Protective Effects Of Erythropoietin On Pancreatic Acinar Cells And Lung Tissues Of Rats

[Objective] To explore a new method on primary culture of pancreatic acinar cells and indentify the nature of the cells, it is the preparation for acute pancreatitis (AP) in vitro. To investigate the protective effects of Erythropoietin (EPO) on pancreatic acinar cells and lung, this will be useful for researching the clinical theoretical basis.[Methods] Pancreatic acinar cells were placed in DMEM-F12 medium after separated and purified. 24 hours later, the ultrastructural changes of pancreatic acinar cells were observed by electron microscopy. PaA antibody was used to identify the properties of the cultured cells. After that, Sprague Dawley (SD) rats were randomly divided into sham operation group(SO), SAP (acute pancreatitis, SAP), EPO1000 group, EPO3000 group, EPO5000 group, Each group contained 18 rats and executed at the time of 24h, 48h and 72h. SAP rats were prepared by retrograde pancreatic duct injection of 5% sodium taurocholate (STC)(0.1ml/100g). Dry-Wet proportion and weight coefficient of lung were measured at the different time points by electronic scale. The levels of amylase (AMS) in serum were tested by artificial iodine colorimetry. The levels of interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) in serum were tested by ELISA kits,and then the relationship would be tested among them. The pathological changes of pancreas and lung were observed by HE staining. The expressions of EPOR were tested by immunohistochemical method and ELISA kits.[Results]①Fresh isolated pancreas acinar cells suspended in the medium, the shape of the cells were irregular. It was large of the proportion of cytoplasm and nuclear. Most of the separated and purified acinar cells were round or oval.②The nuclear was round, leaned to one side and impurities in the intercellular were less. The number of the cells was increased after 24 hours. It was obviously gathered together. After 2~3 days, changed the medium and the observed the activities of the cells. The number of the cells was significantly increased and passaged well.③The viability of the acinar cells was greater than 96% as determined by trypan blue assay and results were in accordance with experimental requirements.④Under the electron microscopy, there are a large number of zymogen granules in the cytoplasm were at the top of the cells, which were rich in rough endoplasmic reticulum. It could be seen the golgi complexes in the region of zymogen granules gethering.⑤It could be found the highly expression of green fluorescence in acini using the antibodies against PaA by immunocytochemistry.⑥The ratio of lung Drought-Wet weight in SAP group was much higher (P < 0.05) than in SO group. After the treatment of EPO, lung and pancreas edema could be easer than before. Lung index of rats were much higher than SO and EPO group (P < 0.05).⑦The levels of AMS and IL-1,IL-6,TNF-αin serum were significantly higher in SAP groups and EPO groups than that in SO groups, however, AMS and IL-1,IL-6,TNF-αwas declined significantly when given EPO intervention for 24h and 48h(P<0.05), but there were no statistics significant difference between EPO3000 and EPO5000 groups. The effects of EPO was time-dependent, it was positive correlation (r=0.534, p=0.02; r=0.584, p=0.04; r=0.820,p=0.00), there is a mutual role in promoting expression.⑧T here are no significant pathological changes or only slight edema of pancreas and lung tissues in SO group. However, varying degrees of edema, exudation, and inflammatory cell infiltration have happed in pancreatic and lung tissues of rats in SAP group, partial pancreatic or lung tissues had happened hemorrhage and necrosis, the structure of which had changed into blur, and even into a slice. The treatment of EPO could improve the pathological damages and the pathological score was significantly lower than SAP group (P<0.05).⑨I t could be seen that EPOR-positive stanining cells, located in the cytoplasm, were scattered in the pancreatic tissue in SO group. The expression of EPOR in pancreas and lung tissue showed large deeply stained EPOR-positive staining cells in different doses of EPO treatment groups.⑩There is no significant EPOR expression changes in SO, SAP, EPO1000 groups measured by ELISA kits. The quantities of the expression of EPOR increased with the increasing doses of EPO. [Conclusion]①Primary pancreatic acinar cells can be successfully cultured by this method, the purity and viability of acinar cells were in line with the vitro experiments.②PaA antibody may be a convenient ang feasible method to identify the pancreatic acinar cells with its high specificity. It can be widely used for identification of pancreatic acinar cells.③It was gradually increased of the levels of AMS, IL-1,IL-6,TNF-αin serum in SAP group. The treatment of EPO may effective reduce the pathological pancreas and lung injury and obviously decreased the levels of AMS, IL-1,IL-6,TNF-αin serum.④Using ELISA and Immunohistochemical methods can detect the expression of EPOR on pancreatic acinar cells. The expression of EPOR increased with the increasing doses of EPO, but there is no significant correlationship with the timing of treatment. We may speculate that EPO might bind with EPOR and activate the pathways of downstream signal transduction, which may play an critical role on anti-inflammatory and anti-apoptosis.