【作者】 陈忠勇；

【导师】 汪仕良；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 烧伤高代谢反应是严重烧伤应激、炎症反应的重要组成部分。适当的高代谢反应对机体有利,过度高代谢反应对机体有害。众所周知,中枢神经系统在烧伤高代谢反应中起重要作用,近年来越来越多的资料阐述神经-免疫-内分泌系统存在相互影响,相互调节,这为探讨烧伤高代谢发生机制及其调理提供了新思路。机体能量平衡的调节机制十分复杂,在中枢神经系统中,烧伤高代谢反应受下丘脑调控,下丘脑在能量平衡调节中起关键作用。二十多年来神经生物学的不断进展,大量与能量平衡有关的下丘脑神经肽及神经递质网络相继被发现,为探索烧伤高代谢反应机制提供了重要的理论基础。合成代谢作用神经肽刺激摄食,降低能量消耗,分解代谢作用神经肽抑制摄食,增加能量消耗。促肾上腺皮质激素释放因子(CRF)系统属分解代谢神经肽,CRF相关肽及其受体在创伤应激及能量代谢中的作用已有一些证据。而且许多其他中枢神经肽均通过CRF系统起作用。因此,我们推测下丘脑CRF相关肽及其受体在烧伤应激及烧伤高代谢反应中可能起着十分重要作用。CRF相关肽包括CRF、UCN(Urocortin)1、UCN2、UCN3等。1981年Vale等首先从羊下丘脑分离出CRF,CRF是41个氨基酸的神经肽,在中枢神经系统CRF通过与CRFR1、CRFR2两种高亲和力受体亚型结合,在应激反应中发挥多种多样的病理生理作用。研究表明CRFR1受体主要介导HPA轴ACTH、糖皮质激素的分泌,CRFR2受体主要介导能量平衡。UCN1是1995年由vaughan等首先报道,是在大鼠脑中发现的一种普遍存在于哺乳动物中的CRF家族的新成员,与CRF有45%的同源性,UCNs是CRFR2受体的主要配体,研究表明,UCNs可能在烧伤后高代谢反应中起重要作用。近年来,CRFR1、CRFR2受体拮抗剂相继被发现,Astressin2-B是CRFR2受体特异拮抗剂,Antalarmin是CRFR1受体特异拮抗剂,同时,反义核酸技术是目前比较成熟的下调基因表达的方法。CRF受体拮抗剂的应用及反义核酸技术为特异受体作用机制研究提供了重要手段。烧伤高代谢主要来自创面,肠道是烧伤高代谢的另一重要来源。肠道作为外科应激的中心器官,肠道在烧伤高代谢反应中的作用已有一些证据。我所通过大量的动物实验及临床研究证实早期肠道营养可降低烧伤高代谢反应,并由此提出“肠源性高代谢”理论。严重烧伤后早期肠道营养通过食物对胃肠道刺激,促进肠道复苏,改善肠道血循,维护肠道结构功能,调节肠道运动、分泌、代谢功能,降低肠道细菌/内毒素移位,从而降低烧伤高代谢反应。但已有的研究局限在早期肠道营养对静息能量消耗及肠道结构功能影响的研究,未涉及中枢代谢相关神经肽的变化情况。因此,我们推测,既然“肠源性高代谢”理论认为肠道参与烧伤高代谢的形成,早期肠道营养可降低烧伤高代谢反应,而中枢神经系统在烧伤高代谢反应中起重要作用,因而可以设想早期肠道营养在降低烧伤高代谢时,也可能存在由体液和神经机制通过中枢神经系统及其代谢相关肽的调节。目前国内外尚未见这方面的资料报道。CRF相关肽及其受体在胃肠道也有相应表达。功能研究显示大鼠外周静脉注射CRF、UCN可促进结肠的推进运动,抑制胃排空,而CRF、UCN不能通过血脑屏障,提示胃肠道局部可能也有CRF相关肽的合成与分泌,胃肠道内存在相应CRF受体。近年来,肠道粘膜、粘膜下层CRF相关肽及受体在肠道应激、肠道炎症反应及肠道粘膜屏障功能中作用研究越来越受重视。但严重烧伤应激后肠道CRF相关肽及其受体的动态变化及早期肠道营养对肠道CRF相关肽及其受体有何调节作用?国内外未见文献报道。本实验应用CRFR1、CRFR2受体的特异性拮抗剂及反义核酸技术,通过阻断CRFR1、CRFR2受体作用及下调下丘脑相应受体的表达,试图探索CRF受体与烧伤高代谢反应的关系;建立烧伤后早期肠道营养与静脉营养模型,观察两种不同的营养支持途径对烧伤大鼠代谢指标及下丘脑CRF相关肽及其受体的影响,为肠源性高代谢理论进一步提供依据;同时,本实验还对烧伤后早期肠道营养对回肠粘膜CRF相关肽及其受体的表达情况进行动态观察,进一步探讨早期肠道营养的作用机制。一、方法1.采用大鼠30%TBSAⅢ度烧伤模型,观察第三脑室置管应用CRFR1、CRFR2受体反义核酸及CRFR1、CRFR2受体特异拮抗剂对烧伤后第7天静息能量消耗(REE)的影响,通过RT-PCR检测应用反义核酸后下丘脑CRFR1mRNA、CRFR2mRNA的表达变化,通过Western blot检测应用反义核酸后下丘脑CRFR1、CRFR2蛋白表达变化。通过免疫组化及免疫荧光观察应用反义核酸烧伤后对下丘脑CRFR2表达变化情况。2.建立大鼠30%TBSAⅢ度烧伤早期肠道营养与静脉营养模型,动物随机分为正常对照组、早期肠道营养组和静脉营养组,观察烧伤后静息能量消耗(REE)变化情况及早期肠道营养对烧伤高代谢反应的影响。检测伤后1、3、5、7天不同时相点血浆ACTH、CORT、CA及血清TNFα、IL-1β、IL-6的变化情况,检测下丘脑CRFR2mRNA及蛋白表达变化。同时检测下丘脑CRFmRNA、UCN1mRNA的变化情况。3.建立大鼠30%TBSAⅢ度烧伤早期肠道营养与静脉营养模型,动物随机分为正常对照组、早期肠道营养组和静脉营养组,观察烧伤后回肠粘膜层CRFR1、CRFR2受体mRNA及蛋白水平表达的变化情况,以及早期肠道营养对回肠CRFR1、CRFR2受体表达的影响。同时观察烧伤后回肠粘膜层CRFmRNA、UCN1mRNA水平表达变化情况,以及早期肠道营养对回肠CRFmRNA、UCN1mRNA表达的影响。通过免疫组化检测分析回肠粘膜层CRFR1、CRFR2受体的表达情况。二、结果1.与CRFR2ODN组比较,CRFR2ASODN可明显降低烧伤静息能量消耗(REE),与CRFR1ODN组比较,CRFR1ASODN无明显降低烧伤高代谢反应作用。Astressin2-B可明显降低严重烧伤高代谢反应,Antalarmin未见明显降低烧伤高代谢反应作用。CRFR1ASODN、CRFR2ASODN明显下调严重烧伤大鼠下丘脑CRFR1mRNA、CRFR2 mRNA及蛋白水平表达。2.烧伤后REE均呈逐渐增高的趋势,EN组REE在伤后第3、5、7天明显低于PN组(P<0.05)。血浆ACTH、CORT烧伤后均明显增高,伤后第三天以后逐渐下降,EN组在伤后第3、5天明显低于PN组(P<0.05)。血浆儿茶酚胺(CA)浓度伤后第1天最高,以后逐渐下降,EN组在伤后第3、5天明显低于PN组(P<0.05)。