【作者】 梁丽娜；

【导师】 战丽彬；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2009， 硕士

【摘要】 目的:糖尿病(diabetes mellitus,DM)是一组慢性血葡萄糖(简称血糖)水平增高为特征的代谢性疾病,是由于胰岛素分泌和(或)作用缺陷所引起的。2型糖尿病以胰岛素抵抗为其特征。糖尿病可以引起多系统的损害,导致心脏、肾脏、神经、视网膜、血管等组织器官的慢性进行性改变,糖尿病脑病(diabetic encephalopathy,DE)是糖尿病慢性并发症之一。其主要表现为轻中度的认知功能障碍、学习和记忆能力下降。研究表明,内质网应激与糖尿病的发生存在着密切的关系。内质网应激(endoplasmic reticulum stress,ERS)是细胞的一种应激反应过程,糖饥饿、钙平衡紊乱、糖基化抑制、二硫键合成减少等情况下,内质网的微环境发生改变,蛋白质折叠受到影响,导致大量未折叠或错误折叠的蛋白质堆积于内质网内,由此引起未折叠蛋白反应(unfolded protein response,UPR)。UPR可以引起分子伴侣葡萄糖调节蛋白78(glucose regulated protein 78, GRP78)的活化,使其与抑制物阻抗性酯酶1(inositol-requiring enzyme,IRE1)、RNA依赖的蛋白激酶样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)和活化的转录因子6(activating transcription factor 6,ATF6)解离,PERK与GRP78分离后低聚化和自身磷酸化并使真核起始因子2的α亚单位(eukaryotic translation initiation factor 2α,eIF2α)磷酸化,引起蛋白质翻译抑制以减少新蛋白质的合成,阻止蛋白的进一步积聚。PERK和eIF2α磷酸化标志着内质网应激的发生。糖尿病脑病属于中医消渴病的合并症,其病位在脑,涉及肝、肾、心、脾诸脏,病性多为本虚标实,阴虚燥热。脾为后天之本,气血生化之源,气血为人体生命活动的物质基础,脾虚一方面引起运化不足,水谷精微和津液不能正常输布排泄,积于体内,发为消渴;另一方面则引起气血生化不足,脑髓失养,神机失调而发病。滋补脾阴方药(ZBPYR)是战丽彬教授根据吴澄《不居集》中资成汤化裁而成。为进一步探讨ZBPYR对脾阴虚糖尿病脑病大鼠海马内质网应激的影响,我们建立脾阴虚糖尿病脑病病证结合模型,应用ZBPYR进行干预,观察模型大鼠学习记忆的行为学变化,并通过Western Blotting的方法探讨ZBPYR对内质网应激相关蛋白表达的影响,旨在从蛋白水平探讨ZBPYR的新靶点,为糖尿病脑病的治疗提供新的治疗线索。方法:1. SPF级健康成年雄性SD大鼠适应性喂养3天,随机分为空白对照组、脾阴虚组、糖尿病脑病组、脾阴虚糖尿病脑病组和脾阴虚糖尿病脑病+ZBPYR治疗组,每组4只。2.适应性喂养结束后,空白对照组和脾阴虚组仍以普通饲料喂养,糖尿病脑病组、脾阴虚糖尿病脑病组和脾阴虚糖尿病脑病+ZBPYR治疗组喂食高脂饲料,4周后大鼠按30mg/kg的标准一次性腹腔注射1%的链脲佐菌素(streptozocin,STZ)溶液,建立糖尿病模型。72h后尾静脉采血测量随机血糖,随机血糖>16.7mmol/L的大鼠确定为糖尿病模型。在STZ注射后的第4、8、12天分别检测空腹血清胰岛素水平(fasting serum insulin,FSI),进行口服葡萄糖耐量实验(oral glucose tolerance test,OGTT)和胰岛素耐量实验(insulin tolerance test,ITT)。糖尿病模型成功建立后,在此基础上通过疲劳过度加饥饱失常的复合因素造模的方法建立脾气虚证模型,共18天。之后造模大鼠灌服伤阴药,共9天,建立脾阴虚糖尿病脑病病证结合模型。脾阴虚糖尿病脑病+ZBPYR治疗组大鼠给予ZBPYR治疗,共16天。治疗结束后断头取脑。造模期间观察各组大鼠的体征变化。3.通过跳台实验和Morris水迷宫实验进行行为学测试,比较各组大鼠空间学习记忆能力的改变。4.通过Western Blotting方法检测各组大鼠海马GRP78、PERK和eIF2α蛋白表达的变化。结果:1. STZ注射后糖尿病模型大鼠随机血糖明显高于非糖尿病模型大鼠(P<0.01)。口服葡萄糖耐量实验结果表明糖尿病模型大鼠各时间点血糖均高于非糖尿病模型大鼠(P<0.05),且出现糖耐量异常;胰岛素耐量实验结果显示糖尿病模型大鼠血糖降低速率比非糖尿病模型大鼠小,说明糖尿病模型大鼠存在胰岛素抵抗现象。空腹血清胰岛素水平糖尿病模型大鼠与非糖尿病模型大鼠无明显差异。2.脾阴虚组表现出饮水量增加(P<0.01),肛温升高(P<0.05)等阴虚内热症状;行为学检测结果显示糖尿病脑病组和脾阴虚糖尿病脑病组学习记忆力降低(P<0.05);脾阴虚糖尿病脑病+ZBPYR组学习记忆能力有所改善(P<0.05)。3. Western Blotting结果显示糖尿病脑病组、脾阴虚糖尿病脑病组大鼠海马GRP78、磷酸化PERK、磷酸化eIF2α水平均明显高于空白对照组和脾阴虚组(P<0.05),脾阴虚糖尿病脑病+ZBPYR组与脾阴虚糖尿病脑病组相比GRP78、磷酸化PERK、磷酸化eIF2α水平明显降低(P<0.05)。PERK、eIF2α总蛋白水平各组间没有差异。结论:1.脾阴虚糖尿病脑病大鼠学习记忆障碍可能与内质网应激PERK信号途径被启动有关。2.脾阴虚糖尿病脑病大鼠学习记忆障碍可能与大鼠海马中GRP78蛋白水平增加、磷酸化PERK以及磷酸化eIF2α的表达增加有关。3. ZBPYR改善脾阴虚糖尿病脑病大鼠学习记忆障碍可能是通过影响内质网应激PERK信号传导实现的。4. ZBPYR改善脾阴虚糖尿病脑病大鼠学习记忆障碍可能与减弱GRP78蛋白表达、减弱磷酸化PERK以及磷酸化eIF2α的表达有关。更多还原

【Abstract】 Objective:Diabetes mellitus(DM) is one of metabolic disorders with chronic blood glucose increased.It is the result of the lack of insulin secretion and (or) the role of insulin. Type 2 diabetes mellitus is associated with insulin resistance. Diabetes mellitus can cause multi-system damages, including hearts, kidneys, nerves, retina, blood vessels and other tissues and organs .Diabetic encephalopathy(DE) is one of chronic complications