【作者】 田惠玉；

【导师】 胡振杰； 张玉想；

【作者基本信息】 河北医科大学 ， 急诊医学， 2008， 硕士

【摘要】 目的:脓毒症合并相对肾上腺皮质功能不全与多器官功能障碍综合征的发生密切相关,显著增加脓毒症患者的病死率。Marik报道危重患者相对肾上腺皮质功能不全(relative adrenal insufficiency,RAI)的总体发病率为30% ~40%,脓毒症休克患者可高达60%,严重影响患者的预后。为此,早期识别及积极干预脓毒症及脓毒症休克合并RAI患者,对改善预后意义重大。但是,目前脓毒症合并RAI的发病机制尚不很清楚,伴随肾上腺皮质功能不全的发生是否存在相应的组织形态学改变,国内外尚无明确报道。小剂量糖皮质激素能显著降低脓毒症休克合并急性肾上腺皮质功能不全患者的病死率,改善预后。但目前糖皮质激素应用的时机、剂量、具体疗程均存在争议。本实验通过观察脓毒症早期大鼠的血清皮质醇浓度、肾上腺组织Toll样受体4(TLR4)、肿瘤坏死因子α(TNF-α) mRNA的表达、组织微观结构变化以及不同时间应用小剂量地塞米松对其干预的影响,判别脓毒症早期是否存在相对肾上腺皮质功能不全,并探讨其可能的发病机制,以及地塞米松替代治疗的时机,以期为临床积极治疗脓毒症及脓毒症休克,降低病死率提供有利的实验依据。方法:脓毒症动物模型采用盲肠结扎穿孔法(cecal liga- tion and puncture,CLP)复制,小剂量促肾上腺皮质激素(ad- renocor- ticotropic hormone,ACTH)刺激试验判断脓毒症早期大鼠肾上腺皮质的功能状态,以ACTH刺激前后血清皮质醇浓度无显著差异,定义为RAI。实验动物选择健康雄性清洁级Wistar大鼠260只(607123),体重250~300g,随机分为5组:(1)正常对照组(n=50);(2)假手术组(n=50):仅打开腹腔,不做CLP;(3)模型组(n=50):仅做CLP ;(4)地塞米松预先给药组(n=50):CLP术前腹腔注射地塞米10mg.kg-1;(5)地塞米松治疗组(n=50):CLP术后7h腹腔注射地塞米松10mg. kg-1。各组均分2h、4h、6h、8h、12h五个时间点,每个时间点10只大鼠。每组按时间点留取肾上腺,液氮保存做半定量RT-PCR检测TLR4、TNF-αmRNA的表达。各组8h、12h时间点动物做ACTH刺激试验,均于术后(CLP/单纯开腹术)6h腹腔注射ACTH 0.8μg.kg-1,于注射前、后30min经颈总动脉测压管采血,分别记做T0、T30,留取血清,-70℃冻存,采用液相平衡放射免疫分析法(radioimmunoa- ssay,RIA)测定血清中皮质醇浓度。此外,随机选取各组大鼠各2只,于CLP /开腹术后12h行1%多聚甲醛经主动脉灌注固定留取肾上腺皮质,做电镜切片。模型组、地塞米松预先给药组和治疗组动物做12h生存率的统计(按时间点处死的动物除外)。用SPSS11.5统计分析软件进行统计学分析,数据用均数±标准差(x±s)表示,计量资料行单因素方差分析及配对t检验,计数资料行多个样本率比较的x2检验,P<0.05为有统计学意义。结果:1各组ACTH刺激前血清皮质醇浓度的比较:模型组(13.84±4.42ng/ml)、地塞米松预先给药组(20.83±6.58ng/ml)和地塞米松治疗组(12.60±3.07ng/ml)ACTH注射前血清皮质醇浓度明显高于正常对照组(9.80±2.27ng/ml)和假手术组(8.32±2.09ng/ml)(P<0.05);而地塞米松预先给药组明显高于模型组和地塞米松治疗组(P<0.05)。2各组ACTH刺激前、后血清皮质醇浓度变化的比较:正常对照组和假手术组腹腔注射ACTH后30分钟血清皮质醇浓度(T30)较注射ACTH前(T0)明显升高(13.91±2.56 ng/ml vs 9.80±2.27ng/ml;9.41±2.83ng/ml vs8.10±1.86ng/ml),具有统计学差异(P<0.05);模型组、地塞米松预先给药组和地塞米松治疗组T30血清皮质醇浓度与T0无明显差异。3各组肾上腺组织TLR4、TNF-αmRNA表达的比较:模型组TLR4、TNF-αmRNA的表达均明显升高,TLR4于4h达高峰,TNF-α于2h即达高峰,12h两者均保持在较高水平。地塞米松预先给药组TLR4、TNF-αmRNA的表达2h均较模型组明显下降(P<0.05),但仍显著高于假手术组(P<0.05), 4h TLR4、TNF-αmRNA的表达与假手术组没有差异, 12h TLR4、TNF-αmRNA的表达明显高于假手术组(P<0.05);地塞米松治疗组TLR4、TNF-αmRNA于8、12h的表达较模型组明显下降,但仍显著高于假手术组(P<0.05)。4各组肾上腺组织超微结构变化的比较:模型组、地塞米松预先给药组和地塞米松治疗组大鼠肾上腺皮质束状带细胞均出现脂质耗减、髓鞘样小体以及管泡状嵴的线粒体嵴和膜部分或全部溶解消失的组织形态结构改变,其中以模型组为著。5各组大鼠12h生存率的比较:地塞米松预先给药组12h生存率(76.92%)明显高于模型组(33.33%)(P<0.01)和地塞米松治疗组(40%)(P<0.05),地塞米松治疗组12h生存率显著高于模型组(P<0.05)。结论:1脓毒症早期即存在相对肾上腺皮质功能不全。2脓毒症早期合并RAI大鼠肾上腺组织TLR4、TNF-αmRNA的表达明显升高,可能与脓毒症伴RAI的发生有关。3脓毒症早期合并RAI大鼠肾上腺组织TNF-αmRNA的表达高峰明显早于TLR4,由此推测,LPS引起炎性因子的释放可能并不完全依靠TLR4的识别。4脓毒症早期合并RAI大鼠肾上腺组织超微结构发生了代谢性改变,影响皮质醇的合成与分泌,可能参与了脓毒症早期相对肾上腺皮质功能不全的发生。5小剂量地塞米松治疗可以降低肾上腺组织TLR4、TNF-αmRNA的表达,减轻肾上腺组织超微结构变化,提高12h生存率,改善预后。6小剂量地塞米松早期应用效果更理想,为有效控制炎性反应,需重复给药。更多还原

【Abstract】 Objective: Sepsis ensuing relative adrenal insufficiency was related closely to multiple organ dysfunction syndromes, increasing significantly case fatality of sepsis. Marik reported that the total morbility of relative adrenal insufficiency of critically ill patients was 30%~40%, while septic shock patients was 60%, influencing prognosis of patients. Therefor, it is important to discriminate and interfere with RAI in sepsis and septic shock patients and improve prognosis. But, now mechanisms of relative adrenal insufficiency with sepsis dot not understand yet, few definite studies both home and abroad