An Experiment About The Protective Effect Of Ulinastatin On The Rats Of Lipopolysaccharide-Induced Acute Lung Injury And Its Mechanism

Backgroud and objectivesAcute lung injury(ALI) refers to clinical manifestations of respiratory distress and refractory hypoxemia.sufferring from severe infection, the wounds, shock, acidosis and harmful gases inhalation factors.Its pathologic characteristics are pulmonary edema and micro atelectasis caused by diffusive alveolar-capillary membrane damage. Common complications of ALI are acute respiratory distress syndrome(ARDS) and multiple organ dysfunction syndrome(MODS).ALI has the high mortality rate and is a difficult problem of modern critical medical.The pathogenesis of ALI has not yet been fully clarified. It is now widespread thought that ALI is a performance in the lungs of systemic inflammatory response syndrome (SIRS), its essence is a kind of incontrollable and excessive inflammation reaction. The difference between ALI and general inflammation is there is imbalance of proinflammatory reaction and anti-inflammatory reaction in ALI.Cytokines play a crucial role in this process.Therefore, research on the balance of cytokine network and the regulation of upstream become focus.It has important significance to reveal the development of ALI as well as the treatment.Cytokines can be divided into proinflammatory and anti-inflammatory cytokines according to their role in inflammatory reaction.Tumor necrosis factor alpha(TNF-a) is the earliest release and most important endogenous medium among proinflammatoty cytokines.It plays a trigger role during inflammation.Interleukin 10 is considered the most important factor of anti-inflammatory cytokines.It acts on the multiple links of inflammation cascade in systemic inflammatory reaction process and plays a protective role.Mitogen activated protein kinase(MAPK) is a kind of serine/threonine protein kinase within the eukaryotic cells.It can phosphorylated other proteins to shift them from the cytoplasm to nuclear and then adjust the activation of transcription factors and gene expression of inflammatory cytokines. Among them,p38 signaling pathway is considered "stress induced MAPK".It is closely related with the mechanism of regulation of inflammatory reaction and may play an important role in lung tissue in early stage of ALI.Ulinastatin is a urine trypsin inhibitor(UTI) developed in recent years and widely used in clinical.Data shows that UTI can inhibit the release of inflammatory medium, prevent cytokines cascade and block the excessive activation of white cell.Therefore UTI plays an important role in the early blocking development of SIRS to MODS by inhibitting vicious circle between cytokines and white cells,but the mechanism has not been clearly showed.The existing research is mainly on the effect of UTI to inflammatory cytokines,little is about the signal transduction mechanism. We can not find relative report about whether the protective of UTI has the relationship with p38MAPK, and its mechanism is expected to study.Endotoxin namely lipopolysaccharide is the main pathogenic componengt on the wall of G negative bacteria.When released into the circulation, LPS can activate toll-like receptor (TLR)-4 which has the signal transduction function, and then activate p38 MAPK signalling pathways in the cells through a series of cascade signals.At last, LPS acts on the gene expression of inflammatory cytokines such as TNF-a and IL-1, and all kinds of adhesion molecules.In this study,we have established the rat model of LPS-induced ALI and intervented it by intravenous injection of UTI.On the one hand,we tried to reveal the protective effect on the lung tissue through observing the ation of lung tissue pathology, wet/dry weight ratio and the changes in serum cytokines; on the other hand,we examined the level of TNF-a mRNA in lung tissue by Real time RT-PCR detection the expression of phosphorylated p38 MAPK in the lung tissue by Western blot and immunohistochemical staining method to reveal the molecular biologic mechanism of action of UTI,we hoped to provide certain theoretical and experimental basis for the clinical application of UTI.Methods1 Establishment and grouping of animal model:6-8-week-aged male Wistar rats were randomly divided intdo:control group, model group, and the intervention group. Control group was divided into three subgroups by 1,3,6h,the model and intervention group were divided into four subgroups by 0.5,1,3, and 6h.There was a total of 11 groups, and each group was 6 rats.Rats in model group were given 5mg/kg LPS through tail intravenous;rats in intervention group were given 50,000 units/kg UTI 30min before 5mg/kg LPS through the tail intravenous; rats in control group were given the equal amount of saline solution(NS) through tail intravenous.The experimental rats were all fed standard feed during experiment,they were no food and free water 12h before experiment.2. Collection of specimen:all the animals in the different groups were given 3% pentobarbital sodium by intraperitoneal injection to anesthesia.When chest cavity was exposed and opened,we observed the pathological changes of lung tissue and got the blood sample by puncturing from left ventricular.After taking blood samples,we got the left lung tissue to calculate the wet/dry weight ratio (W/D),got the right upper lobe fixed in 10% formalin for HE and immunohistochemical staining,at last,we got the right middle and lower lobe freeze-stored in liquid nitrogen and preservated at 80 degrees below zero for Real Time RT-PCR and Western blot inspection.3. Pathology examination by conventional HE staining and to calculate the score of lung tissue pathologic damage.4. The levels of TNF alpha and IL-10 were detected quantitatively by ELISA method.5. The relative expression of TNF alpha mRNA in lung tissue were examined by real time RT-PCR method.6 The expression of phosphorylated p38 MAPK in lung tissue were detected by immunohistochemical staining method.7. The expression of phosphorylated p38 MAPK in lung tissue were detected by western blot method.8. Statistical analysis:all data were