Protective Effects Of Ulinastatin On Lung Injury Caused By Heat Stress In Rats

Study background:The morbidity of heatstroke may increase with global warming and the increasing in the intensity and frequency of heat waves, the heat stroke has become one of common clinical critical disease. If not treated timely and properly, the mortality rates as high as40%to50%. The mechanism of tissue damage and organ dysfunction caused by heat stroke is not fully understood, for decades generally agreed the point that heat stroke is a complex interplay of body temperature regulation obstacles, acute phase reaction and HsP(heat stress protein) abnormal expression. However, recent clinical and experimental evidence suggests that the mechanism of severe heat stroke is not only because of dierect damage of heat stress, but also is stepped by systemic inflammatory response syndrome (SIRS), even by multiple organ dysfunction syndrome (MODS) process.Ulinastatin (UTI) is a kind of the trypsin inhibitor that is separated and purified from human urine, which belong to the Kunitz serine protein hydrolysis enzyme inhibitors.UTI contains a glycosylation area and two Kunitz reactive functional domains, glycosylation area can stable lysosome and suppress excessive release of inflammatory cytokines,two Kunitz reactive functional domains can inhibit a variety of hydrolytic enzymes, such as protease G, elastase, fibrinolytic enzyme. At the same time, the low molecular components of UTI break down also have the function of inhibition hydrolytic enzymes, so the UTI can inhibit protein, sugar and lipid hydrolase activity, removal of oxygen free radicals, stabilize lysosomal membrane, suppress excessive release of TNF-α, IL-1and IL-8, improve the microcirculation, and reduce tissue ischemia-reperfusion injury.UTI has been widely used in the treatment of acute pancreatitis, septic shock and DIC, which played an important role in the protective of multiple organ.Objective:We hope to use an experimental animal model which can imitate severe heat stroke induced by hyperthermia, observe characteristics of lung injury caused by heat stress in rats. On the other hand,we try to explore whether ulinastatin given at different time point can protect lung injury caused by heat stress in rats.To guide the heat stroke in clinical treatment practice.Materials and methods1.Experimental animal and groups60adult male specific pathogen-free SD rats, bodyweight180-220g, were randomly assigned to5groups, each group with12.(1)Group C(normal control group) rats stay at room temperature.(2) Group H (high temperature group) rats treat with high temperature but without UTI.(3) Group HU(high temperature+UTI group) rats are injected with UTI at the beginning of heat stress.(4)Group HU1(inject UTI after high temperature1h group) rats are injected with UTI5×104U/kg after high temperature1h.(5) Group HU2(inject UTI after high temperature2h group) rats are injected with UTI5×104U/kg after high temperature2h.2. The preparation of animal modelsAnesthesia administered by intraperitoneal injection of3%pentobarbital sodium (45mg/kg).after anesthesia success,femoral vein puncture and tube insertion.and then put the rats into the biological oxygen tank which temperature of35℃, humidity60%.rats are injected with UTI or the same amount of saline solution according to the groups at the beginning of heat stress, heat stress1h and heat stress2h,2.5h later, rats are taken out of the biological oxygen tank and given the corresponding processing.3.Indicators observation3.1RR and Tr:Respiratory rate and rectal temperature were recorded every30min, To (at the beginning of the experiment), record6times (T0～T150). extract arterial blood to test at the beginning of heat stress and the end of heat stress.3.2Lung wet/dry(W/D) weight ratiosAt the end of heat stress, six rats in each group were rapid thoracotomy, and taken upper left lung tissue. Used filter paper to absorb the lung tissue surface water and blood, recorded the lung tissue wet weight, Then the lung tissue was put in the constant temperature oven80℃for72h, recorded dry weight. Calculated lung tissue W/D.3.3Pulmonary microvascular permeability determinationSix rats in each group were injected with2%Evans blue3ml/kg from the femoral vein, and taken upper left lung tissue at the end of the experiment. Filter drain liquid surface, put the lung tissue100mg that are filled with lml formamide, then lidded them, Put them into37℃constant temperature water bath tank,48h later, centrifugate them for10min. The extravasation of EB dye in the lung was then measured by spectrophotometer reader at620nm wave length, measured optical density values. According to the standard curve, calculating the EB content.3.4Light microscopy specimen collection and observationAt the end of heat stress, rats were rapid thoracotomy, the upper right lung tissue was put in4%paraformaldehyde