of diabetic rats for 48 hours,it was found that the expression of GS increased and the expression of IL-1beta; decreased significantly(RT-PCR GS:t=3.87,P＜0.01IL-1beta;:t=3.61,P＜0.05;immunohistochemistry:GS:t=3.32, P＜0.05；IL-1beta;: t=2.63,P＜0.05). Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1beta; and increasing expression of GS significantly (RT-PCR：GS: t=2.89, P＜0.05；IL-1beta;: t=3.37,P＜0.05；Western blot：GS:t=2.66,P＜0.05;IL-1beta;:t=3.23,P＜0.05).Apoptosis of Muuml;ller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF (t=3.21,P＜0.05). Conclusions In diabetic rats,PEDF may decrease expression of IL-1beta; in rat retinal Muuml;ller cells, which may result in increasing expression of GS.To some degree,it inhibits possibly the death of retinal ganglion cells.
Objective To evaluate the effects of inflammatory cytokines, including tumor necrosis factorTNF-alpha; and interleukins (IL-6 and IL-8), to the expression of pigment epithelium-derived factor (PEDF) in human retinal pigment epithelium (RPE)cells. Method Cultured primary human RPE cells were treated with 20，2，0.2 , and 0.02 ng/ml of TNF-alpha;, IL-6 and IL-8 separately. The levels of PEDF expression were determined by Western blot of the supernant after 6，12，24 and 48 hours of culture. Results PEDF secretion of RPE cells was inhibited by TNF-alpha;, IL-6 and IL-8 in a time- and dose-dependent fashion. Compared with the controls, the expression of PEDF decreased significantly in 0.02 ng/ml and 6 hours group (F＝7.14, P＜0.05）, 2.00 ng/ml and 48 hours group（F=14.05，P＜0.01) , and 20.00 ng/ml and 24 hours group（F=11.53，P＜0.01). TNF-alpha; was the most strength inhibitor (F＝14,P＜0.01）.Conclusion TNF-alpha;, IL-6, and IL-8 could suppress the expression of PEDF in the cultured human RPE cells.