**Present address: Ophthalmology
Department of Hebei Province People’s Hospital,
*** Present address: Ophthalmology
Department of PLA Hospital，Beijing，100806
*Corresponding author. Tel:
+86-311-86265720; E-mail: Lingyiling @tom.com
Puerarin
decreases lens epithelium cell apoptosis induced partly by peroxynitrite
in diabetic rats
HAO Li-Na*,
LING Yi-Qun, LUO Xiu-Mei, MAO
Yu-Xiang, MAO Qi-Yan, HE Shou-Zhi**, LING Yi-Ling *
Abstract:
The present study was designed to observe if puerarin
decreases lens epithelium cell (LEC) apoptosis induced partly by peroxynitrite (ONOO-). One hundred and eight rats were
randomly divided into control group (n=36), streptozotocin
(STZ) group (n=36) and STZ + puerarin group (n=36).
The animals of control group received 0.5 ml of saline. STZ group and STZ + puerarin group received intraperitoneal
injection of STZ (45 mg/kg). Three days later, the rats in STZ + puerarin group were given puerarin
(140 mg/kg per day, i.p.). On the days 20, 40 and 60
of experiment，morphologic changes of lenses were observed with slit lamp. Then, the animals
were sacrificed for further analysis. The amount and percentage of apoptotic
LEC were determined by flow cytometry. Nitrotyrosine (NT, the foot print of ONOO-) was
examined by immunohistochemistry. Apoptosis-related
genes iNOS, etc. were analyzed by gene array.
The results showed that in thecontrol group, all the
lenses were clear. In STZ group, gradually severe opacity of the lens was
observed on the days 20, 40 and 60. But in STZ + puerarin
group, mild opacity of the lens was observed on the day 20 and more severe on the
day 40, but markedly decreased on the day 60. In the control group, mild
apoptosis of LEC was observered. In STZ group, time-dependent
increase in apoptosis of LEC was observed. In STZ + puerarin
group, mild apoptosis of LEC was observed on the day 20, significantly
increased on the day 40, but markedly decreased on the day 60. There was no
expression of NT in the lens in the control group, but an increased expression
of NT in STZ group. In STZ + puerarin group, mild expression
of NT was observed on the day 20, significantly increased on the day 40, but markedly
decreased on the day 60. There was no expression of iNOS in the lens in the
control group, but continuous
up-regulation of iNOS gene in STZ group. In
STZ + puerarin group, mild expression of iNOS was observed on the day 20, significantly
increased on the day 40, but markedly decreased on the day 60. Except the changes of iNOS
related to NO production, the other apoptosis-related genes, including BCL-2
and SOD, showed down-regulation, while NF-κB and TNFR1-FADD-caspase signal
transduction were also up-regulated in STZ group. The results were opposite in
STZ + puerarin group and control group. These
findings show that NT is expressed in diabetic rat lens. It is suggested that
LEC apoptosis in diabetic lens is partly induced by ONOO- which may
be a new oxidative damage way to form cataract. Puerarin partly decreases
LEC apoptosis induced by ONOO- and is a potential medicine for therapy of
diabetic cataract. The mechanism of puerarin dealing
with diabetic cataract may be related to its direct inhibition of LEC apoptosis
and antagonism of damage of ONOO- to LEC of diabetic rats.
Key
words: puerarin; lens epithelium cell; diabetes mellitus; cataract
葛根素减轻部分由过氧亚硝基阴离子导致的糖尿病大鼠晶状体上皮细胞凋亡
郝丽娜**，凌毅群，罗秀梅**，毛宇湘，毛绮妍，何守志***，凌亦凌*
河北医科大学病理生理教研室，石家庄 050017
摘 要：本研究观察葛根素是否减轻部分由过氧亚硝基阴离子(peroxynitrite,
ONOO－)导致的糖尿病大鼠晶状体上皮细胞(lens epithelium cell, LEC)凋亡。 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。36只大鼠作为对照组，腹腔注射生理盐水；其他72只大鼠腹腔注射STZ (45 mg/kg)后分为STZ组和STZ+葛根素组，每组36只。STZ注射3 d后，STZ+葛根素组大鼠每天腹腔注射葛根素(140 mg/kg)。于实验开始后第20、40和60
天用裂隙灯检查晶状体的形态学变化后处死动物。用流式细胞仪检测LEC凋亡，用免疫组化方法检测晶状体中ONOO－的标志物——硝基酪氨酸(nitrotyrosine, NT)的表达，用基因芯片分析技术检测LEC凋亡相关基因iNOS等的表达。结果发现，对照组大鼠晶状体均透明，各项指标基本正常；STZ 组大鼠第20天时即出现晶状体混浊， 40~60 d期间混浊不断加重；STZ+葛根素组大鼠20~40 d时晶状体混浊呈加重趋势，但40~60 d以后明显减轻。对照组LEC轻度凋亡，而STZ 组凋亡细胞呈持续性增长，STZ+葛根素组大鼠20~40 d时细胞凋亡呈增长趋势，但40~60 d以后明显下降。对照组大鼠晶状体NT未见明显表达；STZ组大鼠NT表达明显加强；STZ+葛根素组大鼠20~40 d时NT表达呈增长趋势，但40~60 d以后明显下降。对照组凋亡相关基因及其他凋亡相关基因未见明显变化，STZ 组凋亡相关基因iNOS表达明显上调。其他凋亡相关基因如BCL-2、SOD表达明显下调，但NF-κB 和 TNFR1-FADD-caspase信号转导途径明显上调；STZ+葛根素组凋亡相关基因则呈相反改变。上述结果表明，在糖尿病大鼠晶状体中有ONOO－的标志物NT表达，提示糖尿病大鼠LEC凋亡部分由ONOO－诱导，这可能是氧化损伤导致白内障形成的新途径。葛根素能够部分逆转ONOO－对于LEC的致凋亡作用，提示葛根素可能是治疗糖尿病性白内障的有效药物，其治疗机制可能与葛根素直接抑制凋亡和对抗ONOO－对糖尿病大鼠LEC的损伤有关。
关键词：葛根素；晶状体上皮细胞；糖尿病；白内障
中图分类号：R683.42