【作者】 何冰；

【导师】 韩萍；

【作者基本信息】 中国医科大学 ， 内科学， 2008， 博士

【摘要】 前言胰岛素抵抗(Insulin Resistance IR)和胰岛素分泌不足是2型糖尿病发病的两个关键因素,但两者发生机制尚未阐明。近年来炎症学说备受关注,该学说认为营养过剩、体力活动减少、吸烟、应激、衰老、遗传等因素,可以促发机体炎症因子活化,炎症因子包括:免疫炎症反应细胞,如白细胞;肝脏产生的急性期反应蛋白如C-反应蛋白;细胞因子如肿瘤坏死因子-α(TNF—α)、白介素系列;脂肪因子如脂联素等。日益增多的证据表明炎症因子可以通过血液和(或)旁分泌的作用影响胰岛素敏感性和胰岛β细胞功能,进而引起胰岛素抵抗和胰岛素分泌障碍,最终导致2型糖尿病的发生。因此使用抗炎症药物,抑制炎症因子活化,就有可能阻断2型糖尿病发病的两个基本环节,降低2型糖尿病的发病风险。近年研究发现,作为经典非甾体抗炎药的水杨酸制剂,可能通过非环氧化酶途径,发挥改善胰岛素抵抗及保护胰岛β细胞功能的作用,机制存在争议。本研究拟观察水杨酸钠对脂肪乳输注诱导的胰岛素抵抗大鼠胰岛素敏感性的影响及其作用机制。方法(1)大鼠清醒状态下输注脂肪乳,制备胰岛素抵抗的动物模型。(2)清醒状态下行高胰岛素正血糖钳夹试验评价大鼠胰岛素抵抗。(3)采用葡萄糖氧化酶法测定血糖,放射免疫法测定胰岛素和C肽;ELISA方法测定血浆中IL-6,TNF-α,HsCRP和脂联素水平;采用比色法测定血浆游离脂肪酸(FFA)、丙二醛(MDA)和肝脏、肌肉组织中的MDA水平及谷胱甘肽过氧化物酶(GSH-PX)活性。(4)采用免疫组化技术观察脂肪细胞、骨骼肌细胞细胞核内核因子-K B(NF—KB)表达;免疫组化技术检测脂肪细胞、骨骼肌细胞和肝细胞中诱导型一氧化氮合酶(iNOS)的表达。(5)采用RT-PCR技术检测脂肪、肌肉和肝脏组织中IL-6、细胞因子信号传导抑制因子-3(SOCS-3)、TNF-α和脂联素mRNA的表达。(6)采用Western blot杂交技术检测肌肉、肝脏中307位丝氨酸磷酸化的IRS-1及总IRS-1水平。结果(1) 2h脂肪乳输注组大鼠较盐水输注组葡萄糖输注率(GIR)下降了近27%,继续输注脂肪乳5h大鼠GIR降低52%,输注脂肪乳7h GIR降低56%,输注脂肪乳48h GIR降低58%,5h、7h及48h脂肪乳输注组间GIR没有统计学差异。(2)输注水杨酸钠7h,可轻度降低血浆FFA水平,血糖下降至脂肪乳组70%,胰岛素和C肽水平也分别下降至脂肪乳输血糖下降至脂肪乳组70%,胰岛素和C肽水平也分别下降至脂肪乳输注组61%、68%。高胰岛素钳夹试验中,脂肪乳输注组大鼠GIR是生理盐水组45%,水杨酸钠的输注可使GIR增加1.3倍。(3)三组大鼠血浆炎症因子水平比较:脂肪乳输注组大鼠血浆IL-6水平是生理盐水输注组大鼠的1.2倍,TNF-α为生理盐水组1.3倍,HsCRP为生理盐水组1.5倍,脂联素为生理盐水组的1.3倍。水杨酸钠+脂肪乳输注组大鼠血浆IL-6水平较脂肪乳输注组下降19%,TNF-α下降20%,HsCRP下降20%,脂联素水平两组大鼠无差异。(4)三组大鼠NF-KB相对表达量比较:脂肪乳输注组大鼠脂肪细胞核内NF-K B相对表达量是生理盐水输注组的1.8倍,水杨酸钠+脂肪乳输注组较脂肪乳输注组下降35%;三组大鼠骨骼肌细胞核中NF-KB相对表达量差异无统计学意义。(5)三组大鼠炎症因子在组织中表达比较:脂肪组织中,脂肪乳输注组大鼠炎症因子IL-6mRNA、SOCS-3mRNA、TNF-αmRNA相对表达量分别是生理盐水输注组的1.9倍、2.6倍和4倍,输注水杨酸钠,IL-6mRNA、SOCS-3mRNA、TNF-αmRNA相对表达量较脂肪乳组分别下降30%、50%和50%;肌肉组织中,脂肪乳输注组大鼠SOCS-3mRNA相对表达量是生理盐水输注组大鼠的3.5倍,水杨酸钠+脂肪乳输注组较脂肪乳输注组降低68%,三组大鼠IL-6及TNF-αmRNA在肌肉组织相对表达量差异无统计学意义;肝脏组织中,输注脂肪乳大鼠IL-6mRNA、SOCS-3mRNA、TNF-αmRNA相对表达量分别较输注生理盐水大鼠升高的1倍、1.9倍和1倍,输注水杨酸钠大鼠IL-6mRNA、SOCS-3mRNA、TNF-αmRNA表达降低38%、40%和50%。(6)脂肪乳输注组大鼠肌肉及肝脏中307位丝氨酸磷酸化的IRS-1分别为生理盐水输注组大鼠的3.8倍和3倍,输注水杨酸钠后,大鼠肌肉及肝脏中307位丝氨酸磷酸化的IRS-1水平下降20%和45%。三组大鼠肌肉、肝脏中总IRS-1含量,差异无显著性。(7)三组大鼠氧化应激水平比较:脂肪乳输注组大鼠血浆MDA水平较生理盐水输注组增加了2倍,水杨酸钠+脂肪乳组较脂肪乳组降低62%;脂肪乳输注组大鼠与生理盐水输注组比较,肌肉组织中MDA含量增加4倍,GSH-PX活性降低46%,肌细胞内iNOS相对表达量增加了1.9倍。水杨酸钠输注使肌肉组织中MDA含量降低66%,GSH-PX活性升高35%,肌细胞内iNOS的表达降低35%;脂肪乳输注组大鼠较盐水输注组大鼠,肝脏中MDA含量增加2倍,GSH-PX活性降低45%,肝细胞内iNOS相对表达量增加4倍,水杨酸钠+脂肪乳与脂肪乳组比较,MDA含量降低63%,GSH-PX活性升高37%,iNOS相对表达量降低70%;脂肪细胞iNOS相对表达量,脂肪乳输注组大鼠是生理盐水组的2.5倍,水杨酸钠+脂肪乳输注组较脂肪乳输注组下降48%。结论(1)给大鼠输注脂肪乳使血浆游离脂肪酸浓度达到基础的3倍左右,可成功复制出脂毒性胰岛素抵抗的动物模型,而且胰岛素抵抗达到一定程度后保持稳定,不再继续增强。(2)短时间输注脂肪乳血浆游离脂肪酸增高,大鼠葡萄糖输注率下降,输注水杨酸钠,大鼠葡萄糖输注率升高,胰岛素抵抗明显减轻,抗炎药物水杨酸钠可以改善高游离脂肪酸引起的胰岛素抵抗。(3)脂毒性引起大鼠胰岛素抵抗的机制及水杨酸钠改善高游离脂肪酸引起的胰岛素抵抗的可能机制:①血浆游离脂肪酸升高,胰岛素作用靶组织脂肪、肌肉及肝脏中NF-κB及下游的炎症因子激活,可能通过血液和(或)旁分泌的作用发挥致胰岛素抵抗的作用,水杨酸钠抑制脂肪、肌肉及肝脏中NF-κB的活性,降低了局部组织炎症因子的表达,同时下调了全身炎症反应水平。②血浆游离脂肪酸升高,胰岛素作用的靶组织肌肉及肝脏组织中IRS-1丝氨酸磷酸化水平升高,引起胰岛素抵抗。抗炎药物水杨酸钠可以逆转肌肉和肝脏组织IRS-1丝氨酸磷酸化水平的升高,减轻了胰岛素抵抗。③血浆游离脂肪酸水平升高引起机体氧化应激增强,可能是导致胰岛素抵抗发生的机制之一,应用水杨酸钠,大鼠氧化应激减弱,胰岛素抵抗改善,抗炎药物水杨酸钠有可能通过降低氧化应激途径而发挥改善胰岛素抵抗的作用。更多还原