have reported that whether the corresponding changes of morphologicfunctional status exsisted following the occurance of organ dysfunction.Low-dose glucocorticoid could degrade obviously case fatality of acute adrenal insufficiency with septic shock patients, improve prognosis.Howere, dispution exist yet on oppor- tunity,dosage, time of glucocorticoid therapy. This study investigate serum cortisol concentration, the expression of Toll-like receptor-4 and tumour necrosis factor-αmRNA, changes of tissue micstruture in adrenal glands in rat in the early phases of sepsis and effects of Dexamethasone with the same dosage (low-dose) at different time, discriminate relative adrenal insufficiency in the early phases of sepsis ,and approach probalble mechanisms of that and time of glucocorticoid replactment therapy, And looking forward to providing favorable exparimental evidence for the clinical treatment ,case fatality degression of sepsis and sepsis shock.Methods: The sepsis animal model used cecal ligation and puncture (CLP) to replicate and adopted the low-dose adrenocorticotropic hormone (ACTH) stimulated test to evaluate adrenal function of sepsis animals,it is difinfy RAI that serum cortisol concentration was not significant deviation between before and after ACTH stimulation. 260 healthy male wistar rats with weight 250~300g were chosen and divided into five groups on random: (1) normal control group (n=50); (2) sham group (n=50): only cut open the celiac without CLP;(3) model group (n=50): only CLP; (4) Dexprevention group (n=50): injected 10mg.kg-1 dexamethasone into the abdominal cavity before CLP; (5) Dextreatment group (n=50): injected 10mg.kg-1 dexamethasone into the abdominal cavity 7 hours after CLP. Every group sets up five time points of 2h, 4h, 6h, 8h and 12h, every of which had 10 rats, collected adrenal according to time points of every group, stored in liquid nitrogen and detected the expressions of TLR4 and TNF-αmRNA with semiquantitative RT-PCR. The rats of time points of 8h and 12h were all injected ACTH (0.8μg.kg-1) into the abdominal cavity 6h after CLP. Before injection and 30min later, collected blood samples from arteria carotis communis catheter as T0, T30. Blood serum was stored in - 70℃and detected cortisol with radioimmuno- assay(RIA). In addition, two rats of every group were chosen on random and fixed with 1% paraformaldehyde by aorta perfusion at 12h after CLP/ open the celiac. And adrenal were taken to be electron microscopic section. Add up the survival rate of 12h of model,Dexprevention and Dextreatment groups.(without rats executed according to points.) Measurement data were presen- ted as?x±s and analyzed with indendpent-sample One- Way ANOVA and paired-sample t test , numeration data analyzed with multiple-sample ratio x2-test using SPSS 11.5 statistical program. A level of P<0.05 was considered to be statistically significant.Results1 Comparion of serum cortisol concentration before ACTH stimulation of rats.The levels of T0 of model(13.84±4.42ng/ml), Dex- prevention(20.83±6.58ng/ml) and Dextreatment groups (12.60±3.07ng/ml) were respectively higher than that of normal control and sham groups; and the levels of