促红细胞生成素对急性高眼压大鼠视网膜的保护作用

The protective function of erythropoietin to the rat retina after acute intraocular hypertension

目的:探讨重组人促红细胞生成素(recombinant human erythropoietin,rhEPO)预处理对大鼠急性高眼压视网膜损伤的保护作用及其机制。方法:选用健康成年雄性Wistar大鼠36只,随机分为正常组4只(不做任何处理直接处死),生理盐水组16只(造模前3hip生理盐水),rhEPO组16只(造模前3hiprhEPO)。采用生理盐水前房加压灌注法升高大鼠眼压至100mmHg并维持60min,于造模成功后6,24,48,72h分别处死动物摘取眼球制作视网膜石蜡切片。HE染色观察视网膜组织的形态学改变;分别采用TUNEL法和免疫组化法观测凋亡细胞和磷酸化蛋白激酶B(p-Akt)在视网膜中的表达和分布,并利用计算机图像分析系统量化检测指标,进行统计学分析。结果:急性高眼压造成大鼠视网膜损伤,生理盐水组大鼠视网膜内层变薄,所占整个视网膜的百分比较正常组下降,rhEPO组视网膜内层所占百分比较生理盐水组大,差异有统计学意义(P<0.01);正常组大鼠视网膜未见TUNEL阳性细胞,生理盐水组和rhEPO组在神经节细胞层、内核层均可见TUNEL阳性凋亡细胞,但rhEPO组与生理盐水组比较,TUNEL阳性细胞减少,差异有统计学意义(P<0.01)。正常组视网膜p-Akt有弱表达,生理盐水组和rhEPO组在神经节细胞层、内网层、内核层均可见p-Akt阳性细胞,但rhEPO组比生理盐水组表达增强,差异有统计学意义(P<0.01)。神经节细胞层与内核层p-Akt的表达较内网层强。结论:rhEPO预处理可减弱高眼压对视网膜造成的损伤,减少和延缓细胞凋亡的发生,其可能的机制是通过激活磷脂酰肌醇3-激酶(PI3K)/Akt信号传导通路上调p-Akt来抑制凋亡的发生。

AIM： To investigate the protective function and mechanism of recombinant human erythropoietin （rhEPO） pretreatment on the rat retina with acute intraocular hypertension injury. METHODS： Thirty-six healthy male Wistar rats were randomly divided into 3 groups： normal group （n =4, killed directly without any treatment）, saline group （n =16, physiological saline was administered i.p. 3 hours before raising intraocular pressure [IOP]）, rhEPO group （n =16, rhEPO was administered i.p. 3 hours before raising IOP）. Either of bilateral eyes was randomly made into the acute high IOP model using the method of saline perfusion into anterior chamber until the IOP reached 100mmHg, then sustained the IOP for 60 minutes. All the rats were killed respectively and the eyeballs were enudeated at different observation periods after the model was successfully established （6, 24, 48 and 72 hour）. The histological changes of retina were observed by HE staining; the expression end distribution of apoptotic cells and p-Akt protein were observed respectively by using TUNEL detection and immunohistochemistry. The data was statistically analyzed. RESULTS： ①The acute high IOP damaged the rat retina, the inner layer of retina got thinner end its percentage of whole retina was decreasing in the saline group, but the percentage of the rhEPO group was bigger than that of the saline group, the difference was significant （P〈0.01）; ②No TUNEL-positive cells were seen in the normal group, the apoptotic cells of the other two groups were observed in the retinal ganglion cell layer （RGL） and inner nuclear layer （INL）. There were less apoptotic ceils in the rhEPO group than those in the saline group, the difference was significant （P〈0.01）;③The minute expression of p-Akt protein was found in the normal group, while the p-Akt positive cells which were expressed in the RGL, inner plexiform layer （IPL） and INL were observed both in the saline group and the rhEPO group. There were stronger expression in the rhEPO group than those in the saline group, the difference was of significance （P〈0.01）. The expression of p-Akt in RGL and INL is stronger than that in IPL. CONCLUSION： The damage caused by the acute intraocular hypertension was mainly occurred in RGL, IPL and INL. while rhEPO pretreatment can lessen the damage and the cell apoptosis. The mechanism of rhEPO＇s protective function may be the activation of PI3K/Akt signaling pathway and up regulation p-Akt in retina, which can restrain cell apoptosis.