| Objective:Sepsis is one of serious complication for critically ill patients with trauma,empyrosis,shock,infection etc and major reason of septic shock and multiple organ dysfunctionsyndrome(MODS).UP to date,Sepsis is one of leading causes of death for critically ill patients and the utmost obstacle for improving successfull treatments.Inflammation reaction mechanism with sepsis people already have studied several decades to offer effective measures for spesis treatment and intervene.However,So far the result cannot be desired.The theory of neuroendocrine-immune regulation network provides an opportunity to further understand it,which the HPA axis specially paid more attention. hypothalamus-pituitary-adrenal(HPA)axis is the hub of neuroendocrine-immune network that plays part in infection,trauma etc stress response.We investigate the changes of the HPA axis in sepsis progress to realize essence of sepsis and open up new approach for clinical treatment of sepsis.Acidoglycoprptein(AGP)secreted by liver,called ulinastatin,is a human endogenous inflammatory inhibitor extracted and purified from human urine.It can inhibit serious hyperfunction protease and inflammatory factors to release,get out of oxyradical,inhibit the production of myocardial depressant factor,control excessive inflammatory reaction and reduce the damage of organism,improve organism recovery.This study adopts cecal ligation perforation (CLP) method copy rats sepsis model,we observe the level changes of adrenocorticotropic horm- one(ACTH),corticosteroids ketone (CORTT grants) in plasma,the change of corticotropin-releasing hormones mRNA(CRHmRNA) in hypothalamus tissue and the change of tissue ultrastructural in early sepsis rats, without and with the use of ulinastatin.The main purpose is to reveal whether ulinastatin affect the adrenocorticotropic hormone,corticotropic cortical hormone in plasma and corticotropin-releasing hormones mRNA in hypothalamus tissue and the mechanism.Methods:In this study, the septic rat models were challenged by CLP,36 Male Wistar (250-300g) rats were randomly divided into 4 groups(1)normal group:do not give any treatment,and directly executed;(2)sham group:the cecum was exposed but not ligated or punctured;(3)sodium chloride group(NS group): treated with saline of 10mL/kg through abdominal injection after CLP;(4)ulinastatin group:treated with ulinastatin of 100.000U/kg through abdominal injection after CLP,the concentation of ulinastatin was 10,000 units/mL.During recovery rats were administered 30mL/kg of normal saline in the abdomen,added the loss of volume and physiological requirements during the surgery.2ml blood was taken from the jugular vein 6 hours after surgery in each rat,the plasma were preserved in -80°C and the hypothalamus tissues were preserved in nitrogen canister.Radioimmunoassay (RIA) was used to detect the levels of the CORTT and ACTH in the plasma,the expression of CRHmRNA in hypothalamus tissues was detected with reverse transcription-polymerase chain raction (RT-PCR) and was semiquantitatively analyzed by Gel-pro Analyzer software.The histopathological morphologic changes in 1mm3 hypothalamus,pituitary and adrenal specimens were determined with transmission electron microscopy technique in Saline and Ulinastatin group.Statistical analysis was performed using SPSS13.0 software package.Data were expressed as mean±SD if meet normality,one-way ANOVA completely was used when homogeneity of variance and nonparametric rank test used when variance not neat.P<0.05 was considered significant for all statistical analyses.Results:1 The general stituation of rats in the experiment:the rats in Sham group analepsiaed earlier than that in other groups and act more actively,no abdominal dropsy and evil smelling were found when their abdomens were opened.The rats in the NS group analepsiaed later than that in the Sham group,and then were depressed with inaction,piloerection,canthus secretions increase,dyspnea and shivering,When the abdomen was opened, bloody ascites and evil smelling were found and bowels looked edema,the cecum was darkened in some rats.Compared with the NS group,the clinical manifestations in the rats of Ulinastatin group were better.2 The ultrastructural changes in hypothalamus, pituitary and adrenal tissues were observed by electron microscope2.1 The ultrastructural changes in hypothalamus tissueIn NS group,rough endoplasmic reticulum(RER)expansion and had degranulation phenomenon,the majority of the mitochondrial cristae and membrane were merged together and blurred,part cristae had ruptured.In Ulinastatin group,the interstitial RERs cells arranged in surrounding and a smaller number arranging disorderly,olgi complex scattered in perinuclear and chondrosome in cytoplasm.2.2 The ultrastructural changes in hypophysis tissueIn NS group,a large number of endocrine granules could be seen in the ACTH-releasing cells and larger nucleus,high electron density,the rough endoplasmic reticulum showed expansion and degranulation phenomenon;The ribosomals were indistinct,Membrane fusion,disappear,edema,cavitiation phenomenon were found in the majority of mitochondrial cristae.In Ulinastatin group,little and little neurosecretory granules could be found in the ACTH-releasing cells and oval-shaped dense core,with low electron density annular halo in part endocrine granule and lamellar mitochondrial.In NS group,the cytoplasmic edema were observed in the fascicular zone of the adrenal cortex and lipid droplets diminished,Adrenal cortical zona fasciculata cells cytoplasmic edema;reduced smooth endoplasmic retic- ulum;mitochondrial edema,fusion and disarray of most of the crista- es,indistinction and fusion of part of the double membrane;the rough endoplasmic reticulum showed expansion and degranulation phenomenon.In Ulinastatin group,the fascicular zone of the adrenal cortex cells were larger and cytoplasmic rich,color shallow and more lipid droplets;air-water bubbly mitochondrial,a lot of smooth endoplasmic reticulum.3 Compared with the normal control group or sham group,the levels ofCRHmRNA in hypothalamus tissue (1.151±0.120VS1.151±0.120,1.151±0.120 VS0.797±0.130,p<0.05)and the ACTH(52.387±5.339pg/mlVS 21.704±0.492pg/ml,52.387±5.339pg/mlVS24.354±1.238pg/ml,P<0.05),CORT(24.446±0.561ng/mlVS12.459±1.472ng/ml,24.446±0.561ng/ml VS 12.486±2.614 ng/ml,P<0.05) in plasma in saline group were increased in the early stage of sepsis.4 Compared with the saline group,the levels of CRHmRNA in hypothalamus tissue (0.603±0.069VS1.151±0.120,p<0.05) and the ACTH(24.832±2.426 pg/mlVS52.387±5.339pg/ml,P<0.05),CORT(19.686±0.498ng/mlVS24.446±0.561ng/ml,P<0.05) in plasma in ulinastatin were decreased in the early stage of sepsis.5 There was indifference of the level of ACTH and CORTT(24.354±1.238pg/mlVS24.354±1.238pg/ml,12.486±2.614ng/mlVS21.704±0.492pg/ml,P>0.05)in plasma between sham group and control group.Compared with the control group,the levels of CRHmRNA in hypothalamus tissue (0.797±0.130 VS0.412±0.038,P<0.05) in sham group were increased. |
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