烧伤后血清肿瘤坏死因子(TNFα)、IL-1β、IL-6浓度呈逐渐升高趋势,EN组在伤后第3、5、7天明显低于PN组(P<0.05)。3.烧伤后下丘脑CRFR2mRNA及蛋白表达均呈明显增加趋势,EN组下丘脑CRFR2mRNA及蛋白的表达在伤后第5、7天明显低于PN组。烧伤后下丘脑CRFmRNA及蛋白表达呈下降趋势,在伤后第三天降低至最低,以后逐渐回升,EN组下丘脑CRFmRNA及蛋白表达在伤后第3、5、7天明显高于PN组。烧伤后下丘脑UCN1mRNA的表达呈逐渐增高趋势,EN组下丘脑UCN1mRNA的表达在伤后第5、7天明显低于PN组。4.正常大鼠回肠粘膜层均有CRFmRNA、UCN1mRNA、CRFR1mRNA、CRFR2mRNA表达,烧伤后CRFmRNA、UCN1mRNA、CRFR1mRNA、CRFR2mRNA表达明显增加,早期肠道营养可下调回肠粘膜CRFmRNA、CRFR1mRNA和蛋白的表达,增加UCN1mRNA、CRFR2mRNA和蛋白的表达。5.免疫组化结果显示正常大鼠回肠肌间神经丛、粘膜下神经丛、粘膜层均有CRFR1、CRFR2表达,烧伤后CRFR1、CRFR2表达明显增加,早期肠道营养可下调回肠粘膜CRFR1的表达,增加CRFR2的表达。三、结论1.下丘脑CRFR2受体可能参与严重烧伤大鼠高代谢反应调节,CRFR1受体在烧伤高代谢反应中作用可能不是主要的。2.早期肠道营养可减低严重烧伤大鼠高代谢反应,可能通过降低烧伤大鼠血浆ACTH、CORT、儿茶酚胺、血清TNFα、IL-1β、IL-6浓度起作用。3.早期肠道营养下调下丘脑CRFR2mRNA及蛋白的表达,早期肠道营养可能通过下调下丘脑CRFR2mRNA及蛋白的表达降低烧伤高代谢反应。下丘脑UCN1可能参与烧伤高代谢反应的形成,下丘脑CRF可能与烧伤急性应激反应有关。4.回肠肌间神经丛、粘膜下神经丛、粘膜层均有CRFR1、CRFR2受体表达。早期肠道营养可明显下调回肠粘膜层CRFmRNA、CRFR1mRNA和蛋白水平的表达,增加回肠粘膜UCN1mRNA、CRFR2mRNA和蛋白水平的表达。更多还原

【Abstract】 The hypermetabolism is an important part of the stess response to burn injury.Suitable response of hypermetabolism is beneficial,and excessive response is harmful to burn organism.The central nervous system play an important role in burn hypermetabolism response.More evidences showed that there were intercommunications among nervous,immune and endocrine system,which gave us new idea of burn hypermetabolism mechanism and metabolism intervention.The regulation mechanism of energy balance is more complicated.The hypothalamus is a center of energy balance regulation.The burn injury hypermetabolism is also regulated by hypothalamus.The hypothalamus play a critical role in energy balance.Along with neurobiology advancement in twenty years,many hypothalamic neuropeptides and neurotransmitters related to energy balance were discovered gradually,which provided us with more importance rationale to search for burn injury hypermetabolism mechanism.The anabolic neuropeptides provoke ingestion,lower energy expenditure,and catabolic neuropeptides restrain ingestion,increase energy expenditure.The corticotrophin releasing factor(CRF) system belongs to catabolic neuropeptides.There were some evidences of CRF related peptides and its receptor roles in stress and energy metabolism. Roles of many other neuropeptides may go through CRF system.We presumed that the hypothalamic CRF related peptides and its receptors may play an important role in burn stress and hypermetabolism response. The hypothalamic CRF related peptides include CRF ,Urocortin1(UCN1),UCN2 and UCN3. Corticotropin-releasing factor (CRF) is a 41-residue peptide characterized from ovine hypothalamus by Vale et al in 1981.Within the brain, CRF binds to two receptor types,referred to as the CRFR1 and the CRFR2 receptors.The corticotrophin releasing factor (CRF) system could play diverse pathophysiologic role