N-乙酰-5-羟色胺对视网膜缺血-再灌注损伤大鼠视网膜Fas、FasL蛋白表达的影响

Effects of N-acetylserotonin on the expression of Fas and FasL protein in rat retina after ischemia-reperfusion injury

目的探讨N-乙酰-5-羟色胺(N-acetylserotonin,NAS)对视网膜缺血-再灌注损伤(retina ischemia-reperfusion injury,RI-RI)大鼠视网膜Fas、FasL蛋白表达的影响。方法取健康成年Sprague Dawley大鼠54只,将大鼠随机分为正常组(6只)、RIRI组(24只)与NAS组(24只);采用高眼压法建立大鼠RIRI模型,依据造模后不同时间点将RIRI组与NAS组大鼠又分为6 h、12 h、24 h及72 h四个亚组。NAS组于造模前30 min腹腔注射NAS(5 mg·kg-1),RIRI组腹腔注射等剂量的生理盐水。通过HE染色在光学显微镜下观察各组大鼠视网膜形态学变化,并记录各组大鼠视网膜厚度及视网膜神经节细胞数,采用免疫组织化学染色法检测NAS对RIRI大鼠视网膜Fas、FasL蛋白表达的影响。结果 HE染色显示,正常组大鼠视网膜各层细胞分界清晰,形态正常,神经细胞排列整齐; RIRI组大鼠再灌注后6 h视网膜各层出现水肿,以神经节细胞层及内核层较显著,神经节细胞数较正常组减少;随后视网膜水肿进一步加重,神经节细胞继续减少; NAS组大鼠在再灌注后6 h、12 h、24 h视网膜水肿程度较RIRI组轻,NAS组在再灌注后72 h视网膜厚度较RIRI组厚,NAS组各时间点神经节细胞数均较RIRI组多,差异均有统计学意义(均为P <0. 05)。免疫组织化学染色显示,正常组几乎未见Fas^+细胞。再灌注后6 h,RIRI组视网膜神经节细胞及内核层开始出现少量Fas^+细胞;再灌注后12 h,RIRI组视网膜Fas^+细胞表达逐渐增多;再灌注后24 h视网膜Fas^+细胞数达到高峰,棕色阳性染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层;再灌注后72 h视网膜Fas^+细胞较再灌注后24 h减少。NAS组在再灌注后6 h、12 h、24 h、72 h视网膜Fas^+细胞数均较RIRI组各时间点减少,再灌注后24 h,Fas^+细胞数达较高水平,随后下降,差异均有统计学意义(均为P <0. 05)。正常组视网膜可见FasL全层低表达。RIRI组再灌注后6 h,视网膜神经节细胞层和神经纤维层存在少量FasL^+细胞;再灌注后12 h FasL蛋白表达逐渐增多;再灌注后24 h FasL^+细胞数达高峰,可见深棕色的细胞膜及细胞质染色细胞分布在视网膜神经节细胞层、内丛状层、内核层及神经纤维层;再灌注后72 hFasL蛋白的阳性表达逐渐减少。NAS组再灌注后6 h、12 h、24 h、72 h视网膜FasL^+细胞数均少于RIRI组各时间点阳性细胞数,差异均有统计学意义(均为P <0. 05)。结论 NAS可通过抑制RIRI大鼠视网膜细胞Fas、FasL蛋白的表达,减轻缺血再灌注对大鼠视网膜细胞造成的损伤。

Objective To investigate the effects of N-acetylserotonin(NAS)on the expression of Fas and FasL in the retina of rats with retina ischemia-reperfusion injury(RIRI).Methods The RIRI rat model was established by ocular hypertension method.Adult male Sprague-Dawley rats were randomly divided into normal control group(n=6),RIRI group(n=24)and NAS group(n=24).The NAS group received intraperitoneal injection of NAS 30 min before RIRI,and the RIRI group received intraperitoneal injection of the same amount of normal saline at the same time.The groups were divided into 6 h,12 h,24 h and 72 h subgroups according to the time of modeling.Morphological changes of retina in each group were observed under light microscope by HE staining,and recorded the retinal thickness and the number of retinal ganglion cells in each group.Immunohistochemical staining was used to detect the effect of NAS on the expression of Fas and FasL protein in the retina of RIRI rats.Results HE staining showed that the cells in the retina of normal rats had clear boundaries,normal morphology,and neatly arranged nerve cells.In the RIRI group,edema appeared in the retina at 6 h after reperfusion,and the ganglion cell layer and inner nuclear layer were more obvious.The number of ganglion cells was lower than that in the normal group.Then the retinal edema was further aggravated and the ganglion cells continued to decrease.The degree of retinal edema in the NAS group was lower than that in the RIRI group at 6 h,12 h and 24 h after reperfusion.The retinal thickness of the NAS group was thicker than the RIRI group at 72 h after reperfusion.The number of ganglion cells in the NAS group was higher than that in the RIRI group at each time point.The difference was statistically significant(all P<0.05).Immunohistochemical staining showed that almost no Fas^+cells were seen in the normal group.At 6 h after reperfusion,a small number of Fas^+cells began to appear in the retinal ganglion cells and inner nuclear layer of the RIRI group.At 12 h after reperfusion,the expression of retinal Fas^+cells in the RIRI group gradually increased.Retinal Fas^+cells reached a peak at 24 h after reperfusion,and brown positive staining cells were distributed in retinal ganglion cell layer,inner plexiform layer,inner nuclear layer and nerve fiber layer.Compared with 24 h after reperfusion,Retinal Fas^+cells was decreased at 72 h after reperfusion.The number of retinal Fas^+cells in the NAS group at 6 h,12 h,24 h,and 72 h after reperfusion was lower than that in the RIRI group.At 24 h after reperfusion,the number of Fas^+cells reached a high level and then decreased.The difference was statistically significant(all P<0.05).FasL full-layer low expression in the normal group.At 6 h after reperfusion in the RIRI group,a small number of FasL^+cells were present in the retinal ganglion cell layer and nerve fiber layer.FasL protein expression increased gradually at 12 h after reperfusion.The number of FasL^+cells reached a peak at 24 h after reperfusion.It was found that dark brown cell membrane and cytoplasmic staining cells were distributed in retinal ganglion cell layer,inner plexiform layer,inner nuclear layer and nerve fiber layer.The positive expression of FasL protein decreased gradually at 72 h after reperfusion.The number of retinal FasL^+cells in the NAS group at 6 h,12 h,24 h,and 72 h after reperfusion was less than that in the RIRI group at each time point.The difference was statistically significant(all P<0.05).Conclusion NAS can attenuate the damage of retinal cells induced by ischemia-reperfusion in rats by inhibiting the expression of Fas and FasL proteins in retinal cells of RIRI rats.