【作者】 郭颖；

【导师】 高毅；

【作者基本信息】 第一军医大学 ， 普通外科学， 2007， 博士

【摘要】 研究背景器官移植是目前治疗终末期器官功能衰竭的重要手段，近年来，随着临床实践的进步和新型免疫抑制剂的不断推出和广泛应用，使得移植术后器官的排斥反应得到了有效的控制，抑制物的存活率有了显著的提高，但术后的各种并发症仍威胁着患者的生命。在移植后多种并发症中，移植后糖尿病(PTDM)是主要并发症之一，大量的临床观察表明，PTDM患者发生移植物失功、感染、微血管并发症(视网膜病变、神经病变和糖尿病。肾病)和大血管并发症(心肌梗塞、脑卒中、冠心病和周围血管病变)等移植物相关的危险性增高。胰岛素抵抗(insulin resistance，IR)是包括移植后糖尿病在内的2型糖尿病、代谢综合症的病理生理学基础，目前认为胰岛素抵抗是一个慢性系统性炎症的过程，许多抗炎药物如阿司匹林抗炎治疗可以逆转胰岛素抵抗，降低血糖和心、脑血管病的发生率。胰岛素抵抗的炎症学说在临床应用的结果令人鼓舞，这个新的机制将对2型糖尿病的治疗模式产生根本性的变革。本研究将利用胰岛素抵抗的小鼠模型，观察几种免疫抑制药物姜黄素(Curcumin)、小白菊内酯(Parthenolide)及雷帕霉素(rapamycin)对胰岛素抵抗的治疗作用，并对其作用机制进行了初步探讨，为临床治疗包括移植后糖尿病在内的2型糖尿病提供依据。本文共包括三部分。第一部分：免疫抑制剂姜黄素逆转胰岛素抵抗的研究目的：姜黄素(curcumin)具有免疫调节、抗氧化、抗炎及抗肿瘤、抗动脉粥样硬化等生理和药理作用。研究发现姜黄素是良好的免疫调节剂，并有结果显示姜黄素可以抑制宿主抗移植物反应，提高受体动物的存活率，本研究部分旨在探讨姜黄素对胰岛素抵抗的治疗作用及其机制。方法：利用FAO细胞和肥胖并极度胰岛素抵抗动物模型(obob小鼠)，观察姜黄素：(1)与NF-kB的量效和时效关系；(2)对小鼠的空腹血糖、胰岛素及葡萄糖耐量曲线水平的影响，研究姜黄素对小鼠胰岛素抵抗的治疗作用；(3)对循环细胞因子IL-1β，IL-6和TNFα水平的影响，探讨姜黄素的可能作用机制；(4)免疫沉淀法分析胰岛素刺激的IRS-1、P85及AKT磷酸化水平，分析obob小鼠对胰岛素敏感性的变化。结果：(1)姜黄素抑制NF-kB的量效和时效关系。姜黄素有效剂量30μM对NF-κB的抑制作用在30-60分钟时最强；(2)姜黄素治疗后小鼠的空腹血糖水平没有明显下降，但空腹胰岛素的水平下降约30％，葡萄糖耐量曲线的水平也明显下降；(3)循环中TNFα(34.5±3.4 Vs31.3±1.1pg／ml)水平没有明显改变，而IL-1β(33.9±3.2 Vs 15.6±1.1pg／ml)和IL-6(72.1±9.8 Vs47.2±5.3 pg／ml)水平明显被抑制；(4)胰岛素刺激的IRS-1酪氨酸的磷酸化水平明显升高，而胰岛素受体的磷酸化水平没有明显改变。与IRS-1酪氨酸的磷酸化水平平行，P85的磷酸化水平也明显升高，AKT的磷酸化的水平也明显升高。结论：姜黄素能提高obob小鼠胰岛素敏感性，有治疗改善肥胖诱导的胰岛素抵抗的作用，可能是通过抑制NF-wJ3来改善胰岛素敏感性的。第二部分：免疫抑制剂小白菊内酯逆转胰岛素抵抗的研究目的：小白菊内酯(Parthenolide)是良好的免疫调节剂，通过抑制NF-kappa B通道而发挥各种生物活性作用：包括抗炎及抗肿瘤、抗动脉粥样硬化等生理和药理作用，还可以提高器官移植患者的生存率，并抑制宿主的免疫排斥反应，本研究部分旨在探讨小白菊内酯对系统性胰岛素抵抗的逆转作用及分子机制。方法：利用FAO细胞和肥胖并极度胰岛素抵抗动物模型(obob小鼠)，观察小白菊内酯：(1)与NF-kB的量效和时效关系；(2)对小鼠的空腹血糖、胰岛素及葡萄糖耐量曲线水平的影响，研究小白菊内酯对小鼠胰岛素抵抗的治疗作用：(3)对循环细胞因子IL-1β，IL-6和TNFα水平的影响，探讨小白菊内酯的可能作用机制。(4)免疫沉淀法分析胰岛素刺激的IRS-1、P85及AKT磷酸化水平，分析obob小鼠对胰岛素的敏感性。结果：(1)小白菊内酯有效剂量10-20mM在20分钟时可对NF-κB的活性发挥最大的抑制作用；(2)小白菊内酯治疗组有空腹血糖和胰岛素水平计算的HOMA指数明显下降，葡萄糖耐量曲线的水平也明显下降，部分小鼠接受了胰岛素钳夹试验，治疗组基础肝糖输出较对照组明显被抑制，整体葡萄糖的摄取和葡萄糖的输注速率在胰岛素钳夹状态较对照组分别增加了40％；(3)发现小白菊内酯能有效抑制细胞模型NF-κB活性，逆转高脂肪饮食和肥胖诱导的胰岛素抵抗，降低循环中细胞因子TNF，IL-6和MCP-1的水平。经小白菊内酯治疗的动物肝糖输出的水平明显抑制，而在高胰岛素钳夹稳态时的组织葡萄糖的利用率(Rd)明显升高；(4)循环中IL-1β，IL-6，TNFα水平在小白菊内酯治疗后被抑制约50％。除此之外，PAI-1(1.75±0.20 Vs 1.14±0.15 ng／ml)和MCP-1(1.48±0.18 Vs 0.97±0.14，p=0.03)水平也明显下降，但是Leptin和Resistin的水平没有明显改变；(4)小白菊内酯可明显抑制色氨酸S964和1148两个位点的磷酸化。结论：小白菊内酯对系统性炎症有抑制作用，并通过抑制NF-κB来得到改善胰岛素敏感性，具有治疗2型糖尿病的潜在活性。第三部分：新型免疫抑制剂雷帕霉素逆转胰岛素抵抗的研究目的：雷帕霉素(rapamycin，RAPA)是美国Home Products公司研制开发的新型免疫抑制剂，属大环内酯类，是一种作用机制不同于钙调磷酸酶抑制剂和抗代谢类药物的新型免疫抑制剂，现在已作为移植术后减少或避免肾脏毒性的基础免疫抑制剂而广泛应用于临床。本研究部分旨在利用肥胖并极度胰岛素抵抗模型小鼠(Obob小鼠)探讨雷帕霉素对胰岛素抵抗的作用及可能机制。方法利用肥胖胰岛素抵抗动物模型(obob小鼠)，观察雷帕霉素对(1)小鼠空腹血糖和胰岛素水平的影响，研究雷帕霉素对小鼠胰岛素抵抗的治疗作用；(2)对循环细胞因子IL-1β，IL-6和TNFα水平的影响，探讨雷帕霉素的可能作用机制。结果雷帕霉素治疗后(1)小鼠的空腹血糖水平明显下降，与对照组比较有统计学意义，空腹胰岛素的水平也下降，曲线下面积的比较有显著差异(8.90±0.95Vs 5.27±0073，p＜0.05) (2)循环中TNFα(31.7±3.4 Vs37.7±1.1pg／ml)和IL-1β(18.0±3.2 Vs 29.5±1.1pg／m1)水平虽也下降，但没有统计学差异，与之对应，IL-6(46.2±9.8 Vs85.6±5.3 pg／ml)的水平明显降低(p＜0.05)。结论雷帕霉素能提高obob小鼠胰岛素敏感性，有治疗改善肥胖诱导的胰岛素抵抗的作用。更多还原

