胰岛素改善脓毒症大鼠生长激素抵抗机理的研究

  本文关键词：胰岛素改善脓毒症大鼠生长激素抵抗机理的研究，由笔耕文化传播整理发布。

        在目前的营养支持领域,高分解代谢严重影响脓毒症患者的预后,是目前治疗的难点之一。白20世纪80年代起,使用相关激素等药物调整代谢激素合成与分泌,进而促进合成代谢并抑制分解代谢的代谢调理治疗逐渐受到广泛的重视。作为合成激素的代表之一,生长激素(GrowthHormone),具有明显的刺激组织生长、促进机体内氮质潴留进而促蛋白质合成等作用,另有研究发现生长激素具有免疫调节等作用,其曾被人们在调整代谢激素合成与分泌,进而促进合成代谢并抑制分解代谢的代谢调理治疗寄予较大期望。然而,应用生长激素治疗脓毒症患者的过程中,常并发获得性生长激素抵抗(acquired growth hormone resisitanceAGHR),主要表现为：脓毒症机体血浆中生长激素水平升高,胰岛素样生长因子-1(IGF-1)减低及外源性生长激素的促合成作用减弱。生长激素抵抗(AGHR)是脓毒症患者应用生长激素后,促合成代谢效应减低、不良反应增加的重要可能机制。脓毒症机体之所以出现生长激素抵抗,究其原因,可能为：泛素-蛋白酶体途径过度激活导致生长激素受体水平下降；炎性细胞因子过度释放导致受体后的细胞内信号通路受阻抑。作为另一种重要的合成激素胰岛素具有抗炎、抑制泛素-蛋白酶体途径及提高生长激素受体水平等作用。且我们前期以及国内外关于支持胰岛素具有抗炎和抑制泛素—蛋白酶体途径活性的证据在不断增多。有趣的是Kin-Chuen等通过体外研究发现,胰岛素能促进肝细胞GHR的合成、减少GHR的降解；同时还能促进GHR胞内摄作用。当然,此研究仅为正常培养条件下针对肝细胞的研究结果,对于脓毒症状态下,胰岛素对骨骼肌有无类似作用尚需进一步研究证实。目前有研究表明强化胰岛素治疗能降低脓毒症等危重病人骨骼肌蛋白分解,促进合成代谢,不过该研究未能明确具体原因到底是控制血糖的作用还是胰岛素的直接作用。另外,Orellana等研究发现,胰岛素能直接增加新生儿内毒素血症时的骨骼肌蛋白合成。脓毒症状态下,生长激素抵抗(AGHR)的发生主要与生长激素受体(GHR)合成减少和降解增加(泛素-蛋白酶体的过度活化参与此过程)以及受体后信号传导受阻(可能因炎症介质过度释放引起)有关；胰岛素对脓毒症机体具有抗炎、抑制泛素—蛋白酶体途径活性、上调生长激素受体及促进蛋白合成等作用。据此,我们推论：胰岛素联合生长激素可改善脓毒症大鼠的生长激素抵抗。目前,国内外尚无胰岛素协同GH治疗改善脓毒症机体代谢的相关研究。有鉴于此,本研究通过腹腔注射内毒素(LPS)建立大鼠脓毒症模型,观察胰岛素能否改善脓毒症状态下的生长激素抵抗。第一部分检测相关药物治疗后血中胰岛素、生长激素、胰岛素样生长因子-1,检测肝脏生长激素受体mRNA表达变化(RT-PCR法)、肝脏中IGF-1的-nRNA表达变化(RT-PCR法),检测生长激素受体以及pJAK2、pSTAT5b、JAK2及tSTAT5b蛋白含量(Western-blotting法)。验证胰岛素能否改善脓毒症状态下的生长激素抵抗(AGHR)。第二部分研究中,在第一部分实验基础上,通过阻断泛素-蛋白酶体途径及PI3K-Akt通路后,检测血中相关指标(同第一部分),从而进一步探讨胰岛素联合生长激素改善脓毒症大鼠的生长激素抵抗的可能信号通路。第一部分胰岛素改善脓毒症大鼠获得性生长激素抵抗的研究目的：通过建立脓毒症模型,明确胰岛素联合生长激素治疗能否减轻脓毒症所致的生长激素抵抗。方法：SD成年雄性大鼠,禁食12h,自由饮水,腹腔注射脂多糖(LPS)方法造模,腹腔注射LPS (1mg/Kg)1小时后存活的大鼠为造模成功者。随机将30只造模成功的大鼠分为5组,即对照组(control)腹腔注射等渗盐水；脓毒症组(sepsis)腹腔注射LPS；胰岛素组(insulin)腹腔注射LPS (1mg/Kg)+尾静脉注射胰岛素(5u/kg.d);生长激素(GH)组腹腔注射LPS (1mg/Kg)+尾静脉注射生长激素(1IU/kg.d);联合组(IG)腹腔注射LPS (1mg/Kg)+尾静脉注射生长激素(1IU/kg.d)+尾静脉注射胰岛素(5u/kg.d)组。血糖控制在4.4-6.1mmol/L,血糖过低示静脉给予50%葡萄糖,其余组给予等量生理盐水。于给药24h时应用氯胺酮(1mg/kg)麻醉,并留取血浆、肝脏(取大鼠肝右叶组织)的标本,置于液氮中。数据用SPSS19.0软件作单因素方差分析(LSD),检验水准a=0.05。结果：2.1放免法测血浆中胰岛素、生长激素浓度和ELISA法测血浆中IGF-1浓度我们通过放免法测得血浆胰岛素浓度,脓毒症组,因为腹腔注射内毒素导致机体应激反应较正常组显著增高(P<0.01)；胰岛素组与联合组相比,胰岛素浓度不具有显著性差异(P>0.05)(见图1)。同时,我们通过放免法测得血浆中生长激素浓度,脓毒症组较正常组增高并且有统计学意义(P<0.01)；生长激素组与联合组相比,生长激素水平不具有显著性差异(P>0.05)(见图2)。我们通过ELISA法测得血中IGF-1浓度,在脓毒症组浓度较正常组低,有统计学意义(P<0.01)；联合组IGF-1浓度较脓毒症、胰岛素、生长激素组升高有显著性意义(P<0.01)。(见图3)。RT-PCR结果示：脓毒症组肝脏组织中IGF-1mRNA含量显著低于正常组(P<0.01)；生长激素组IGF-1mRNA含量较脓毒症组无显著升高(P>0.05)；联合组IGF-1mRNA含量较脓毒症、胰岛素、生长激素组显著升高(P<0.01)(见图4)。RT-PCR示脓毒症组肝脏组织中GHR mRNA含量较正常组显著降低(P<0.01)；生长激素组较脓毒症组无显著升高(P>0.05)；联合组其肝脏中GHRmRNA含量显著高于脓毒症组、胰岛素组、生长激素组,但显著低于正常组(P<0.01)；脓毒症组,肝脏组织中GHRmRNA含量较正常组显著降低(P<0.01)；胰岛素组肝脏中GHRmRNA含量较脓毒症组显著升高(P<0.01),但低于正常组和联合组,具有统计学意义(P<0.01)(见图5)。WB结果示：联合组肝脏中GHR的蛋白含量显著高于脓毒症组、胰岛素组、生长激素组(P<0.01),但显著低于正常组(P<0.01)；脓毒症组,肝脏组织中GHR蛋白含量较正常组显著降低(P<0.01)；胰岛素组肝脏中GHR的蛋白含量较脓毒症组显著升高(P<0.01),但显著低于正常组和联合组(P<0.01)(见图6)。联合组肝脏中pJAK2/JAK2水平高于脓毒症组、胰岛素组、生长激素组(P<0.01),但显著低于正常组(P<0.01)；脓毒症组,肝脏组织中pJAK2/JAK2水平较正常组显著降低(P<0.01)；胰岛素组、生长激素组肝脏中pJAK2/JAK2水平较脓毒症组显著升高(P<0.01),但低于正常组和联合组,具有统计学意义(P<0.01)(见图7)。