【作者】 宋玉萍；

【导师】 刘志民；

【作者基本信息】 第二军医大学 ， 内科学（专业学位）， 2013， 博士

【摘要】 乙酰化酶（SIRT）是NAD依赖的组蛋白去乙酰化酶，属于沉默信息调节因子家族，参与能量生成，脂肪酸代谢，细胞周期和应激反应的调节，以及密切相关，细胞的生命。在哺乳动物中sirtuins有7个（SIRT1-7），其功能主要取决于其在细胞内的定位。SIRT3在哺乳动物线粒体中是一种重要的去乙酰化酶，高表达于骨骼肌中。线粒体S1RT3在线粒体基础生物学，包括代谢、能量生成、细胞内信号控制和细胞凋亡等方面功能发挥了重要的作用。活性氧自由基（ROS）主要在线粒体产生，SIRT3能减少细胞内ROS水平，更关键的是，SIRT3很大程度地增强超氧化物歧化酶-2（SOD₂）的活性，降低了细胞内活性氧自由基水平，增强细胞对氧化应激的耐受性。机体在遭受各种有害刺激或病理状态时体内高活性分子时会产生氧化应激，假如活性氧自由基或者是活性氮自由基产生过多，那么抗氧化酶的活性将受到影响，氧化的程度如果超出机体对氧化物的清除能力，抗氧化系统和氧化系统就会失衡，会导致组织损伤。以往动物实验及临床试验均显示氧化应激在2型糖尿病及其并发症中起了重要作用。活性氧自由基参与胰岛素抵抗（IR）的整个过程。糖脂代谢紊乱和多种心脑血管疾病的共同的病理生理学基础是胰岛素抵抗，这与高血糖、高血压以及血脂异常等心脑血管疾病有着密切的关系，同时也是2型糖尿病和单纯性肥胖症的重要诱发因素。骨骼肌是机体处理葡萄糖最为重要的组织，是外周胰岛素作用的主要靶组织。近几年文献报道，骨骼肌胰岛素抵抗形成的重要机制之一则是线粒体功能障碍。津力达颗粒主要由人参、、地黄、苍术（炒）、制何首乌、山茱萸、黄精、苦参、麦冬茯苓、佩兰、知母、黄连、淫羊藿（炙）丹参、荔枝核、葛根、地骨皮等组成，具有益气养阴，健脾运津的作用。有临床研究表明津力达颗粒对治疗糖尿病及其并发症具有很好的作用，还能增强2型糖尿病患者的胰岛素敏感性，保护胰岛β细胞功能，但其药理学机制尚有待进一步明确。二甲双胍是目前治疗2型糖尿病的一线药物，二甲双胍不仅是理想的降糖药，尤其是肝脏和骨骼肌：可以抑制肝脏糖异生，而且还能增强胰岛素的敏感性，对防治胰岛素抵抗综合征（IRs）有独特功效，能显著降低空腹血浆胰岛素水平，提高周围组织对胰岛素的敏感性（比如：骨骼肌和脂肪组织），从而提高外周葡萄糖的利用率。先前的研究表明，二甲双胍能改善周围组织胰岛素抵抗并且能降低由长期高脂饮食诱导的胰岛素抵抗大鼠血清和脑的氧化应激水平。二甲双胍不仅影响线粒体的生物合成，而且还能影响线粒体氧化机制的功能。但其具体机制仍需要进一步的研究。综上所述，探讨SIRT3在糖尿病大鼠骨骼肌组织中的作用及津力达颗粒和二甲双胍改善骨骼肌胰岛素抵抗的机制，有助于阐明胰岛素抵抗机理，并为临床上寻找新的治疗靶点及拓展津力达颗粒及二甲双胍药物的应用提供坚实的理论依据。本研究拟通过观察津力达颗粒及二甲双胍对糖尿病大鼠骨骼肌组织SIRT3及氧化应激水平的影响，并通过建立L6骨骼肌胰岛素抵抗细胞模型，进一步研究SIRT3在二甲双胍改善骨骼肌胰岛素抵抗中的作用，探讨其可能的作用信号通路。实验一共分为三个部分：第一部分，建立2型糖尿病大鼠模型，给予津力达颗粒及二甲双胍干预，观察津力达颗粒及二甲双胍对其氧化应激指标的影响及对骨骼肌的保护作用；第二部分，观察津力达颗粒及二甲双胍对2型糖尿病大鼠骨骼肌组织SIRT3表达及氧化应激水平的影响；第三部分，建立L6骨骼肌胰岛素抵抗细胞模型，观察二甲双胍在SIRT3过表达和敲除时对葡萄糖吸收的影响，探讨其可能的信号通路。本研究发现：在高糖高脂饮食加腹腔一次性注射小剂量链脲佐菌素（STZ）制备的2型糖尿病大鼠模型骨骼肌中SIRT3表达减少；津力达颗粒及二甲双胍对糖尿病大鼠骨骼肌组织具有一定的保护作用，且能改善糖尿病大鼠的氧化应激水平；能使糖尿病大鼠骨骼肌组织SIRT3表达增加。在体外，过表达SIRT3能改善L6骨骼肌细胞及L6胰岛素抵抗细胞的胰岛素抵抗，骨骼肌细胞葡萄糖消耗增加，ROS产生减少；敲除SIRT3时，骨骼肌细胞葡萄糖消耗减少，ROS产生增加；二甲双胍上调L6及L6-IR骨骼肌细胞SIRT3表达，而且，过表达SIRT3时，增强二甲双胍促进骨骼肌细胞对葡萄糖的消耗及ROS产生减少的作用；敲除时，则减弱这种作用。说明二甲双胍改善骨骼肌胰岛素抵抗的药理机制可能部分是通过上调SIRT3的表达以及同时使NF-κB、JNK1表达下调，SOD2表达上调，从而减弱氧化应激，最终改善胰岛素抵抗。第一部分津力达颗粒及二甲双胍对糖尿病大鼠氧化应激的影响及对骨骼肌组织的保护作用目的：观察津力达颗粒及二甲双胍对2型糖尿病大鼠氧化应激相关指标的影响及其对骨骼肌组织超微结构的保护作用。方法：利用随机数字表将雄性Sprague-Dawley（SD）大鼠分为：糖尿病模型组70只和正常对照组（NC组）10只。NC组给予普通饲料，糖尿病模型组给予高脂饲料喂养，4周后，一次性小剂量左侧腹腔注射链脲佐菌素（STZ）（30mg·kg﹣1）制备2型糖尿病大鼠模型， HOME-IR确认2型糖尿病大鼠模型。模型成功后，再次将模型成功大鼠随机分为：津力达低剂量组(JL组，0.75g·kg^-1·d^-1)、津力达中剂量组(JM组，1.5g·kg^-1·d^-1)、津力达高剂量组(JH组，3.0g·kg^-1·d^-1)、α-硫辛酸组(ALA组，50mg·kg^-1·d^-1)、胰岛素组(INS组，1.5U·kg^-1·d^-1)和二甲双胍治疗组(MT组，50mg·kg^-1·d^-1)和模型对照组（DM）。8周后，测定大鼠FBG、FINS计算HOME-IR，全自动生化分析仪测定其血脂水平，ELISA检测大鼠血浆SOD、ROS、GSH-Px的活性和MDA的含量。透射电镜观察2型糖尿病大鼠骨骼肌组织超微结构的病理变化。结果：与NC组大鼠比较，糖尿病模型组（DM）大鼠FBG、FINS、TC、TG、LDL、MDA、ROS升高（p<0.05），而血清GSH、SOD、HDL及ISI降低（p<0.05）；与DM组相比，JH及MT组的大鼠FBG、FINS、TC、TG、LDL、MDA、ROS降低（p<0.05），而血清GSH、SOD、HDL及ISI升高（p<0.05）。透射电镜，NC：大鼠骨骼肌组织横纹较清晰，部分肌丝断裂，部分线粒体损伤，胞浆内有空泡。DM：肌组织横纹较清晰，部分肌丝断裂，大部分线粒体破坏，胞浆内有空泡。JM：肌组织横纹不清晰，部分线粒体损伤，胞浆内有空泡。JH：部分线粒体损伤，胞浆内有空泡，可见神经肌接头，终板较完整。ALA：部分线粒体损伤，胞浆内有空泡，可见神经肌接头，终板部分损伤。INS：肌组织横纹较清晰，部分肌丝断裂，部分线粒体损伤，胞浆内有空泡。MT:肌组织横纹较清晰，部分肌丝断裂，部分线粒体损伤，胞浆内有空泡。