【作者】 徐杰；

【导师】 高峰； 张星；

【作者基本信息】 第四军医大学 ， 生理学， 2014， 硕士

【摘要】 研究背景应激性高血糖（stress hyperglycemia）是危重病患者死亡的独立危险因素，合理控制血糖已成为危重医学领域的共识。然而临床上发现，常规剂量的胰岛素往往不能控制应激性高血糖。最近有学者提出，强化胰岛素治疗将血糖控制到正常水平，可有效降低ICU中危重病患者的死亡率。但强化胰岛素治疗过程中产生的低血糖事件制约了其广泛应用。本课题组前期研究发现，大鼠在重度烧伤后可发生急性胰岛素抵抗（acute insulinresistance, AIR），其发生是应激性高血糖难以控制的重要原因。阐明危重病AIR的产生机制并寻找新的方法减轻AIR成为目前亟待解决的问题。糖基化终末产物（advanced glycation endproducts,AGEs）最早发现于糖尿病患者体内，其与胰岛素敏感性降低密切相关。AGEs通过与其跨膜受体（receptor foradvanced glycation endproducts, RAGE）结合发挥作用。细胞外液中存在可溶性RAGE（soluble receptor for advanced glycation endproducts, sRAGE）。因其含有与AGEs结合的肽段，sRAGE可以阻止AGEs与细胞膜上RAGE结合，从而阻断了AGEs的生物学效应。近年研究发现，以羧甲基赖氨酸（carboxymethyllysine, CML）为代表的AGEs可以在缺血再灌注和脑卒中等危重病中产生。然而，在重度烧伤情况下是否会产生AGEs，以及AGEs与AIR的关系尚未见报道。针对AGEs进行干预是否能减轻AIR亦不清楚。实验目的1.探讨重度烧伤后AIR的发生机制，尤其是烧伤后是否会导致AGEs产生及后者在AIR发生中的作用。2.明确烧伤早期用胰岛素控制血糖能否减少大鼠AGEs的产生，进而影响AIR的发生和大鼠烧伤后的死亡率。实验方法采用成年雄性SD大鼠重度烧伤模型（烧伤面积40%）。利用小动物PET/CT检测葡萄糖摄取。通过IPGTT实验检测大鼠烧伤后糖耐量变化。培养H9C2细胞，观察AGEs在细胞水平对胰岛素敏感性的影响。提取组织和细胞蛋白，通过蛋白免疫印迹技术检测pAkt/Akt、pGSK-3β/GSK-3β、CML等分子的表达水平。采用ELISA方法检测血清中sRAGE和CML的水平。实验结果1.与假烧伤大鼠（sham）相比，大鼠烧伤后3h的胰岛素敏感性显著降低。表现为：出现明显的高血糖（10.0±1.0mM vs.6.5±0.1mM. n=31,与sham相比,**P<0.01），胰岛素刺激的糖摄取能力受到抑制，心肌和骨骼肌中pAkt/Akt和pGSK-3β/GSK-3β下降。2.重度烧伤后大鼠血清中CML水平在0.5h即有增高（14.61±0.65ng/ml vs.11.60±0.72ng/ml. n=6,与sham相比,*P<0.05），1h达到峰值（18.61±1.34ng/ml vs.10.32±0.94ng/ml. n=6,与sham相比,**P<0.01），而后逐渐下降。在骨骼肌中同样发现CML在1h明显增高，而后逐渐下降。3.为降低CML的水平，我们在烧伤后立即给予尾静脉注射外源性sRAGE（3μg/kg）发现：sRAGE注射组烧伤后1h血清中CML的水平显著降低（12.64±0.69ng/mlvs.19.11±1.22ng/ml. n=6,与烧伤组大鼠相比，**P<0.01）；3h的血糖峰值显著降低（7.40±0.47mM vs.10.05±0.92mM. n=15,与burn相比,**P<0.01）；心肌和骨骼肌中pAkt/Akt升高；IPGTT实验显示3h时的糖耐量得到明显提高。4.在正常大鼠中，给予尾静脉一次性注射丙酮醛（Methylglyoxal，MG；AGEs的一种前体），发现：2h后血清中CML水平增高（13.63±0.32ng/ml vs.12.23±0.31ng/ml. n=6,与control相比,*P<0.05）；心肌和骨骼肌中pAkt/Akt显著降低。给予MG1.5h后检测IPGTT发现糖耐量受到明显抑制。说明AGEs对大鼠胰岛素敏感性具有抑制作用。5. CML-BSA处理1h可抑制H9C2细胞Akt的基础磷酸化水平（0.19±0.11vs.0.66±0.17. n=4,与control相比,**P<0.01）及胰岛素刺激的Akt磷酸化水平（0.40±0.12vs.1.70±0.29. n=4,与control相比,**P<0.01），说明AGEs对胰岛素信号有抑制作用。6.烧伤后立即给予胰岛素（2.5U/kg）发现：烧伤后的应激性高血糖消失；1h AGEs的峰值降低（9.34±0.90ng/ml vs.18.32±1.72ng/ml. n=6,与烧伤组相比,**P<0.01）。提示早期给予胰岛素干预通过控制烧伤后应激性高血糖，降低AGEs的产生，进而减轻烧伤后3h的AIR。7.早期给予sRAGE或胰岛素干预均可提高大鼠重度烧伤后的生存率（92%vs.71%,*P<0.05, n=27；95%vs.71%,*P<0.05, n=43）。结论1.大鼠重度烧伤后早期（1h）即可致血清AGEs的水平增加，后者是导致AIR发生的因素之一。2.烧伤后即刻给予sRAGE或胰岛素，可降低烧伤大鼠血清中AGEs的水平，减轻重度烧伤后AIR，提高烧伤大鼠的生存率。更多还原

