| Objective:Severe acute pancreatitis is one of the most dangerous acute abdominal diseases, with extremely high mortality and early concurrent organ failure. Acute lung injury is the leading cause of severe acute pancreatitis patients with early high mortality, which induce systemic inflammation, and even lead to acute respiratory distress syndrome to cause death. Acute lung injury is pathologically characterized by diffuse pulmonary microvascular endothelial injury, alveolar epithelial injury, inflammatory cells (neutrophils and macrophages, etc.) infiltration, pulmonary hemorrhage and edema, etc. Cell apoptosis plays a very important role in cell survival and death balance, on one hand, neutrophil apoptosis in lung injury can prevent the excessive release of inflammatory factor, thus protecting the lung tissue; Apoptosis pathway, on the other hand, can also induce alveolar epithelial cells and pulmonary vascular endothelial cell apoptosis, and cause the destruction of the alveolar walls structural integrity, thereby increasing the degree of lung injury. Alveolar epithelial type Ⅱ is the original stem cell of alveolar epithelial cells, which may not only be supplement and differentiation of alveolar epithelial cells of the type Ⅰ to have the repairing effect of lung tissue injury, but also can synthesize and secrete pulmonary surfactants, to maintain the stability of alveolar, maintain the balance of the alveolar surface water electrolyte, participate in the immune regulation. Alveolar epithelial cells in normal lung tissue, can be self-renewal and repair its normal apoptosis; In acute lung injury, alveolar type Ⅱ epithelial cells become one of the main damage on its target cell that affect its differentiation and apoptosis. At present, the study of alveolar type Ⅱ epithelial cell apoptosis, mainly involves three classic apoptosis pathways (death receptor pathway, mitochondrial pathway and endoplasmic reticulum pathway), three classic apoptosis pathways are independent and relative: Fas triggered early death receptor mechanism of apoptosis and closely related to the rise of intracellular Ca2+concentration. Calcium as an important second messenger in cells, it may overload in the mitochondria mediated apoptosis pathway and plays an important role. Endoplasmic reticulum Ca2+is the main repository. Endoplasmic reticulum Ca2+homeostasis imbalance can activate Caspase-3and induce cell apoptosis.Patch clamp technique is a kind of technology by recording a single whole-cell ion channels current and directly reflect the activity of ion channel. The technology can distinguish the single ion channels current directly, in order to observe the opening and closing process of ion channels, and used in the detection of intracellular calcium ion concentration change and influence.The objective of this study is to establish mature and stable SAP-associated lung injury rats model to further explore the main pathogenesis of severe acute pancreatitis-associated acute lung injury. We investigate the effects of Qingyitang、nifedipine、dexamethasone on the apoptosis of alveolar type Ⅱ epithelial cells of severe acute pancreatitis intracellular free calcium concentration of alveolar type Ⅱ epithelial cells of severe acute pancreatitis, expression level of apoptosis-related gene regulation system of Caspase-8, Bax mRNA of alveolar type Ⅱ epithelial cells of severe acute pancreatitis, expression level of mitochondria apoptosis-related gene regulation system of Bcl-2, Bax, Caspase-3, Caspase-8, Caspase-9in lung tissue of severe acute pancreatitis,express ion level of proinflammatory cytokine of severe acute pancreatitis. Using patch clamp technique to observe the calcium channel current changes in nifedipine and lipopolysaccharide (LPS) treated A549cell. Our research will demonstrate the overload of intracellular free calcium of alveolar type Ⅱ epithelial cells associated with apoptosis, apoptosis-mediated mechanism of severe acute pancreatitis-associated lung injury, protection mechanism of different western drugs and Chinese medicine and provide a theoretical guidance and experimental basis.Methods:Healthy male SPF SD rats were selected to induce SAP for establishing mature and stable rat model by retrograde infusion of15g/L sodium deoxycholic acid (sodium deoxycholate) into the bili-pancreatic duct. Based on the successful rat SAP model,60healthy male SD rats were randomly divided into5groups, with12SD rats in each group:sham opration group (SHAM group), model group (SAP group), Qingyitang group (QYT group), N ifedipine group (NIF group), dexamethasone group (DEX group). The SHAM group was only flipped the pancreas lightly several times after laparotomy. Drug treatment was given to the rats at the specific time. NIF group and DEX group were done with injection immediately and6h、12h after operation once again, inject rat0.3ml/min, respectively dose:2ml/kg,0.5ml/lkg. Qingyitang was given to the QYT group rats by