本文關(guān)鍵詞：胰島素改善膿毒癥大鼠生長激素抵抗機(jī)理的研究，由筆耕文化傳播整理發(fā)布。

        在目前的營養(yǎng)支持領(lǐng)域,高分解代謝嚴(yán)重影響膿毒癥患者的預(yù)后,是目前治療的難點(diǎn)之一。白20世紀(jì)80年代起,使用相關(guān)激素等藥物調(diào)整代謝激素合成與分泌,進(jìn)而促進(jìn)合成代謝并抑制分解代謝的代謝調(diào)理治療逐漸受到廣泛的重視。作為合成激素的代表之一,生長激素(GrowthHormone),具有明顯的刺激組織生長、促進(jìn)機(jī)體內(nèi)氮質(zhì)潴留進(jìn)而促蛋白質(zhì)合成等作用,另有研究發(fā)現(xiàn)生長激素具有免疫調(diào)節(jié)等作用,其曾被人們在調(diào)整代謝激素合成與分泌,進(jìn)而促進(jìn)合成代謝并抑制分解代謝的代謝調(diào)理治療寄予較大期望。然而,應(yīng)用生長激素治療膿毒癥患者的過程中,常并發(fā)獲得性生長激素抵抗(acquired growth hormone resisitanceAGHR),主要表現(xiàn)為：膿毒癥機(jī)體血漿中生長激素水平升高,胰島素樣生長因子-1(IGF-1)減低及外源性生長激素的促合成作用減弱。生長激素抵抗(AGHR)是膿毒癥患者應(yīng)用生長激素后,促合成代謝效應(yīng)減低、不良反應(yīng)增加的重要可能機(jī)制。膿毒癥機(jī)體之所以出現(xiàn)生長激素抵抗,究其原因,可能為：泛素-蛋白酶體途徑過度激活導(dǎo)致生長激素受體水平下降；炎性細(xì)胞因子過度釋放導(dǎo)致受體后的細(xì)胞內(nèi)信號通路受阻抑。作為另一種重要的合成激素胰島素具有抗炎、抑制泛素-蛋白酶體途徑及提高生長激素受體水平等作用。且我們前期以及國內(nèi)外關(guān)于支持胰島素具有抗炎和抑制泛素—蛋白酶體途徑活性的證據(jù)在不斷增多。有趣的是Kin-Chuen等通過體外研究發(fā)現(xiàn),胰島素能促進(jìn)肝細(xì)胞GHR的合成、減少GHR的降解；同時(shí)還能促進(jìn)GHR胞內(nèi)攝作用。當(dāng)然,此研究僅為正常培養(yǎng)條件下針對肝細(xì)胞的研究結(jié)果,對于膿毒癥狀態(tài)下,胰島素對骨骼肌有無類似作用尚需進(jìn)一步研究證實(shí)。目前有研究表明強(qiáng)化胰島素治療能降低膿毒癥等危重病人骨骼肌蛋白分解,促進(jìn)合成代謝,不過該研究未能明確具體原因到底是控制血糖的作用還是胰島素的直接作用。另外,Orellana等研究發(fā)現(xiàn),胰島素能直接增加新生兒內(nèi)毒素血癥時(shí)的骨骼肌蛋白合成。膿毒癥狀態(tài)下,生長激素抵抗(AGHR)的發(fā)生主要與生長激素受體(GHR)合成減少和降解增加(泛素-蛋白酶體的過度活化參與此過程)以及受體后信號傳導(dǎo)受阻(可能因炎癥介質(zhì)過度釋放引起)有關(guān)；胰島素對膿毒癥機(jī)體具有抗炎、抑制泛素—蛋白酶體途徑活性、上調(diào)生長激素受體及促進(jìn)蛋白合成等作用。據(jù)此,我們推論：胰島素聯(lián)合生長激素可改善膿毒癥大鼠的生長激素抵抗。目前,國內(nèi)外尚無胰島素協(xié)同GH治療改善膿毒癥機(jī)體代謝的相關(guān)研究。有鑒于此,本研究通過腹腔注射內(nèi)毒素(LPS)建立大鼠膿毒癥模型,觀察胰島素能否改善膿毒癥狀態(tài)下的生長激素抵抗。第一部分檢測相關(guān)藥物治療后血中胰島素、生長激素、胰島素樣生長因子-1,檢測肝臟生長激素受體mRNA表達(dá)變化(RT-PCR法)、肝臟中IGF-1的-nRNA表達(dá)變化(RT-PCR法),檢測生長激素受體以及pJAK2、pSTAT5b、JAK2及tSTAT5b蛋白含量(Western-blotting法)。驗(yàn)證胰島素能否改善膿毒癥狀態(tài)下的生長激素抵抗(AGHR)。