黄芪黄酮对糖尿病周围神经病变大鼠背根神经节的保护作用及机制

Protective effect and mechanism of astragalus flavone on dorsal root ganglion in diabetic rats with peripheral neuropathy

目的探讨黄芪黄酮对糖尿病周围神经病变大鼠背根神经节氧化应激及细胞凋亡的影响及机制。方法将40只雄性SD大鼠随机分为正常对照组、模型组、黄芪黄酮组和阳性对照组,每组10只。模型组、黄芪黄酮组和阳性对照组腹腔注射2%的链脲佐菌素制备糖尿病周围神经病变大鼠模型,正常对照组腹腔注射等量枸橼酸盐缓冲液。造模成功后,分别给予黄芪黄酮组、阳性对照组大鼠灌胃黄芪黄酮、金芪降糖片,给予其他两组灌胃等量蒸馏水。造模30 d时,采用血糖仪及Von Frey仪分别测量各组大鼠血糖及机械痛阈值。取各组大鼠的背根神经节组织,检测细胞凋亡率、膜电位水平,并测定背根神经节组织的活性氧簇(ROS)、超氧化物歧化酶(SOD)、丙二醛含量以及细胞色素C(Cyt-C)、B细胞淋巴瘤-2(Bcl-2)、B细胞淋巴瘤-2相关X蛋白(Bax)和活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved Caspase3)、p38和磷酸化p38(p-p38)的蛋白表达水平。结果 (1)造模30 d时,与正常对照组比较,模型组大鼠的血糖水平、背根神经节细胞凋亡率升高,背根神经节组织的ROS和丙二醛含量以及Cyt-C、Bax、cleaved Caspase3、p38、p-p38蛋白表达水平均增加,而机械痛阈值及背根神经节组织的膜电位水平、SOD含量、Bcl-2蛋白表达水平均降低(均P<0.05)。(2)造模30 d时,与模型组比较,黄芪黄酮组和阳性对照组大鼠的血糖水平、背根神经节细胞凋亡率均降低,背根神经节组织的ROS和丙二醛含量以及Cyt-C、Bax、cleaved Caspase3、p38、p-p38蛋白表达水平均下降,机械痛阈值及背根神经节组织的膜电位水平、SOD含量、Bcl-2蛋白表达水平均升高(均P<0.05);且与阳性对照相比,黄芪黄酮组背根神经节细胞凋亡率下降,背根神经节组织的ROS和丙二醛含量以及Cyt-C、Bax、p38、p-p38蛋白表达水平均降低,机械痛阈值、背根神经节组织的膜电位水平、SOD含量均升高(均P<0.05)。结论黄芪黄酮可抑制糖尿病大鼠背根神经节组织氧化应激反应、细胞凋亡以及p38丝裂原活化蛋白激酶信号通路,从而减轻周围神经损伤。

Objective To investigate the effect and mechanism of astragalus flavone on oxidative stress and apoptosis of dorsal root ganglion in diabetic rats with peripheral neuropathy.Methods Forty male SD rats were randomly divided into normal control group, model group, astragalus flavone group and positive control group, with 10 rats in each group. The model group, the astragalus flavone group and the positive control group were intraperitoneally injected with 2% streptozotocin to prepare diabetic rat models of peripheral neuropathy, while the normal control group was intraperitoneally injected with the same amount of citrate buffer. After the successful modeling, rats in the astragalus flavone and positive control groups were given intragastric administration of astragalus flavone and Jinqi Jiangtang Tablets, respectively, whereas the other two groups were given intragastric administration of isometric distilled water. On day 30 of the modeling, the blood glucose and mechanical pain thresholds of rats in each group were measured with blood glucose meter and Von Frey meter, respectively. Rats′ dorsal root ganglion tissues were harvested from each group to detect the apoptosis rate and membrane potential levels. Additionally, a detection was conducted for dorsal root ganglion tissues′ contents of reactive oxygen species(ROS), superoxide dismutase(SOD), and malondialdehyde, as well as the expression levels of cytochrome C(Cyt-C), B-cell lymphoma-2(Bcl-2), B-cell lymphoma-2-associated X protein(Bax), cleaved cysteinyl aspartate specific proteinase 3(cleaved Caspase3), p38, and phosphorylated p38(p-p38) proteins. Results(1) On day 30 of the modeling, as compared with the normal control group, rats in the model group exhibited an elevated blood glucose level, a higher apoptosis rate of dorsal root ganglion cells, increased contents of ROS and malondialdehyde in dorsal root ganglion tissues, higher expression levels of Cyt-C, Bax, cleaved Caspase3, p38, and p-p38 proteins in dorsal root ganglion tissues, lower mechanical pain thresholds, and reductions in the membrane potential level, SOD content, and expression level of Bcl-2 protein in dorsal root ganglion tissues(all P<0.05).(2) On day 30 of the modeling, as compared with the model group, rats in the astragalus flavone and positive control groups reported a decreased blood glucose level, a lower apoptosis rate of dorsal root ganglion cells, reduced contents of ROS and malondialdehyde in dorsal root ganglion tissues, lower expression levels of Cyt-C, Bax, cleaved Caspase3, p38, and p-p38 proteins in dorsal root ganglion tissues, higher mechanical pain thresholds, and increases in the membrane potential level, SOD content, and expression level of Bcl-2 protein in dorsal root ganglion tissues(all P<0.05);moreover, by comparison with the positive control group, the astragalus flavone group had a decreased apoptosis rate of dorsal root ganglion cells, lower contents of ROS and malondialdehyde in dorsal root ganglion tissues, reduced expression levels of Cyt-C, Bax, p38, and p-p38 proteins in dorsal root ganglion tissues, higher mechanical pain thresholds, and increases in the membrane potential level and SOD content in dorsal root ganglion tissues(all P<0.05). Conclusion Astragalus flavone can inhibit oxidative stress and cell apoptosis in dorsal root ganglion tissues as well as suppress p38 mitogen-activated protein kinase signaling pathway in diabetic rats, thus alleviating peripheral nerve injury.