intragastric administration before operation and6h,12h after operation with dose lOmL/kg. Alveolar type Ⅱ epithelial cells were isolated and cultured24hours after operation, apoptosis was detected by flow cytometry(FCW). Intracellular concentration of free calcium ion of alveolar type Ⅱ epithelial cells was detected by laser scanning confocal microscopy; intracellular concentration of free calcium ion of alveolar type Ⅱ epithelial cells was detected by patch clamp technique; genetic transcription of caspase-8、Bax apoptosis were determined by reverse transcription polymerase chain reaction; protein expression of caspase-8and Bax were examined by immunohistochemical method; protein expression of Bcl-2, Bax, Caspase-3, Caspase-9were examined by western blot, alveolar type Ⅱ epithelial cells were identified by transmission electron microscope and the mitochondrial ultras truc ture changes of alveolar type Ⅱ epithelial cells were observed.The lung tissue and pancreatic histopathological detection were observed with light microscopy; the ratio between Wet and Dry weight of lung tissue were calculated by vacuum drying method;blood gas were analyzed by biochemical method. Patch clamp technique was adopted to detect calcium channel current in A549individual cell treated with different concentration of lipopolysaccharide (LPS)and nifedipine to records of nifedipine’s role.Results:(1)Compared with the SHAM group, the result of HE staining of lung tissue showed changes of ALI in SAP group; lung wet/dry ratio was significantly increased in SAP group (P<0.01); the arterial PaO2was significantly decreased in SAP group(P<0.01); the arterial PaCO2was significantly increased in SAP group(P<0.01); serum TNF-α and AMY were significantly increased in SAP group (P<0.01); transmission electron microscope examination showed that in alveolar type Ⅱ epithelial cells, mitochondria morphology were destroyed, showed mitochondria double membrane structure fuzzy, mitochondria swelling, crest fracture; alveolar type Ⅱ epithelial cell apoptosis was significantly higher in SAP group by flow cytometry(P<0.01); intracellular concentration of free calcium ion in alveolar type Ⅱ epithelial cells was significantly higher in SAP group by laser scanning confocal microscopy(P<0.01); intracellular concentration of free calcium ion in alveolar type Ⅱ epithelial cells was significantly higher in SAP group by patch clamp technique(P<0.01); RT-PCR results showed that gene expression of apoptosis in alveolar type Ⅱ epithelial cells were significantly increased in SAP group(P<0.01); immunohistochemical results showed that the protein expression of apoptosis gene was significantly higher in SAP group(P<0.01). Compared with SAP group, the indicators decreased at different degrees after three different drug treatment,(2) Compared with the SHAM group, the whole cell patch clamp recording results showed no significant changes in cell peak current after treated with the5[ig/mL LPS (n=10, P>0.05); but10μg/mL LPS induced significant changes, the peak current increased (n=10, P<0.05);15μg/mL LPS had induced the peak current significant decreased (n=10, P <0.05); Nifedipine caused90%calcium current blocked in A549cell treated with10μg/ml LPS, which further proved that this electric current was the calcium ion current.Conclusion:(1) The apoptosis of alveolar type Ⅱ epithelial cells participate in severe pancreatitis-associated lung injury. (2) In severe pancreatitis-associated lung injury, serum TNF-α concentration obviously increased.(3) The overload of intracellular calcium ion may participate in apoptosis of alveolar type II epithelial cells.(4) Apoptosis genes mRNA expression of caspase-8,Bax in the alveolar type Ⅱ epithelial cells and apoptosis genes proteins expression of Bcl-2, Bax, Caspase-3, Caspsase-8, Caspase-9in lung tissues were increased in severe pancreatitis-associated lung injury. The mitochondrial pathway and death receptor pathway may be involved in alveolar type Ⅱ epithelial cells apoptosis and may play a regulatory role.(5) Qingyitang, Nifedipine, Dexamethasone can significantly reduce the degree of lung tissue damage, decrease concentration of serum TNF-α, reduce the apoptosis of alveolar type Ⅱ epithelial cells and Caspsase-8,Bax mRNA expression, inhibit the Bax, Caspase-3, Caspsase-8, Caspase-9proteins expression of lung tissue in severe acute pancreatitis.(6) The current clinical use of pure western medicine treatment has many side effects, influence the treatment and recovery of disease; combined traditional Chinese medicine and western medicine may be an effective way to prevent and treat severe acute pancreatitis associated lung injury.(7) The patch clamp technology used for ion channel current detection can directly reflect cell ion channels permeability changes after drug treatment, which has a good application value in revealing the cellular e lee trophysio logy mechanism. |
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