Observation Of The Biological Effects Of S1P Induced Epithelial-myofibroblast Transition Of Aecii And Exploration Of The Involved Mechanics

Part1: Observation of the biological effects of S1 P induced EMT of A549 cells Backgroud and ObjectiveA variety of different reasons can cause pulmonary interstitial fibrosis,which is common outcome of most interstitial lung disease and important causes of respiratory failure.It is minly manifested as alveolar unit structure disorder,pathological changes with a large number of fibroblasts,external matrix deposition（ECM）and associated with structural damage caused by inflammatory lesions.Its pathogenesis is extremely complex,involving many network systems and regulation of signal molecules.Among them,a large number of studies have confirmed that alveolar epithelial mesenchymal transition（EMT）in the formation and progression of pulmonary interstitial fibrosis plays an important biological role.EMT refers to the process of epithelial cells with polarities that are transformed into mesenchymal fibroblasts or myofibroblasts under various factors and are involved in the process of fibrosis after pulmonary interstitial injury.In recent years,it has been found that the expression of sphingosine 1-phosphate sphingosine（S1P）has a significant increase in tissue fibrosis,and can promote the formation of fibrosis by EMT formation through a variety of signal transduction pathways.Purpose of this study is toobserve the biological effects of S1 P on EMT in A549 cellsand analyze whether S1 P was involved in the formation of pulmonary interstitial fibrosis through EMT.MethodsTo examine the effects of S1 P on EMT of A549 cells.The cultured A549 cells were divided into three groups: a,negative control;b,treated by S1 P with different concentration(10^-8,10^-7,10^-6,10^-5mol/L)for 72 h,respectively;c,treated with S1 P at different time point（12,24,48,72h）,respectively.The morphology was assessed by phase-contrast microscopy.The protein and m RNA expression of α-SMA,FN and E-cad were assessed by Western Blot and RT-PCR,respectively.ResultsMorphological changes in A549 cells began at a S1 P concentration of10^-7mol/L,which induced a more fibroblast-like morphology with reduced cell-cell contact as assessed by phase contrast light microscopy.By Western Blot and RT-PCR analysis,α-SMA,FN expression significantly increased and E-cad significantly decreased in A549 cells treated with S1 P compared with negative controls（P<0.05）in a time-and concentration-dependent manner.ConclusionsThe results documented that S1 P can induce EMT in A549 cells in vitro in a time-and concentration-dependent manner,which confirmed that S1 P induced idiopathic pulmonary fibrosis（IPF）in alveolar type Ⅱ cells by EMT.Part 2:Cross-communication on EMT between S1 P and TGF-β1 signaling by SPHK1/S1P_2-3 Backgroud and ObjectiveThe synthesis and catabolism of S1 P is regulated by a number of enzymes such as sphingosine kinase（SPHKs）and sphingosine lyase（S1PL）.The resulting S1 P binds to G-protein coupled receptors（GPCRs）to activate a series of signal transduction molecules,triggering the corresponding molecular network system to regulate EMT formation.In fact,the S1 P molecular network system can also be interacted with other cytokine network systems to complete corresponding biological effects.Transforming growth factor（TGF-β1）is a class of cytokines with multiple biological activities,which is also a classical factor to promote the formation and progression of pulmonary interstitial fibrosis.It mainlymediates EMT through Smad and non-Smad ways to regulate the formation of pulmonary interstitial fibrosis.The aim of this study was to compare the biological effects of S1 P and TGF-β1 and to investigate whether TGF-β1 participates in S1 P network through receptors（S1PRs）and related enzymes.MethodsThe study includes five experiments.1 To compare the effects between S1 P and TGF-β1.The cultured A549 cells were divided into three groups: a,negative