Effects Of Ulinastatin On Intestinal Mucosa Barrier In Mice With Severe Heatstroke

BACKGROUND ANDAIMSBy far,it was widely acceptedthat,the pathological physiology response of heatstroke is not only caused by direct damage expose to heat,but more dependent on the process of SIRS which secondary to thermal injury then trigger MODS.Previous studies have suggested that intestinal is the body's largest "bacteria storage" and"libraryofendotoxin",participateinthe pathophysiologic process of SIRS and MODS with its own unique physiological characteristics,and hence considered as the"motive power of MODS".The intestinal mucosal barrier function could change by various factors,the stimulus caused the dysfunction of the intestinal mucosal barrier,which render the translocation of intestinal bacterial,lead to endotoxin,bacteria,antibody medium continuously into the blood and lymph,resulted in a variety of release of inflammatory mediators and finally initiate and aggravate the SIRS.SIRSin turn exacerbate the intestinal injury and so it goes on,finally cause MODS.Gastrointestinal lesions have been found in the study of the severe heatstroke,including the intestinal mucosa damage,apoptosis of intestinal epithelial cells,increased intestinal permeability and so on.Lambert studied the responses of rat intestinal(including duodenum,jejunum,ileum and colon)during high fever hit,the intestinalepithelial cells damage was found when the core temperature reach 42.5 ?,including loss of microvilli,tight junction open,the mitochondrial swelling,cavitation and permeability of Fluorescein is thiocyanate(FITC)-glucan increased.The main symptoms including gastrointestinal hemorrhage and endotoxemia were always seen in the clinical cases died of heat stroke,which means the dysfunction of intestinal mucosa induced by heatstroke has close relationship with SIRS.It has also been proved later by a series of research that heatstroke has something to do with intestinal derived LPS,remove of LPS or the therapy of endotoxemia could has a better outcome.These results suggest that inflammatory cytokines in endotoxemia could in turn exacerbate the intestinal injury and so it goes on.Therefore,to prevent the intestinal endotoxemia and bacterial translocation during heatstroke,"the second critical point "aim at improving the intestinal mucosal barrier function may also be emphasized besides reduction of the coretemperature.UTI is a urinary trypsin inhibitor isolated and purified from male urine and widely used in clinical,it can inhibit phospholipase A2,trypsin,elastase and other hydrolytic enzymes;in addition,it could improve blood circulation,inhibit the generation of myocardial depressant factor and the release of inflammatory mediator,besides,it can also stabilize the lysosomal membrane.It has been proved in recent studies that UTI has protective effect on the damage of intestinal mucosa induced by different diseases,but whether it has the same protective effect during heatstroke is rarely reported at present.Based on those studies,we use thermal heat animal model in mice to observe the effect of heatstroke on Intestinal mucosal barrier function and its relationship with SIRS;Furthermore,we use UTI pretreatment the animal before heatstroke and study the effect of UTI on severe heatstroke-induced Intestinal mucosal barrier function.METHODS AND RESULTSMethods:To established the thermal heat animal model,six to eight weeks of SPF male BALB/c mice were randomly divided into groups as follows:a control group that did not expose to heat stress,40 ? and 42 C heat stress group;to observe the effect of the drug treatment,animals were randomly divided into 42 ? heat stress group and two groups that respectively received 5000 or 10000U/kg of UTI before 42 C heat stress.The control/sham group animals keeps at room temperature(25±0.5?),while heat stress group keeps at simulation of climate chamber with temperature(35.5±0.5)? and humidity(60±5)%.The rectal temperature in mice was measured by anal rectal temperature table every 30 min,when the temperature reach 41.5 ? rectal temperature was measured every 10 min.The heat exposure was stopped when the temperature in different group arrive at their own level(40? or 42?),and the animals kept at 37? for each observation time point as follows:0?1?3?6?12?24h.In medical treatment group,the animals received daily intraperitoneal administration of UTI(5000U/kg)or(10000U/kg)for 3 days before heatstroke,while the sham group and the HS group received daily intraperitoneal