in stress response. The CRFR1 is the primary receptor that activates the secretion of ACTH and cortisone of hypothalamic-pituitary-adrenal(HPA) axis in response to a stressor,CRFR2 receptor regulates energy balance.Urocortin1 is a 40-amino acid peptide expressed in the mammalian brain.Urocortins show a particularly high specificity for CRFR2 receptor.Recent investigations have demonstrated that urocortins could play a significant role in the regulation of burn injury hypermetabolism.Recently,CRFR1,CRFR2 receptor specific antagonist had been discovered successively,astressin2-B was specific antagonist of CRFR2 receptor,antalarmin was specific antagonist of CRFR1 receptor.Meanwhile,antisense nucleic acids technique was a good way which can lower gene expression.This specific antagonist of CRF receptors and antisense nucleic acid technique had become a valuable tool for in vivo characterization of CRFR2 receptor function.Hypermetabolism of burn injury is mainly from wound,the gut is another importance source.The intestinal tract is a central organ to manifest surgical stress.The view that hypermetabolism is regulated by gut has gained acceptance in recent years.On the basis of many animal experiments and clinical studies in our institute,we concluded that early enteral nutrition can lower burn injury hypermeatbolism,and proposed“Enterogenous Hypermetabolism”theory.Early enteral nutrition is an essential stimulant to the growth and renewal of the mucosal cells of gastrointestinal tract.It could regulate the gastrointestinal tract movement,secretion and cell metabolism,lower intestinal ischemia injury,preserve the structure and function of gastrointestinal tract,reduce intestinal bacteria/endotoxin translocation,decrease hypermetabolism response.But,studies now only refer to the effects of early enteral nutrition on REE and the structure and function of gastrointestinal tract.As is known to all that hypermetabolism is regulated by central nervous system,there were no any data of central neuropeptides related to burn hypermetabolism by gut and the effect of early enteral nutrition on burn hypermetabolism.We suppose that the effect of early enteral nutrition on REE is through central neuropeptides related to metabolism.CRF related peptides and its receptors were also discovered expressing in gastrointestinal tract.Functional studies demonstrated that peripheral injection of CRF or Ucn 1 can also stimulate colonic transit and defecation,inhibit gastric emptying in rats.CRF or UCN 1 didn’t pass through blood-brain barrier,it indicated that there were peripheral CRF,UCN 1,CRFR1 and CRFR2 receptors in gastrointestinal tract.Recently,some published data show that CRF related peptides and its receptors in gastrointestinal tract mucosa,submucous layer play an