重组人促红细胞生成素对大鼠视网膜缺血-再灌注损伤的保护作用

Neuroprotective effect of recombinant human erythropoietin on retinal ischemia reperfusion induced retinal neuron injury in rats

目的制作Sprague-Dawley(SD)大鼠视网膜缺血-再灌注(RIR)损伤模型,探讨腹腔内注射重组人促红细胞生成素(rHuEPO)对急性RIR损伤所致的大鼠视网膜神经元损伤的保护作用及其对热休克蛋白72(HSP72)表达的影响。方法采用前房灌注的方式建立RIR损伤模型,灌注压110mmHg(1mmHg=0.133kPa),缺血时间1h;腹腔注射rHuEPO。78只SD大鼠随机分组:正常组6只,EPO组、EPO+槲皮黄酮组、RIR组各24只,均以右眼为实验眼。采用免疫组织化学法和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法(TUNEL)分别测定正常对照组和各实验组大鼠再灌注24h、48h、72h和1周视网膜中HSP72及凋亡细胞的表达,观察各组大鼠视网膜病理学改变。结果①正常组大鼠视网膜中HSP72表达微弱,各实验组大鼠视网膜中HSP72表达自再灌注12h开始增强,24h达到高峰,随后逐渐减弱,72h时表达稍高于正常。再灌注后各时间段,EPO组大鼠视网膜中HSP72表达均高于RIR组、EPO+槲皮黄酮组(P<0.05)。②正常大鼠视网膜中几乎没有凋亡细胞。再灌注后12h,各实验组大鼠视网膜中可见凋亡细胞,24h达高峰,48h后凋亡细胞数逐渐减少;再灌注后各组大鼠视网膜中凋亡细胞数比正常组多(P<0.05)。③再灌注后,RIR组,EPO+槲皮黄酮组大鼠内层视网膜明显水肿,炎性细胞侵入,膜结构逐渐破坏;EPO组大鼠视网膜结构保持相对完整,炎性细胞相对较少。结论①HSP72在正常大鼠视网膜中表达微弱,RIR损伤后表达增多。腹腔注射EPO可以明显诱导大鼠视网膜中HSP72的表达增多。②EPO可以减少大鼠RIR损伤后视网膜细胞凋亡,减少视网膜内炎性细胞的浸润,保护视网膜结构,对视网膜具有明显的保护作用。其机制可能与使HSP72表达上调有关。

Objective To observe the protective effect of erythropoietin（EPO） on retinal neurons, and the ex- pression of heat shock protein72 （HPS72） in retinal of rats with ischemia-reperfusion （RIR） injury. Methods The model of RIR injury was induced by increasing intraocular pressure via an intracameral catheter. The pressure was 110 mm Hg（1 mm Hg =0. 133 kPa） lasting for 60 min. EPO was injected intraperitoneally. Totally 78 SD rats were divided randomly into normal control group （ n = 6 eyes）, RIR group （ 24 eyes ）, EPO treated group （24 eyes ） and quereetin group（24 eyes）, with the right eye being the experimental eye. Immunohistoehemical technique and TdT-mediated bio-tin-dUTP nick end labeling（TUNEL） staining technique was used to examine the expression of HPS72 and the apoptosis of retinal ganglion cells （RGCs） respectively. The architecture of the retinal was measured by hematoxylin eosin staining before and 12,24,48,72 hours after reperfusion. Results ①The expression of HSP72 in the retinal of normal control group was weak. After RIR the HSP72 expression began to increase at 12 h of reperfusion and peaked at 24 h, then gradually decreased. The expression of HSP72 in EPO group was higher than either RIR group or quercetin group at 12, 24, 48 h after reperfusion. ②Very few apoptotic cells were seen in normal control rat retinal. In every experimental group, the apoptotic cells began to increase at 12 h after reperfusion and peaked at 24 h, then gradually decreased after48 h. The apoptotic cells were more than normal control group at each time of reperfusion, and the apoptotic cells of EPO treated group were less than RIR group or quercetin group at each time of reperfusion （ P 〈 0.05 ）. ③After reperfusion, the internal retina became edematic in RIR group and quercetin group. The structure of retina was destroyed gradually while the retina in EPO treated group was relatively complete. Conclusions ① The expression of HSP72 in the retina of normal control group was very weak. Ischemia-reperfusion injury could induce HSP72 expression. Intraperitoneal injec-tion of EPO to the rat could induce the expression of HSP72 expression in retina of rat. ②EPO could reduce apoptotic cell which was induced by the injury of RIR, and sustain the retinal morphous. EPO could protect the retina by increasing the expression of HSP72. （ Chin J Ophthalmol and Otorhinolaryngo1,2012 ,12 ：30-35 ）