【Abstract】 IntroductionInsulin resistance and insulin deficiency are major factors in the pathogenesis of type 2 diabetes,but the mechanism by which they occur is unknown.There has been a recent explosion of interest in the notion that chronic low-grade inflammation and activation of innate immune system are closely involved in the pathogenesis of type 2 diabetes.It says that many factors such as nutrition excess、age、smoking、inactivity、stress、genetics can activate markers of inflammation,which include immune cells、acute-phase protein as C-reactive protein、cytokine as TNF-α.Growing evidence show that inflammatory mediators can influence the sensitivity of insulin and the function ofβcell in pancreas through circulation or paracrine,furthermore cause insulin resistance and insulin deficiency,this eventually mediate incurrence of type 2 diabetes.These findings provide insights that anti-inflammatory agents may prevent the activation of inflammatory mediators and reduce the risk of developing type 2 diabetes.Recently Yuan et al hypothesized that salicylate may improve insulin resistance and protectβcell opposed to inhibition of cyclooxygenases,but the mechanism is still unclear.Our study aim at examining the influence of sodium salicylate on insulin sensitivity in lipid infusion rats and the mechanism.Methods(1) Infusion of saline、intralipid、intralipid +sodium salicylate for 7 hours in awake rats.(2) Hyperinsulinemic-euglycemic clamp in awake rats to estimate insulin resistance.(3) Plasma glucose was measured at the bedside with a Beckman Instruments Inc.BIOSEN5030,Germany.Plasma insulin and C-peptide were determined by RIA. We determined plasma concentration of cytokines,including IL-6、TNF-α、HsCRP and adiponectin using enzyme-linked immunosorbent assay.Plasma free fatty acids and MDA in liver,muscle and the activity of GSH-PX in liver and muscle were detected by colorimetric method.(4) NFκB in lipocytes、skelecton muscle cells and iNOS in hepatocytes、lipocytes、skelecton muscle cells were measured by immunhistochemistry methods.(5) We examined the gene expression of IL-6、TNF-α、SOCS-3 and adiponectin in liver、skelecton muscle、white adipose tissue by RT-PCR.(6) The levels of serine phosporylation of insulin receptor substrate 1 and insulin receptor substrate 1 in liver and skelecton muscle were detected by Western blotting.Results(1) Compared with SAL group,there was elevated plasma FFA levels by 2 times in 2h IH group while GIR decreased 27%,GIR decreased 52%in 5h IH group, GIR decreased 56%in rats with IH for 7h,GIR decreased 58%in 48h IH group, however there was no significant difference among 5h、7h and 48h IH group.(2) Infusion of sodium salicylate for 7 hours in rats can decrease plasma FFA levels gently.Compared with SAL group,there was decreased 30%in glucose,39% in insulin and 32%in C-peptide in rats with SI.During hyperinsulinemic-euglycemic clamp test,infusion of intralipid resulted in a 55% reduction in GIR,while Compared with IH group,GIR increased 1.3 times in rats with infusion of sodium salicylate.(3) Compared with SAL group,there were elevated plasma IL-6 levels by 1.2 times and were elevated plasma TNF-αlevels by 1.3 times,and HsCRP levels by 1.5 times,adiponectin levels by 1.3 times in IH group.The levels of IL-6、TNF-α、HsCRP in rats with infusion of sodium salicylate decreased 19%、20%、20% respectively compared with rats in IH group,however there was no significant difference in adiponectin levels between IH and SI group.(4) Compared with SAL group,NF-κB relative expression in lipocyte elevated nearly 1 time in SI group,sodium salicylate treatment resulted in a 35% reduction.There was no significant difference of NF-κB relative expression in skelecton muscle cell among three groups.