| Objective:The purpose of this study is to investigate the main pathogenesis of severepancreatitis-associated lung injury. we investigate the effects of Qingyitang、dexamethasone、 verapamil on the apoptosis of alveolar type II epithelial cells,intracellular free calcium concentration, apoptosis-related gene regulation system ofBax、Caspase-8and express level of proinflammatory cytokine and to reveal overload ofintracellular free calcium associated with apoptosis, apoptosis-mediated mechanism oflung injury, protection mechanisms of drugs and provide an experimental basis andtheoretical guidance.Methods:(1)60healthy SD rats (male) were randomly divided into5groups, with12rats ineach group: model group (SAP), normal control group (CON), Qingyitang treatmentgroup (QYT), dexamethasone treatment group (DEX), verapamil treatment group(VER).Using pancreatic duct retrograde injection through the duodenal papilla toestablish the SAP model was induced by retrograde injection through biliopancreaticduct with1.5%sodium deoxycholate solution at1ml/kg in SD rats. The control groupwas only flipped the pancreas gently several times after laparotomy. Drug treatment wasgiven to the rats at the specific time. DEX group and VER group were done withinjection immediately after modeling the first and6h、12h once again, inject rat0.3ml/min, respectively dose:0.2ml/100g,0.05ml/100g. Qingyitang was given inQingyitang group by intragastric administration before modeling the first and after6h、12h once again.(2)24hours after making models, alveolar type II epithelial cells were isolated andcultured, apoptosis was detected by flow cytometry; Intracellular concentration of free calcium ion was detected by laser scanning confocal microscopy; Bax、Caspase-8apoptosis genetic transcription were determined by reverse transcription polymerasechain reaction; The lung tissue and pancreatic histopathological detection were observedwith light microscopy; Bax and Caspase-8protein expression were examined byimmunohistochemical method; Serum cytokine were determined by radio-immunitymethod; Blood gas are analyzed by biochemical method; The ratio between Wet andDry weight of lung were calculated by vacuum drying method.Results:Compared with normal control group, the result of HE staining of lung tissueshowed changes of ALI in SAP group; The arterial PaO2was significantly decreased inSAP group(P <0.01); Lung wet/dry ratio was significantly increased in SAP group (P<0.01); Serum TNF-α and AMY were significantly increased in SAP group (P <0.01);Type Ⅱ alveolar epithelial cell apoptosis was significantly higher in SAP group by flowcytometry (P <0.01); Alveolar type II epithelial cells intracellular concentration of freecalcium ion was significantly higher in SAP group by laser scanning confocalmicroscopy (P <0.01); RT-PCR results showed that alveolar type II epithelial cellsapoptosis gene expression was significantly increased in SAP group (P <0.01);Immunohistochemical results showed that the protein expression of apoptosis gene wassignificantly higher in SAP group (P <0.01). After drug treatment, compared withmodel group, the indicators decreased at different degrees.Conclusion:(1) The apoptosis of alveolar type II epithelial cells participate in severepancreatitis-associated lung injury.(2) In ALI, serum cytokine TNF-α concentration obviously increased.(3) The damage of intracellular calcium ion homeostasis participate in apoptosis ofalveolar type II epithelial cells.(4) Bax, Caspase-8, apoptosis genes mRNA expression of the alveolar type IIepithelial cells, were increased in ALI. The mitochondrial pathway and death receptorpathway may be involved in alveolar type II epithelial cells apoptosis and play aregulatory role.(5)Dexamethasone, Qingyitang, verapamil can significantly reduce the degree oflung tissue damage, decrease concentration of serum TNF-α, reduce the apoptosis ofalveolar type II epithelial cells and Bax, Caspsase-8mRNA and proteins expression.(6) Combined traditional Chinese medicine and western medicine may be an effective way to prevent and treat severe acute pancreatitis and associated lung injury. |
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