联合组肝脏中pSTAT5b/tSTAT5b水平高于脓毒症组、胰岛素组、生长激素组,但低于正常组,有统计学差异(P<0.01)；脓毒症组,肝脏组织中pSTAT5b/tSTAT5b水平较正常组显著降低(P<0.01)；胰岛素组、生长激素组肝脏中pSTAT5b/tSTAT5b水平较脓毒症组无显著升高(P>0.05),较正常组和联合组显著降低(P<0.01)(见图8)。高效液相色谱法示：联合组趾长伸肌中酪氨酸和3-MT水平显著低于脓毒症组、胰岛素组、生长激素组,但显著高于正常组(P<0.01)；脓毒症组,趾长伸肌中酪氨酸和3-MT水平较正常组显著升高(P<0.01)；胰岛素组趾长伸肌中酪氨酸和3-MT水平较脓毒症组显著降低(P<0.01),但高于正常组和联合组,具有统计学意义(P<0.01)(见图9、10)。结论：脓毒症状态下,胰岛素联合生长激素改善生长激素抵抗。第二部分胰岛素发挥生长激素协同作用的胞内信号传导路径研究目的：通过阻断泛素蛋白酶体及PI3K-Akt通路后,应用激素,明确胰岛素联合生长激素治疗改善脓毒症所致的生长激素抵抗的可能信号通路。方法：SD成年雄性大鼠,禁食12h,自由饮水。我们采用腹腔注射脂多糖(LPS)方法造模,腹腔注射LPS(1mg/Kg)1小时后存活的大鼠为造模成功者。随机将42只造模成功的大鼠分为5组,即对照组(control)腹腔注射等渗盐水；脓毒症组腹腔注射LPS；胰岛素(insulin)组腹腔注射LPS(1mg/Kg)+尾静脉注射胰岛素(5u/kg.d);生长激素组(GH)腹腔注射LPS(1mg/Kg)+尾静脉注射生长激素(1IU1kg.d);联合组(IG)腹腔注射LPS(1mg/Kg)+尾静脉注射生长激素(1IU/kg.d)+尾静脉注射胰岛素(5u/kg.d);LY294002组静脉注射LY294002+腹腔注射LPS(1mg/Kg)+尾静脉注射生长激素(1IU/kg.d)+尾静脉注射胰岛素(5u/kg.d);MG-132组静脉注射MG-132+腹腔注射LPS(1mg/Kg)+尾静脉注射生长激素(1IU/kg.d)+尾静脉注射胰岛素(5u/kg.d)。血糖控制在4.4-6. lmmol/L,血糖过低示静脉给予50%葡萄糖,其余组给予等量生理盐水。于给药24h时应用氯胺酮(1mg/kg)麻醉,并留取肝脏(取大鼠肝右叶组织)的标本,置于液氮中。数据用SPSS19.0软件作单因素方差分析(LSD),检验水准α=0.05。结果：通过放免法测得血浆胰岛素浓度,在脓毒症组,因为腹腔注射内毒素机体应激反应而增高,显著高于正常组(P<0.01)；在胰岛素组、联合组、LY294002组和MG-132组之间,胰岛素浓度不具有显著性差异(P>0.05)(见图1)。同时,通过放免法测得血浆中生长激素浓度,在脓毒症组显著增高(P<0.01)；在生长激素组、联合组、LY294002组和MG-132组之间,生长激素浓度不具有显著性差异(P>0.05)(见图2)。通过ELISA法测得血浆中IGF-1浓度,在脓毒症组浓度较正常组显著降低(P<0.01)；胰岛素组、生长激素组、LY294002组和MG-132组IGF-1浓度较脓毒症组均无显著升高(P>0.05)；联合组IGF-1浓度较胰岛素组、生长激素组、LY294002组和MG-132组显著升高(P<0.01)(见图3)。RT-PCR结果示：肝脏组织中IGF-1mRNA含量在脓毒症组浓度较正常组显著降低(P<0.01)；生长激素组IGF-1mRNA含量较脓毒症组无显著升高(P>0.05)；联合组IGF-1mRNA含量较胰岛素组、生长激素组、LY294002组和MG-132组显著升高(P<0.01)(见图4)。RT-PCR示肝脏组织中GHRmRNA在脓毒症组浓度较正常组显著降低,有统计学意义(P<0.01)；胰岛素组较脓毒症组显著升高(P<0.01)；生长激素组较脓毒症组无显著升高(P>0.05)；联合组GHRmRNA较胰岛素组、生长激素组、LY294002组和MG-132组显著升高(P<0.01)(见图5)。Western-blot结果示：脓毒症组,肝脏组织中GHR蛋白水平较正常组显著降低(P<0.01)；联合组较胰岛素组、生长激素组、LY294002组和MG-132组,肝脏中GHR的蛋白水平显著升高(P<0.01),但低于正常组,具有显著性意义(P<0.01)(见图6)。联合组肝脏中pJAK2/JAK2水平显著高于脓毒症组、胰岛素组、生长激素组、LY294002组和MG-132组,但低于正常组,有统计学差异(P<0.01)；脓毒症组,肝脏组织中pJAK2/JAK2蛋白水平较正常组显著降低(P<0.01)；胰岛素组、生长激素组、LY294002组和MG-132组,肝脏中pJAK2/JAK2水平显著低于正常组和联合组(P<0.01)(见图7)。联合组肝脏中pSTAT5b/tSTAT5b水平显著高于脓毒症组、胰岛素组、生长激素组、LY94002组和MG-132组,但显著低于正常组(P<0.01)；脓毒症组,肝脏组织中pSTAT5b/tSTAT5b水平较正常组显著降低(P<0.01)(见图8)。高效液相甘色谱法示：联合组趾长伸肌中酪氨酸和3-MT水平显著低于脓毒症组、胰岛素组、生长激素组、LY294002组和MG-132组,但高于正常组(P<0.01)；脓毒症组,趾长伸肌中酪氨酸和3-MT水平较正常组显著升高(P<0.01)；胰岛素组趾长伸肌中酪氨酸和3-MT水平较脓毒症组显著降低(P<0.01),但高于正常组和联合组,具有统计学差异(P<0.01)。结论：胰岛素可以改善脓毒症大鼠生长激素抵抗,可能与其抑制泛素蛋白酶体路径有关,PI3K途径可能参与此过程；胰岛素联合生长激素能提高IGF-I的水平；胰岛素联合生长激素能改善脓毒症大鼠高分解代谢情况。

    Hypercatabolism appearing in septic state is one of challenges in the field of nutritional support. Currently, which affects the prognosis of sepsis patients seriously. Since the1980s, regulating metabolic hormone secretion, with the aim of inhibiting catabolic metabolism and promoting anabolic metabolism, has been intensely investigated.Great attention has once been paid to growth hormone (Growth Hormone), one of the representatives of the synthetic hormone which has significantly