of diabetes mellitus. Reflected that learning and memory decline.Some researches have shown that there is a close relationship between the endoplasmic reticulum stress(ERS) with the diabetes mellitus. Endoplasmic reticulum stress is the response of the cell when the stress is actived. The environmental changes of endoplasmic reticulum such as sugar starvation, disorders of calcium balance, inhibition of glycosylation and reduction of disulfide bond’s synthesis could affect the folding of protein, resulting in a large number of accumulation of unfolded or misfolded protein in the endoplasmic reticulum,then unfolded protein response (UPR) is induced. UPR signaling pathways elicit the molecular chaperone glucose regulated protein 78 (GRP78), and then GRP78 was separated from inositol-requiring enzyme(IRE1), protein kinase-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). It leads to the oligomerization and autophosphorylation of PERK ,and then induces the phosphorylation of eIF2α.The process could reduce the synthesis of new proteins and prevent the accumulation of proteins. The phosphorylation of PERK and eIF2αindicates that endoplasmic reticulum stress is provoked. Diabetic encephalopathy belongs to the complications of XiaoKe in the Traditional Chinese Medicine.The location of this disease is brain, referring to liver, kidney, heart and spleen. The nature of the disease is root deficiency and branch excess with yin deficiency induced dryness and heat.The spleen provides the materiel basis of the acquired postnatal, which is the source of growth and development of qi and blood.Qi and blood is the material basis of life. Spleen deficiency induces the disorder of transportion and transformation,leading to nutrient and body fluid distributing abno- mally.This is the main reason of XiaoKe.On the other side, the deficiency of qi and blood induces lack of nutrients in the brain,leading to DE. Nouri- shing spleen yin recipe(Zibu Piyin Recipe,ZBPYR), created by Professor Libin Zhan, could reinforce spleen and nourish spleen yin. To research the effects of ZBPYR on ERS in the hippocampus of spleen-yin deficiency diabetic encephalopathy rats ,we established spleen-yin deficiency diabetic encephalopathy rat models.We Observed the changes of behavior of the rat models treated by ZBPYR,and investigated the effects of ZBPYR on the proteins which are related with ERS by Western Blotting.We want to found the new target of ZBPYR in the protein level,and provide a new clue for the treatment of DE.Methods: 1. Healthy adult male Sprague Dawley( SD) rats were divided randomly into control group, spleen-yin deficiency group ,diabetic encephalopathy group, spleen-yin deficiency diabetic encephalopathy group and spleen-yin deficiency diabetic encephalopathy +ZBPYR group, four rats each group.2. After the acclimatization, the control group and spleen-yin deficiency group were feed with foundational diet. Other groups were feed with high fat diet.4 weeks later,the rats which