important role in gut stress response,inflammatory reaction and mucosal barrier function.But until now,there is no data showing that the changes of CRF related peptides and its receptors and effects of early enteral nutrition on CRF related peptides and its receptors in gastrointestinal tract after burn injury.ObjectivesTo investigate the relation between CRF receptors and burned hypermetabolism response,and observe the effects of early enteral nutrition on hypermetabolism,hypothalamus and ileum mucosa CRF related peptides and its receptors.Methods1. In this study we employed a stainless-steel cannula implanted into the 3rd ventricle animal model, after at least one week to recover from these operations, SD rats were inflicted with 30% TBSA full thickness flame burn on the back,to observe the effects of CRFR1ASODN,CRFR2ASODN and astressin2-B,antalarmin on resting energy expenditure(REE) at PBD7.The hypothalamic CRFR1mRNA,CRFR2mRNA expression level were determined by RT-PCR at PBD7 after CRFR1ASODN,CRFR2ASODN injection.The changes of hypothalamic CRFR1,CRFR2 were determined at PBD7 after CRFR1ASODN, CRFR2ASODN injection by Western blot and immunohistochemistry.2. The animal model of early enteral nutrition and parenteral nutrition was employed,SD rats were divided randomly into control group,parenteral nutrition(PN) group and early enteral nutrition(EN) group,and inflicted with 30% TBSA full thickness flame burn on the back.The resting energy expenditure(REE) were determined at PBD1, 3, 5, 7 respectively,and plasma ACTH,CORT,CA,and serum TNFα,IL-1β,IL-6 were detected at 1d, 3d, 5d, 7d postburn respectively.The hypothalamic CRFR2mRNA,CRFmRNA and UCN1mRNA were detected respectively by RT-PCR.The hypothalamic CRFR2 and CRF were detected respectively by Western blot.3. We employed early enteral nutrition and parenteral nutrition animal model, SD rats were divided randomly into control group,parenteral nutrition(PN) group and early enteral nutrition(EN) group,and inflicted with 30% TBSA full thickness flame burn on the back.The ileum mucosal CRFR1mRNA,CRFR2mRNA,CRFmRNA and UCN1mRNA were detected respectively by RT-PCR.The ileum mucosal CRFR1,CRFR2, CRF were detected respectively by Western blot.The ileum CRFR1 and CRFR2 immunohistochemistry expression were investigated.Results1. REE in CRFR2ASODN group were significantly lower than that of CRFR2ODN group.There was no significant difference of REE between CRFR1ASODN group and CRFR1ODN group.Compared with burn control group,astressin2-B lowered REE significantly.There was no significant difference of REE between antalarmin group and burned control group.CRFR1ASODN,CRFR2ASODN significantly lowered hypothalamic CRFR1mRNA,CRFR2 mRNA and protein level expression in burned rats.2. There was an obvious increasing trend