promote bone and soft tissue growing, body nitrogen retention, protein synthesis, and immunomodulatory role. To date, growth hormone (GH) has been an unsatisfactory therapeutic in critically ill patients with severe sepsis or hypercatabolic diseases. Recent evidence suggests that acquired growth hormone resistance (AGHR), which inhibits the anabolic effects of exogenous growth hormone, may be responsible for the ineffectiveness of GH therapy in these patients. AGHR performance mainly as follows:elevated growth hormone, insulin-like growth factor-1(IGF-1) reduction and exogenous growth hormone anabolic weakened. The reasons for AGHR in sepsis is:ubiquitin-proteasome pathway overactivation of the growth hormone receptor levels; excessive inflammatory cytokine release signaling pathway inside the cell after the receptor blocked suppression.Insulin,another important synthetic hormone,has anti-inflammatory action, inhibiting the ubiquitin-proteasome pathway and enhancing the role of the growth hormone receptor level. Evidence about that continues to grow in our study as well as other domestic and foreign studies. Accordingly, we infer:combined insulin and growth hormone can improve growth hormone resistance in septic rats. We designed this experiment to verify this inference that has not been reported at home and abroad.There is no study about combined insulin with GH to improve AGHR in sepsis.But the studies which provide some theoretical support about this is increasing in recent years. First, GHR synthesized reduction as well as post-receptor signaling blocked in sepsis leaded to AGHR. Insteretingly,Kin-Chuen found that insulin regulates hepatic GHR biosynthesis and surface translocation in a reciprocal manner. The divergent actions of insulin appear to be mediated by the mitogenactivatedprotein kinase and phosphatidylinositol3-kinase pathways, respectively. Of course, there is no similar effects for insulin to skeletal muscle in sepsis requiring further studies,though there are studies about liver cells under normal culture conditions.Second, important reasons for AGHR contain the ubiquitin-proteasome excessive activation, release of inflammatory mediators and so on. In addition, studies at home and abroad supportting insulin has anti-inflammatory and inhibition of the ubiquitin-proteasome pathway activity results in increased. Studies have shown intensive insulin therapy in critically ill patients can reduce skeletal muscle protein breakdown> promote anabolic,but the study failed to make it clear the effect is to control blood sugar or insulin directly. Orellana etc. found that insulin can directly promote skeletal muscle protein synthesis in neonatal endotoxemia recently. These results support our previous findings and may be complement with those, collaborative GH to improve sepsis patients metabolic provide theoretical support for insulin.In view of this, in order to consure