administration of equal normal saline(volume)for 3 days.Animals in the successfully established heatstroke models in medical treatment group were sacrificed after rewarming 12h.Mice intestinal mucosal morphology change in different group was observed by histopathologic and electron microscope.Bacterial content in peripheral tissue and the frequency of intestinal bacteria translocation to mesenteric lymph nodes,liver,spleen and kidney was measured by bacteriologic method.Limulus reagent colorimetric method was used to detect the serum endotoxin,plasma LPS,IL-6,and the content of TNF-a in vena cava were measured by ELISA,the apoptosis rate of intestinal mucosal epithelial cell was tested by TUNEL method,and expression of apoptosis-relative protein caspase-3 was measured by immunohistochemistry.Statistical analysis:All data were analyzed for statistical significance using SPSS 13.0 software,Data were expressed as means±SD,statistical comparisons of the results were made using analysis of One-way ANOVA,linear regression and correlation analysis(Pearson test),p<0.05 was considered statistically significant,and p<0.01 the difference was very obvious.Results:1.By established heatstroke models,Limulus reagent colorimetric method and ELISA were used to study the intestinal mucosa changes on the influence of early systemic inflammatory response after heatstroke.The change of survival time and survival rate of the mice after heatstroke was evaluated by Kaplan-Mier and Log Rank test.The results showed as follows:1.1 The core temperature appear as a "twin peaks" change during the process of heatstroke,15min before exposed to heatstroke,the temperature of the mice rise rapidly reach 39 ?,and Quickly fell to nearly 38 ?after 15?30min,after then,the temperature rise gradually with the time of heatstroke,before it reach 41 ? it had a long stage that temperature rise steadily,but after it reach 41?,the rising range increase quickly,and rapidly rise above 42 ?.When the temperature reach 42 ?,the vital signs are extremely unstable,and the temperature could rise sharply to 43 ? above in just 10?30 min and result in death of the mice.In this study,we chose 40 ? and 42 ? as the target core temperature for rewarming observation,and found that the rewarming temperature after different degrees of heatstroke drop rapidly in the short time(30min),the heavier the heatstroke degree is,the lower the rewarming temperature is.1.2 The observation of SIRS in mice the plasma LPS,IL-6,and the content of TNF-ain vena cava are related to the degree of heatstroke and rewarming time.Compare to sham group,the plasma LPS began to rise in 40 ? group after rewarming 1h,after rewarming 3h,it rose significantly and reaches its peak at 24h;while in 42 ?group,the plasma LPS rose markedly after heatstroke,the rising range is more obvious than 40?group.Compare to control and 40? group,the plasma concentration of IL-6 andTNF-ain 42°C group rise more obviously and appear as the rewarming time dependence,the difference between three groups had significant statistical significance.Compare to control group,the plasma concentration of IL-6 and TNF-a in 40? group also has a significant increase,and this difference shows statistical significance(p<0.05)1.3 The survival analysis of the mice In this study,the survival time of the mice in both group(control and 40? heatstroke group)is longer than 72h,while in 42? heatstroke group,after rewarming 72h,all mice were dead,the mortality was 100%.And most of them die within 24 h;the median survival time is 18h2.1 HE staining to observe the intestinal mucosa damage In 40? heatstroke group,the thickness of the ileum mucosa is well-distributed,the intestinal epithelium was relatively complete,the microvilli ranged regularly and there was a bit loosen inlamina propria with the infiltration of a few lymphocytes and inflammatory cells.Intestinal mucosa atrophy was obviously in 42?group,there has severe separation in mucosa lamina propria,the microvilli was almost fall out completely,the lamina propria was uncovered and hydropic with infiltration of lymphocytes and neutrophil granulocytes.It has significant statistical significance in villus height,thickness and surface area of small intestinal between control and heatstroke group(p<0.05)2.2 Electron microscope observation of intestinal mucosa damageIn 40? heatstroke group,the microvilli on the surface of intestinal mucosal epithelial cell ranged regularly,Part of the epithelial cell had light swelling,the mitochondrial swelling