of REE after severe burn injury.REE in EN group was significantly lower than that of PN group on PBD3,5,7 respectively (P<0.05).Plasma ACTH and CORT increased and peaked at PBD3,and subsided gradually after PBD3.Plasma ACTH and CORT in EN group were significantly lower than that of PN group on 3d,5d postburn respectively(P<0.05).Plasma CA increased and peaked at PBD1,and subsided gradually after PBD1.Plasma CA in EN group was significantly lower than that of PN group on 3d,5d postburn respectively(P<0.05).Serum TNFα,IL-1β,IL-6 increased gradually after burn injury, Serum TNFα,IL-1β,IL-6 in EN group were significantly lower than that of PN group on 3d,5d,7d postburn respectively(P<0.05).3. The hypothalamic CRFR2mRNA and protein expression increased gradually after burn injury. The hypothalamic CRFR2mRNA and protein expression in EN group were obviously lower than that of PN on 5d,7d postburn respectively.The hypothalamic CRFmRNA and protein expression decreased gradually after burn injury and peaked at PBD3.The hypothalamic CRFmRNA and protein expression in EN group were obviously higher than that of PN on 3d 5d,7d postburn respectively.The hypothalamic UCN1mRNA expression increased gradually after burn injury ,and UCN1mRNA expressions in EN group were obviously lower than that of PN on 5d,7d postburn respectively.4. The ileum mucosa CRFmRNA,UCN1mRNA,CRFR1mRNA,CRFR2mRNA expression in normal control group rats were lower and increased gradually after burn injury.Early enteral nutrition lowered obviously ileum mucosa CRFmRNA, CRFR1mRNA and protein expressions, and increased UCN1mRNA,CRFR2mRNA and protein expressions.5. The immunohistochemistry results also indicated that CRFR1 and CRFR2 receptor expressed in myenteric nerve plexus,submucous layer and mucosa of normal rats and increased significantly after burn injury.Early enteral nutrition lowered CRFR1 expression and increased CRFR2 expression in ileum mucosa.Conclusions1. The hypothalamic CRFR2 receptor is a main regulator and CRFR1 receptor may be of no importance in severely burned hypermetabolism response.2. Early enteral nutrition can lower severely burned hypermetabolism and plays very important roles in mediating hypermetabolism by decreasing plasma ACTH, CORT,CA and serum TNFα,IL-1β,IL-6.3. Early enteral nutrition plays important roles in lowering hypermetabolism by decreasing hypothalamic CRFR2mRNA and protein expression.The hypothalamic UCN1 may take part in hypermetabolism response, hypothalamic CRF may relate to burn stress response.4. CRFR1,CRFR2 expression in rat myenteric nerve plexus, submucous layer and mucosa were discovered.Early enteral nutrition lowered obviously ileum mucosa CRFmRNA,CRFR1mRNA and protein expressions,and increased UCN1mRNA, CRFR2mRNA and protein expressions.更多还原