whether insulin could improve growth hormone resistance in septic rats,we designed this study by intraperitoneal injection of endotoxin (LPS) to establish a rat model of sepsis. In the first part of this study, we detected the consentration of insulin growth hormone、insulin-like growth factor-1in serum, the mRNA levels of liver growth hormone receptor expression and IGF-1 mRNA expression (RT-PCR method) in liver, growth hormone receptor、pJAK2、 pSTAT5b、tJAK2and tSTAT5b protein content (Western-blotting method) to verify our assume. On the basis of the first part of the experiment, in the second part of the study, we investigated the possible signal pathways that the treatment of combined insulin with growth hormone could improve growth hormone resistance in sepstic rats by blocking the ubiquitin-proteasome pathway and PI3K-Akt pathway.PartⅠInsulin improves growth hormone resistence in septic ratsObjective:To observe whether insulin can improves growth hormone resistence in septic rats.Methods:This study used30adult male Sprague-Dawley rats, weighing200±10g, from the animal center of Jinling Hospital. The Institutional Animal Care Committee approved the study protocol. The Association accredits the animal care facility for Assessment and Accreditation of Laboratory Animal Care. Rats were housed in mesh cages at25℃under alternating12h light-dark cycles. Animals were acclimated in the facility for7d before the study. They were provided with standard rodent chow and water ad libitum.Rats were anesthetized by intraperitoneal (i.p.) injection of sodium phenobarbital (60mg/kg) and catheters (PE-50or PE-10; Becton-Dickinson, Sparks, MD) were implanted into the right jugular vein and the left carotid artery as previously described. A solution of insulin and dextrose was infused through the right jugular vein using a micro-pump (provided by the Research Center for Analytical Instrument, Zhejiang University) and blood glucose measurements were performed by an Elite glucometer (Bayer, Elkhart, IN) on blood from the left carotid artery. The catheters were filled with saline containing sodium heparin.Rats were withheld food for12h and divided randomly into the following seven treatment groups (n=6per group)Rats in the insulin, IG groups received continuous insulin infusion (Humulin R, EliLilly&Co., Indianapolis, IN) at a rate of about4.8mU/min/kg for1h (5u/kg/24h)after LPS stimulation. Rats that did not receive insulin received a sham infusion of sterile saline instead. Rats in the GH, IG groups were subcutaneously injected with GH (1IU/kg, Novo Nordisk, A/S)20min before harvesting while in other groups received a sham injection of sterile saline. Blood glucose was maintained between4.4-6.1mmol/l.The control group received only sham treatments of sterile saline in place of LPS injection, insulin infusion, and GH injection. In the LPS group, rats were injected with LPS.Rats were then infused with