| Acute lung injury (ALI) concurrent with severe acute pancreatitis (SAP) in the early stage is the leading cause of death in patients. The mechanism of acute pancreatitis-associated ALI is complex, and it is thought to be related with the activation of systemic inflammatory response and the release of diverse cytokines and inflammatory factors, which further affects the permeability of pulmonary microvasculature and water transport across alveolar epithelial cells.Tumor necrosis factor alpha (TNFa) is an important inflammatory factor in the process of acute pancreatitis-associated ALI, and its level in serum and lung tissue is closely related with ALI. The biological functions of TNFa are mediated by TNF receptor-1 (TNFR-1) which is located in caveolae.Caveolae are a subset of lipid rafts characterized by flask-shaped invagination of 50-100 nm in diameter in plasmalemma of most tissue cells. Caveolin-1 (Cav-1) is the principle structural and functional component of caveolae participating signal transduction, cholesterol trafficking, endocytosis, tumorigenesis and so on. TNFa is the main factor regulating the expression of Cav-1. TNFR-1 appears in caveolae and interacts with Cav-1 by caveolin binding motif. Therefore, TNFa would affect the expression and function of caveolae/Cav-1 through TNFR-1 and further regulate the activities of diverse signaling pathways to produce different effects. Previous studies have shown that the expression of Cav-1 could be down-regulated by TNFa released by alveolar macrophage through TNFR-1.Then multi-signaling pathways related with inflammatory reaction could be activated to lead to lung injury. However, so far, the expression and significance of Cav-1 and TNFR-1 in the process of ALI induced by SAP has not yet been elucidated.The transendothelial transport of water and electrolytes is also regulated by caveolae. The water permeability of caveolae microdomains is probably due to abundant aquaporins (AQPs) in caveolae. AQPs are composed of a family of transmembrane proteins which facilitate water transport across cellular membrane. Some AQP subtypes can interact with lipid rafts and influence water permeability. Previous studies have shown that AQP1 and AQP5 are related with the elimination of excessive water in lung tissue. But the research coverage of the locations and expressions of AQP1 and AQP5 in caveolae is rather sparse.At present, the clinical treatment of ALI induced by SAP is comprehensive, and traditional Qingyitang has got good clinical effect. Though a lot of research work has been carried out on the therapeutic mechanism of Qingyitang, it is unreported on the effect of Qingyitang on the expression of Cav-1 in lung tissue.Rat model of acute pancreatitis-associated ALI was established by intraductal injection of deoxycholate. Wistar rats were randomly divided into sham operation (SHAM) group, ALI group, dexamethasone (DEX) group and Qingyitang (QYT) group. DEX (2 mg/kg) was injected once through sublingual vein immediately after pancreatitis. Qingyitang (10 ml/kg) was admistrated by lavage as soon as the rats become sober and repeat again 12 h later. Blood and lung tissues were drawn after 24 h. Serum amylase, lung wet/dry (W/D) ratio and pathological section were detected to evaluate the degree of injury. Immunoradiosassay was taken to detect serum TNFa. RT-PCR and immunohistochemistry were taken to detect mRNA and protein expression of Cav-1, TNFR-1, AQP1 and AQP5 respectively. Lipid rafts were prepared by sucrose density gradient centrifugation for detecting the distribution and expression of Cav-1, TNFR-1, AQP1 and AQP5 by Western Blotting.The results are as follows:1. The establishment of rat model with acute pancreatitis-associated ALI and the therapeutic role of DEX and QYT.Compared with SHAM group, the concentration of serum amylase, TNFa and the value of W/D increased significantly (P<0.01) in ALI rats. Microscopically, interstitial congestion, edema, infiltration of inflammatory cells could be seen. The alveolar septa significantly thickened and alveolar cavity partially fused to form bullae. Capillary vessels dilated with focal hemorrhages. Compared with ALI rats, the concentration of serum amylase, TNFa, W/D and pathological inflammatory reaction decreased obviously in DEX or QYT treated rats.2. The expressions of Cav-1 and TNFR-1 in the lung of acute pancreatitis-associated ALI rats and the potential therapeutic mechanism of Qingyitang.RT-PCR and immunohistochemical results showed that the mRNA and protein expression levels of Cav-1 were obviously reduced in the lung tissue of ALI rats (P<0.01), while the expression of TNFR-1 was enhanced (P<0.01). But in QYT or DEX treated rats, the expression of Cav-1 increased and TNFR-1 lowered compared with ALI rats (P<0.01).Western Blotting results showed that Cav-1 and TNFR-1 were distributed in both lipid rafts and non-lipid rafts, especially in lipid rafts. Compared with SHAM rats, the expression of Cav-1 in both lipid rafts and non-lipid rafts was decreased in the lung tissue of ALI rats, while the expression of TNFR-1 in both fractions was increased, especially in lipid rafts. The alteration of TNFR-1 expression suggested that tanslocation of TNFR-1 to lipid rafts might occur in ALI rats. After treatment with DEX or QYT, the increased expression of Cav-1 and the reduced expression of TNFR-1 in the lung were detected compared with ALI rats.3. The expressions of Cav-1, AQP1 and AQP5 in the lung of acute pancreatitis-associated ALI rats and the potential therapeutic mechanism of Qingyitang.The mRNA and protein expression levels of Cav-1, AQP1 and AQP5 were obviously reduced in the lung tissues of ALI rats detected by RT-PCR and immunohistochemistry (P<0.01), while the expression of them was enhanced in QYT or DEX treated rats (P<0.01).Western Blotting results showed that Cav-1 was distributed mainly in lipid rafts. AQP1 was almost only located in lipid rafts with non-glycosylated and glycosylated forms and AQP5 only appeared in non-lipid rafts. Compared with SHAM rats, the expression of Cav-1 in both lipid rafts and non-lipid rafts was decreased in the lung tissue of ALI rats. The expression of AQP1 and AQP5 was also obviously reduced. But in DEX or QYT treated rats, the expression of Cav-1, AQP1 and AQP5 was enhanced significantly.Conclusions:1. Cav-1, TNFR-1 and AQP1 are mainly expressed in lipid rafts, while AQP5 is distributed in non-lipid rafts in the lung of acute pancreatitis-associated ALI rats.2. The expression of Cav-1 is reduced while TNFR-1 is enhanced with translocation to lipid rafts in the lung of acute pancreatitis-associated ALI rats. It is suggested that TNFa may suppress the expression of Cav-1 through TNFR-1 and then activates multiple signal pathways related with inflammatory reactions and participates in ALI.3. The expression of Cav-1, AQP1 and AQP5 is decreased, which suggests that the reduced expression of Cav-1 may impair the ability to remove excess liquid from lung tissue and get pneumonedema grow worse by affecting the expression of AQP1 in lipid rafts in acute pancreatitis-associated ALI rats. The reduced expression of AQP5 maybe caused by other mechanisms than Cav-1.4. Qingyitang can effectively reduce acute pancreatitis-associated ALI, for one thing, inhibiting lung injury probably by down-regulating the expression of TNFR-1 and then increasing the expression of Cav-1, for another thing, accelerating the elimination of excess liquid in the lung by up-regulating the expression of AQP1 and AQP5. The results provide theoretical basis for the clinical application of Qingyitang in the treatment of SAP.
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