| Objective: Sepsis is systemic inflammatory response syndrome(SIRS)which caused by infection and one of the most common complicationsdeveloping from serious trauma,empyrosis,shock,major surgery.Thepathogenesy is complicated,the mortality is very high and no specific therapyfor treating by now.For quite some time, the pathogenesis of sepsis have beenstudied to find out the effective intervention measures, but the result waslamentable. The theory of nerve-endocrine-immunity network,in which thehypothalamic-pituitary-adrenal axis (HPAA) was under the spotlight,provided an opportunity to further investigate the pathogenesis of sepsis. As ahub of the nerve-endocrine-immunity network, HPAA is activated after stressresponses such as infection and trauma. The hypothalamic paraventricularnucleus (PVN) which in addition to participate in the neuroendocrineregulation,also have a hand in the regulation of the autonomic nerves system,is the starting point of the HPAA and integration center forneuroimmunomodulation. Previous studies have shown that nitric oxidesynthase(NOS), immediate early genes(c-fos) and corticotropin releasinghormone(CRH)coexist in the PVN. Nitric oxide (NO) catalyzed by NOS maybe involved in the regulation of c-fos, CRH and then activate the HPAA.Investigating the changes of the HPAA in sepsis contribute to realize essenceof sepsis and provide new avenues for exploring clinical interventions forsepsis.Ulinastatin purified from freshurine of normal adult male is glyco-protein.It can suppress the activity of many proteases, reduce inflammatory mediators,restrain excessive inflammatory responses, removed oxyradical, inhibitmyocardial depressant factor production, reduce the injury of the tissues and restore human to health.In this study, the septic rat models used cecal ligation and puncture (CLP)to replicate. We survey the changes of NOS expression in PVN in the earlystage of septic rats, investigate the effect and mechanism of action of NO onthe HPAA, observe the effect of ulinastatin on the expression of NOS in PVNand further explore the regulating action of ulinastatin on HPAA in the earlystage of septic rats.Methods: In this study, the septic rat models used CLP to replicate.40healthy male Wistar rats of clean grade with weight200～250g wererandomly divided into4groups:(1) Control group: do not give any disposal,directly put to death;(2) Sham group: only open surgery, pull out and turn thececum, do not ligate or puncture, sew up the cut at last.(3) NS group: treatedwith saline of10ml/kg through intraperitoneal injection after CLP;(4) UTIgroup: treated with ulinastatin of100,000U/kg through intraperitonealinjection after CLP, the concentration of ulinastatin was10,000U/ml.Cephalic artery intubatton was to observe blood pressure and heart ratebefore CLP in Sham group, NS group and UTI group. Saline of30ml/kg wereinjured under the skin of thigh after CLP to replenish the volume of fluid lossand physiological minimum requirement during oper-ation in each group. Thesedated rats were killed by decollation six hours after CLP surgery in eachgroup. The tissues of hypothalamic paraventri-cular nucleus were removedand wrapped in foil on ice table with stereoscopic microscope, and then frozenin liquid nitrogen. The expressions of nNOSmRNA, iNOSmRNA,c-fosmRNA, CRHmRNA in PVN were detected with reverse transcriptionpolymerase chain raction (RT-PCR). The expressions of nNOS, iNOS, c-fos,CRH protein in PVN were detected with Western blot.Statistical analysis was performed using SPSS13.0software package(SPSS Company, Chicago, Illinois, USA). Data were expressed as mean±SD(x±s), when the data fit a normal distribution. One-way ANOVA was used tocompare the means among three groups, when homogeneity of variance, if not,non-parametric test was used. The multiple comparison was performed with least-significant differenc(eLSD)method. A level of P<0.05was considered tobe statistically significant.Results:1The general condition of rats after CLPIn Sham group, the time of analepsia of narcosis was earlier than that inother groups, and limb movements were as usual. In NS group, the time ofanalepsia of narcosis was later than that in other groups, and spirits bad,activity decreased obviously, canthal secretions increased, piloerection,tachypnea. Dissection of a rat revealed bloody ascites, gut edema, darkenedcecum, foul