insulin1h after injection of LPS and received a sham injection of sterile saline in place of GH20min prior to euthanasia. In the insulin group, rats were injected with LPS. In the GH group, rats were injected with LPS, and then received an injection of GH20min before euthanasia. In the IG group, rats were given LPS. They were then infused with a combination of insulin and GH.The consentration of insulin、growth hormone、insulin-like growth factor-1in serum, the mRNA levels of liver growth hormone receptor expression and IGF-1mRNA expression (RT-PCR method) in liver, growth hormone receptor、pJAK2、pSTAT5b、tJAK2and tSTAT5b protein content (Western-blotting method).Data are expressed as means±standard error (SE). All data were analyzed with SPSS software (Version19.0, SPSS, Chicago, IL). Statistical analyses entailed ANOVA using the Tamhane’s T2M test for post-hoc analysis. P<0.05was considered statically significant.Results:Insulin and GH concentrations in serum were shown by RIA to significantly increase after LPS injection. Additionally, serum insulin levels in septic rats increased gradually (Fig.1)15.25±0.40mU/L vs.16.84±0.64mU/L, P=0.00after insulin administration and GH levels in septic rats increased gradually (Fig.2)1.71±0.85vs.1.90±0.03, P=0.04)after GH administration. ELISA showed that serum IGF-1levels were significantly reduced after LPS injection (Fig.3)(18.13±0.26vs.12.14±0.85, P=0.00). However, serum IGF-1levels increased following the combination treatment with insulin and GH (Fig.3) compared to GH alone (16.63±0.47VS.12.89±0.83, P=0.00). GHR and IGF-1mRNA expression in liver. RT-PCR showed that GHR and IGF-1mRNA decreased significantly in the liver after LPS injection(99.49±0.69vs.64.38±0.72, P<0.01;99.50±0.84vs.52.91±0.88, P<0.01) After administration of both insulin and GH in septic rats, GHR and IGF-1mRNA levels significantly increased compared to GH alone (Figs.4and5)(85.81±2.66vs.68.65±0.83P<0.01;73.53±0.19vs.52.67±0.31, P<0.01. Western blot analysis showed that LPS injection reduced levels of GHR, pJAK2, and pSTAT5b. After insulin treatment however, GHR levels increased (64.38±0.72vs.76.18±0.89, P<0.01). When both insulin and GH were administered, levels of GHR, pJAK2, and pSTAT5b were increased compared to GH alone (Figs.6,7and8)(86.09±3.09vs.64.61±0.52, P=1.00;83.00±1.79vs.49.83±2.23, P<0.01;85.77±2.22VS65.95±1.92, P<0.01).Conclusion:insulin can improves growth hormone resistence in septic rats. Part IIThe mechanism of insulin improves growth hormone resistence in septic ratsObjective:by blocking the PI3K-Akt pathway or the ubiquitin-proteasome system, to observe whether insulin can improves growth hormone resistence in septic rats septic rat.Methods:This study used42adult male Sprague-Dawley rats, weighing200±10g, from the animal center of Jinling Hospital. The Institutional Animal Care Committee approved the study protocol. The Association accredits the animal