结论：津力达及二甲双胍明显改善2型糖尿病大鼠血糖、胰岛素、胰岛素敏感指数、血脂及氧化应激等指标的水平，且对糖尿病大鼠骨骼肌组织超微结构的损伤具有一定的保护作用。第二部分津力达颗粒及二甲双胍对糖尿病大鼠骨骼肌SIRT3及氧化应激水平的影响目的：观察津力达颗粒及二甲双胍对2型糖尿病大鼠骨骼肌SIRT3及氧化应激水平的影响，探讨津力达颗粒及二甲双胍改善糖尿病大鼠骨骼肌胰岛素抵抗的可能机制。方法：采用随机数字表法将SD大鼠分为：糖尿病模型组30只和正常对照组（NC组）10只。NC组予以普通饲料，糖尿病模型组喂以高脂饲料，4周后，一次性小剂量左侧腹腔注射链脲佐菌素（STZ）（30mg·kg﹣1）制备2型糖尿病大鼠的模型,检测空腹血糖及空腹胰岛素并计算HOME-IR确立2型糖尿病大鼠模型。模型成功大鼠，再将其随机分成津力达颗粒高剂量组(JH组，3.0g·kg^-1·d^-1)、二甲双胍治疗组(MT组，50mg·kg^-1·d^-1和模型对照组（DM）。8周后，ELISA方法检测各组大鼠骨骼肌组织超氧化物歧化酶（SOD）、活性氧（ROS）和谷胱甘肽过氧化物酶（GSH-Px）的活性及丙二醛（MDA）的含量。应用实时定量PCR（Real-time PCR）及蛋白印迹（Western Blotting）的方法检测糖尿病大鼠及经津力达颗粒和二甲双胍干预后大鼠骨骼肌中SIRT3mRNA及蛋白质的表达水平变化。结果：与空白对照组相比，模型对照组的大鼠骨骼肌SIRT3mRNA（p<0.01）及GSH、SOD、SIRT3protein表达水平降低（p<0.05），MDA和ROS水平升高（p<0.05）；与模型对照组的大鼠相比，津力达及二甲双胍组大鼠骨骼肌组织GSH、SOD、SIRT3mRNA及蛋白质表达增加（均p<0.05），MDA和ROS水平降低（p<0.05）。结论：津力达颗粒及二甲双胍能有效改善糖尿病大鼠骨骼肌氧化应激状态，其机制可能是通过上调糖尿病大鼠骨骼肌SIRT3mRNA及蛋白质的表达，从而提高糖尿病大鼠的胰岛素敏感性，延缓糖尿病的病程。第三部分二甲双胍对L6胰岛素抵抗细胞SIRT3表达的影响及机制探讨目的：研究SIRT3在L6大鼠骨骼肌细胞胰岛素抵抗中的作用，探讨二甲双胍改善骨骼肌胰岛素抵抗可能的信号通路。方法：利用棕榈酸（PA）诱导造大鼠L6肌细胞胰岛素抵抗模型，在L6正常细胞及L6胰岛素抵抗细胞中过表达和干扰SIRT3基因。考马斯亮蓝染色的方法观察L6细胞肌管结构情况，葡萄糖氧化酶-过氧化酶法检测细胞上清中葡萄糖浓度，利用荧光探针DCFH-DA检测ROS水平。用Western blot检测蛋白质SIRT3、NF-κB、JNK1及SOD2表达水平的变化。结果： L6胰岛素抵抗细胞比L6正常细胞中葡萄糖吸收率降低，而ROS生成增多；SIRT3过表达后，L6细胞对葡萄糖的吸收率升高，ROS生成减少，相反，干扰时，L6细胞对葡萄糖的吸收率降低，ROS生成增多；二甲双胍能使L6细胞SIRT3和SOD2表达增加，同时NF-κBP65和JNK1表达降低，葡萄糖吸收率升高，而ROS的产生减少；当过表达SIRT3时，二甲双胍的这种作用增强，敲除时减弱。结论：SIRT3能明显提高L6胰岛素抵抗细胞的葡萄糖的吸收率,减少其ROS的生成；二甲双胍可促进SIRT3对细胞的作用，考虑二甲双胍可能通过SIRT3-ROS-NF-κB P65信号通路，改善骨骼肌氧化应激状态，从而提高骨骼肌组织的胰岛素敏感性。更多还原

【Abstract】 Sirtuins (SIRT) are NAD-dependent histone deacetylases, belonging to the silentinformation regulator factor family, involved in energy production, fatty acid metabolism,cell cycle and the regulation of the stress response, closely related with the life span ofcells.. There are7sirtuins in mammals (SIRT1-7) whose functions are postulated to belargely determined by their intracellular localization. SIRT3is a crucial regulator ofmitochondrial function, controlling global acetylation of the organelle. SIRT3is importantin mammalian mitochondrial deacetylase and highly expresses in skeletal muscle. SIRT3can reduce the level of intracellular ROS and, more importantly, the SIRT3greatlyenhanced superoxide dismutase-2, reducing cells radicals and promoting the tolerancecapacity of oxidative stress.Oxidative stress (OS) can be defined as the steady state condition where the freeradical/reactive oxygen species (ROS) flux is balanced by antioxidant defenses. Theupregulation of OS, which mainly occurs in conditions of increased flux of free radicals/ROS and/or reduced antioxidant levels, is now accepted to play a critical role in thepathogenesis of obesity, atherosclerosis, T2D and IR. Skeletal muscle is the major organinvolved in postprandial glucose uptake and metabolism and peripheral insulin sensitivity.Changes in mitochondrial mass and oxidative phosphorylation have been shown to occurin muscle of individuals with diabetes and