The Study Of The Effect Of Surfactant Protein A And Secreted Phospholipase A₂-Ⅱ In Lung Injury Induced By Severe Acute Pancreatitis In Rats And The Influence Of Qingyitang

Objectives:The purpose of our study is to investigate the expression of surfactant protein A (SP-A) and secreted phospholipase A₂-II (sPLA₂-II) in rats with acute lung injury (ALI) caused by severe acute pancreatitis (SAP), observe the morphological and functional alteration of alveolar type II cells (ATII cells) and the therapeutic effect of Qingyitang. In this study we also created an ALI model of cultured AT II cells in vitro, which is generated by treating AT II cells with pancreatitis-associated ascitic fluid (PAAF). Based on this model, we investigated the secretary function alteration in AT II cells. We use Emodin, one of major compounds of traditional medicine pieplant, to interfere the injury process in vitro to evaluate its therapeutic effect.Methods:Part 1:Sixty male SD rats were randomly divided into 6 groups: sham operation group (SO, n=10),SAP model group (SAP, n=10), Qingyitang group (QYT, n=10) , Dexamethasone group (DEX, n=10), Sandostatin group (SS, n=10) and 5-(4-benzyloxyphenyl)- 4S- (phenyl-heptanoylamino) - pentanoic acid group (KH064, n=10). Severe acute pancreatitis was induced by inversely injecting 1.5% sodium deoxycholate into the common bile-pancreatic duct of rats for SAP and interference groups. Sham operation group was performed operation only. Qingyitang (10 ml/kg) was intragastric administrated 30 minutes and 12 hours after SAP was induced in QYT group.Dexamethasone (10mg/kg) was injected intravenously 30 minutes and 12 hours after SAP was induced in DEX group. Octreotide (50μg/kg) was subcutaneously injected 30 minutes and 12 hours after SAP was induced in SS group. KH064(5mg/kg) was intragastric administrated 30 minutes and 12 hours after SAP was induced in KH064 group. Serum amylase (AMY),sPLA₂ levels, PaO₂,PaCO₂,pH and lung wet/dry ratio (W/D) were recorded. The SP-A and sPLA₂ mRNA expression levels in lung tissue were detected by reverse transcription polymerase chain reaction (RT-PCR). The SP-A protein level in lung tissue was detected by both Western-blot and immunohistochemical methods. The pathological changes of pancreas, lung and AT II cells were observed 24 hours after the SAP model was established.Part 2: AT II cells were isolated by Dobbs's method which had been proved to be effective. Then, cells were purified by using rat IgG coated dishes and were characterized by using electronic microscope and SP-A immunohistochemical staining. The cellular ultra-structure of AT II cells was observed by using electronic microscope.Part3: 1.5% sodium deoxycholate (1ml/kg)was inversely injected into the common bile-pancreatic duct of SAP rat model. Pancreatitis-associated ascitic fluid (PAAF) was obtained from the SAP rats. The activity of sPLA₂ in ascites was detected by using sPLA₂ kits. AT II cells were previously cultured in vitro and exposed to medium with different PAAF concentrations at 12.5μl/ml, 25μl/ml, 50μl/ml and 100μl/ml. The morphology and activity of those cells were measured by using inverted microscope and MTT method at 6,12 and 24 hours post treatment.Cultured ATII cells were treated by KH064 with the final concentration at 0μg /ml,0.5μg/ml,1μg/ml, 2μg/ml and 4μg/ml at the presence of 25μl/ml PAAF for 24 hours. Another group of ATII cells were exposed to Emodin with the final concentration at 0μg/ml,10μg/ml,20μg/ml,40μg/ml and 80μg/ml at the presence of 25μl/ml PAAF for 24 hours. Cell survival rate was detected with MTT assay. Then Cultured AT II cells were exposed to medium containing PAAF at final concentrations of 12.5μl/ml, 25μl/ml, 50μl/ml and 100μl/ml for 24 hours, and cells were exposed to 25μl/ml PAAF with KH064 (1μg/ml,2μg/ml) or Emodin (10μg/m , 20μg/ml). Cells were harvested and extracted for total RNA isolation with Trizol reagent, mRNA level of SP-A was detected by using reverse transcription polymerase chain reaction (RT-PCR). The expression of SP-A protein was detected by Western blot. AT II cells were observed by using electron microscope.Results: Serum AMY level, PaCO₂, W/D of lung in SAP group were found significantly higher than other groups (P<0.05). Serum sPLA₂ level in SAP was significantly higher than all other groups (P<0.05), and PaO₂ level in SAP was lower than SO and interference group (P<0.05). The SP-A mRNA and SP-A expression level in lung tissue of SAP group was significantly decreased than both SO and interference groups (P<0.05). The mRNA levels of sPLA₂-II and SP-A in lung tissue of SAP group were significantly lower than SO and interference groups (P<0.05). There were milder pathological changes of pancreas, lung tissue and AT II cells in either interference group and SO group than SAP group.There were collected 5×10⁸ cells isolated from ascitic fluid and only 1.6×10⁷ cells were harvested after purification. The trypan blue staining showed the cell viability is above 95 percent. Immunohistochemical staining using anti-SP–A demonstrated that the purity of AT II cell is 92%. Morphologic manifestation of AT II cell was changed with PAAF concentrations and time points. PAAF can induce AT II cells damage measured by morphology and death detected by cell viability. The cell viability detected with MTT in PAAF groups were significantly lower than in control group. SP-A expression decreased at both transcription and translation levels in PAAF intervention groups.Emodin and KH064 can effectively prevent the damage induced by PAAF on AT II cells and increase SP–A expression in both transcription and translation levels. The pathological changes of AT II cells induced by KH064 and Emodin were milder than PAAF group.Conclusion: The cellular structure disorganization of AT II cells was observed in lung tissue of SAP rats with lung injury. SP-A decreased and sPLA₂-II increased significantly during ALI induced by SAP. Qingyitang can lessen the lung injury by protecting AT II cells, up regulating SP-A expression and down regulating sPLA₂-II expression. The balance between SP-A and sPLA₂-II is crucial in acute pancreatitis-associated lung injury (APALI). Dexamethasone can up regulate SP–A expression and down regulate sPLA₂-II expression in injured lung. This effect might involve alleviating inflammatory reaction. The content of hemodiastase was much higher in SS group, while the change of SP-A and sPLA₂-II expression in lung of this group was not found as significant as in SO group.In AT II cells isolation, purification and identification procedures, optimizing the concentration of digest enzyme and time can improve the cell vitality. Purification of AT II cell with IgG coated plate is a better method than other reported methods. AT II cell could be characterized through detection of the lamellar bodies with electronic microscope.PAAF can damage AT II cell in vitro. It might be due to induce cell necrosis and decrease expression of SP-A. However, both KH064 and Emodin can prevent PAAF induced damage in ATII cell culture system through up-regulation of SP-A expression level.