Protective Effects And Mechanism Of Ulinastatin On Acute Lung Injury In Septic Young Rats

Objectives:1. To observe lung injury and inflammatory mediators release in young rats after sepsis by establishing the model of young rats with CLP.2. To study the existence of the activation of JAK-STAT signal pathway in lung injury of young rats with sepsis, and the relationship between JAK-STAT signal pathway with TNF-α、HMGB-1 release.3. To study the effect of UTI on acute lung injury in young rats with sepsis, and to explore its possible mechanism.Methods:1. Grouping and model building:Totally 128 Sprague-Dawley(SD) rats of 4week-old were randomly divided into 4 groups(n=32 in each group): 1.Ctrl group,2.Sham-operated group, 3.CLP group, 4.UTI-treated sepsis group, each group was further randomly divided into four time points at 6h, 12 h, 24 h, 48h(n=8 at each time point). Ctrl group: Injecting the same dose of NS(5 ml/kg) at 0 h, 24 h without other treatment; Sham-operated group: Only cut open enterocoelia and suture after finding the cecum but without any surgery. then, injected NS(30 ml/kg) from the medial femoral subcutaneous for fluid resuscitation, and the same dose of NS(5 ml/kg) at 0h,24 h instead of UTI Immediately; CLP group: Establishing the Sepsis model of young rats with CLP method, and then, injected NS(30 ml/kg) from the medial femoral subcutaneous for fluid resuscitation, and the same dose of NS(5 ml/kg) at 0h,24 h instead of UTI Immediately; UTI group: Establishing the Sepsis model of young rats with CLP method, and then, injected NS(30 ml/kg) from the medial femoral subcutaneous for fluid resuscitation, and the same dose of UTI(5 ml/kg) at0 h, 24 h. Then the blood and lung tissue samples were obtained in batches at four time points, which were used for the detection of relevant index.2. Sample collection:Rats in each group were quickly thoracotomy at 6 h, 12 h,24 h, 48 h time points, collected blood from heart for determing serum TNF-a by ELISA. Wet weight was measured immediately and dry weight was measured after drying specimens at 60 ℃ for 48 h to constant weight.The lung wet/dry weight ratio was then calculated. Lung tissue was then placed in 4 % neutral formalin-fixed and paraffin-embedded, for HE and immunohistochemical staining.3. Observational index: Detected the level of lung W/D. Observed the pathological changes of lung under optical microscope after HE staining. Detected the expression of serum TNF-α. Detected the expression of lung HMGB-1、P-JAK2 and P-STAT3 by immunohistochemical staining.Results:1. General performance: All of the young rats in CLP group got serious sepsis symptom-activity decreased obviously, appetite decreased obviously, lethargic,breathless, cyanosis. After putting the rats to death and paunch, we could see bloody exudate in enterocoelia, caecum was tumid and black and sticky, intestinal canal of jejunum was flatulence. The sepsis’ s symptom of UTI-treated sepsis group was better than CLP group. The activity of young rats in Sham-operated group was normal after anesthesia awake.2. Lung W/D determination: Compared with Sham-operated group, the lung W/D in CLP group and UTI- treated sepsis group rised at different time points(P <0.01). Compared with CLP group, the index of lung in UTI-treated sepsis group reduced at each time points(P < 0.01)4. The pathological changes of lung: Compared with Sham-operated group, the pathologic changes in CLP group was more obvious—alveolar septum was bigger,edema and congestion happen in pulmonary alveolar space, and infiltration of inflammatory cells could be seen in pulmonary mesenchyme. Compared with the CLP group at same time point, alveolar septum widened slightly, edema and congestion of stroma and infiltration of inflammatory cells could be alleviated in UTI-treated sepsis group.5. The level of TNF-α in serum: Compared with Sham-operated group, the level of TNF-α in CLP group rised at different time points(P < 0.01),it reached thepeak at 12 h and continued to go down until 48 h. Compared with CLP group, the level of TNF-α in UTI-treated sepsis group reduced observably(P < 0.01).6. The expressions of Lung HMGB-1 protein: Compared with Sham-operated group, the expressions of Lung HMGB-1 protein increased significantly at different time points in CLP group and UTI-treated sepsis group(P < 0.01), it rised at 6 h, and continued to go up until 48 h. Compared with the CLP group, it decreased significantly at each time point in UTI-treated sepsis group(P < 0.01).7. The expressions of Lung P-JAK2 、 P-STAT3 protein: Compared with Sham-operated group, the expressions of Lung P-JAK2 、P-STAT3 protein increased significantly at different time points in CLP group and UTI-treated sepsis group(P <0.01), it rised at 6h, reached the peak at 24 h, and was still higher than normal at 48 h. Compared with the CLP group, it decreased significantly at each time point in UTI-treated sepsis group(P < 0.01).8. The correlation analysis between the expression of Lung P-JAK2、P-STAT3 and HMGB-1:the expression of Lung P-JAK2、P-STAT3 was positively correlated with the HMGB-1 at 6 h,12 h, 24 h and 48 h after sepsis.Conclusions:1. The model of sepsis in rats can be established by cecal ligation and puncture(CLP) successfully.2. The release of inflammatory mediators such as TNF-α、HMGB-1 takes part in the pathogenesis of acute lung injury by sepsis.3. There exists the activation of JAK-STAT signal pathway in acute lung injury of young rats after sepsis.4. UTI plays a protective effect on acute lung injury of young rats after sepsis by:1) interferencing the activation of JAK-STAT signal transduction pathway to reduce the release of inflammatory medium such as TNF-α and HMGB-1;2)directly inhibiting the expression of TNF-α and HMGB-1 and relieving their direct toxic effect, so as to prevent the occurrence of acute lung injury by sepsis.