control;b,treated with TGF-β1（5ng/ml）for 72 h as positive control;c,treated with S1P(10^-5 mol/L)for 72 h.By indirect immunofluorescence,α-SMA and E-cad expression were analyzed.Besides,the protein and m RNA expression of α-SMA,FN and E-cad were assessed by Western Blot and RT-PCR,respectively.2 To explore the related receptors.The cultured A549 cells were divided into five groups: a,negative control;treated with S1P(10^-5 mol/L)for 72 h as positive control;c,co-treated with W146(10^-6mol/L)and S1P(10^-5 mol/L),for 30 min and 72 h respectively;d,co-treated with JTE013(10^-6mol/L)and S1P(10^-5 mol/L),for 30 min and 72 h respectively;e,co-treated with CAY10444(10^-5mol/L)and S1P(10^-5 mol/L),for 30 min and 72 h respectively.The protein and m RNA expression of α-SMA,FN and E-cad were assessed by Western Blot and RT-PCR,respectively.3 To explore whether S1 P can promote the secretion and expression of TGF-β1 by binding to S1 PRs.The groups are the same with the former.The secretion of TGF-β1 was detected by ELISA,and the protein and m RNA expression of TGF-β1 were assessed by Western Blot and RT-PCR,respectively.4 To examine if TGF-β1 unregulated the expression of SPHK.The cultured A549 cells were divided into two groups: a,negative control;b,treated by TGF-β1 with different concentration（0.5,2,5,10ng/ml）for 72 h,respectively.The protein expression of SPHK was assessed by Western Blot.5 To analyze whether TGF-β1 mediates EMT responses through S1 PRs.The cultured A549 cells were divided into five groups: a,negative control;b,treated with TGF-β1（5ng/ml）for 72 h as positive control;c,co-treated with W146(10^-6mol/L)and TGF-β1（5ng/ml）,for 30 min and 72 h respectively;d,co-treated with JTE013(10^-6mol/L)and TGF-β1（5ng/ml）,for 30 min and 72 h respectively;e,co-treated with CAY10444(10^-5mol/L)and TGF-β1（5ng/ml）,for 30 min and 72 h respectively.The m RNA expression of α-SMA,FN and E-cad were assessed by RT-PCR.Results1、By indirect immunofluorescence,Western Blot and RT-PCR analysis,the expression of α-SMA,FN and E-cad of A549 treated with S1 P were not changed compared with the positive control（P>0.05）.2、The protein and m RNA expression of α-SMA and FN of cells in JTE013 and CAY10444 groups significantly decreased,whereas the expression of E-cad significantly increased compared with positive controls（P<0.05）.However,the expression of all related markedmolecules did not change significantly when the cells were co-treated with W146 and S1P（P>0.05）.3、The secretion of TGF-β1 was dramatically decreased when cells are were co-treated with JTE013 and S1 P,or CAY10444 and S1 P compared with positive controls（P<0.05）.And the secretion of TGF-β1 in W146 group was not significant（P>0.05）.Similarly,the protein and m RNA expression of TGF-β1 of cells in JTE013 and CAY10444 groups significantly downregulated（P<0.05）.However,the expression of TGF-β1 did not display obvious change when the cells were co-treated with W146 and S1P（P>0.05）.4、TGF-β1 unregulated the protein expression of SPHK1 in a concentration-dependent manner（P<0.05）.Notably,we did not detect the evident expression of SPHK2（P>0.05）.5、the m RNA expression of α-SMA and FN of cells in JTE013 and CAY10444 groups significantly lessened,whereas the expression of E-cad significantly increased compared with positive controls（P<0.05）.However,the expression of α-SMA,FN and E-cad did not change obviously when the cells were co-treated with W146 and S1P（P>0.05）.Conclusions1、TGF-β1 and S1 P display the similar biological effects,and S1 P can induce EMT of alveolar epithelial cells in vitro coupled with S1P_2-3.2、Based on the mediation of S1P_2-3,S1 P increased the expression and secretion of TGF-β1,which amplified the S1 P signaling system.3 、 TGF-β1 unregulated the protein expression of SPHK1 in a concentration-dependentmanner and inhibition of S1P_2-3 suppressed the TGF-β1-induced EMT in A549 cells,suggesting that cross-communication on EMT between S1 P and TGF-β1 signaling by SPHK1/S1P_2-3.