Calpain-2在运动改善肝脏内质网应激中的作用及相关机制研究

发布时间：2018-04-04 01:41

本文选题：肝脏内质网应激　切入点：钙激活中性蛋白酶2　出处：《上海师范大学》2017年硕士论文

【摘要】：内质网应激(endoplasmic reticulum stress,ERS)是指由于各种原因导致的细胞内质网(endoplasmic reticulum,ER)功能发生紊乱,蛋白质合成受到影响的病理、生理过程。而肝脏作为人体内重要的代谢器官,其出现ERS可诱发一系列代谢性疾病,肝脏ERS已成为诸多疾病的重要风险因素,危害到人类健康。Calpain-2是钙激活中性蛋白酶,最新的研究表明,Calpain-2在人体内的激活可能参与到某些病理活动中去,如肝脏ERS。但Calpain-2在肝脏ERS中的作用及相关机制目前尚不清楚。研究目的:本研究通过对SD大鼠进行8周高脂饮食喂养后成功建立大鼠肝脏ERS动物模型,并对高脂饮食组大鼠实施8周游泳运动干预,探究Calpain-2在运动改善肝脏ERS中的作用及相关机制,为运动作为非药物疗法治疗相关疾病提供理论依据。研究方法:30只雄性SD大鼠随机分成对照组(C组)、高脂饮食ERS模型组(HF组)、高脂饮食ERS模型运动组(HE组),每组10只。其中,C组大鼠用基础饲料(碳水化合物65%,蛋白质22%,脂肪13%)喂养,HF组和HE组大鼠均使用高脂饲料喂养(碳水化合物33%,蛋白质22%,脂肪45%),HE组大鼠在喂养高脂饲料的同时施以8周无负重游泳运动干预。8周后对三组大鼠进行取材,用比色法(enzymatic colorimetric assay)检测大鼠血清游离脂肪酸(free fatty acids,FFAs)含量;用免疫印迹法(westernblotting)检测大鼠肝脏Calpain-2、自噬相关基因7(autophagy related gene,Atg7)的蛋白表达变化;用实时荧光定量PCR(RT-qPCR)检测大鼠肝脏Calpain-2、Atg7、CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer binding protein homologus protein,Chop)、葡萄糖调节蛋白78(glucose regulated protein78,Grp78)的基因表达水平。研究结果:(1)与C组相比,HF组大鼠肝脏组织中Grp78、Chop、EIF2αmRNA显著升高(p0.01),说明大鼠肝脏ERS模型建立成功。(2)与C组相比,HF组大鼠血浆FFAs含量显著增加,肝脏组织中Calpain-2mRNA及蛋白表达水平显著增加,Atg7 mRNA及蛋白表达水平显著降低(p0.01),说明高脂饮食可通过上调肝脏中Calpain-2的表达,抑制肝脏中Atg7的表达水平进而诱导ERS的标志物Grp78、Chop及EIF2α的出现。(3)与HF组相比,HE组大鼠血清FFAs含量显著下降,肝脏组织中Calpain-2mRNA及蛋白表达水平显著降低,Atg7 mRNA及蛋白表达水平显著升高(p0.01),继而使得ERS标志物Grp78、Chop及EIF2αmRNA表达水平下降,说明游泳运动可有效缓解肝脏ERS。结论:(1)8周高脂饮食成功建立大鼠肝脏ERS模型。(2)运动可有效缓解肝脏ERS。Calpain-2在运动缓解肝脏ERS中发挥重要作用,其可能机制是:运动降低血清中FFAs含量,进而下调肝脏组织中Calpain-2的表达水平,恢复肝脏中Atg7的表达,Atg7表达上调可使ERS标志物Grp78、Chop及EIF2α的mRNA表达水平降低,说明肝脏ERS得到有效缓解。
[Abstract]:Endoplasmic reticulum stress (ERS) is a pathological and physiological process in which the endoplasmic reticulum (ER) function is disturbed and the protein synthesis is affected.As an important metabolic organ in human body, liver ERS can induce a series of metabolic diseases. Liver ERS has become an important risk factor for many diseases. Calpain-2 is a calcium activated neutral protease, which is harmful to human health.Recent studies suggest that the activation of Calpain-2 in the human body may be involved in certain pathological activities, such as liver ERSs.However, the role of Calpain-2 in liver ERS and its related mechanisms are unclear.Objective: to establish a rat liver ERS model after feeding SD rats with high fat diet for 8 weeks, and to interfere with swimming exercise for 8 weeks in high fat diet group.To explore the role of Calpain-2 in the improvement of liver ERS by exercise and its related mechanism, and to provide theoretical basis for the treatment of related diseases by exercise as a non-drug therapy.Methods Thirty male Sprague-Dawley rats were randomly divided into three groups: control group (n = 10), high-fat diet ERS model group (n = 10), high-fat diet ERS model group (n = 10) and high-fat diet ERS model exercise group (n = 10).The rats in group C were fed with basic diet (carbohydrate 65, protein 22, fat 13), HF group and HE group were fed with high fat diet (carbohydrate 33, protein 22, fat 4530%) while feeding high fat diet.The rats in the three groups were selected after 8 weeks of weightless swimming intervention.The content of free fatty acid free fatty acids#en0# in serum was detected by colorimetric method, and the protein expression of Calpain-2 and 7(autophagy related gene Atg7 in rat liver was detected by Western blotting method.The expression of CCAAT-2Ag7 / enhancer binding protein homologus protein Chop#en0# and 78(glucose regulated protein 78 in rat liver were detected by real-time fluorescence quantitative PCR- RT-qPCR.The results showed that the expression of CCAAT-2CCAAT-7 / enhancer protein homologus protein and 78(glucose regulated protein 78-Grp78 were detected by real-time fluorescence quantitative PCR.Results compared with group C, the level of plasma FFAs in HF group was significantly higher than that in group C (P < 0.01).The expression of Calpain-2mRNA and protein in liver tissue increased significantly, and the expression of Atg7 mRNA and protein decreased significantly, indicating that high fat diet can up-regulate the expression of Calpain-2 in liver.Inhibiting the expression of Atg7 in liver and inducing the appearance of ERS markers Grp78 Chop and EIF2 伪.) compared with HF group, the serum FFAs content in HE group was significantly lower than that in HF group.The expression of Calpain-2mRNA and protein in liver tissue decreased significantly, and the expression of Atg7 mRNA and protein increased significantly, and the expression level of ERS marker Grp78 Chop and EIF2 伪 mRNA decreased, which indicated that swimming exercise could effectively alleviate liver ERSs.Conclusion ERS model of rat liver was successfully established by high-fat diet for 8 weeks. Conclusion exercise can effectively alleviate the role of liver ERS.Calpain-2 in relieving liver ERS. The possible mechanism is that exercise can reduce the content of FFAs in serum.Furthermore, down-regulation of Calpain-2 expression and up-regulation of Atg7 expression in liver could decrease the mRNA expression of ERS marker Grp78 Chop and EIF2 伪, which indicated that liver ERS was effectively alleviated.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R455

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