were feed with high fat diet are injected with low dose STZ(30mg/kg) to establish diabetes mellitus models. The rats whose the radom blood glucose was more than 16.7mmol/L were identified as diabetes mellitus models after 72h. We detected the level of fasting serum insulin(FSI),oral glucose tolerance tes(tOGTT)and insulin tolerance test(ITT)after intraperitoneal injection with 1% STZ on the 4th、8th、12nd day respectively.Spleen-yin deficiency diabetic encephalopathy model rats were induced by defatigation、hungry-full diet and drenching the drug of damaged yin.ZBPYR group were given by the intervention of ZBPYR in 16 days and decapitated for collecting Hippocampus.3. Morris water maze test and step-down test were used to evaluate the behavioral changes.4. Using Western blotting method to detect protein expression of GRP78,PERK,phospho-PERK,eIF2α,phospho-eIF2αin rat hippocampus.Results:1. After injection of STZ,the blood glucose of the rats of diabetes mellitus were higher than nondiabetes rats(P<0.01) In the oral glucose tolerance test,the blood glucose of diabetes mellitus rats were higher than nondiabetes rats on every time point,and presented impaired oral glucose tolerance (P<0.01), in the insulin tolerance test, the rate of decline in blood glucose of diabetes rats were more slowly than nondiabetes rats,indicating impaired insulin tolerance.Fasting serum insulin levels were similar between diabetes rats and non-diabetes rats(P>0.05).2. The spleen-yin deficiency group presented endogenous heat symptomes caused by yin-deficiency,such as the increment of water drink- ing(P<0.01),obvious elevation of the rectal temperature(P<0.05). The results of Morris water maze test and step-down test showed that the learning and memory ability of the diabetic encephalopathy group and the spleen-yin deficiency diabetic encephalopathy group were reduced signi- ficantly(P<0.05), but that of the ZBPYR treatment group were increased significantly(P<0.05).3. Western Blotting results indicated the levels of GRP78, phospho- PERK,phospho-eIF2αin hippocampus of the diabetic encephalopathy group and spleen-yin deficiency diabetic encephalopathy group were significantly increased ,and that of spleen-yin deficiency diabetic encephalopathy +ZBPYR group was signigicantly decreased.Total protein levels of PERK and eIF2αwere not changed.Conclusion:1. The impaired learning and memory ability of spleen-yin deficiency diabetic encephalopathy may relate to the initiating of the PERK signaling pathway of ERS.2. The high expressions of GRP78, phospho-PERK and phospho-eIF2αwere involved in the pathogenesis of spleen-yin deficiency diabetic ence- phalopathy 3. ZBPYR may improve the learning and memory ability of spleen-yin deficiency diabetic encephalopathy rats by affecting on the PERK signaling pathway of ERS.4. ZBPYR may improve the learning and memory ability of spleen-yin deficiency diabetic encephalopathy rats by inhibiting the expressions of GRP78,phospho-PERK and phospho-eIF2α更多还原