Evaluation of inhibitory effects of neuroprotective treatment on diabetic retinopathy

为了研究尼帕地洛尔和拉坦前列素是否对视网膜神经节细胞（RGCs）具有抗凋亡作用，我们采用血清抽吸法诱导RGC-5细胞凋亡，用链脲佐菌素（STZ）诱导糖尿病大鼠视网膜细胞凋亡。采用活化caspase-3免疫组化方法对视网膜冷冻切片或末端dUTP缺口末端标记染色进行细胞凋亡定量。尼帕迪洛尔和拉坦前列酸抑制RGC-5细胞凋亡呈剂量依赖性（p=0.001），而去硝基尼帕迪洛尔则无此作用。抑制剂研究表明，尼普拉地尔的抗凋亡作用依赖于蛋白激酶A和g，而拉坦前列酸的抗凋亡作用依赖于mapk。转染CREB显性阴性突变体也阻断了尼帕地洛尔的拯救作用。尼普拉地洛尔和拉坦前列素灌注5 d后，4周和12周糖尿病视网膜视网膜神经节细胞层凋亡细胞均较对照组减少（p<0.05和p<0.0001）。地硝尼普地洛尔滴眼液没有任何效果。拉坦前列素滴眼液显著增加大鼠视网膜MAPR磷酸化。这些结果表明，尼帕地洛尔和拉坦前列素通过不同的细胞内信号通路拯救糖尿病诱导的rgc细胞凋亡

In order o investigate if nipradilol and latanoprost have anti-apoptotic effects in retinal ganglion cells (RGCs), apoptosis was induced in RGC-5 cells by withdrawing serum and in rat retina by inducing diabetes with streptozotocin (STZ) injection. Apoptosis was quantified by activated caspase-3 immunohistochemistry on retinal cryosection or terminal dUTP nick end labeling staining. Nipradilol and latanoprost acid suppressed apoptosis of RGC-5 cells with dose dependency (p=0.001), whereas desnitro-nipradilol did not. Inhibitor studies showed that the anti-apoptotic effects of nipradilol were protein kinase A and G-dependent, whereas those of latanoprost acid were MAPK-dependent. Transfection of dominant negative mutants of CREB also blocked the nipradilol rescue effect. When instilled for 5 days, both nipradilol and latanoprost reduced apoptotic cells in retinal ganglion cell layer of 4-week and 12-week diabetic retina than controls (p<0.05 and p<0.0001, respectively). Desnitro-nipradilol eye drops did not show any effects. Latanoprost eye drops significantly increased MAPR phosphorylation in rat retina .These results indicated that nipradilol and latanoprost rescue RGCs from apoptosis induced by diabetes through distinct intracellular signaling pathways