care facility for Assessment and Accreditation of Laboratory Animal Care. Rats were housed in mesh cages at25℃under alternating12h light-dark cycles. Animals were acclimated in the facility for7d before the study. They were provided with standard rodent chow and water ad libitum.Rats were anesthetized by intraperitoneal (i.p.) injection of sodium phenobarbital (60mg/kg) and catheters (PE-50or PE-10; Becton-Dickinson, Sparks, MD) were implanted into the right jugular vein and the left carotid artery as previously described. A solution of insulin and dextrose was infused through the right jugular vein using a micro-pump (provided by the Research Center for Analytical Instrument, Zhejiang University) and blood glucose measurements were performed by an Elite glucometer (Bayer, Elkhart, IN) on blood from the left carotid artery. The catheters were filled with saline containing sodium heparin.Rats were withheld food for12h and divided randomly into the following seven treatment groups (n=6per group)Rats in the insulin, IG groups received continuous insulin infusion (Humulin R, EliLilly&Co., Indianapolis, IN) at a rate of4.8mU/min/kg for1h after LPS stimulation. Rats that did not receive insulin received a sham infusion of sterile saline instead. Rats in the GH, IG groups were subcutaneously injected with GH (1IU/kg, Novo Nordisk, A/S)20min before harvesting while in other groups received a sham injection of sterile saline. Blood glucose was maintained between4.4-6.1mmol/l.The control group received only sham treatments of sterile saline in place of LPS injection, insulin infusion, and GH injection. In the LPS group, rats were injected with LPS and then injected with LY294002or MG-132through the tail vein at the beginning Rats were then infused with insulin1h after injection of LPS and received a sham injection of sterile saline in place of GH20min prior to euthanasia. In the insulin group, rats were injected with LPS followed by an injection of LY294002or MG-132through the tail vein prior to insulin infusion. In the GH group, rats were injected with LPS followed by LY294002or MG-132through the tail vein, and then received an injection of GH20min before euthanasia. In the IG group, rats were given LPS followed by LY294002or MG-132through the tail vein. They were then infused with a combination of insulin and GH.The LY294002group received an injection of LY294002(1.4mg/Kg) through the tail vein followed by infusion of insulin and GH. The MG-132group received an injection of MG-132(30mg/Kg) into the tail vein followed by infusion of insulin and GH. the consentration of insulin、growth hormone、insulin-like growth factor-1in serum, the mRNA levels of liver growth hormone receptor expression and IGF-1mRNA expression (RT-PCR method) in liver, growth hormone receptor、pJAK、pSTAT5b、 UAK2and tSTAT5b protein content (Western-blotting method).Data are expressed as means±standard error (SE). All data