黄芪多糖对急性高眼压诱导大鼠视网膜神经节细胞的保护作用

背景青光眼患者视神经保护的问题日益引起关注。黄芪多糖（APS）是黄芪的主要活性成分，可增加再生神经蛋白的表达并促进损伤的周围神经修复，但其对视网膜神经节细胞（RGCs）再生作用的研究少见报道。目的探讨APS对急性高眼压状态下RGCs的保护作用。方法采用抽签法将40只SPF级sD大鼠随机分为正常对照组、模型对照组、低剂量APS组和高剂量APS组，每组各10只。低剂量APS组和高剂量APS组大鼠自实验开始每日分别给予APS500mg／kg、2000mg／kg（均溶于2．5ml生理盐水）灌胃，模型对照组仅给予2．5ml生理盐水灌胃，正常对照组不做任何处理。用药2周后，除正常对照组外，其余3个组均抽取0．2ml房水继而单眼前房注射等体积甲基纤维素使眼压升高至22mmHg（1mmHg=0．133kPa）以上制作急性高眼压模型。造模后5d，过量麻醉法处死动物，摘除该眼球制作视网膜石蜡切片，常规组织病理学观察视网膜的形态结构变化，采用免疫组织化学法检测caspase-3蛋白在大鼠视网膜中的表达，采用TUNEL染色法观察和计算各组大鼠RGCs的凋亡率。ImageProPlus5．1软件测量各组大鼠视网膜厚度和神经纤维层厚度。结果大鼠成模后5d，模型对照组、低剂量APS组和高剂量APS组大鼠的眼压均明显高于正常对照组，差异均有统计学意义（t=-8．900、-10．700、-11．300，P〈0．01）。正常对照组大鼠视网膜形态正常；模型对照组大鼠视网膜水肿，细胞排列紊乱；低剂量APS组视网膜可见空泡样变性，但视网膜细胞排列较模型对照组整齐，视网膜水肿减轻；高剂量APS组视网膜水肿较明显。低剂量APS组视网膜厚度、外颗粒层及视神经纤维层厚度值均明显低于模型对照组，差异均有统计学意义（t=-23．700、-14．770、-11．640，P〈0．01），但高剂量APS组外颗粒层及视神经纤维层厚度与模型对照组比较差异均无统计学意义（t=-0．780、-0．460，P〉0．05）。低剂量APS组大鼠caspase-3蛋白阳性RGCs百分比及RGCs凋亡百分率均明显低于模型对照组（caspase-3蛋白：F=87．710，P=0．001；RGCs凋亡：F=272．840，P〈0．01），差异均有统计学意义（t=-11．700、-8．600，P〈0．01），高剂量APS组与模型对照组比较caspase-3蛋白阳性RGCs百分比及RGCs凋亡百分率的差异均有统计学意义（t=-7．900、-6．400，P〈0．05）。结论500mg／kgAPS可有效抑制急性高眼压模型大鼠的视网膜水肿及RGCs的凋亡率，对急性高眼压大鼠的RGCs有保护作用。

Protective effect of astragalus polysaccharides on retinal ganglion cell in acute ocular hypertension rat model

Background More efforts have been made in the functional protection of the glaucoma ganglion ceils （RGCs） nowadays. As main ingredient,astragalus polysaecharides （APS） enhances neuron regeneration protein expression and promotes peripheral nerve recovery. But whether APS has a protecting effect on RGCs is incompletely clear. Objective The purpose of this study was to evaluate the neuroprotective effect of APS on the RGCs in a rat model of experimental glaucoma. Methods Forty-four SPF SD rats were divided into 4 groups randomly as follows ： normal control group, negative control group, low dose APS group and high dose APS group, with 10 rats for each group. APS of 500 mg/kg or 2000 mg/kg （2.5 ml） was administered by gavage feeding once daily for 2 weeks in low dose or high dose of APS group, respectively, and the same volume of normal saline solution was applied instead of APS in the model control group. Two weeks later, aspirate 0.2 ml aqueous followed by methyleellulose injected into the anterior chamber to create the acute ocular hypertension model in the three groups above. No any intervention was performed in the normal control group. The rats were sacrificed on the fifth day after model established to take a retinal section. Ocular hypertension-induced damage was evaluated by regular retina histopathologic examination.Immunolhistochemistry for caspase-3 and TUNEL kits were used to determine the expression of caspase-3 protein in retina and apoptosis rate of RGCs. Retinal cross-sections were analyzed by Image Pro Plus 5. 1 software to determine the thickness of various retinal layers and the positive staining cell density in the retinal ganglion cell layer （ RGCL）. Results On the fifth day after establishment of models,intraocular pressure （lOP） was significantly elevated in the model control group, low dose APS group and high dose APS group in comparison with the normal control group （t=-8. 900,-10. 700 ,-11. 300,P〈0.01 ）, Retinal morphology was normal in the rats of the normal control group, but in the model control group, rat retina was significantly thickened from severe retinal edema and cell arrangement disorder. Mild retinal abnormality was seen in the low dose APS group;while obvious retina edema was in high dose APS group. The entire retinal thickness, outer nuclear layer thickness and retinal nerve fiber thickness values were lower in the low dose APS group than those of model control group （ t = - 23. 700, - 14. 770, - 11. 640, P 〈 0.01 ）. However,no difference was found in outer nuclear layer thickness and retinal nerve fiber thickness values between high dose APS group and normal control group （ t = -0. 780, -0. 460, P〉0.05 ）. Percentage of positive RGCs for easpase-3 protein and rate of apoptotic RGCs were significantly reduced in low dose APS group compared with model control group （caspase-3：F=87.710,P=0.001;RGCs apoptosis：F=272.840,P〈0.01）. Conclusions 500 mg/kg APS can protect retina and RGCs against ocular hypertension-induced damage. The protection of APS is non-dosedependent.