【Abstract】 BackgroundOrgan transplant is an important means of treating end stage organ failure at present. In recent years, with the progress of clinical practice and extensive application of new-type immune inhibitors, rejection of transplanted organ has been effectively controlled. The survival rates have been improved greatly. But many kinds of post-operation complications are still threatening the patient’s life. Post transplant diabetes mellitus (PTDM) is one of the main complications. A large number of clinical observations indicate that the risk of transplanted organ dysfunction、infection, microvascular complication (retinopathy, neuropathy and nephropathy) and macrovascular complication (myocardial infarction, cerebral apoplexy, coronary artery disease, peripheral angiopathy) in PTDM patients are increasing. Insulin resistance (IR) is one of the common pathophysiological features of type 2 diabetes mellitus and metabolic syndrome after transplantation. Now it is considered that insulin resistance is a chronic systemic inflammation. A few anti-inflammatory treatment such as aspirin can reverse insulin resistance, and decrease blood sugar and incidence of cardiovascular and cerebrovascular disease. The results from clinical practice of IR inflammatory hypothesis are encouraging. The new mechanism will lead to dramatic fundamental change in type 2 diabetes treatment regimes. In this study, we have observed the effects of several irnmunosuppressive drugs including curcumin, parthenolide and rapamycin in the treatment of IR mouse models, and have carried out the preliminary discussion about related mechanisms. It will provide new insights in further intervention of type 2 diabetes pertaining to post-transplantation diabetes and insulin resistance. This paper includes three parts as follows.Section one: Research on Immunodepressant Curcumin to Reverse Insulin ResistanceObjective: Curcumin has physiological and pharmacological functions such as immunoregulation, antioxidation, anti-inflammation, tumor resistance and anti-artherosclerosis. Studies have found that curcumin is a good immunomodulator. It can inhibit host versus graft reaction and improve the survival rate of the recipients (animals). The aim of this study is to investigate the therapeutic action of curcumin and its mechanism on insulin resistance.Methods: To observe the effects of curcumin on FAO cells and obese insulin resistant animal model (obob mouse) (1) The dose/time- dependent effects of Curcumin on NF-κB inhibition; (2)To investigate therapeutic action of curcumin on insulin resistance in obob mice and observe fasting plasma glucose levels、insulin and glucose tolerant tests. (3) Investigate influence of curcumin on the circulating cytokines included IL-1, IL-6 and TNFαlevels and possible mechanism. (4) To analyze insulin stimulated IRS-1, P85 and AKT phosphorylation levels with immuno-co-precipitation, and observe influence of curcumin on insulin sensitivity in obob mice.Results: (1)The dose/time dependent inhibition of