(5) In white adipose tissue,inflammation cytokines including IL-6、SOCS-3、TNF-αgene expression increased 0.9、1.6 and 3 times respectively in IH group compared with SI group,infusion of sodium salicylate resulted in 30%、50%and 50% decrease.In skelecton muscle tissue,SOCS-3mRNA levels in IH group is 3.5 times compared with SAL group,There was no significant difference of IL-6 and TNF-αmRNA relative expression in skelecton muscle among three groups.In liver,the relative expression of IL-6、SOCS-3、TNF-αmRNA increased 1、1.9 and 1 times respectively in IH group compared with SI group,infusion of sodium,the relative expression ofIL-6、SOCS-3、TNF-αmRNA salicylate decrease 38%、40%and 50% respectively.(6) The levels of serine phosporylation of insulin receptor substrate 1 in liver and skelecton muscle increased 2.8 and 2 times in rats with IH compared with SAL group,sodium salicylate treatment resulted in 20%and 45%reduction,and there was no significant difference of insulin receptor substrate 1 in liver and skelecton muscle among three groups.(7) Compared with SAL group,there were elevated 2-4 times of MDA in plasma、liver、muscle and pancreas in rats with IH,and the activity of GSH-PX in liver、muscle and pancreas decreased 45%-50%.Compared with IH group,the levels of MDA in plasma,liver,muscle and pancreas decreased 62%-66%,the activity of GSH-PX increased 35-38%in rats with SI.Conclusion(1) The model of insulin resistance of lipotoxicity may be established after the concentration of FFA reached 3 times of basic level that induced by intravenously infused intralipid,and insulin resistance would keep steady until it reached some degree.(2) The elevation of FFA can cause a reduction of GIR,sodium salicylate treatment resulted in a increase of GIR,these data support the hypothesis that anti-inflammatory drug sodium salicylate can improve insulin resistance induced by intravenously infused intralipid.(3) The mechanism of insulin resistance induced by intravenously infused intralipid and the mechanism of sodium salicylate action:(4) There have been activation of NF-κB and downstream cytokine production in liver、skelecton muscle、white adipose tissue,which induced by intravenously infused intralipid.This cause the insulin resistance both locally and systemically,sodium salicylate can suppress the activation of NF-κB and downstream cytokine production in liver、skelecton muscle、white adipose tissue,at same time decrease the levels of inflammatory reactions systemically.(5) The increase of serine phosporylation of insulin receptor substrate 1 in liver and skelecton muscle which induced by intravenously infused intralipid result in the insulin resistance,sodium salicylate can decrease insulin resistance by reversing the elevation of the serine phosporylation of insulin receptor substrate 1 in liver and skelecton muscle(6) Oxidative stress may be one of the mechanisms of insulin resistance induced by elevation of FFA,and there were decrease of oxidative stress and insulin resistance in rats with sodium salicylate.Perhaps sodium salicylate improved insulin resistance and protectedβcell through decreasing oxidative stress in rats.更多还原