insulin resistance.Jinlida granules consist of ginseng, sophora, ophiopogon, cornus, poria, perrin,berberine, Anemarrhena Epimedium (Sunburn) Salvia, arrowroot, litchi seed and so on.Clinical studies have shown that jinlida granules have protection on the treatment ofdiabetes and its complications, reducing the insulin resistance of type2diabetes andprotect β-cell function, but its pharmacological mechanism remains to be further clear.Metformin is currently the drug of first choice for the treatment of T2D, in which itsmechanisms are to reduce hepatic glucose output and to increase the insulin mediatedutilization of glucose. Metformin is also described as an insulin-sensitizer, leading toreduction in insulin resistance and a significant decrease in plasma fasting insulin levels,been reported to have numerous cellular effects in multiple tissues. Both experimental andclinical studies indicate that oxidative stress plays a major role in the development andcomplications of type2diabetes. Previous studies demonstrated that metformin attenuatedperipheral insulin resistance and decreased plasma and brain oxidative stress ininsulin-resistant rats caused by long-term HFD consumption. Metformin not only influences mitochondrial biogenesis but also the function of mitochondrial oxidativemachinery, but the mechanism still needs further research. Therefore, to explore the role ofSIRT3in skeletal muscle tissue diabetic rats and the mechanisms of improving insulinresistance in skeletal muscle, which elucidate the mechanism of insulin resistance, andprovide a solid theoretical basis for finding new therapeutic targets in clinical andexpanding the application of metformin.The aim of this study is to clarify the exact role for SIRT3in insulin resistance ofskeletal muscle in diabetic rats. This study is consisted of three main parts：The first part isto investigate the effect of jinlida granules and metformin on oxidative stress instreptozotocin induced diabetic rats, the second is to see the effect of jinlida granules andmetformin on SIRT3and oxidative stress of skeletal muscle in diabetic rats. Finally, theeffect of SIRT3on glucose uptake and ROS production in L6cells, further investigationunderlying molecules mechanism of insulin resistance.In this study, we found that SIRT3mRNA and protein expression is decreased in HFDand STZ induced T2DM animal model; Jinlida granules and metformin had a certainprotective effect on diabetic rats skeletal muscle, could improve the oxidative stress levelsin diabetic rats, and could increase SIRT3expression of skeletal muscle in diabetic rats. Invitro, over expression of SIRT3alleviated insulin resistance of L6and L6-IR cells,Metformin increased the expression of SIRT3and SOD2, as well as down regulation ofNF-κBP65and JNK1. When SIRT3knocked down, could reverse the decrease ofNF-κBP65and JNK1, and increase of SOD2that induced by