第二部分研究中,在第一部分實(shí)驗(yàn)基礎(chǔ)上,通過阻斷泛素-蛋白酶體途徑及PI3K-Akt通路后,檢測血中相關(guān)指標(biāo)(同第一部分),從而進(jìn)一步探討胰島素聯(lián)合生長激素改善膿毒癥大鼠的生長激素抵抗的可能信號通路。第一部分胰島素改善膿毒癥大鼠獲得性生長激素抵抗的研究目的：通過建立膿毒癥模型,明確胰島素聯(lián)合生長激素治療能否減輕膿毒癥所致的生長激素抵抗。方法：SD成年雄性大鼠,禁食12h,自由飲水,腹腔注射脂多糖(LPS)方法造模,腹腔注射LPS (1mg/Kg)1小時(shí)后存活的大鼠為造模成功者。隨機(jī)將30只造模成功的大鼠分為5組,即對照組(control)腹腔注射等滲鹽水；膿毒癥組(sepsis)腹腔注射LPS；胰島素組(insulin)腹腔注射LPS (1mg/Kg)+尾靜脈注射胰島素(5u/kg.d);生長激素(GH)組腹腔注射LPS (1mg/Kg)+尾靜脈注射生長激素(1IU/kg.d);聯(lián)合組(IG)腹腔注射LPS (1mg/Kg)+尾靜脈注射生長激素(1IU/kg.d)+尾靜脈注射胰島素(5u/kg.d)組。血糖控制在4.4-6.1mmol/L,血糖過低示靜脈給予50%葡萄糖,其余組給予等量生理鹽水。于給藥24h時(shí)應(yīng)用氯胺酮(1mg/kg)麻醉,并留取血漿、肝臟(取大鼠肝右葉組織)的標(biāo)本,置于液氮中。數(shù)據(jù)用SPSS19.0軟件作單因素方差分析(LSD),檢驗(yàn)水準(zhǔn)a=0.05。結(jié)果：2.1放免法測血漿中胰島素、生長激素濃度和ELISA法測血漿中IGF-1濃度我們通過放免法測得血漿胰島素濃度,膿毒癥組,因?yàn)楦骨蛔⑸鋬?nèi)毒素導(dǎo)致機(jī)體應(yīng)激反應(yīng)較正常組顯著增高(P<0.01)；胰島素組與聯(lián)合組相比,胰島素濃度不具有顯著性差異(P>0.05)(見圖1)。同時(shí),我們通過放免法測得血漿中生長激素濃度,膿毒癥組較正常組增高并且有統(tǒng)計(jì)學(xué)意義(P<0.01)；生長激素組與聯(lián)合組相比,生長激素水平不具有顯著性差異(P>0.05)(見圖2)。我們通過ELISA法測得血中IGF-1濃度,在膿毒癥組濃度較正常組低,有統(tǒng)計(jì)學(xué)意義(P<0.01)；聯(lián)合組IGF-1濃度較膿毒癥、胰島素、生長激素組升高有顯著性意義(P<0.01)。(見圖3)。RT-PCR結(jié)果示：膿毒癥組肝臟組織中IGF-1mRNA含量顯著低于正常組(P<0.01)；生長激素組IGF-1mRNA含量較膿毒癥組無顯著升高(P>0.05)；聯(lián)合組IGF-1mRNA含量較膿毒癥、胰島素、生長激素組顯著升高(P<0.01)(見圖4)。RT-PCR示膿毒癥組肝臟組織中GHR mRNA含量較正常組顯著降低(P<0.01)；生長激素組較膿毒癥組無顯著升高(P>0.05)；聯(lián)合組其肝臟中GHRmRNA含量顯著高于膿毒癥組、胰島素組、生長激素組,但顯著低于正常組(P<0.01)；膿毒癥組,肝臟組織中GHRmRNA含量較正常組顯著降低(P<0.01)；胰島素組肝臟中GHRmRNA含量較膿毒癥組顯著升高(P<0.01),但低于正常組和聯(lián)合組,具有統(tǒng)計(jì)學(xué)意義(P<0.01)(見圖5)。WB結(jié)果示：聯(lián)合組肝臟中GHR的蛋白含量顯著高于膿毒癥組、胰島素組、生長激素組(P<0.01),但顯著低于正常組(P<0.01)；膿毒癥組,肝臟組織中GHR蛋白含量較正常組顯著降低(P<0.01)；胰島素組肝臟中GHR的蛋白含量較膿毒癥組顯著升高(P<0.01),但顯著低于正常組和聯(lián)合組(P<0.01)(見圖6)。