Efficacy Of Ulinastatin For Pulmonary Capillary Leakage In Septic Rats And Its Mechanism Of Action

Background:Patients with sepsis often show respiratory failure early after onset of the disease,in which,destruction of the alveolar-capillary barrier plays a major role.Destruction of the barrier causes pulmonary capillary leakage and subsequent alveolar and interstitial edema,resulting in decreased lung compliance,ventilation/perfusion mismatch,and ultimately respiratory failure.It has been found that ulinastatin(UTI)can significantly ameliorate acute lung injuries induced by burns and intraperitoneal injection of lipopolysaccharide(LPS)in mice.However,the mechanism underlying this phenomenon and the connection between doses and effects and have largely remained elusive.Previous in vitro experiments by our research group showed that UTI could inhibit LPS-induced TNF-asecretion by pulmonary microvascular endothelial cells and reduce the destruction of tight junction proteins(Occludin、Claudin-5、ZO1).However,the finding has not yet been verified in animal models.Based on the background information above,we proposed the following scientific hypothesis:UTI inhibits the production of TNF-α in lung tissue of the animal models of sepsis and reduces its destruction of pulmonary capillary endothelial tight junction protein,thereby improving pulmonary capillary leak and oxygenation.At the same time,sepsis models are basic materials for sepsis research,but the existing studies have shown that cecal ligation and perforation(CLP)model is prone to encapsulation in the ligation and perforation area,resulting in poor model stability and homogeneity,and ultimately making it difficult to implement research findings in clinical practice for lack of evaluation of organ functions in the test models.This study intends to establish and evaluate a septic rat model using the modified cecal ligation and puncture(mCLP)method.In this study,by giving different doses of UTI,we aim to elucidate the mechanism of ulinastatin on the protection of pulmonary capillary leak in septic rats from perspectives of pathological anatomy,oxygenation function,immunohistochemistry,and molecular biology,and explore the relationship between its dose and efficacy to provide a scientific basis for the treatment of clinical lung injury resulting from sepsis.Methods:1)The septic rat model was established by modified CLP.Post-operative condition,mortality,serum creatinine,total bilirubin,arterial partial pressure of oxygen were compared with the traditional CLP.The time when postoperative oxygenation dysfunction happens was identified.The encapsulation of the perforation area,systemic inflammatory response and organ dysfunction in the modified sepsis model were rated.2)Based on the clinical application dose of UTI,we calculated the doses given to the rats with reference to their body surface area,and three different UTI dose treatment groups,low(10,000 IU/kg),medium(50,000 IU/kg),and high(100,000 IU/kg),were set up and the tested rats are injected intravenously in two divided doses.Eighty-eight male Sprague-Dawley rats were divided into 3 groups:Sham operation group,mCLP1 group,mCLP group+low/medium/high dose UTI treatment group,and UTI treatment group.Their arterial partial pressure of oxygen was monitored.Levels of pulmonary capillary leak were assessed by Evans blue method and hematoxylineosin staining.Pathological injuries of lung tissue were scored,and structures of pulmonary capillary endothelial cells were observed in the scanning electron microscope.3)Thirty rats were divided into 3 groups:Sham operation group,mCLP group,and mCLP+UTI treatment group,and the animals were sacrificed 48 hours following the surgery to detect the TNF-α content in their lung tissues by enzymelinked immunosorbent assay,and the expression of tight junction proteins ZO-1,claudin-5,and occludin in the lung tissues by Western blot and immunofluorescence techniques.4)PMVEC cells were cultured and divided into 3 groups:CON,LPS,and LPS+TNF-α receptor antagonist groups,and the effects of TNF-α receptor antagonists on occludin,claudin-5,and ZO-1 expression in LPS-treated PMVEC were examined by Western blot.Results:1)Compared with the traditional CLP group,the survival rate 72 hours following the surgery in the septic rat model established by the modified CLP method was 40%(40%vs.50%,P=0.751).Respiratory rate(160±5 vs.127±18,P=0.033),incidence of encapsulation in the cecal ligation and puncture area(15%vs.55%,P=0.019)serum creatinine(108.5±5.6 vs.98.8±6.3,P=0.023),serum total bilirubin(2.87±0.25 vs.2.15±0.2,P=0.016),arterial lactate(3.25±0.64 vs.2.15±0.2,P=0.016)were significantly increased,and arterial partial pressure of oxygen(68.8±2.8 vs.82.1±6.2,P=0.002)was significantly decreased in septic rats.Oxygenation dysfunction in septic rats occurred 48 hours after CLP;2)Compared with the mCLP model group,the mCLP+medium-dose UTI treatment group had lower Evans blue content in lung tissue(129.8±15.7 vs 188.7±16.6,P=0.001),a lower score of lung injury(7.560±0.250 vs.9.826±0.660,P<0.001),more intact alveolar-capillary barrier structure under electron microscope,and higher the arterial partial pressure of oxygen(79.3±1.9 vs.69.7±3.3,P=0.004).Compared with the mCLP+low-dose UTI group,the PaO2(79.3±1.9 vs.75.6±4.2,P=0.036;78.5±3.6 vs.75.6±4.2,P=0.048)was improved,and the Evans blue content in lung tissue(129.8±15.7 vs.155.4±18.8,P=0.002;104.4±20.4 vs.155.4±18.8,P<0.001)and lung injury score(7.560±0.250 vs.8.293±0.101,P=0.037;7.360±0.451 vs.8.293±0.101,P=0.012)were significantly reduced in the medium and high dose groups.3)Compared with the CLP group,the TNF-α content in the lung tissue of the mCLP+medium-dose UTItreated group(0.483±0.063 vs.0.762±0.087,P=0.003)was lower;Western blot experiments showed that tight junction protein ZO-1(0.792±0.102 vs.0.473±0.065,P=0.001),claudin-5(0.730±0.090 vs.0.433±0.062,P<0.001),and occludin(1.092±0.150 vs.0.793±0.120,P=0.001)expression in the lung tissue were significantly improved,and the same trend was observed by immunofluorescence experiments.In LPS-treated PMVEC cells,TNF-α receptor antagonists markedly decreased LPSinduced disruption of occludin,claudin-5,and ZO-1 expression.Conclusion:(1)The modified CLP method in rats can stably reproduce the sepsis model,and there are no significant differences in the mortality rate 48 hours after the surgery compared with the traditional CLP method.The ligation and puncture area is not prone to encapsulation,the systemic inflammatory response,organ dysfunction,and tissue hypoperfusion are more serious,and the model is more stable,which is highly consistent with the pathophysiological process of clinical sepsis.(2)UTI at 1-100,000 IU/kg·d can dose-dependently reduce pulmonary capillary leak in septic rats;UTI at 1-50,000 IU/kg·d can dose-dependently improve oxygenation in sepsis rats.(3)Ulinastatin inhibited TNF-α expression in the lung tissue of septic rats,reduced destruction of tight junctions between pulmonary capillary endothelial cells,preserved the normal structure of pulmonary capillary endothelial cells,and thus reduced pulmonary capillary leakage and improved oxygenation in these rats.