expressed as means±S.D and analyzed by statistical package SPSS 13.0 for windows.One-way analysis of variance (ANOVA) was used to determine statistically significant differences among different groups and followed pairwise comparison by LSD inspection.A level of p< 0.05 was condidered statistically significant.Results1. General health state:rats in control group all survived and breathed smoothly, could escape quickly when grabbed.The lungs were pink without of abnormal changes after anatomy. Rats in model group were shortness of breath(>100/min),lip cyanosis,listlessness,less food and dynamic,could not to escape when grabbed.Two rats in 6h group and 3h group died,they appeared dying twitching, incontinence,deep breathing and stiff before death.The lungs increased in size, visceral pleura was with high tension,surface were dark red,include subcapsular pointlike, flake bleeding,and the dissection was loose with yellow or pink liquid spilled. Performance of rats in intervention group was relatively light, all animals were alive, could escape when grabbed.Changes of lungs were similar with those of lungs of model rats after anatomy, but relatively light.2.Changes of lung tissue wet/dry weight ratio (W/D):the lung tissue W/D weight ratio of rats in 3h and 6h were significantly higher in model group than control group(p< 0.01), the lung tissue W/D weight ratio of rats in intervention group at the above timepoint were lower than the model group(p< 0.05).The lung tissue W/D weight ratio of rats in control group?1h model group and 1h intervention group has no change.3. Pulmonary pathologic examination3.1 HE staining:lung tissue structure of rats in control group was complete, the alveolar cavity was clear without exudate, the alveolar walls were not thickening and no inflammatory cells infiltrated in the alveolar interstitial.The lung tissue structure of rats in model group were damaged, the alveolar cavity were filled with bleeding, inflammatory cells and liquid.Large gap surrounding vessels widened to pale pink mesh structure. There were many inflammatory cells infiltratde in the alveolar space, mainly neutrophils, lymphocytes, monocyte macrophages, and so on.Part of lung interval breakaged to be emphysema.Compared with the model group, damage of lung tissue of rats in the intervention group were relative light.3.2 Results of pathologic damage score:the pathologic damage score of rats in model group (5.90±0.48)was obviously higher than that in the control group(1.60±0.28),and reduced after intervention UTI(3.62±0.46),the difference was statistically significant (p< 0.01).4. Serum concentrations of cytokines4.1 Changes of TNF alpha concentration in serum:the serum concentrations of TNF alpha of rats in model group were significantly higher in each timepoint than control group, peaked at 3h and began to decline at 6h.After UTI injection, the serum concentrations of TNF alpha of rats in intervention group were below in each timepoint than model group (p< 0.01).4.2 Changes of IL-10 concentration in serum:the serum concentrations of IL-10 of rats in control group were below the level, after LPS injection, concentrations of IL-10 increased, arrived the highest level at 6h.The serum concentrations of IL-10 of rats in interention group were lower than those in model group (p< 0.01).5.Expression of lung tissue TNF alpha mRNA:the relative expression of lung tissue TNF alpha mRNA of rats in model group were higher than those in the control group, while the most obvious, was 128 folds with control group at lh.After the injection of UTI,the relative expression of TNF alpha mRNA decreased,the highest level was 58 folds with control group.Compared with the model group, the relative expression level of TNF alpha mRNA of rats in intervention group reduced at each timepoint,the difference was statistically significant (0.5 h, 1h p< 0.01,3h p< 0.05).6. Expression of lung tissue phosphorylated p38 MAPK protein6.1 Results of immunohistochemical staining:p-p38 MAPK in lung tissue of rats in control group were negatively expression.While in model group,p-p38 MAPK in lung tissue expressed as nuclei (or merge the cytoplasm) yellowish-brown particles,positive cells mainly conclude neutrophils and macrophages in the pulmonary intersitital and alveolar epithelial cells and vascular endothelial cells.Expression of p-p38 in UTI intervention group was significantly reduced.6.2 Results of Western blot detection:the expression of p-p38 MAPK in control group was very weak, after injection of LPS, the expression of p-p38 MAPK increased obviously, peaked at 1h, then began to decline, there were significantly difference (p< 0.01) at each timepoint compared with controls.After UTI intervention, the expression of p-p38 MAPK were lower than model group at each timepoint(p< 0.01).Conclusions1. The concentrations of both proinflammatory cytokines and anti-inflammatory cytokines elevated in the early stage of lipopolysaccharide-induced ALI.Compared to the concentrations of proinflammatory cytokines, the concentrations of anti-inflammatory cytokines were lower and later, there was imbalance between inflammatory reaction and anti-inflammatory reaction.2. It could reduce the pathological damage and pulmonary edema of LPS-induced ALI by injection UTI intravenously.3. UTI inhibited proinflammatory cytokines and apronounced anti-inflammatory cytokines, it played its protective role through regulate the imbalance between inflammatory reaction and anti-inflammatory reaction.4. P38 MAPK signaling pathways involved in the process of LPS-induced ALI. 5. UTI suppressed the proinflammatory cytokine TNF alpha at transcription level.6. UTI may be reduce the TNF alpha mRNA through inhibitting p38 MAPK signaling pathway.Innovation and significance1. We described the protective role of UTI in LPS-inducde ALI in this study, which provided a new idea for clinical therapy method.2. We clarified the feasible molecular biological machaniam of UTI inhibition TNF alpha, it may be through inhibitting the p38 MAPK signaling pathway, which provided a theoretical and experimental basis for the clinical application of UTI.