聯(lián)合組肝臟中pJAK2/JAK2水平高于膿毒癥組、胰島素組、生長激素組(P<0.01),但顯著低于正常組(P<0.01)；膿毒癥組,肝臟組織中pJAK2/JAK2水平較正常組顯著降低(P<0.01)；胰島素組、生長激素組肝臟中pJAK2/JAK2水平較膿毒癥組顯著升高(P<0.01),但低于正常組和聯(lián)合組,具有統(tǒng)計(jì)學(xué)意義(P<0.01)(見圖7)。聯(lián)合組肝臟中pSTAT5b/tSTAT5b水平高于膿毒癥組、胰島素組、生長激素組,但低于正常組,有統(tǒng)計(jì)學(xué)差異(P<0.01)；膿毒癥組,肝臟組織中pSTAT5b/tSTAT5b水平較正常組顯著降低(P<0.01)；胰島素組、生長激素組肝臟中pSTAT5b/tSTAT5b水平較膿毒癥組無顯著升高(P>0.05),較正常組和聯(lián)合組顯著降低(P<0.01)(見圖8)。高效液相色譜法示：聯(lián)合組趾長伸肌中酪氨酸和3-MT水平顯著低于膿毒癥組、胰島素組、生長激素組,但顯著高于正常組(P<0.01)；膿毒癥組,趾長伸肌中酪氨酸和3-MT水平較正常組顯著升高(P<0.01)；胰島素組趾長伸肌中酪氨酸和3-MT水平較膿毒癥組顯著降低(P<0.01),但高于正常組和聯(lián)合組,具有統(tǒng)計(jì)學(xué)意義(P<0.01)(見圖9、10)。結(jié)論：膿毒癥狀態(tài)下,胰島素聯(lián)合生長激素改善生長激素抵抗。第二部分胰島素發(fā)揮生長激素協(xié)同作用的胞內(nèi)信號傳導(dǎo)路徑研究目的：通過阻斷泛素蛋白酶體及PI3K-Akt通路后,應(yīng)用激素,明確胰島素聯(lián)合生長激素治療改善膿毒癥所致的生長激素抵抗的可能信號通路。方法：SD成年雄性大鼠,禁食12h,自由飲水。我們采用腹腔注射脂多糖(LPS)方法造模,腹腔注射LPS(1mg/Kg)1小時(shí)后存活的大鼠為造模成功者。隨機(jī)將42只造模成功的大鼠分為5組,即對照組(control)腹腔注射等滲鹽水；膿毒癥組腹腔注射LPS；胰島素(insulin)組腹腔注射LPS(1mg/Kg)+尾靜脈注射胰島素(5u/kg.d);生長激素組(GH)腹腔注射LPS(1mg/Kg)+尾靜脈注射生長激素(1IU1kg.d);聯(lián)合組(IG)腹腔注射LPS(1mg/Kg)+尾靜脈注射生長激素(1IU/kg.d)+尾靜脈注射胰島素(5u/kg.d);LY294002組靜脈注射LY294002+腹腔注射LPS(1mg/Kg)+尾靜脈注射生長激素(1IU/kg.d)+尾靜脈注射胰島素(5u/kg.d);MG-132組靜脈注射MG-132+腹腔注射LPS(1mg/Kg)+尾靜脈注射生長激素(1IU/kg.d)+尾靜脈注射胰島素(5u/kg.d)。血糖控制在4.4-6. lmmol/L,血糖過低示靜脈給予50%葡萄糖,其余組給予等量生理鹽水。于給藥24h時(shí)應(yīng)用氯胺酮(1mg/kg)麻醉,并留取肝臟(取大鼠肝右葉組織)的標(biāo)本,置于液氮中。數(shù)據(jù)用SPSS19.0軟件作單因素方差分析(LSD),檢驗(yàn)水準(zhǔn)α=0.05。結(jié)果：通過放免法測得血漿胰島素濃度,在膿毒癥組,因?yàn)楦骨蛔⑸鋬?nèi)毒素機(jī)體應(yīng)激反應(yīng)而增高,顯著高于正常組(P<0.01)；在胰島素組、聯(lián)合組、LY294002組和MG-132組之間,胰島素濃度不具有顯著性差異(P>0.05)(見圖1)。同時(shí),通過放免法測得血漿中生長激素濃度,在膿毒癥組顯著增高(P<0.01)；在生長激素組、聯(lián)合組、LY294002組和MG-132組之間,生長激素濃度不具有顯著性差異(P>0.05)(見圖2)。通過ELISA法測得血漿中IGF-1濃度,在膿毒癥組濃度較正常組顯著降低(P<0.01)；胰島素組、生長激素組、LY294002組和MG-132組IGF-1濃度較膿毒癥組均無顯著升高(P>0.05)；聯(lián)合組IGF-1濃度較胰島素組、生長激素組、LY294002組和MG-132組顯著升高(P<0.01)(見圖3)。