【作者】 刘馨；

【导师】 刘学政；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2013， 博士

【摘要】 目的：目前临床上治疗糖尿病的药物主要有胰岛素及一些口服降糖。这些药物在治疗糖尿病的同时往往也带来了许多不良反应。因此，寻找高效低毒的抗糖尿病及其并发症的药物目前仍然是医药工作者面临的重大课题。本研究旨在观察五味子油对2型糖尿病大鼠相关血清学生化指标的影响，明确其在2型糖尿病治疗中的保护作用。并进一步分析其对脂肪组织中多种脂肪细胞因子及炎症因子表达以及对胰腺组织凋亡的影响，以阐明五味子油抗2型糖尿病的潜在机制。材料与方法：第一部分：五味子油对2型糖尿病大鼠生化指标的影响。首先采用高脂饲料喂养SD大鼠4周，然后按30mg/kg(0.24ml/100g)的剂量一次性腹腔注射链脲佐菌素(Streptozotocin, STZ)溶液的方法来制备2型糖尿病大鼠模型。将模型大鼠随机分为模型组、低剂量五味子油组、高剂量五味子油组及罗格列酮阳性对照组，另设空白对照组及五味子油对照组。每组8只，分别按照分组给予不同处理因素：正常对照组继续以普通饲料喂养；五味子油对照组在进行普通饲料喂养的同时，每天以五味子油1.0mg/kg经灌胃给药；模型组继续喂以高脂饲料；五味子油低剂量组在喂以高脂饲料的同时，每天以五味子油0.5mg/kg经灌胃给药；五味子油高剂量组在喂以高脂饲料的同时，每天以五味子油1.0mg/kg经灌胃给药；罗格列酮阳性对照组在喂以高脂饲料的同时，每天以罗格列酮30mg/kg经灌胃给药。各组SD大鼠经处理6周后，每组大鼠随机抽取6只用于采集数据，各组大鼠禁食不禁水12h，用血糖仪检测其空腹血糖；采用乙醚麻醉，颈动脉取血5mL，离心收集血清，检测血脂及胰岛素水平，并计算胰岛素敏感指数。第二部分：五味子油对模型动物脂肪组织中与胰岛素抵抗相关的各种炎症因子及脂肪细胞因子mRNA及蛋白表达的影响。该部分动物饲养、建立动物模型、分组及处理因素与第一部分相同。于用药处理6周后麻醉取腹膜脂肪组织，分别利用RT-PCR及Western blot方法检测胰岛素抵抗相关的炎症因子：白细胞介素6(Interleukin-6, IL-6)、核因子κB(Nuclear factor-κB, NF-κB)、白细胞介素1β(Interleukin-1β,IL-1β)、C反应蛋白(C-reactive protein, CRP)、肿瘤坏死因子α(Tumor necrosis factor-α, TNF-α)及单核细胞趋化蛋白-1(Monocytechemoattranctant protein-1, MCP-1)和脂肪细胞因子：葡萄糖转运体4(Glucosetransporter-4, GLUT-4)、脂联素(Adiponectin)及视黄醇结合蛋白-4(Retinol bindingprotein-4, RBP-4)在转录和翻译水平的表达；第三部分：五味子油对模型动物胰腺组织形态学及凋亡相关蛋白Bcl-2、Bax和cleaved Caspase-3蛋白表达的影响。该部分动物饲养、建立动物模型、分组及处理因素等与第一部分相同。于用药处理6周后麻醉摘取SD大鼠的胰腺组织，用HE染色观察各组大鼠胰腺组织的形态学改变；采用Western blot检测各组大鼠胰腺组织的中凋亡相关蛋白Bcl-2、Bax和cleaved Caspase-3蛋白的表达水平。结果：1五味子油对2型糖尿病大鼠生化指标的影响1.1五味子油对2型糖尿病大鼠一般状况的影响空白对照组大鼠精神状态良好，皮毛有光泽，反应灵敏，体重维持稳定上升，未见死亡。五味子油对照组大鼠与对照组大鼠情况基本类似。与空白对照组大鼠相比，模型组大鼠饮食和饮水均增加，尿多并且消瘦，精神萎靡不振，反应迟钝，皮毛无光泽。五味子油低剂量及高剂量组，经五味子油治疗6周后，糖尿病大鼠的上述症状均有所好转，且高剂量组的治疗效果显著优于低剂量组。高剂量五味子油的效应与阳性对照药物罗格列酮类似。1.2五味子油对2型糖尿病大鼠空腹血糖的影响与空白对照组相比，糖尿病模型组大鼠空腹血糖(fasting blood glucose, FBG)水平明显升高(P<0.01)，五味子油对照组大鼠的FBG表达水平相当，未见明显差异。五味子油低剂量及高剂量组，FBG均明显低于模型组(低剂量组P<0.05；高剂量组P<0.01)，而五味子油高剂量组对FBG的影响与罗格列酮类似，血糖几乎恢复正常值。1.3五味子油对2型糖尿病大鼠血脂水平的影响与空白对照组大鼠相比，模型组大鼠血清中总胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)TG、低密度脂蛋白胆固醇（Low density lipoproteincholesterol, LDL-C）水平均显著升高(P<0.01)，而高密度脂蛋白胆固醇（Highdensity lipoprotein cholesterol, HDL-C）水平显著降低(P<0.01)；对于五味子油对照组与空白对照组相比血脂水平无明显差异(P>0.05)，与模型组相比，五味子油低剂量和高剂量组TC、TG、LDL-C水平均显著降低，(P<0.05，P<0.01)，而HDL-C水平则显著升高(P<0.05，P<0.01)，且高剂量组对改善脂类代谢异常作用显著优于低剂量组。五味子油高剂量组对血脂水平的影响与阳性对照药罗格列酮类似。1.4五味子油对2型糖尿病大鼠空腹胰岛素水平（fasting plasma insulin level,FINS）和胰岛素抵抗指数（insulin resistance index, HOMA-IR）的影响与空白对照组相比，五味子油对照组FINS和HOMA-IR均无显著差异(P>0.05)，而糖尿病模型组FINS和HOMA-IR水平显著升高(P<0.01)。在用药6周后，与模型组相比，五味子油低剂量、高剂量组均能降低FINS和HOMA-IR水平(P<0.05，P<0.01)，且高剂量组改善FINS和HOMA-IR作用显著优于低剂量组。五味子油高剂量组和罗格列酮组对FINS和HOMA-IR水平的影响类似。2五味子油对模型动物脂肪组织中与胰岛素抵抗相关的炎症因子及脂肪细胞因子mRNA及蛋白表达的影响利用RT-PCR检测各组大鼠脂肪组织中脂肪因子(GLUT-4、Adiponectin及RBP-4)和炎症因子(IL-6，CRP，MCP-1，IL1β，TNF-α、NF-κB)mRNA的表达。结果显示，与空白对照组相比，模型组脂肪因子GLUT-4、Adiponectin的表达水平明显降低(P<0.01)，而脂肪因子RBP-4的表达显著升高(P<0.01)，各种炎症因子IL-6，CRP，MCP-1，IL1β，TNF-α、NF-κB的表达均明显升高(P<0.01)。