论文部分内容阅读
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3d的STZ腹腔注射(每天45mg/kg)。第4天起,葛根素组每天腹腔注射葛根素(140mg/kg),直至第60天实验结束。腹腔注射STZ后20、40和60d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(ni-trotyrosine,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20d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40d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。
The purpose of this study was to investigate the effect and mechanism of puerarin on the apoptosis of retinal pigment epithelial (RPE) cells in diabetic rats. Animal models of diabetes were established by intraperitoneal injection of streptozotocin (STZ) in rats. One hundred and eighty Sprague-Dawley rats were randomly divided into three groups: control group (intraperitoneal injection of normal saline), STZ group and puerarin group, n = 36 in each group. STZ group and puerarin group received 3d STZ intraperitoneal injection (45mg / kg per day). From the fourth day onward, puerarin (140 mg / kg) was intraperitoneally injected into the puerarin group until the end of the experiment on the 60th day. Animals were sacrificed at 20, 40 and 60 days after intraperitoneal injection of STZ, and RPE cells were isolated. The expression of peroxynitrite (ONOO?), Nitrotyrosine (NT) and Fas / FasL protein was detected by Western blotting, and the apoptosis of RPE cells was detected by DNA laddering The expression of iNOS was detected by RT-PCR and the expression of Fas / FasL signal transduction pathway was detected by immunohistochemistry. The results showed that a small amount of NT and iNOS mRNA and Fas / FasL were expressed in RPE cells in the whole experiment group, and other indexes (blood glucose, body weight and apoptosis) were basically normal. STZ group showed obvious apoptosis 20 days after STZ injection, The expression of NT, iNOS mRNA and Fas / FasL increased significantly compared with the control group. The above indexes increased with the prolongation of experimental time. The apoptosis of puerarin group was significantly reduced compared with that of STZ group. The expressions of NT, iNOS mRNA and Fas / Although the expression of FasL was still higher than that of the control group, the expression of FasL was significantly lower than that of STZ group at 20 or 40 days after STZ injection. The above results suggest that puerarin can reduce the apoptosis of RPE cells in diabetic rats by down-regulating the ONOO level and inhibiting the expression of iNOS, which may be used as a potential drug for the treatment of diabetic retinopathy.