积雪草酸对原代大鼠视网膜神经节细胞低氧损伤的保护作用研究  

Neuroprotection of asiatic acid to the hypoxic injury in rat primary retinal ganglion cells

作　　者：胡方园 黄婉静 吴继红 陈君毅 HU Fang-yuan;HUANG Wan-jing;WU Ji-hong;CHEN Jun-yi(Eye Institute,Eye Ear Nose and Troat Hospital of Fudan University,Shanghai 200031,China)

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科研究院,上海200031

出　　处：《中国眼耳鼻喉科杂志》2018年第4期240-244,共5页Chinese Journal of Ophthalmology and Otorhinolaryngology

基　　金：国家自然科学基金(81470624;81770925)

摘　　要：目的研究积雪草酸(AA)在体外大鼠视网膜神经节细胞(RGC)低氧损伤中的保护作用,并探讨其作用机制。方法选取出生1~3 d的新生SD大鼠,解剖游离出视网膜,利用Thy1.1抗体包被法纯化获取RGC,定量聚合酶链反应(q PCR)鉴定RGC的纯度。体外给予原代RGC不同时长(0、12、24、48 h)的低氧(1%O2)损伤刺激,TUNEL检测RGC的凋亡情况。预先给予原代RGC不同浓度(0、5、10、20、50μmol/L)的AA干预后,再以1%O_2培养48 h,通过TUNEL凋亡检测、CCK-8细胞增殖检测研究AA在RGC凋亡中的作用。利用Wes全自动蛋白质印迹定量分析系统对凋亡相关蛋白c-Abl进行检测。结果低氧条件下(1%O2),RGC的TUNEL凋亡阳性细胞数随着低氧刺激时间的递增呈梯度增加。低氧环境下(1%O2),给予不同浓度的AA干预,TUNEL结果显示10μmol/L和20μmol/L AA处理后,RGC凋亡细胞数显著减少(P值均<0.01)。同时CCK-8结果显示10μmol/L和20μmol/L AA干预后,RGC的增殖活力明显升高(分别为P<0.05,P<0.01)。Wes全自动蛋白质印迹定量分析系统结果表明,AA在原代RGC低氧损伤中可以明显抑制c-Abl的磷酸化水平。结论体外低氧刺激时,AA可以减少RGC的凋亡,对原代RGC的低氧损伤有一定的保护作用,其作用机制与抑制c-Abl的磷酸化激活相关。Objective To elucidate the neuroprotective effects of asiatic acid（ AA） on rat primary retinal ganglion cells（ RGCs） under hypoxic conditions. Methods RGCs were purified by Thy1. 1 antibody from newborn（ 1-3 days after born） SD rats. q PCR was performed to detect the purity of RGCs. The apoptosis of primary RGCs was examined by TUNEL assay under hypoxia（ 1% O2） from 0 to 48 h. Next,primary RGCs were pre-treated with different concentrations of AA（ 0,5,10,20 and 50 μmol/L） and cultured under hypoxia（ 1% O2） for 48 h. Then,TUNEL assay and CCK-8 assay were performed to detect RGCs apoptosis. The expression of c-Abl was determined by Wes automatic protein quantitative analysis system. Results RGCs with TUNEL positive were increased in a time-dependent manner induced by hypoxic stimulation. AA significantly inhibited RGCs apoptosis under hypoxia（ 1% O2） at the concentrations of 10 μmol/L and 20 μmol/L（ P 〈 0. 01）. Moreover,the Cell Counting Kit-8 assay showed that AA enhanced the proliferation ability of RGCs at the concentrations of 10 μmol/L and 20 μmol/L（ P〈 0. 05,P 〈 0. 01,respectively）.Furthermore,Wes protein analysis system indicated that AA markedly inhibited the phosphorylation levels of c-Abl in primary RGCs under hypoxia（ 1% O2）. Conclusions AA significantly reduced RGCs apoptosis through inhibiting the phosphorylation of c-Abl.

关 键 词：大鼠原代视网膜神经节细胞 积雪草酸 C-ABL 神经保护 

分 类 号：R285.5[医药卫生—中药学]