五味子油能显著逆转上述蛋白表达的改变，且高剂量组优于低剂量组。五味子油高剂量可达到药性对照药罗格列酮的水平。五味子油本身不影响对上述因子表达。利用免疫印迹法检测各组大鼠脂肪组织中脂肪因子(GLUT-4、Adiponectin及RBP-4)和炎症因子(IL-6，CRP，MCP-1，IL1β，TNF-α、NF-κB)翻译水平的表达。结果显示，与空白对照组相比，模型组脂肪因子GLUT-4、Adiponectin的表达水平明显降低(P<0.01)，而脂肪因子RBP-4的表达显著升高(P<0.01)，对于脂肪组织中炎症因子IL-6，CRP，MCP-1，IL1β，TNF-α、NF-κB的表达均明显升高(P<0.01)。五味子油能显著逆转上述蛋白表达的改变，且高剂量组优于低剂量组。五味子油高剂量可达到药性对照药罗格列酮的水平。五味子油本身不影响对上述因子表达。3五味子油对模型动物胰腺组织形态学及凋亡相关蛋白Bcl-2、Bax和cleavedCaspase-3蛋白表达的影响经HE染色，镜下观察各组SD大鼠胰腺组织及胰岛细胞的形态，空白对照组及五味子油对照组大鼠胰岛形状规则，呈圆形或椭圆形团索状，边界清晰，胰岛内β细胞分布均匀，排列紧密，胞浆丰富。糖尿病模型组大鼠胰岛体积变小，明显萎缩，结构不清、边缘不整。胰岛内胰岛β细胞细胞数明显减少，排列稀疏，部分细胞肿胀、坏死，有空泡变性发生。经五味子油治疗6周后，与模型组相比，胰岛细胞形态更趋于完整，胰岛边界渐清晰，β细胞数目增加，细胞形态较好，无明显的肿胀及坏死，高剂量组对胰腺组织的保护作用可达到阳性对照药罗格列酮的水平。经蛋白质免疫印迹检测各组大鼠脂肪组织中Bcl-2、Bax和cleaved Caspase-3蛋白的表达水平。结果显示，与空白对照组相比，模型组的cleaved Caspase-3的表达水平明显升高(P<0.01)，Bcl-2的表达水平显著降低，而Bax表达情况未见明显改变，五味子油对照组中Bcl-2、Bax和cleaved Caspase-3蛋白的表达水平均无明显改变。经五味子油治疗6周后，与模型组相比，五味子油低剂量和高剂量组糖尿病大鼠胰腺组织cleaved Caspase-3的表达均显著降低，而Bcl-2的表达水平均显著升高。且高剂量五味子油组的治疗效果显著优于低剂量组。高剂量五味子油的效果与阳性对照药物罗格列酮相似。结论：通过五味子油对2型糖尿病大鼠模型进行干预，并以罗格列酮作为阳性对照药物。本研究得出如下实验结论：1.五味子油对高脂饮食饲养加小剂量STZ注射诱导的2型糖尿病大鼠血糖、血脂及胰岛素的抵抗状态都有显著的改善作用。2.五味子油治疗纠正脂肪组织中胰岛素抵抗相关的脂肪因子及炎症因子的表达，进而改善胰岛素抵抗。证实五味子油具有显著的抗炎活性，该活性可能是其发挥抗糖尿病作用的重要机制。3.五味子油还可以保护2型糖尿病模型鼠受损的胰腺组织，抑制胰腺细胞凋亡。表现为：促进抑凋亡基因Bcl-2的表达；抑制凋亡执行蛋白cleaved-caspase-3的表达。更多还原

【Abstract】 Purpose：Present therapy for diabetes mellitus (DM) comprehend insulin and someoral hypoglycemic medications including sulphonylurea derivatives, α glucosidaseinhibitors, biguanides as well as thiazolidinediones. Although efficient, theses drugsgenerally have many undesirable adverse reactions such as constipation, obesity, drymouth, valvular heart disease and high blood pressure, etc, and some agents like thefirst-generation and second-generation sulphonylurea derivatives even destroy β cellof Langerhans islands when producing glycemic efffect, all of these side effectsgreatly limit their utilization in the treatment of DM. To date, traditional herbal agentshave received increased attention due to their well-known limited adverse effects.Therefore current work was carried out to examine the beneficial effects ofSchizandrae Fructus oil (SFO), an oily extract of Schisandra chinensis (Turcz.) Baill.,on the serological indicators of rats with type2diabetes, and furtherly, explore itspotential mechanism on two major grounds, i.e., insulin resistance and pancreatictissue apoptosis.Materials and Methods: Part I: Effects of Schizandrae Fructus oil on theserological indicators of rats with type2diabetes. Through high-fat diet plus low-dosetail vein injection of streptozotocin (STZ), type2diabetic rats were developed andwere divided into the following groups: model group, low-dose SFO group, high-doseSFO group and rosiglitazone group. Additionally, control group and SFO controlgroup, in