and had focal cavitation,rough endoplasmic reticulum had slight dilation;in 42? heatstroke group,the microvilli are sparse,ranged irregularly and lodging,with part absence,gap of intercellular space widened and formation of a large number of digitation,part of the tight junction broadening or open,vacuole bodies appeared in part of the epithelial cytoplasm,endoplasmic reticulum to expand,chondriosome to swell.Compare to control group,theultra-microstructure of the intestinal mucosa show significant change in heat stress group.2.3 The bacteria detection in different organs of mice after heatstroke There was no difference between control and 40'C heatstroke group(p>0.05),the detection in 42 ? heatstroke group is significantly greater than control and 40 ?heatstroke group(p<0.05)2.4 The dysfunction of intestinal mucosa during the early stage of heatstroke Compare to control group,the activity of DAO slightly elevated in 40? heatstroke group,but prominently in 42? heatstroke group,it has significant statistical significance compare to control and 40? heatstroke group(p<0.05).The variance of D-lactic acid concentration after heatstroke was different to the activity of DAO,there was no difference between control and 40? heatstroke group,thelevel was significantly higher in 42? heatstroke group and it has significant statistical significance compare to control and 40? heatstroke group(p<0.05).2.5 The correlation analysis of intestinal barrier dysfunction and systemic inflammatory response linear regression and line correlation analysis were made to study the relationship between the indicator of intestinal barrier dysfunction(activity of DAO and concentration of D-lactic acid)and SIRS(TNF-a?IL-6 and LPS).Research findings show as follows:Theplasmmarkerof inflammatory(INF-a?IL-6 and LPS)was significantly positively related to the activity of DAO and the concentration of D-lactic acid,which means the dysfunction of intestinal mucosa plays a very important role in heatstroke induced SIRS.2.6 Intestinal mucosa cells apoptosis in situ were detected by tunnel In 42?heatstroke group,there were many apoptotic cells in villus tips and recess.The Apoptosis rate were 3.95±0.96,9.55±2.20 and 33.72±6.14 in control,40? and 42? group,compare to control,the apoptosis rate in heatstroke group was statistically significant(p<0.05).2.7 The correlational analysis was conducted in intestinal mucosa epithelial cells apoptosis index and intestinal mucosa dysfunction and SIRS,research findings show as follows:1.AI is highly correlated with the activity of DAO and the concentration of D-lactic acid.2.AI has significantly positive correlation with the level of TNF-?and IL-6.3.We use the method mentioned above to observe the protective effect of UTI pretreatment towards intestinal mucosal barrier during early stage of heatstroke.Survival analysis was made between severe heatstroke mice.We found that compare to heatstroke group,the morphology and ultrastructure of mice intestinal mucosa and intestinal barrier function had significant improvement in UTI pretreatment group.The bacterial metathesis rate?LPS and inflammatory cytokines IL-6 and TNF-a showed significantly decrease,systemic inflammatory response decreased significantly in severe heatstroke mice and intestinal mucosa epithelial cells apoptosis index decreased markedly;At the same time,the survivalanalysis indicatedthat the survival time and survival rate of severe heatstroke mice had significant improvement in UTI pretreatment group.Moreover,the improvement is more obvious in high dose group(10000U/kg).conclusion:In this study,we successfully replicated the heatstroke animal model established by our prophase research,and elaborated the mechanism of intestinal mucosal barrier dysfunction during early stage of heatstroke and its relationship with SIRS in vivo,and gives a preliminary discussion on the protective effect and mechanisms of-Through research we found that:(1)The temperature changes and survival time of the heatstroke animal related to the core body temperature of the mice.(2)Intestinal mucosa dysfunction after heatstroke may be the main pathogenic mechanism of SIRS or even of the MODS,among them,large amount of apoptotic intestinal epithelial cells may be the important factor of intestinal mucosal barrier dysfunction.(3)UTI could effectively reduce the apoptosis of intestinal epithelium during severe heatstroke,thus alleviate the intestinal mucosa injury and systemic inflammatory response,And Improve Prognosis of the animal.