【Abstract】 BackgroundThe stress hyperglycemia occurred in critical illness has been an independent riskfactor which is related with the mortality of patients, thus hyperglycemia control has beendoctors’ common sense in critical care medicine. However, it’s showed that regular doseof insulin usually can not control the stress hyperglycemia in critical illness. Recently,intensive insulin therapy was recommended to control hyperglycemia and showed brightprospect in decreasing mortality in ICU. To our disappointment that the hypoglycemiaaccident during this procedure limits the widespread use of intensive insulin therapy.Our team found that there was acute insulin resistance (AIR) following severe burnwhich is the primary reason for the difficulty of stress hyperglycemia control. Therefore,elucidation the mechanism of AIR and finding new ways to alleviate AIR in critical illness have already been urgent issues.Advanced glycation endproducts (AGEs) were found in diabetic patients whichparticipated in the damage of insulin sensitivity. AGEs recognize the transmembranereceptor (receptor for advanced glycation endproducts, RAGE) and initiate theintracellular signaling that disrupts celluar function. sRAGE, the extracellular ligandbinding domain of RAGE, soluble receptor for advanced glycation endproducts (sRAGE),blocks AGEs from binding to RAGE and disrupts the biological effect of AGEs. Recentstudies have showed that carboxymethyllysine (CML), a dominate AGEs in vivo,participats in myocardial ischemia/reperfusion and stroke. But the roles of AGEs in burninjury and AIR have not been reported. The treatment targeting AGEs is necessary toameliorate AIR and improve the prognosis of patients.Aims1. To investigate the mechanism of AIR following severe burn, especially the productionof the AGEs postburn and it’s role in AIR.2. To explore whether early insulin treatment decreases the level of AGEs, inhibits AIRand increases the survival rate postburn.MethodsYoung adult (180-200g) SD rats were subjected to40%burn area to build the severeburn model. PET/CT was used to determine the glucose uptake. IPGTT test was used toassess the glucose tolerance. H9C2cell were cultured to investigate the in vitro insulinsensitivity interrupted by AGEs. The tissue and cell proteins were extracted and westernblotting was used to evaluate pAkt/Akt, pGSK-3β/GSK-3β, and CML expression levels.sRAGE and CML in serum were detected by ELISA kit.Results1. Insulin sensitivity decreased at3h following severe burn evidenced byhyperglycemia (10.0±1.0mM vs.6.5±0.1mM. n=31/group, compared with sham,**P<0.01), inhibition insulin stimulated glucose uptake of heart and decreasedpAkt/Akt and pGSK-3β/GSK-3β in heart and skeletal muscle.2. The CML level increased at0.5h （14.61±0.65ng/ml vs.11.60±0.72ng/ml. n=6/group, compared with sham,*P<0.05）and reached a peak at1h (18.61±1.34ng/ml vs.10.32±0.94ng/ml. n=6/group, compared with sham,**P<0.01) followingsevere burn in serum as well as in skeletal muscle.3. Early exogenous sRAGE treatment (3μg/kg) decreased the CML peak at1h followingburn (12.64±0.69ng/ml vs.19.11±1.22ng/ml. n=6/group, compared with burngroup,**P<0.01), inhibited the hyperglycemia at3h (7.40±0.47mM vs.10.05±0.92mM. n=15, compared with burn,**P<0.01) and increased the pAkt/Akt in heart andskeletal muscle. IPGTT test showed that the glucose tolerance increased at3hpostburn compared with the vehicle treatment group.4. Insulin sensitivity was compromised in vivo2h after intravenous injectionMethylglyoxal (MG, an AGEs precursor) evidenced by decreased pAkt/Akt in heartand skeletal muscle. IPGTT test showed that the glucose tolerance was inhibited afterMG injection.5. Insulin sensitivity was compromised in vitro by CML-BSA treatment for1h in H9C2cell evidenced by decreased both basal (0.19±0.11vs.0.66±0.17. n=4, comparedwith control,**P<0.01) and insulin-stimulated Akt activation (0.40±0.12vs.1.70±0.29. n=4, compared with control,**P<0.01).6. Early insulin treatment after burn decreased the AGEs level at1h (9.34±0.90ng/mlvs.18.32±1.72ng/ml. n=6/group, compared with burn group,**P<0.01) andhyperglycemia within12h. IPGTT test showed that the glucose tolerance wasincreased by insulin treatment. These results showed that insulin alleviated AIR at3hby inhibiting AGEs production following severe burn.7. Early sRAGE or insulin treatment improved the survival rate of rats following severeburn (92%vs.71%,*P<0.05, n=27；95%vs.71%,*P<0.05, n=43).Conclusions1. Severe burn-induced acute insulin resistance (AIR) was mediated by elevatedproduction of advanced glycation endproducts (AGEs) at1h postburn.2. Early sRAGE or insulin treatment after burn decreased AGEs level, ameliorated AIRand increased survival rate following burn.更多还原