Role Of Tec Non-receptor Kinase In The Production Of Pro-inflammatory Cytokines In A549 Alveolar Epithelial Cells Induced By LPS

I.Research BackgroundAcute lung injury refers to an incomplete acute hypoxic respiratory function caused by the diffused pulmonary interstitium and alveolar edema due to the injury of alveolar epithelial cells and capillary endothelial cells caused by the various direct and indirect inducing factors.The pathophysiologic features include reduction of lung volume,decline of lung compliance,and imbalance of ventilation-perfusion,and hyoxemia and respiratory distress are manifested clinically.In severe phase,this symptom is called acute respiratory distress syndrome(ARDS).Various inflammatory cells will be activated after serious burn,and excessive inflammatory response will occur,leading to systemic inflammatory response syndrome.ALI or ARDS could be regarded as the manifestation of systemic inflammatory response syndrome.Tec is a non-receptor tyrosine kinase of cytoplast with key function discovered in the liver cancer.It mainly exists in the liver,T cells,B cells and hematopoietic cells,regulating the differentiation and development of T cells and B cells.Tec also participates in various signal transduction pathway,including the signal transduction pathway mediated by IL-6,GM-SF,and EPO cell factors,indicating that Tec kinase is,to some extent,related to inflammatory response.The latest research discovers that Tec has promoted the expression of pro-inflammatory factor of monocyte-macrophages in sepsis,but few studies are conducted on the effect of Tec kinase in acute lung injury.A549 alveolar epithelial cells were adopted as research object in this experiment.Endotoxin LPS was used to stimulate A549 cells to simulate acute lung injury.The expression and activation rule of Tec in alveolar epithelial cells were observed,and Tec kinase inhibitor LFM-A13 and Tec-si RNA were applied for intervention in an attempt to observe the role that Tec kinase played in stimulating alveolar epithelial cells to produce pro-inflammatory cytokines IL-8,and to understand the role and mechanism of Tec kinase in producing inflammatory mediator for alveolar epithelial cells during acute lung injury.Therefore,this study aims to further understand the occurrence mechanism of acute lung injury and provide a new method for its prevention.II.Purpose of Research 1.Probe into the influence of LPS on the expression level of Tec kinase in A549 alvelor epithelial cells.2.Study the effect of Tec kinase in stimulating the production of IL-8 in A549 alveolar epithelial cells.3.Learn the influence of Tec kinase inhibitor and si RNA on the secretion and MAPK pathway after using them,expect to further understand the potential mechanism of Tec kinase in the occurrence and development of acute lung injury and provide a new target for the treatment.III.Research Content Part I The Influence of LPS on the Expression of TEC Kinase in A549 Alveloar Epithelial Cells Study was conducted on the dosage effect and time effect when routine culture was done in human alveolar epithelial cells A549.(1)Study on dosage effect: add different concentrations of LPS(0.01,0.1,1,10,100?g/ml)for stimulation for 24 h.(2)Study on time effect: Add 1?g/ml LPS for stimulation for 0,30 min,45min,1h,2h and 3h respectively;Western Blot was adopted to detect the expression of Tec kinase in cells.(3)Research on inhibitor: LPS(1?g/ml)with different concentration of LFM-A13(25?M,75?M,100?M)were combined for pre-treatment for 1h.Cellular protein was extracted and Western Blot was applied to detect the expression of Tec Kinase.Part II The Function of TEC Kinase in Inducing the Production of Pro-inflammatory Cytokines in Alveolar Epithelial Cells1.A549 alveolar epithelial cells that were cultured in the 6-pore plate were divided into6 groups,4 pores of cell in each group,according to the random number table method.1.1 Different concentrations of LPS(0.01,0.1,1,10,100?g/ml)were added respectively for stimulation for 24 h and blank control group was prepared.1.2 1?g/ml LPS was added respectively for stimulation for 0,30 min,45min,1h,2h and3 h respectively and blank control group was prepared.1.3(1)Blank control group: Routine culture was conducted on 1640 culture solution with volume fraction of 10% FBS for 2h;(2)LFM-A13 Group: 75 ?mol/L Tec Kinase specific inhibitor LFM-A13 was adopted for cell pretreatment for 1h,and routine culture for 1h;(3)LPS Group: Routine culture was conducted on 1640 culture solution with 10%FBS of volume fraction for 1h,and 1?g/m L LPS for stimulation for 1h;(4)LPS+LFM-A13 Group: 25?mol/L,75?mol/L,100?mol/L LFM-A13 was respectively used to treat cells for 1h,and 1?g/ml LPS for stimulation for 1h.ELISA method was adopted to determine the IL-8 content of culture supernatant fluid in each group;in the meantime,Western Blot was adopted to detect the activity of Tec Kinase,p38 MAPK and ERK MAPK in cells of 1.3.3.Tec Kinase and si RNA interfered A549 cell specific silencing and Tec kinase gene expression.After transient transfection,LPS1?g/ml was added to stimulate A549 alveolar epithelial cells for 1h.ELISA was used to determine the IL-8 content in culture supernatant fluid;Western Blot was adopted to determine the activity of Tec Kinase,p38 MAPK and ERK MAPK in cells.IV.Research Results Part I(1)In a certain range(<10?g/ml),the expression nonreceptor Tec kinase induced by LPS in A549 alveolar epithelial cells in a dose-dependent manner.LPS of 10?g/ml induce the peak of Tec kinase expression,and then that reduced gradually.(2)The expression of Tec kinase in a time-dependent manner when LPS(1?g/ml)induced A549 alveolar epithelial cells.The expression of macrophage TEC kinase reached to the peak at 1h,then decreased.(3)LPS-induced Tec expression was prevented by LFM-A13 at25?M,75?M and 100?M.Part II(1)ELISA has showed that the release of IL-8 in A549 cell increased by different concentrations of LPS and different induced time.(2)ELISA has showed that LFM-A13 significantly reduced the release of IL-8.Tec-si RNA can evidently abrogate the expression of Tec protein,p38 and ERK phosphorylated protein measured by western blot.V.Research Conclusion1.LPS could induce the expression of Tec kinase in A549 alveolar epithelial cells,and is in dose-dependent relation with LPS;the change of Tec kinase level is in time-dependent relation with LPS;Tec kinase inhibitor LFM-A13 and Tec-si RNA could significantly reduce the expression of Tec kinase stimulated by LPS in A549 alveolar epithelial cells.2.Tec kinase has mediated the the production and release of pro-inflammatory cytokines IL-8 in A549 alveolar epithelial cells through the p38 MAPK and ERKMAPK signal pathway.The production of Tec kinase gene and IL-8 has declined gradually.