which high dose of SFO was applied on normal rat were establishedaccordingly.8rats were included in each group and treated according to the protocolas belows: control group received a normal chow diet; SFO control group were treatedwith SFO when fed with a normal chow diet; model group continued on its high-fatdiet; low-dose SFO group and high-dose SFO group were treated with0.5and1.0mg/kg SFO, respectively; and rosiglitazone group recived30mg/kg rosiglitazone.SFO and rosiglitazone were both administered via epi-gastric route using a feeding needle. All the treatment was given daily for6weeks and subsequently,6rats pergroup were sampled and fasted up to12h for the examination of diverse serologicalindicators, including fasting blood glucose (FBG), lipidemic and insulinemic level.Furthermore, index of insulin resistance was estimated and calculated by thehomeostasis model assessment (HOMA). Part II: Effects of Schizandrae Fructus oilon the expresion of insulin resistance related adipokines and inflammatory markers inthe adipose tissue. Procedures including rat feeding, model developing, grouping andtreatment protocol were all in agreement with Part I. After6week treatment,6ratsper group were sampled and peritoneal adipose tissue were taken and subsequentlyreverse transcript polymerase chain reaction (RT-PCR) and westen blotting were usedto examine the expression of diverse insulin resistance related adipokines andinflammatory markers at the transcription and translation level, respectively. Thosesadipokines include glucose transporter-4(GLUT-4), adiponectin and retinal bindingprotein-4(RBP-4), and inflammatory makers are interleukin-6(IL-6), nuclear factorκB (NF-κB), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), C-reactiveprotein (CRP) and monocyte chemoattanctant protein-1(MCP-1). Part III: Effects ofSchizandrae Fructus oil on the pancreatic tissue morphology and its apoptosis relatedprotein expression, including cleaved caspase-3, Bcl-2and Bax. Procedures includingrat feeding, model developing, grouping and treatment protocol were all in agreementwith Part I. After6week treatment,6rats per group were sampled and pancreatictissue was removed for the subsequent test of pancreatic mophorlogy. Meanwhile,Gene expression of cleaved caspase-3, Bcl-2and Bax was also carried out by usingwestern blotting.Results:1Effects of Schizandrae Fructus oil on the serological indicators of rats withtype2diabetes.1.1Effects of Schizandrae Fructus oil on the general status of type2diabetic rats.Rats in control group were in great form with silky coats, quick reflex and bodyweight steadily creeping up. Rats in Schizandrae Fructus oil control group shared similar status with those in control group. Compared with control group, model grouprats exhibited such classical symptoms of DM as