■本研究旨在研究Raf-1激酶抑制蛋白(RKIP)对链脲佐菌素治疗的大鼠模型和高糖治疗的大鼠Müller细胞中糖尿病视网膜神经变性的影响。
■对照和链脲佐菌素治疗的大鼠玻璃体内注射生理盐水,RKIP基因过表达慢病毒(oeRKIP)或阴性对照慢病毒(RKIP-载体)。正常或高葡萄糖处理的Müller细胞用盐水转染,RKIP基因过表达慢病毒或阴性对照慢病毒。免疫印迹法和免疫荧光法评价RKIP对RKIP表达的影响。p38丝裂原活化蛋白激酶(p38-MAPK),谷氨酸/天冬氨酸转运蛋白(GLAST),谷氨酰胺合成酶(GS),胶质纤维酸性蛋白(GFAP)和半胱氨酸-天冬氨酸蛋白酶-3(caspase-3)。采用谷氨酸检测试剂盒检测视网膜样品中的谷氨酸水平。通过膜联蛋白-V/PI染色和流式细胞术确定Müller细胞的凋亡。
■高糖处理的Müller细胞表现出促进的凋亡,而RKIP在高糖处理的Müller细胞中的过表达下调了增强的凋亡。与注射生理盐水的大鼠相比,链脲佐菌素治疗的高血糖大鼠显示p38-MAPK和GFAP的免疫反应性以及p38-MAPK和caspase-3的蛋白表达增强。引人注目的是,在高血糖大鼠中玻璃体内注射RKIP基因过表达慢病毒逆转了上述增强的免疫反应性和蛋白质表达。同时,RKIP在高血糖大鼠中的过表达提高了RKIP的免疫反应性和蛋白表达,GS和GLAST。此外,RKIP下调高血糖大鼠视网膜谷氨酸水平。
■玻璃体内注射RKIP基因过表达慢病毒可能通过抑制p38-MAPK通路在糖尿病大鼠模型中预防糖尿病视网膜神经变性中起作用。
UNASSIGNED: This study aimed to investigate the effect of Raf-1 kinase inhibitory protein (RKIP) on diabetic retinal neurodegeneration in streptozotocin-treated rat model and high glucose-treated rat Müller cells.
UNASSIGNED: Control and streptozotocin-treated rats were intravitreally injected with saline, RKIP gene overexpression lentivirus (oeRKIP) or negative control lentivirus (RKIP-vector). Normal or high glucose-treated Müller cells were transfected with saline, RKIP gene overexpression lentivirus or negative control lentivirus. Western blotting and immunofluorescence assay were utilized to evaluate the function of RKIP on the expression of RKIP, p38 mitogen-activated protein kinase (p38-MAPK), glutamate/aspartate transporter (GLAST), glutamine synthetase (GS), glial fibrillar acidic protein (GFAP) and cysteine-aspartic acid protease-3 (caspase-3). A glutamate assay kit was adopted to detect glutamate level in retina samples. Apoptosis of Müller cells was determined by Annexin-V/PI staining and flow cytometry.
UNASSIGNED: High glucose-treated Müller cells exhibited promoted apoptosis, while RKIP overexpression in high glucose-treated Müller cells down-regulated the enhanced apoptosis. Compared with rats injected with saline, streptozotocin-treated hyperglycemic rats displayed enhancement in the immunoreactivities of p38-MAPK and GFAP as well as in the protein expression of p38-MAPK and caspase-3. Strikingly, intravitreal injection of RKIP gene overexpression lentivirus in the hyperglycemic rats reversed the augmented immunoreactivities and protein expression mentioned above. Meanwhile, RKIP overexpression in the hyperglycemic rats improved the immunoreactivities and protein expression of RKIP, GS and GLAST. Besides, RKIP down-regulated the increased level of retinal glutamate in the hyperglycemic rats.
UNASSIGNED: Intravitreal injection of RKIP gene overexpression lentivirus functioned in preventing diabetic retinal neurodegeneration in a rat model of diabetes presumably by inhibiting p38-MAPK pathway.