养阴益气活血法调控MAPK信号通路阻断糖尿病大血管病变GK大鼠“代谢记忆”的实验研究

发布时间：2018-10-30 15:56

【摘要】：目的：本研究从氧化应激为入口,观察不同时间窗下具有养阴益气、活血化瘀功效的参芪复方对糖尿病大血管病变GK大鼠血糖、血清CHOL、8-iso-PGF2α;胸主动脉匀浆SOD、MDA;胸主动脉病理形态、细胞凋亡的影响,进行量-时-效关系分析,推测其可能通过抑制氧化应激、阻断“代谢记忆”途径来保护大血管。并应用基因芯片技术寻找参芪复方防治糖尿病大血管病变的分子靶点,探讨养阴益气活血法阻断“代谢记忆”的生物学作用及其机理,为临床推广中医药防治糖尿病大血管病变提供理论依据。方法：160只GK大鼠适应性饲养1周后,依据随机血糖水平由高到低排列编号,随机分为模型组、参芪复方高剂量组(参高组)、参芪复方中剂量组(参中组)、参芪复方低剂量组(参低组)、二甲双胍组(西药组),每组32只,给予高脂饲料饲养；Wistar组(空白组)32只给予普通饲料饲养；连续喂养12周,建立糖尿病“代谢记忆”模型。造模完成后,模型组和空白组给予5ml/kg.d生理盐水灌服,参高组给予2.88g/kg.d参芪复方浸膏灌服,参中组给予1.44g/kg.d参芪复方浸膏灌服,参低组给予0.72g/kg.d参芪复方浸膏灌服,西药组给予0.1g/kg.d二甲双胍灌服,治疗干预16周。实验期间从整体水平观察各组大鼠的一般情况,监测空腹血糖变化；各组大鼠分别在灌胃后第8、16周末处死,检测血清CHOL、胸主动脉匀浆SOD、MDA等指标；从组织水平,检测胸主动脉细胞凋亡情况,HE染色对胸主动脉进行形态学观察；观察不同时间窗下,不同干预方式对模型大鼠“代谢记忆”的影响效果。借助基因芯片技术对胸主动脉组织进行基因表达谱检测,筛选出差异表达基因,进行GO注释,Pathway分析,解释生物学意义。结果：参中组的作用优于参高组和参低组,能够明显改善GK大鼠的一般状况、血糖、血脂、氧化应激指标、胸主动脉病理形态及细胞凋亡。参中组与模型组差异表达基因,经GO注释、Pathway分析,主要涉及大分子代谢过程、细胞死亡、信号转导、细胞迁移和MAPK信号通路、刺猬信号转导通路、Notch信号通路、胰岛素信号转导等通路：涉及到Csnk1d、Lama4、Elavl1、Abi1、Anxa1、 Nsg1等基因。结论：参中组可以更好地改善糖尿病大血管病变GK大鼠的一般状态,降低血糖,改善脂代谢紊乱,减轻氧化应激,保护大鼠血管内皮损伤,阻断“代谢记忆”。其作用机理可能是通过调控大分子代谢过程、细胞死亡、信号转导、细胞迁移和MAPK信号通路、刺猬信号转导通路、Notch信号通路、胰岛素信号转导等通路及Csnk1d、Lama4、Elav11、Abi1、AnxaK1、Nsg1等基因,促进糖尿病大血管病变早期受损血管内皮细胞修复,改善氧化应激对血管的损害。其中,参芪复方可能通过调控MAPK这条重要的信号通路,减轻氧化应激,抑制血管内皮细胞凋亡,消除炎症反应,阻断糖尿病大血管病变“代谢记忆”,从而防治糖尿病大血管病变。
[Abstract]:Objective: to observe the effects of Shenqi compound on blood sugar, serum CHOL,8-iso-PGF2 伪 and thoracic aortic homogenate SOD,MDA; in GK rats with diabetic vascular disease under different time window, which can nourish yin and invigorate qi and promote blood circulation and remove blood stasis. The effects of pathological morphology and apoptosis of thoracic aorta were analyzed by dose-time-effect relationship. It was speculated that the aorta might protect large vessels by inhibiting oxidative stress and blocking "metabolic memory" pathway. The molecular target of Shenqi compound for prevention and treatment of diabetic macrovascular disease was found by using gene chip technology, and the biological effect and mechanism of the method of nourishing yin, supplementing qi and activating blood circulation to block "metabolic memory" were discussed. To provide a theoretical basis for the prevention and treatment of diabetic macroangiopathy with traditional Chinese medicine. Methods: one week after adaptive feeding, 160 GK rats were randomly divided into model group, Shenqi compound high dose group and Shenqi compound medium dose group according to the random blood glucose level from high to low. Shenqi compound low-dose group (Shen-low group), metformin group (western medicine group), 32 rats in each group, were fed with high-fat diet; Wistar group (blank group) was fed with normal diet for 12 weeks to establish diabetes "metabolic memory" model. After the model was made, the model group and blank group were given 5ml/kg.d saline, the Shengao group was given 2.88g/kg.d Shenqi compound extract, and the Shenzhong group was given 1.44g/kg.d Shenqi compound extract. Shen-low group was given 0.72g/kg.d Shenqi compound extract and western medicine group was given 0.1g/kg.d metformin for 16 weeks. During the experiment, the general situation of the rats in each group was observed at the whole level, and the changes of fasting blood glucose were monitored. The rats in each group were killed at the end of 816 weeks after gavage, and the serum SOD,MDA of thoracic aorta homogenate of CHOL, were detected. The apoptosis of thoracic aorta was detected at the tissue level, the morphology of thoracic aorta was observed by HE staining, and the effect of different intervention methods on "metabolic memory" in model rats was observed under different time windows. The gene expression profiles of thoracic aorta were detected by gene chip technique. The differentially expressed genes were screened out, and the biological significance was explained by GO annotation and Pathway analysis. Results: the effect of Shenzhong group was better than that of Shengao group and Shen-low group, and could obviously improve the general condition, blood glucose, blood lipid, oxidative stress index, pathological morphology of thoracic aorta and apoptosis of GK rats. By GO annotation and Pathway analysis, the differentially expressed genes in Shenzhong group and model group were mainly involved in macromolecular metabolic process, cell death, signal transduction, cell migration and MAPK signaling pathway, hedgehog signal transduction pathway and Notch signal pathway. Insulin signal transduction pathway: involved in Csnk1d,Lama4,Elavl1,Abi1,Anxa1, Nsg1 and other genes. Conclusion: Shenzhong group can better improve the general state of GK rats with diabetic macrovascular disease, decrease blood glucose, improve lipid metabolism disorder, alleviate oxidative stress, protect vascular endothelial injury and block "metabolic memory". Its mechanism may be by regulating macromolecular metabolic process, cell death, signal transduction, cell migration and MAPK signaling pathway, hedgehog signal transduction pathway, Notch signal pathway, insulin signal transduction pathway and Csnk1d,Lama4,Elav11,Abi1,AnxaK1,. Nsg1 and other genes can promote the repair of vascular endothelial cells in the early stage of diabetic macrovascular disease and improve the oxidative stress damage to the blood vessels. Among them, Shenqi compound may reduce oxidative stress, inhibit apoptosis of vascular endothelial cells, eliminate inflammatory reaction and block "metabolic memory" of diabetic macrovascular disease by regulating the important signal pathway of MAPK. In order to prevent and treat diabetic macrovascular disease.
【学位授予单位】：成都中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
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本文编号：2300459