The Intervention Effect Of PNS On A549 Cell Epithelial Mesenchymal Transition And Its Influence On P38MAPK Signaling Pathways

ObjectiveExploring the performance and function mechanism of human alveolar epithelial cells Epithelial-mesenchymal transition(EMT)induced by TGF-?1 of Panax Notoginseng Saponins(PNS),to provide experimental basis for application of PNS treatment of pulmonary fibrosis in the future.MethodsHuman alveolar epithelial cells A549 in vitro culture were divided into five experimental groups:the control group,TGF-?1 induced group(TGF-?1 induced final concentration of 5 ng/ml,induction of 24 h),PNS with low?medium and high 3 doses of intervention groups(PNS 50ug/ml+TGF-?1 5ng/ml in low dose group,PNS 1OOug/ml+TGF-?1 5ng/ml in medium group,the high dose group of PNS 200ug/ml+TGF-?1 5ng/ml).1.Cell observation under the inverted phase contrast microscope including the growth and change form after photographic record,and the quantitative analysis of cell circular degree value.2.MTT method to detect each group of four periods of absorbance value(OD value),to calculate its inhibition rate and draw diagrams.3.Western blot method to detect E-cad?Vim?P-P38 MAPK protein expression and its changes.4.ELISA method to detect the expression of COL 1.5.Cell ultrastructure observation observing these ultrastructural changes under transmission electron microscope.Resultsl.Cell observation under Inverted microscope:A549 cells were induced by the final concentration of 5ng/mL of TGF-?1,cells morphology changed obviously and into long shuttle,phenotype similar to that of fibroblasts,some cells extended pseudopodia andmixed growth,the distance between cells were widened and the connection became relatively loose,cells of circular degree value is reduced,the obvious difference with the normal control group(P<0.05).But with PNS low,medium and high dose after the intervention,cells changed shorter and smoother,some cells even returned into pebbles,and with higher doses of PNS intervention,the cells restored increasingly.2.MTT results:A549 cells were induced after using TGF-?1,the TGF-?1 and PNS together after the intervention,the drug groups compared with the TGF-?1 induced group were statistically significant(P<0.05),at the same time,with the increase of PNS drug dose,cell growth inhibition rate subsequently heighten.And when the dose must be as the extension of drug effect time,cell growth inhibition rate increased at the same time.3.Western blot results show:Cells were induced after using TGF-?1,reduced the volume of E-cad expression in cells,Vim,P-P38MAPK increased expression and has significant difference compared with normal control group(P<0.05),after the PNS intervention,E-cad increased expression and Vim,P-P38MAPK expression decreased,compared with the control group each group were statistically significant(P<0.05).4.ELISA results show:A549 cells were induced by TGF-?1,significantly increased the content of COL1,there was statistical significance compared with the control group(P<0.05),while with PNS after the intervention,COL1 expression suppressed obviously,compared with the TGF-?1 induced group,there existed certain differences(P<0.05).5.Cell ultrastructure observation of transmission electron microscope show that cells were induced by TGF-?1,compared with the control group number of microvilli on the cell membrane surface decreased significantly,the connection between adjacent cells became loose,cytoplasm eosinophilic osmium sexual lamellar corpuscle in vacuoles degeneration and decrease in the number,and had a relatively small amount of mitochondria,part of mitochondria swelled,disappeared or ruptured,and the endoplasmic reticulum and golgi apparatus was very rich and less autophagy body,with PNS after the intervention,the microvilli on the cell membrane surface quantity and close connection between adjacent cells gradually restored,lamellar corpuscle in cytoplasm gradually closed to normal,less free bubble phenomenon,gradually enriched and a small amount of endoplasmic reticulum and mitochondria multiple autophagy.Conclusion1.TGF-?1 can induced human alveolar epithelial cells into mesenchymal cells.2.PNS can suppressed the EMT process induced by TGF-?1,and inhibited the proliferation of A549 cells and mesenchymal cells to the epithelial cells,and with higher doses of PNS intervention,increase the number of cells back to epithelial cells increased.3.TGF-?1 induced A549 cells,epithelial marker protein E-cad expression decreased,interstitial marker protein Vim.COL1 expression increased,with PNS(50 ug/mL,l0Oug/mL,200ug/mL)together after the intervention,E-cad expression increased but Vim?COL1 expression decreased.4.PNS could inhibit the phenomenon of TGF-?1 induced A549 cells EMT,and its mechanism may be related to regulating the TGF-?1/P38MAPK signaling pathway.