were analyzed with SPSS software (Version19.0, SPSS, Chicago, IL). Statistical analyses entailed ANOVA using the Tamhane’s T2M test for post-hoc analysis. P<0.05was considered statically significant.Results:Serum levels of insulin, GH, and IGF-1 Insulin and GH concentrations in serum were shown by RIA to significantly increase after LPS injection. Additionally, serum insulin levels in septic rats increased gradually (Fig.1)15.25±0.40mU/L vs.16.84±0.64mU/L, P=0.00after insulin administration and GH levels in septic rats increased gradually (Fig.2)1.71±0.85vs.1.90±0.03, P=0.04)after GH administration. ELISA showed that serum IGF-1levels were significantly reduced after LPS injection (Fig.3)(18.13±0.26vs.12.14±0.85, P=0.00). However, serum IGF-1levels increased following the combination treatment with insulin and GH (Fig.3) compared to GH alone (16.63±0.47VS.12.89±0.83, P=0.00). In the PI3K group,serum IGF-1levels were significantly lower than the IG group (16.63±0.47vs.12.53±0.03, P<0.01). GHR and IGF-1mRNA expression in liverRT-PCR showed that GHR and IGF-1mRNA decreased significantly in the liver after LPS injection(99.49±0.69vs.64.38±0.72, P<0.01;99.50±0.84vs.52.91±0.88, P<0.01) After administration of both insulin and GH in septic rats, GHR and IGF-1mRNA levels significantly increased compared to GH alone (Figs.4and5)(85.81±2.66vs.68.65±0.83P<0.01;73.53±0.19vs.52.67±0.31, P<0.01. In the PI3K roup, levels of GHR and IGF-1mRNA were significantly lower than the IG group (85.81±2.66vs.68.64±0.00, P<0.01;73.53±0.19vs.52.69±0.41, P<0.01) Western blot analysis showed that LPS injection reduced levels of GHR, pJAK2, and pSTAT5b. After insulin treatment however, GHR levels increased (64.38±0.72vs.76.18±0.89, P<0.01). When both insulin and GH were administered, levels of GHR, pJAK2, and pSTAT5b were increased compared to GH alone (Figs.6,7, and8)(86.09±3.09vs.64.61±0.52, P=1.00;83.00±1.79vs.49.83±2.23, P<0.01;85.77±2.22VS65.95±1.92, P<0.01).In the PI3K roup, the levels of GHR, pJAK2, and pSTAT5b were significantly lower than the IG group (86.09±3.09vs.64.19±0.66, P<0.01;83.00±1.79vs.50.00±1.41, P<0.01;85.77±2.22vs.64.69±2.06, P<0.01). In the MG-132group, levels of GHR, pJAK2, and pSTAT5b were also significantly lower than in the IG group (86.09±3.09vs.64.19±0.66, P<0.01;83.00±1.79vs.50.00±1.41, P<0.01;85.77±2.22vs.64.69±2.06, P<0.01). Conclusion:Insulin can improve growth hormone resistence in septic rats septic rat.The ubiquitin-proteasome system and PI3K-Akt pathway may involve this process.

胰岛素改善脓毒症大鼠生长激素抵抗机理的研究

缩略词表5-7摘要7-13Abstract13-20前言21-24    参考文献22-24第一部分 胰岛素改善脓毒症大鼠获得性生长激素抵抗的研究24-43    1 材料和方法24-32    2 结果32-38    3 讨论38-40    4 小结40-41    参考文献41-43第二部分 胰岛素发挥生长激素协同作用的胞内信号传导路径研究43-64    1 材料和方法43-51    2 结果51-57    3 讨论57-59    4 小结59-60    参考文献60-64全文总结64-65    本研究通过动物实验研究证实64    本研究创新性64-65课题综述65-74    参考文献71-74发表文章及获奖情况74-75致谢75-76

  本文关键词：胰岛素改善脓毒症大鼠生长激素抵抗机理的研究，由笔耕文化传播整理发布。