Acute lung injury (ALI) and its more severe stage of acute respiratory distress syndrome (ARDS) are caused by a variety of reasons both within and outside the lung characterized by progressive dyspnea and refractory hypoxemia. They are acute syndromes caused by body excessive inflammatory response. Endothelial cell damage and dysfunction are important pathological features of ALI / ARDS . It is manifested as extensive damage of pulmonary vascular endothelial cells and alveolar epithelial as well as increase of pulmonary vascular permeability. Ulinastatin is a urinary trypsin inhibitor isolated from male urine. It is a glycoprotein with typical Kuniz protease inhibitor structure. It has two completely non-overlapping active function areas and both have a broad spectrum of enzyme inhibition activity. It has been confirmed that ulinastatin can simultaneously inhibit trypsin, hyaluronidase, elastase, phospholipase A2 and other varieties of hydrolytic enzymes . It can also inhibit the release of inflammatory mediators and reduce the damage of inflammatory factor on target organs. Ulinastatin intervention of ALI is a research focus in recent years and studies have shown that ulinastatin can reduce symptoms of ALI, but its mechanism of action is unclear.
320-slice CT perfusion scan is a noninvasive functional imaging method . CTP images were achieved by intravenous infusion of contrast agent and dynamic scanning to a particular level. Perfusion parameters such as rBF, rBV and rPS were obtained by computer processing and they can reflect hemodynamic changes in the capillary level to assess tissue and organ perfusion stage. In this paper, a single intraperitoneal injection of paraquat aqueous solution was used to establish ALI models and ulinastatin was used for ultra-early intervention. 320-Slice CT perfusion technology was applied for scan observation of blood flow changes in the early stages of ALI. Changes in serum VEGF levels and pathology detection indicators were combined to understand microvascular changes after ulinastatin intervention at ALI ultra-early stages and to explore early protective effect of ulinastatin on PQ-induced ALI in rabbits.
During experiments, we found that paraquat group animals appeared quiet without much movement, malaise, anorexia, shortness of breath, rapid heartbeat and other behavioral changes after exposure, in line with paraquat poisoning signs. Two, four, and six hours after exposure, lipiodol perfusion was followed by 320-slice CT scan of the chest. There was distinct color change of perfusion images with time in paraquat group, indicating that lung perfusion changed significantly over time, suggesting possible heavy lung tissue damages result in poor lung perfusion. Determination of CTP parameters found that in paraquat group rBF and rBV decreased with time, while rPS gradually increased with time. rBF reduction indicated blood rate declined in lung tissue; rBV reduction indicated blood capacity within the lung tissue vasculature decreased; rPS elevation suggested the rate of blood unidirectionally going into the tissue space through capillary endothelial cells increased. Three parameter values at the same time point showed a significant difference (P <0.05) compared with the control group. The results verified the changes in the perfusion image, indicating poor lung perfusion at early ALI stage. This revealed ultra early hemodynamic characteristics of acute lung injury induced by paraquat. Ulinastatin group images showed little difference at the 2h time point from the control group, while significant difference at 4 and 6 hour time points. The former may be because paraquat absorption was small and ulinastatin produced direct effect right after entering into the blood. In the latter case, paraquat absorption increased with time and enhanced lung tissue damage, while ulinastatin content in blood reduced due to metabolism and lung damage gradually intensified. Compared with paraquat group, ulinastatin group still showed significantly better image changes. The magnitude of rBF, rBV decline and rPS increase were smaller and there were significant differences (P <0.05) compared with the paraquat group. The changes on the imaging suggest some treatment effect of ulinastatin.
Previous studies have shown that VEGF is a multifunctional cytokine and can regulate endothelial cell survival, proliferation, migration, angiogenesis, vascular permeability and mononuclear cell recruitment. Under normal state VEGF expresses abundantly in alveolar epithelium, bronchial epithelium and bronchial gland cells and the level of VEGF in normal human respiratory alveolar fluid is 500 times more than in serum, but under normal circumstances it is not released directly into blood. The increasing effect of VEGF on vascular permeability is extremely strong and its effect is 20,000 times more powerful than histamine . Therefore, VEGF is one of the markers to determine the degree of endothelial cell injury and vascular permeability. In this experiment, VEGF mass concentration of paraquat group elevated sharply over time. Compared with control group, there was a significant difference (P <0.05), indicating that endothelial cell damage occurs at ALI ultra-early stage and vascular permeability increases. After ulinastatin intervention, VEGF mass concentrations increased to a lesser extent. Compared with paraquat group there was significant differences (P <0.05), suggesting ulinastatin can reduce the damage of endothelial cells, inhibit the release of VEGF and reduce vascular permeability, which verified image changes from the cellular level.
Pathological observation showed that in paraquat group animals lung tissue had significant acute inflammatory changes, while ulinastatin intervention group had less inflammatory injury, which is consistent with pathology detection indicators. Pathology results also well explained the changes in the imaging and VEGF mass concentration. From comprehensive analysis, pathophysiological change at ALI ultra-early stage is vascular endothelial injury, and the exposed endothelial can easily form a local microthrombi which affects blood flow and blood volume, expressed as the decline in rBF and rBV. Endothelial injury prompts VEGF release in large quantities into the blood. The concentration, vascular permeability and rPS increase, resulting in a large number of liquid into the tissue space, alveolar septum widened and various excessive inflammatory cell infiltration to the local tissue and aggravating tissue damage. The experiments showed that early application of ulinastatin can significantly change the status of ALI ultra-early pathophysiology. It is expressed as significant improvement of vascular permeability at ALI early stage, suggesting that its mechanism should be related to reducing endothelial cell damage and lowering serum VEGF content. This is consistent with findings of Cai Shi Xia et al about ulinastatin on pulmonary microvascular permeability in septic rats.
From experimental results, at ultra-early ALI stage, ulinastatin can mitigate vascular endothelial injury, reduce serum VEGF content and elevation level of pulmonary vascular permeability, lower migration and infiltration of inflammatory cells, thereby reducing the degree of lung injury and playing a role in lung protection. Studies[13, 14] also showed that ulinastatin can directly inhibit the release of neutrophils and other inflammatory mediators, thereby reducing damage to the endothelial cells and reducing capillary permeability. Therefore, it is further speculated that ulinastatin on one hand can protect endothelial cells and reduce the expression of VEGF in lung tissue to inhibit neutrophil infiltration, on the other hand can directly inhibit damage of neutrophils to endothelial cell to reduce VEGF release. They promote and influence each other and the specific mechanism still needs further study.