RT-PCR結(jié)果示：肝臟組織中IGF-1mRNA含量在膿毒癥組濃度較正常組顯著降低(P<0.01)；生長激素組IGF-1mRNA含量較膿毒癥組無顯著升高(P>0.05)；聯(lián)合組IGF-1mRNA含量較胰島素組、生長激素組、LY294002組和MG-132組顯著升高(P<0.01)(見圖4)。RT-PCR示肝臟組織中GHRmRNA在膿毒癥組濃度較正常組顯著降低,有統(tǒng)計(jì)學(xué)意義(P<0.01)；胰島素組較膿毒癥組顯著升高(P<0.01)；生長激素組較膿毒癥組無顯著升高(P>0.05)；聯(lián)合組GHRmRNA較胰島素組、生長激素組、LY294002組和MG-132組顯著升高(P<0.01)(見圖5)。Western-blot結(jié)果示：膿毒癥組,肝臟組織中GHR蛋白水平較正常組顯著降低(P<0.01)；聯(lián)合組較胰島素組、生長激素組、LY294002組和MG-132組,肝臟中GHR的蛋白水平顯著升高(P<0.01),但低于正常組,具有顯著性意義(P<0.01)(見圖6)。聯(lián)合組肝臟中pJAK2/JAK2水平顯著高于膿毒癥組、胰島素組、生長激素組、LY294002組和MG-132組,但低于正常組,有統(tǒng)計(jì)學(xué)差異(P<0.01)；膿毒癥組,肝臟組織中pJAK2/JAK2蛋白水平較正常組顯著降低(P<0.01)；胰島素組、生長激素組、LY294002組和MG-132組,肝臟中pJAK2/JAK2水平顯著低于正常組和聯(lián)合組(P<0.01)(見圖7)。聯(lián)合組肝臟中pSTAT5b/tSTAT5b水平顯著高于膿毒癥組、胰島素組、生長激素組、LY94002組和MG-132組,但顯著低于正常組(P<0.01)；膿毒癥組,肝臟組織中pSTAT5b/tSTAT5b水平較正常組顯著降低(P<0.01)(見圖8)。高效液相甘色譜法示：聯(lián)合組趾長伸肌中酪氨酸和3-MT水平顯著低于膿毒癥組、胰島素組、生長激素組、LY294002組和MG-132組,但高于正常組(P<0.01)；膿毒癥組,趾長伸肌中酪氨酸和3-MT水平較正常組顯著升高(P<0.01)；胰島素組趾長伸肌中酪氨酸和3-MT水平較膿毒癥組顯著降低(P<0.01),但高于正常組和聯(lián)合組,具有統(tǒng)計(jì)學(xué)差異(P<0.01)。結(jié)論：胰島素可以改善膿毒癥大鼠生長激素抵抗,可能與其抑制泛素蛋白酶體路徑有關(guān),PI3K途徑可能參與此過程；胰島素聯(lián)合生長激素能提高IGF-I的水平；胰島素聯(lián)合生長激素能改善膿毒癥大鼠高分解代謝情況。

    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.

胰島素改善膿毒癥大鼠生長激素抵抗機(jī)理的研究

縮略詞表5-7摘要7-13Abstract13-20前言21-24    參考文獻(xiàn)22-24第一部分 胰島素改善膿毒癥大鼠獲得性生長激素抵抗的研究24-43    1 材料和方法24-32    2 結(jié)果32-38    3 討論38-40    4 小結(jié)40-41    參考文獻(xiàn)41-43第二部分 胰島素發(fā)揮生長激素協(xié)同作用的胞內(nèi)信號傳導(dǎo)路徑研究43-64    1 材料和方法43-51    2 結(jié)果51-57    3 討論57-59    4 小結(jié)59-60    參考文獻(xiàn)60-64全文總結(jié)64-65    本研究通過動物實(shí)驗(yàn)研究證實(shí)64    本研究創(chuàng)新性64-65課題綜述65-74    參考文獻(xiàn)71-74發(fā)表文章及獲獎情況74-75致謝75-76

  本文關(guān)鍵詞：胰島素改善膿毒癥大鼠生長激素抵抗機(jī)理的研究，由筆耕文化傳播整理發(fā)布。