fixed24h, conventional dehydration with asperse essence, paraffin embedding, ultrathin slices, HE dye, then observe the pathological changes under optical microscope and take some pictures.3.5Electron microscopic specimen collection and observationsix rats in each group were rapid thoracotomy, the lower right lung tissues were cut into the volume of1mm×1mm×1mm small pieces.Immersed in2%glutaraldehyde,2%osmic acid fixed,Gradient of ethanol dehydration,epoxy resin embedding, ultrathin sectioning, lead acetate dyeing of uranium, Observation under transmission electron microscopy (sem) and take some pictures.4. Statistical analysisSPSS13.0were used to analyze the data, measurement data were reported as the mean±standard deviation(x±s). Analysis of one-way ANOVA was used to evaluate the data among groups. multiple comparison among groups were analyzed by using LSD test. the analysis of variance of repeated measurement data was used to analyze the breathing rate and rectal temperature at each time point, arterial blood gas data were analysed with paired t test, P<0.05were accepted as significant.Result:1. There is no statistical difference among groups in body weight, baseline rectal temperature and baseline breathing frequency(P>0.05).2. Breathing rate and rectal temperatureThe RR in heat treatment groups were accelerated in the heating process, there were significant difference compared with group C(P<0.05).At the time point of T6o, T90, T120and T150, the RR in group HU was lower than the group H, there was significant difference between group HU and group H(P<0.05). At the time point of T90, T120and T150, there was no significant difference between group HU and group HU1. T0～Ti50, there was no significant difference between group HU2and group H.(P>0.05). The rectal temperature in each heat treatment group was increased in the heating process, there were significant difference compared with group C(P<0.05),But there were no significant difference among heat treatment groups.3. Lung W/D and EB extravasationThe lung W/D and EB extravasation of lung in the heat treatment groups were increased in the heating process, there were significant difference compared with group C(P<0.05). The lung W/D, EB extravasation of lung in group HU and group HU1were lower than group H, and with significant difference(P<0.05), there was no significant difference between group HU2and group H(P>0.05).4.Arterial blood gasThere was significant difference in PH, PaO2and PaCO2between T150and To, Tiso.the PH、PaO2was decreased and PaCO2was increased. At the time point of To, there was significant difference in PH, PaO2and PaCO2among five groups. T150, PaCO2in group H and group HU2were higher than group HU and group HU1(P<0.05), and there was no significant difference between group HU and group HU1(P>0.05). There were no significant difference in PH and PaCO2among the heat treatment groups.5. Lung tissue pathology change under optical microscopeIn group C,The structure of alveoli was normal, bronchial wall without the congestion and edema, and there was no effusion in alveolar space. Compared with group C, different degree of pathological changes of lung tissue were appeared in heat stress groups, the most serious in group H. In group H, alveolar walls were thickened and twisted, pulmonary interstitial hyperemia, atelectasis, pulmonary hemorrhage and pulmonary emphysema. In group HU and group HU1, the surrounding tissue of bronchial was loose, alveolar walls mild edema, and there was no obvious exudation in alveolar space. In group HU2, the alveolar epithelium swelling, alveolar walls become thinner or fracture. 6.Changes in the lung tissue ultrastructure under electron microscopeIn group C, type II epithelial cells of alveolar were intact, the nuclear membrane is complete, the nucleus chromatin evenly. Mitochondria, rough endoplasmic reticulum, golgi apparatus,lysosome,and parallel arrangement of lamellar corpuscle were seen in the cytoplasmic. In group H, the mitochondrias of type II epithelial cells of alveolar were swelling, alveolar walls were thickened, neutrophil infiltration, and a large number of red blood cell debris were deposited in pulmonary alveolus. In group HU and group HU1, type II epithelial cells of alveolar were intact, cytoplasmic organelles were normal. In group HU2, the type II epithelial cells of alveolar left basement membrane, Lamellar corpuscle decrease and microvilli increased.Conclusion:(1) Heat stress can result in pulmonary capillary permeability increase, pulmonary edema and lung tissue pathology change in rats.(2) Ulinastatin can reduce the pulmonary edema and lung tissue cell damage caused by heat stress in whole-body hyperthermic rats, has lung protective effects.(3) The effect of early using ulinastatin in reducing the lung tissue damage caused by heat stress in rats is better than that of late using ulinastatin.