metformin. While, overexpression of SIRT3promoted the effect of metformin on NF-κB P65, JNK1and SOD2.Metformin increased glucose uptake of L6-IR cells, Knocked down expression of SIRT3diminished the glucose uptake effect that induced by metformin, while over expression ofSIRT3increased the glucose uptake induced by metformin. Accordingly, ROS productionwas decreased by metformin, and ROS production was increased when SIRT3wasknocked down, and ROS production was decreased when SIRT3was over expressed.These results demonstrated the pharmacological effect of metformin in promoting glucoseuptake may partly through SIRT3. Part I Effects of jinlida granules and metformin onoxidative stress and skeletal muscle in HFD andstreptozotocin-induced diabetic ratsObjective: To observe the impact of jinlida granules and metformin on oxidativestress indicators and its protective effect on skeletal muscle tissue ultrastructure in HFDand streptozotocin-induced diabetic rats.Methods: Eighty male Sprague-Dawley (SD) rats were divided into diabetic modelgroup (70) and normal control group (NC)(10) by using a random number table. Diabetesrats were induced a single intraperitoneal injection of streptozotocin (STZ) at a dose of30mg kg-1body weight dissolved in citrate buffer after four weeks. Type2diabetic ratmodels were tested by HOME-IR3days after injection were included in the study andwere randomly divided into seven groups：DM control group, JL, JM, JH, ALA, INS, MTgroups. Rats’ plasma SOD, ROS, GSH-Px activity and MDA content were detected byELISA after eight weeks. The cellular ultrastructure of the skeletal muscle was observedthrough light microscope.Results: Compared with NC group, FBG, FINS, TC, TG, LDL, ROS and MDA wereincreased in DM group,(p<0.05), while serum GSH, SOD, HDL and ISI were decreased(p<0.05); Compared with DM group, in the rats of MT and JH groups, FBG, FINS, TC,TG, LDL, ROS and MDA were decreased (p<0.05), but serum GSH, SOD, HDL and ISIwere increased (p<0.05). Obvious chromosome aberrations, dilatate, widened synapticclefts as well as disappeared mitochondriales crista were also observed in the diabetic rats’skeletal muscle by TEM.Conclusion: Jinlida granules and metformin significantly improves fasting bloodglucose, fasting insulin, and insulin sensitivity index, blood lipids, and oxidative stressindex levels in type2diabetes rats, having a protective effect on skeletal muscle in diabeticrats. Part II Effect of jinlida granules and metformin onSIRT3and oxidative stress of skeletal muscle in HFDand streptozotocin-induced diabetic ratsObjective: To observe the effects of jinlida granules and metformin on oxidativestress and SIRT3of skeletal muscle in diabetic rats, explore the possible mechanism ofmetformin improves skeletal muscle insulin resistance.Methods: Rats’ skeletal muscle superoxide dismutase (SOD), reactive oxygenspecies (ROS) and glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA)content were detected by ELISA after eight weeks. Real-time quantitative PCR andWestern Blotting were used to detect the expression of SIRT3mRNA and SIRT3protein ofskeletal muscle in diabetic rats.Results: Compared with NC group, the expression of SIRT3mRNA (p<0.01) andSIRT3protein (p<0.05) of skeletal muscle were decreased in DM group rats; comparedwith DM group rats, the expression of SIRT3mRNA (p<0.01) and SIRT3protein (p<0.05)of skeletal muscle were increased in MT and JH group (p<0.05). Compared with NC group,GSH and SOD of skeletal muscle were decreased in DM group rats (p<0.05)，MDA andROS increased. Compared with DM group, MDA and ROS of skeletal muscle weredecreased in MT and JH groups (p<0.05), but GSH and SOD of skeletal muscle wereincreased (p<0.05).Conclusion: Metformin and jinlida granules are effective in improving oxidativestress in skeletal muscle in diabetic; the mechanism may be through increasing theexpression of SIRT3mRNA and protein in skeletal muscle, therefore enhancing insulinsensitivity in rats with diabetes, and delaying the development of diabetes. Part Ⅲ Impact of metformin on SIRT3in L6insulinresistance cellsObjective: To study the role of SIRT3in insulin resistance of L6skeletal muscle cells,explore possible signaling pathway metformin to improve insulin resistance in skeletalmuscle.Methods: Differentiated L6skeletal muscle cells were treated with750μM ofpalmitic acid for L6insulin resistance cell model. A plasmid with Flag tagged SIRT3wasobtained from Addgene (plasmid13814). L6cells in24-well plates were transfected with200ng of SIRT3plasmid or with empty pcDNA3.1control plasmid using Fugene HD(Roche) according to the manufacturer’s protocol. For knocking down human SIRT3,specific siRNA (5’-GCC CAA CGT CAC TCA CTA CTT-3’) targeting SIRT3as published.The transfection of siRNA into L6cells using Fugene HD according to the manufacturer’sinstructions (Roche). Glucose uptake and ROS were measured in the L6cells, L6insulinresistance cells, SIRT3-/-cells and SIRT3+/+cells. Anti-SIRT3, anti-NF-κB P65, anti-JNK1,anti-SOD2and anti-GAPDH were analyzed by Western blot.Results: Glucose uptake was significantly decreased in insulin resistant L6cells. ROSlevel in insulin resistant L6cells were significantly increased than L6cells. Overexpression of SIRT3could dramatically decreased ROS production in L6-IR cells,metformin increased the expression of SIRT3and SOD2, as well as downed regulation ofNF-κB P65and JNK1, when SIRT3knocked down, could reverse the decrease of NF-κBP65and JNK1, and increase of SOD2that induced by metformin.Conclusion: SIRT3significantly improve glucose absorption rate and reducegeneration of ROS in L6insulin resistance cells, metformin may improve cellularoxidative stress state by upregulating SIRT3expression, thereby strengthening insulinsensitivity of cells.更多还原