polyuria, polydipsia, polyphagia andweight loss. Moreover, these rats were slouchy, slow on the draw and had dim anddark fur. Compared with control group, the performance of rats in low-dose orhigh-dose Schizandrae Fructus oil was greatly improved, with high-dose group muchbetter than low-dose. The effect of high-dose Schizandrae Fructus oil treatment wascomparable to rosigilitazone treatment.1.2Effects of Schizandrae Fructus oil on fasting blood glucose of type2diabetic rats.Compared with control group, model group had markedly elevated fasting bloodglucose, which was significantly decreased by Schizandrae Fructus oil dosedependently. The fasting blood glucose level in high-dose Schizandrae Fructus oilgroup was similar to rosigilitazone group, almost declined to the normal level.1.3Effects of Schizandrae Fructus oil on lipidemic level of type2diabetic rats.Compared with control group, model group had markedly elevated levels of totalcholesterol, triglyceride, low density lipoprotein cholesterol as well as decreased levelof high density lipoprotein cholesterol, which were significantly reversed bySchizandrae Fructus oil dose dependently. The fasting lipidemic level in high-doseSchizandrae Fructus oil group was comparable to rosigilitazone group.1.4Effects of Schizandrae Fructus oil on FINS and HOMA-IR of type2diabetic rats.Compared with control group, while no significant changes of FINS and HOMA-IRwere observed in Schizandrae Fructus oil group, model group exhibited markablelyelevated FINS and HOMA-IR. These abnormal changes were significantlyameliorated, to varying degrees, by application of low or high dose of SchizandraeFructus oil or resiglitazone. The effects of high-dose Schizandrae Fructus oil werecomparable to resiglitazone and better than low-dose Schizandrae Fructus oil.2Effects of Schizandrae Fructus oil on the expression of insulin resistancerelated adipokines and inflammatory markers in the peritoneal adipose tissue.Reverse transcriptional polymerase chain reaction (RT-PCR) was introduced toexamine the influence of Schizandrae Fructus oil on the expression of variousadipokines and inflammatory markers in the gene (mRNA) transcription level. The results revealed that peritoneal adipose tissue from diabetic model group rats hadhigher mRNA expression of all inflammatory markers including IL-6, CRP, MCP-1,IL1β, TNF-α and NF-κB. Meanwhile, the expression Adipoctokines including andGLUT-4and Adiponectin were also decreased in mRNA level in the peritonealadipose tissue of diabetic model rats, whose expression in RBP-4were, however,lower than that from the control group. These abnormal changes were significantlyreversed, to varying degrees, by application of resiglitazone or low or high dose ofSchizandrae Fructus oil, which itself had no any influence on various geneexpressions. The effects