不同剂量N-乙酰-5-羟色胺(NAS)对视网膜缺血-再灌注损伤大鼠视网膜细胞凋亡的影响

Effects of different doses of NAS on retinal apoptosis in rats with retinal ischemia-reperfusion injury

目的探讨不同剂量N-乙酰-5-羟色胺(N-acetylserotonin,NAS)对视网膜缺血-再灌注损伤(retinal ischemia-reperfusion injury,RIRI)大鼠视网膜细胞凋亡的影响。方法成年SD大鼠随机分为正常对照组、RIRI组和NAS组。采用高眼压法建立RIRI大鼠模型。NAS组按照给药剂量的不同分为NAS低剂量组(5 mg·kg-1)、NAS中剂量组(10 mg·kg-1)和NAS高剂量组(20 mg·kg-1),造模前后30 min腹腔注射NAS。HE染色法观察各组大鼠视网膜形态学的改变,免疫组织化学法检测各组大鼠视网膜中活性Caspase-3的表达,TUNEL法检测各组视网膜细胞凋亡的变化。结果 HE染色结果显示,NAS中剂量组大鼠视网膜各层细胞排列最整齐,视网膜内层厚度[(53.24±1.68)μm]显著薄于RIRI组[(60.54±2.52)μm]及NAS低剂量组[(56.78±1.78)μm](均为P<0.01),NAS中剂量组视网膜神经节细胞数[(1113.65±74.40)个·mm^-2]显著多于RIRI组[(719.89±83.67)个·mm^-2]及NAS低剂量组[(882.09±55.62)个·mm^-2](均为P<0.01)。免疫组织化学结果示,视网膜中活性Caspase-3阳性细胞数RIRI组[(246.08±19.23)个·mm^-2]及NAS低、中、高剂量组[(196.95±19.83)个·mm^-2、(142.77±18.25)个·mm^-2、(133.10±15.19)个·mm^-2]均显著多于正常对照组[(95.37±10.93)个·mm^-2](均为P<0.01);NAS中剂量组视网膜中活性Caspase-3阳性细胞数量显著少于RIRI组及NAS低剂量组(均为P<0.01)。TUNEL检测结果显示,TUNEL阳性细胞数RIRI组[(225.45±18.93)个·mm^-2]及NAS低、中、高剂量组[(175.06±17.69)个·mm^-2、(108.85±13.41)个·mm^-2、(100.37±13.53)个·mm^-2]均多于正常对照组[(81.98±11.29)个·mm^-2](均为P<0.01);NAS中剂量组视网膜TUNEL阳性细胞数显著少于RIRI组及NAS低剂量组(均为P<0.01)。结论腹腔注射NAS治疗可减轻RIRI大鼠视网膜细胞凋亡,具有神经保护作用,中剂量NAS治疗方案最佳。

Objective To observe the effect of different doses of N-acetylserotonin(NAS) on retinal cell apoptosis in rats with retina ischemia-reperfusion injury(RIRI).Methods Adult male SD rats were randomly divided into the normal control group,RIRI group and NAS group.The RIRI rat model was made by elevating the intraocular pressure method.The NAS group was divided into three subgroups,the low-dose NAS group(5 mg·kg-1),the medium-dose NAS group(10 mg·kg-1) and the high-dose NAS group(20 mg·kg-1),according to the different dose of NAS of NAS.And NAS was intraperitoneally injected within 30 min before and after modeling.Hematoxylin eosin(HE) staining was used to evaluate the morphological changes of retina in each group.The cleaved Caspase-3 positive cells in retinas of each group were examined by immunohistochemistry staining and the changes of retinal apoptotic cells in each group were examined by TUNEL method.Results HE staining results showed that the retinal cells in medium-dose NAS group were most neatly arranged,the thickness of retinal inner layer [(53.24±1.68)μm] was obviously thinner than that of the RIRI group [(60.54±2.52)μm] and the low-dose NAS group [(56.78±1.78)μm](both P<0.01).The number of retinal ganglion cells(RGCs) in the middle-dose NAS group [(1113.65±74.40)cells·mm^-2] was significantly higher as compared with the RIRI group [(719.89±83.67)cells·mm^-2] and low-dose NAS group [(882.09±55.62)cells·mm^-2](both N-acetyl-5-hydroxytryptamine;retinal ischemia-reperfusion;cleaved caspase-3;retinal cellsPN-acetyl-5-hydroxytryptamine;retinal ischemia-reperfusion;cleaved caspase-3;retinal cells(all P<0.01).Immunohistochemical results showed that the number of retinal cleaved caspase-3 positive cells in the RIRI group [(246.08±19.23)cells·mm^-2],low-dose,medium-dose and high-dose NAS groups [(196.95±19.83)cells·mm^-2,(142.77±18.25)cells·mm^-2,(133.1±15.19)cells·mm^-2 ] were all significantly higher as compared with the normal control group [(95.37±10.93)cells·mm^-2](all P<0.01).Compared with the RIRI group and the low-dose NAS group,there were fewer retinal cleaved Caspase-3 positive cells in the medium-dose NAS group(both P<0.01).TUNEL staining results showed that the number of retinal TUNEL positive cells in the RIRI group [(225.45±18.93)cells·mm^-2],low-dose,medium-dose and high-dose NAS groups [(175.06±17.6)cells·mm^-2,(108.85±13.41)cells·mm^-2,(100.37±13.53)cells·mm^-2 ] were apparently higher than in the normal control group [(81.98±11.29)cells·mm^-2](all P<0.01);but there were fewer TUNEL positive cells in medium-dose NAS group than in the RIRI group and low-dose NAS group(both P<0.01).Conclusion NAS therapy by intraperitoneal injection could alleviate retinal cell apoptosis in rats with RIRI and has neuroprotective effects,NAS therapy with medium dose is the best therapeutic regimen.