curcumin on NF-κB activation. The curative dose of curcumin was 30μM, which had most effective inhibition on NF-κB during 30-60 minutes. (2)The fasting plasma glucose level was not obviously decreased after the treatment of curcumin, but the level of fasting insulin decreased by about 30%. Furthermore, the level of glucose tolerance curve dropped significantly. (3)The level of TNFα(34.5±3.4 Vs31.3±1.1pg/ml) in circulation was not obviously changed, but the level of IL-1 ( 33.9±3.2 Vs 15.6±1.1pg/ml) and IL-6 (72.1±9.8 Vs47.2±5.3 pg/ml) was suppressed dramatically. (4)IRS-1 tyrosine Phosphorylation levels stimulated by insulin obviously increased, but the phosphorylation level of insulin receptor did not change. Being Parallel with Tyrosine phosphorylation levels of IRS-1, the phosphorylation levels of P85 and AKT increased accordingly.Conclusions: Curcumin can improve obob mouse insulin sensitivity, such effects of reversing insulin resistance may relate to its immunomodulating on cytokines and other inflammatory components.Section two : Research on limmunodepressant Parthenolide to Reverse Insulin ResistanceObjectives: Parthenolide is an another good immunomodulator. Its various biological activities are related to inhibit NF-κB, thus, Parthenolide has physiological and pharmacological actions such as anti-inflammation, inhibition of tumor growth and attenuation of artherosclerosis. It can also improve the survival rates of the recipients by inhibiting the host’s immunological rejection. The aim of this study is to investigate the therapeutic action of parthenolide and its mechanism on insulin resistance.Methods: To observe the effects of parthenolide on FAO cells and obese insulin resistant animal model (obob mouse). (1) The dose/time dependent effects of parthenolide on NF-κB inhibition; (2) To investigate therapeutic action of parthenolide on insulin resistance in obob mice and observe fasting plasma glucose levels、insulin and glucose tolerant tests. (3) To investigate influence of parthenolide on the circulating cytokines, i.e., IL-1, IL-6 and TNFαlevels and possible mechanism. (4) To analyze insulin stimulated phosphorylation of IRS-1, P85 and AKT levels with immuno-coprecipitation, and observe influence of parthenolide on insulin sensitivity in obob mice.Results: (1) The curative dose ofparthenolide was 10-20mmol/1, which had most effective inhibitory action on the NF-kB at 20 minutes. (2) The homeostasis model assessment (HOMA) indexes, which were calculated with fasting plasma glucose and insulin levels, had obviously decreased after the treatment of parthenolide. The levels of glucose tolerant curve dropped significantly. As to parameters during euglycemic hyperinsulinemic clamps, the basic hepatic glucose production in treatment group was significantly inhibited when compared with those in control group. Glucose uptake and glucose infusion rates under the euglycemic hyperinsulinemic clamped condition