of high-dose Schizandrae Fructus oil on the mRNAexpression were comparable to resiglitazone, oral hypoglycemic agent belonging tothiazolidinediones, and better than low-dose Schizandrae Fructus oil.Subsequently, Western blot was employed to examine the influence of SchizandraeFructus oil on the expression of various adipokines and inflammatory markers in theprotein translation level. The results demonstrate that all the alteration of theadipokines and inflammatory markers in protein (translation) were reconcilable withtheir changes in mRNA (transcription) level. That is, peritoneal adipose tissue fromdiabetic model group rats had higher protein expression of all inflammatory markersincluding IL-6, CRP, MCP-1, IL1β, TNF-α and NF-κB. Meanwhile, the expressionsof adipoctokines including GLUT-4and Adiponectin were also decreased in proteinlevel in the peritoneal adipose tissue of diabetic model rats, whose expression inRBP-4was, however, lower than that from the control group. These abnormalchanges were significantly reversed, to varying degrees, by application ofresiglitazone or low or high dose of Schizandrae Fructus oil, which itself had no anyinfluence on various gene expressions. The effects of high-dose Schizandrae Fructusoil on the protein expression were comparable to resiglitazone, oral hypoglycemicagent belonging to thiazolidinediones, and better than low-dose Schizandrae Fructusoil. 3Effects of Schizandrae Fructus oil on the pancreatic tissue morphology and itsapoptosis related protein expression.Application of Schizandrae Fructus oil could take a series of positive influence on thepathological changes including restore shrinked pancrease tissue and decreasednumber of β cells. Besides, Schizandrae Fructus oil greatly protected pancrease fromapoptotic damage, as demonstrated by diverse apoptosis relavant protein expression.Although Bax protein expression was unchanged, model group rats had higher proteinexpression of cleaved caspase-3and lower Bcl-2than control group. Both weregreatly corrected by application of low or high dose of Schizandrae Fructus oil orresiglitazone. The effects of high-dose Schizandrae Fructus oil were comparable toresiglitazone and better than low-dose Schizandrae Fructus oil. Schizandrae Fructusoil itself had no effect on caspase-3and lower Bcl-2expression.Conclusion:1. Schizandrae Fructus oil treatment could improve insulin resisrance, attenuatehyperglycemia and hyperlipidemia, and hyperinsulinemia in fat-fed stepzotocininduced type2diabetic rat model.2. Schizandrae Fructus oil treatment could produce beneficical effects on diabetes bycorrecting insulin resisrance associated gene expression.3. Schizandrae Fructus oil treatment could preserve pancreatic morphology, decreasepancreatic apoptosis by reducing cleaved caspase-3expressin and increasing Bcl-2.更多还原