Synergy Therapeutic Effect Of Qingyitang And Bone Marrow Derived Mesenchymal Stem Cells(BMSCs)attenuate Severe Acute Pancreatitis Induced Acute Lung Injury In Rat

Objective:To observe the influence of Bone marrow mesenchymal stem cells and the Chinese medicine Qingyi Decoction in severe acute pancreatitis induced acute lung injury(SAP-ALI) in rat,then to move forward a single step to research the synergy therapeutic effect and underlying mechanism of Qingyi Decoction and BMSCs on SAP-ALI.Methods:Vitro study:BMSCs of SD rats were isolated 、cultured and proliferated. We examined mesenchymal stem cell morphology,growth and proliferation by optic microscopy. Cell surface markers were identified with CD29-PE,CD34-FITC,CD45-FITC,CD90-FITC antibody by flow cytometry. At the same time,we constructed lung tissue cells co-culture system directly, used LPS to injury lung cell, then collected CCM、NCM、ICM、QCM and IQCM to culture different group’s cells respectively. Following 10 days, we identified differentiation of BMSCs into alveolar type II cells and alveolar type I cells with Rabbit anti-rat SP-C, Rabbit anti-rat AQP-5 antibody by immunocytochemistry in each groups. Vivo study: One hundred and twenty five health SD rats were randomly divided into the following5 groups : Sham group(n=25),SAP group(n=25),BMSCs group(n=25),QYD group(n=25),BMSCs+ QYD group(n=25).we had used a method retrograde biliary and pancreatic duct injection for preparation severe acute pancreatitis induced lung injury model。24 hours after operation and treatment, we observed rat behavior characteristics, lung tissue pathology change, lung weight wet/dry ratio, serum amylase and the content of SOD and MDA, detected serum and lung tissue homogenate IL – 6 and TNF-ɑ by ELISA, and tested alveolar cell SP-A、SP-B、SP-C and water channel protein m RNA expression level 5(AQP- 5) by PCR and SP-C、AQP-5 protein expression by Western blotting in each group.Results:1、microscopic cell morphology with long spindle-based, abundant cytoplasm,nucleus cytoplasm ratio was normal. Cells presented colonies, radial, spiral distribution. The results of flow cytometry were show that surface molecules CD34,CD45 expression were negative, CD29, CD90 expression were positive.Immunocytochemistry assay showed that the CCM、NCM and QCM group were not bone marrow mesenchymal stem cells differentiate into SP-C-positive cells and AQP-5-positive cells, meanwhile ICM and IQCM group showed SP-C, AQP-5positive cells. The percentage of BMSCs differentiation to alveolar Ⅰ、Ⅱ cell in IQCM group were significantly higher than ICM group(P < 0.01)2、Compared with SAP group, the mortality of severe acute pancreatitis in rats(p < 0.05), Pathological change of lung tissue,serum amylase activity, MDA, IL– 6 and TNF-α were significantly decreased in QYD and BMSCs group. The SOD 、SP-C and AQP – 5 proteins were markedly higher in treatment groups than SAP group. The indexes of BMSCs + Qingyi decoction group were better than the BMSCs group( P < 0. 05) and Qingyi decoction(P < 0. 05) respectively.Conclusion:1、LPS can induced rat BMSCs to alveolar Ⅰ、 Ⅱ cell differentiation under damage lung tissue cells in vitro.2.Qingyi decoction which may inhibit excessive inflammation, reduce the release of inflammatory mediators and improve the micro environment in lung, has a certain therapeutic effect against acute lung injury.3.Qingyi decoction which coordinate exogenous BMSCs to participate in the treatment of lung injury and promote rat BMSCs under the condition of lung injury to alveolar Ⅰ, Ⅱ cell differentiation, enhance the treatment effect of BMSCs.