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葛根素通过下调过氧亚硝基阴离子水平和诱导型一氧化氮合酶表达来减少糖尿病大鼠视网膜色素上皮细胞凋亡

郝丽娜^*, 王敏, 马军玲, 杨涛

河北省人民医院眼科，石家庄 050051；河北省邯郸县医院眼科，邯郸 056001；河北医科大学第一医院内科，石家庄 050031

摘要

本研究旨在观察葛根素对糖尿病大鼠视网膜色素上皮(retinal pigment epithelial, RPE)细胞凋亡的影响及其机制。采用大鼠腹腔注射链脲佐菌素(streptozotocin, STZ)的方法建立糖尿病动物模型。108 只Sprague-Dawley (SD)大鼠纳入实验，随机分为三组：对照组(腹腔注射生理盐水)、STZ组和葛根素组，每组n = 36。STZ组和葛根素组大鼠均接受3 d的STZ腹腔注射(每天45 mg/kg)。第4天起，葛根素组每天腹腔注射葛根素(140 mg/kg)，直至第60天实验结束。腹腔注射STZ后20、40和60 d分批处死动物，分离出RPE细胞。用免疫印记方法检测过氧亚硝基阴离子(peroxynitrite, ONOO−)的标志物——硝基酪氨酸(nitrotyrosine, NT)和Fas/FasL蛋白表达情况，用DNA laddering检测RPE细胞的凋亡情况，用RT-PCR检测iNOS的表达，用免疫组化方法检测Fas/FasL信号转导途径的表达。结果显示，实验全程对照组RPE细胞中有少量NT、iNOS mRNA及Fas/FasL的表达，其它各项指标(血糖和体重、凋亡)基本正常；STZ组在STZ注射后20 d时凋亡明显，NT、iNOS mRNA及Fas/FasL的表达相对对照组均有显著的上升，以上指标随实验时间的延长呈上升趋势；葛根素组的凋亡相对STZ组有明显减轻，NT、iNOS mRNA及Fas/FasL的表达虽仍高于对照组，但在STZ注射后20或40 d时均显著低于STZ组。以上结果提示，葛根素能够通过对ONOO−水平的下调和iNOS表达的抑制来减少糖尿病大鼠RPE细胞的凋亡，其可作为治疗糖尿病视网膜病变的潜在药物。

关键词： 葛根素; 视网膜色素上皮细胞; 氧化; 细胞信号转导; 糖尿病

分类号：R683.42

Puerarin decreases apoptosis of retinal pigment epithelial cells in diabetic rats by reducing peroxynitrite level and iNOS expression. Hao LN, Wang M, Ma JL, Yang T.

HAO Li-Na^*, WANG Min, MA Jun-Ling, YANG Tao

Ophthalmology Department of Hebei Province People’s Hospital, Shijiazhuang 050051, China； Ophthalmology Department of Handan County Hospital, Handan 056001, China； Internal Department of First Hospital Affiliated to Hebei Medical University, Shijia-zhuang 050031, China

Abstract

The purpose of this study was to investigate the protective effect of puerarin on retina pigment epithelial (RPE) cells of diabetic rats against apoptosis. One hundred and eight Sprague-Dawley (SD) rats were randomly divided into 3 groups: control group, streptozotocin (STZ) group and puerarin group. STZ and puerarin groups received 3 d of STZ injection (45 mg/kg per day, i.p.). Additionally, puerarin groups were treated with puerarin (140 mg/kg, i.p.) from the 4th day to the end of experiment. The rats from different groups were sacrificed on 20, 40 and 60 d after STZ injection for harvesting RPE cells. Western blot analysis, DNA laddering, RT-PCR and immunohistochemistry were used for determining the expression of nitrotyrosine (NT, the foot print of peroxynitrite), cell apoptosis, iNOS mRNA and Fas/Fas ligand (FasL) signal transduction in RPE cells, respectively. The results showed that control group maintained low apoptosis level and little NT, iNOS mRNA, Fas/FasL protein expressions, as well as normal blood glucose and body weight during 60 d of the experiment. Compared with control group, STZ group showed obvious apoptosis and higher NT, iNOS mRNA, Fas/FasL protein expressions from 20 d after STZ injection. Puerarin relieved apoptosis of RPE cells and decreased NT, iNOS mRNA, Fas/FasL protein expressions in puerarin group 20 or 40 d after STZ injection, compared with STZ group. These results suggest puerarin can decrease RPE cells apoptosis in diabetic rats by reducing peroxynitrite level and iNOS expression, thus being a potential therapeutic agent in controlling of diabetic retinopathy.

Key words: puerarin; retinal pigment epithelial cells; oxidative; cell signal; diabetes

收稿日期：2011-02-26　　录用日期：2011-07-21

通讯作者：郝丽娜　　E-mail: jhaolina@yahoo.com.cn

引用本文：

郝丽娜, 王敏, 马军玲, 杨涛. 葛根素通过下调过氧亚硝基阴离子水平和诱导型一氧化氮合酶表达来减少糖尿病大鼠视网膜色素上皮细胞凋亡[J]. 生理学报 2012; 64 (2): 199-206.

HAO Li-Na, WANG Min, MA Jun-Ling, YANG Tao. Puerarin decreases apoptosis of retinal pigment epithelial cells in diabetic rats by reducing peroxynitrite level and iNOS expression. Hao LN, Wang M, Ma JL, Yang T.. Acta Physiol Sin 2012; 64 (2): 199-206 (in Chinese with English abstract).