odor in some rats. The general conditions of rats in UTI groupwere better than that in NS group.2Changes of CRH, c-fos, iNOS, nNOSmRNA expression in PVN of rats ineach group:In NS group,the level of iNOSmRNA was higher than that of Sham group,Control group and UTI group obviously (1.25±0.05VS0.85±0.05,P<0.05;1.25±0.05VS0.77±0.07,P<0.05;1.25±0.05VS0.57±0.04, P<0.05,respectively), In Sham group,the level of iNOSmRNA was higher than that ofControl group(0.85±0.05VS0.77±0.07, P<0.05)(Fig.3；Fig.10；Table1).In NS group,the level of nNOSmRNA was lower than that of Sham group,Control group and UTI group obviously(0.95±0.03VS1.04±0.02, P<0.05;0.95±0.03VS1.39±0.03,P<0.05;0.95±0.03VS1.44±0.03, P<0.05,respectively), In Sham group, the level of nNOSmRNA was lower than that ofControl group(1.04±0.02VS1.39±0.03, P<0.05)(Fig.4；Fig.10；Table1).In NS group,the level of c-fosmRNA was higher than that of Sham group,Control group and UTI group obviously (1.08±0.08VS0.68±0.09,P<0.05;1.08±0.08VS0.31±0.1, P<0.05;1.08±0.08VS0.82±0.12, P<0.05,respectively), In Sham group,the level of c-fosmRNA was higher than that ofControl group(0.68±0.09VS0.31±0.1,P<0.05)(Fig.5； Fig.9；Table1).In NS group,the level of CRHmRNA was higher than that of Sham group,Control group and UTI group obviously (0.72±0.06VS0.39±0.06,P<0.05;0.72±0.06VS0.33±0.04, P<0.05;0.72±0.06VS0.48±0.05, P<0.05,respectively),but there was no significant difference between Shamgroup and Control group(0.39±0.06VS0.33±0.04,P＞0.05)(Fig.6； Fig.9；Table1).3Changes of CRH, c-fos, iNOS, nNOS protein expression in PVN of rats ineach group:In NS group,the level of iNOS protein was higher than that of Sham group,Control group and UTI group obviously(1.73±0.18VS1.28±0.06,P<0.05;1.73±0.18VS0.76±0.05,P<0.05;1.73±0.18VS0.91±0.06, P<0.05,respectively), In Sham group,the level of iNOS protein was higher than thatof Control group(1.28±0.06VS0.76±0.05,P<0.05)(Fig.7；Table2).In NS group,the level of nNOS protein was lower than that of Controlgroup and UTI group obviously(0.46±0.05VS0.54±0.05, P<0.05;0.46±0.05VS0.66±0.05, P<0.05, respectively), but there was no significant differencebetween NS group and Sham group(0.46±0.05VS0.52±0.04,P＞0.05),aswell as Sham group and Control group(0.52±0.04VS0.54±0.05,P＞0.05)(Fig.7；Table2).In NS group,the level of c-fos protein was higher than that of Sham group,Control group and UTI group obviously(0.77±0.02VS0.64±0.05,P<0.05;0.77±0.02VS0.33±0.04,P<0.05;0.77±0.02VS0.71±0.04, P<0.05,respectively), In Sham group,the level of c-fos protein was higher than that ofControl group(0.64±0.05VS0.33±0.04,P<0.05)(Fig.8；Table2).In NS group,the level of CRH protein was higher than that of Sham group,Control group and UTI group obviously(1.41±0.10VS0.99±0.11,P<0.05;1.41±0.10VS0.84±0.11,P<0.05;1.41±0.10VS1.21±0.15, P<0.05,respectively), In Sham group,the level of CRH protein was higher than that ofControl group(0.99±0.11VS0.84±0.11, P<0.05)(Fig.8；Table2).Conclusions:1In this study, the rat model of sepsis was duplicated by cecal ligation andpuncture(CLP)successfully.2In the early stage of sepsis, the expressions of iNOSmRNA, c-fosmRNA andCRHmRNA in PVN of rats were increased, on the contrary,nNOSmRNA was decreased, and protein expression was consistent with gene expression,which indicate that NO in PVN has a dual role: on the one hand, c-fos,induced by NO which was catalyzed by iNOS, induced the production of CRHas a third messenger, and then generated the overactivation of the HPAA；onthe other hand,NO which was catalyzed by nNOS could suppress the activityof HPAA. The expression of nNOS was decreased in trauma or sepsis, and theinhibition of NO to the HPAA got weaker. The HPAA was under a state ofoveractivation.3Protein and gene expressions of iNOS in PVN of septic rats were decreasedwith ulinastatin injection, which could down-regulate the expressions of c-fosand CRH. By contrast, protein and gene expressions of nNOS were increasedand the inhibition of NO to the HPAA got intense with ulinastatin injection.All of these indicate that ulinastatin could down-regulate the overactivation ofthe HPAA and improved significantly the clinical manifestations and signs inthe early stage of septic rats. This study has promulgated the centralanti-inflammatory action and possible mechanisms of ulinastatin in treatingsepsis for the first time. |
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