that of Dexprevention group was higher than that of model and Dextreatment groups respectively(P<0.05).2 Comparion of changes of serum cortisol concentration of rats between before and after ACTH stimulation.The cortisol levels of blood serum at 30min after ACTH stimulation(T30) of normal control and sham groups were higher than that of before ACTH stimulation(T0)(13.91±2.56 ng/ml vs 9.80±2.27 ng/ml;9.41±2.83 ng/ml vs8.10±1.86 ng/ml), there was statistical significance(P<0.05); While there was no statistical significance to differencite the cortisol levels of blood serum at T30 after ACTH stimulation and that at T0 of model, Dexprevention and Dextreatment groups.3 Comparion of the expressions of TLR4 and TNF-αmRNA in adrenal glands of rats.The expressions of TLR4 and TNF-αmRNA of model group obviously elevated, TLR4 reached the peak 4 hours after CLP, TNF-αreached the peak 2 hours , maintaining high level at 12 hours after CLP.the expressions of TLR4、TNF-αmRNA of Dexprevention group both were lower significantly than model group at 2h(P<0.05),but was higher than sham group, there was no statistics difference witn sham group at 4h, it was higher than sham group at 12h too(P<0.05); The expressions of TLR4、TNF-αmRNA of Dextreatment group both degraded obviously than model group at 8h and 12h,but it was higher than that of sham group (P<0.05).4 Comparion of the changes of ultramicrostructure of adrenal cortex of rats.In the groups of model, Dexprevention and Dextreatment, adrenocortical fascicular zone of rats presented the histological changes of lipid droplets lossing and myelin bodies as well as the mitochondrial tracheid crista and intima partly or completely dissolved and disappeared. especially model group.5 Comparion of survival rate of 12h of rats.The survival rate of 12h in Dexprevention group(76.92%) was higher than Dextreatment group(40%) and Model group(33.33%) (P<0.05),Dextreatment group was higher than model group(P<0.05).Conclusion:1 The rats had adrenal insufficiency at early stage of sepsis.2 The expressions of TLR4、TNF-αmRNA in adrenal glands of rats with early stage of sepsis complicating adrenal insufficiency evidently ascended, was concerned with the occurrence of relatve adrenal insufficiency with sepsis possibly.3 The peak of expression of TNF-αmRNA is higher obviously than TLR4 of rats with early stage of sepsis complicating relative adrenal insufficiency, it is supposed that releasing of inflammatory factor by LPS didn,t at all dependent on identification by TLR4.4 Adrenal gland would have metabolic structural changes in the rats with early stage of sepsis complicating relative adrenal insufficiency, influence synthesis and secretion of cortisol, participate in the occurrence of relative adrenal insufficiency at early stage of sepsis.5 low-dose Dexamethasone therapy could effectively increase the survival rate of 12 hours, improve outcome through down-regulating the expression changes of TLR4,TNF-αmRNA and lessening changes of ultramicro-structure of adrenal glands.6 Low-dose Dexamethasone application early showed best effects, in order to control effectively inflammatory reaction, this need to dosage again and again.更多还原