increased by 40% respectively as compared to control group. (3) Parthenolide inhibited NF-κB activity in cells and reversed insulin resistance that was induced by high fat diet and obesity. Hepatic glucose production of mice was significantly inhibited with treatment of parthenolide. The utilization rates of glucose were significantly elevated under the euglycemic hyperinsulinemic clamped condition. (4) Parthenolide inhibited circulating levels of TNFα, IL-6 and MCP-1. Consistent with findings in curcumin experiments, the cytokine levels of TNFα, IL-6 in circulation was inhibited by about 50% with treatment of parthenolide. The level of PAI-1 (1.75±0.20 Vs 1.14±0.15 ng/ml) and MCP-1 (1.48±0.18 Vs 0.97±0.14, p=0.03) was obviously attenuated as well. In contrast, the levels of Leptin and Resistin did not changed obviously. (5) The parthenolide inhibited two site-specific phosphorylations of Serine residues S964 and 1148, which are related to insulin resistance. Conclusion: The parthenolide has inhibitory effects on the systemic inflammation. Down-regulating NF-k B related pathways related to improving the insulin sensitiveness and was indicated as potential approach for type 2 diabetes treatments.Section three: Research on New-type Immunodepressant Rapamycin to Reverse Insulin ResistanceObjectives: Rapamycin (RAPA), a member of macrolides developed by Home Products Company in USA, was demonstrated as a new-type immunodepressant as compared to calcineurin inhibitors and antimetabolitas. As a basic immunodepressant it has already been used extensively in clinical to reduce or avoid immuno-pathological nephrotoxicity in post-transplantation patients. The aim of this study is to investigate the therapeutic action of rapamycin and its possible mechanism on insulin resistance in obese and insulin resistant animal model (obob mouse).Methods: To observe the effects of rapamycin on obese and insulin resistant animal model (obob mouse) (1) To investigate therapeutic action of rapamycin on insulin resistance in obob mice and observe its effects on fasting plasma glucose and insulin levels. (2) To investigate influence of rapamycin on the circulatory cytokines of IL-1, IL-6 and TNFαlevels and possible mechanism.Result:(1) The fasting plasma glucose level was obviously decreased after the treatment of rapamycin. As compare with control group, the result was statistically significant. The levels of fasting insulin were showing similar pattern, and the area under curve had remarkable difference as compared with control group (8.90±0.95 Vs 5.27±0.73, p＜0.05). (2) The levels of TNFα(31.7±3.4 Vs37.7±1.1pg/ml) and IL-1 (18.0±3.2 Vs 29.5±1.1pg/ml) in circulation were decreased as well, but they did not reach statistical difference. The level of IL-6 (46.2±9.8 Vs85.6±5.3 pg/ml) was obviously decreased. (p＜0.05).Conclusion: Rapamycin can improve insulin sensitivity in obob mice and the detail mechanism is still not clearly demonstrated.更多还原