摘要
目的 探讨黄芪发酵物通过调节脂肪酸合成酶(Fas)信号通路对碘缺乏大鼠海马损伤和学习记忆功能的影响,以期选出药效最佳的黄芪制剂.方法 60只Wistar大鼠随机分为对照组、模型组、黄芪发酵提取液4.00、2.00、1.00 g·kg-1组和阳性药(左甲状腺素钠9×10-3 mg·L-1)组.除对照组外,各组大鼠喂养碘缺乏饲料(碘含量为20 μg·kg-1)90 d构建甲状腺肿大鼠模型.检测各组大鼠血清中游离三碘甲腺原氨酸、游离甲状腺素、促甲状腺激素水平显示造模成功.跳台实验、Morris水迷宫实验检测各组大鼠的学习和记忆水平;酶联免疫吸附试验检测血清乙酰胆碱转移酶、乙酰胆碱酯酶、过氧化氢酶活性和乙酰胆碱、丙二醛含量;观察断头缺氧后存活时间和脑指数;苏木精-伊红染色、末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色观察海马组织病理学变化;同时,定量逆转录聚合酶链式反应检测Fas、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA的相对表达;最后,蛋白质印迹法检测、Caspase-3、Bax、Bcl-2蛋白的表达.结果 与对照组比较,模型组大鼠学习记忆能力明显下降,脑组织中乙酰胆碱水平、抗氧化活力降低,海马组织出现不同程度损伤和细胞凋亡,神经细胞排列紊乱、Fas、Caspase-3、Bax基因及蛋白表达增加,Bcl-2表达降低.与模型组相比,黄芪发酵物和阳性药组大鼠跳台潜伏期、错误次数和Morris水迷宫实验逃避潜伏期均明显减少,目标象限停留时间百分率和平台穿越次数均增加;血清乙酰胆碱转移酶、过氧化氢酶活性和乙酰胆碱含量升高,乙酰胆碱酯酶活性和丙二醛含量降低;缺氧后存活时间延长,脑指数增加;海马组织病理损伤得到明显改善;Fas、Caspase-3、Bax mRNA相对表达量减少,Bcl-2表达量增加,Bax/Bcl-2降低;蛋白质印迹法显示,Fas、Caspase-3、Bax蛋白表达量显著减少,Bcl-2蛋白表达增加,具有剂量依赖性.结论 黄芪发酵物能够提高碘缺乏大鼠学习记忆水平,可能与抑制Fas信号通路及调节乙酰胆碱代谢、改善能量代谢、减轻氧化应激损伤和抑制凋亡效应有关.
Abstract
Objective To explore the effect of Astragali radix fermentation on hippocampal damage and learing and memory function in iodine-deficient rats by regulating fatty acid synthetase(Fas)signaling pathway,in or-der to select its best preparation.Methods Sixty Wistar rats were randomly divided into a normal control group,model control group,4.00,2.00,1.00 g·kg-1 group,and positive drug(levothyroxine sodium 9×10-3 mg·kg-1)group.Except for the normal control group,rats were fed an iodine-deficient diet(iodine content of about 20 μg·kg-1)for 90 days to replicate an iodine-deficient model.The levels of free triiodothyronine,free tetraiodo-thyronine and thyroid stimulating hormone in the serum of each groupwere tested and they showed successful modeling.The learning and memory levels were tested by the platform and Morris water maze;the activities of choline acetyltransferase,acetylcholinesterase,catalase and the contents of acetylcholine and malondial-dehyde in serum were detected by enzymoly-linked immunosorbent assay.The survival time and brain index after hypoxia were observed.Hematoxylin-eosinand terminal deoxynucleotide transferase mediated dUTP notch end labeling(TUNEL)staining were used to observe the histopathological changes in hippocampus.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression of Fas,Caspase-3,B lymphoblastoma-2 gene(Bcl-2)and Bcl-2 associated X protein(Bax);and the expressions of Fas,Caspase-3,Bax and Bcl-2 proteins were detected by Western Blotting.Results Compared with the normal group,the model rats underwent a significant decrease in learning and memory ability,a decrease in Ach level and antioxidant vitality in brain tissue,different degrees of damage and apoptosis,disordered arrangement of nerve cells,an increased expression of Fas,Caspase-3,Bax genes and protein,and decreased expression of Bcl-2.Compared with the model group,the latency and number of errors in Morris water maze,the percentage of residence time in the target quadrant and the number of platform crossings were increased;activities of choline acetyltransferase,catalase viability and acetylcholine content,acetylcholines-terase vitality and malondialdehyde content decreased;prolonged survival time after hypoxia,and brain index increased;the histopathological damage of the hippocampus was significantly improved;the relative expres-sion levels of Fas,Caspase-3,and Bax mRNA were reduced,the expression level of Bcl-2 increased and the Bax/Bcl-2 decreased;the relative expression levels of Fas,Caspase-3 and Bax proteins were also significantly decreased while the Bcl-2 protein was increased.The results were related to the doses given.Conclusion Astrag-alus fermentation can improve the learning and memory dysfunction in iodine-deficient rats,and the mecha-nism may be related to the inhibition of Fas signaling pathway and the regulation of acetylcholine metabolism,thus improving energy metabolism and alleviating oxidative stress damage and inhibition of apoptotic effects.
国家科技期刊平台
NSTL
万方数据