Effect Of P38MAPK Signaling Pathways On IL-1β-induced ICAM-1Expression In Lung Fibroblasts

Objective: The mainly pathogenesis of pulmonary fibrosis is fibroblastproliferation and intercellular adhesion molecule-1(ICAM-1) expression. P38mitogen-activated protein (MAP) kinase signal transduction pathway whichplays a crucial role in intracellular signaling. P38MAPK may be involved inthe formation and development of pulmonary fibrosis. In this study culturesthe lung fibroblasts of rat’s embryo in vitro, We investigated the effect ofIL-1β on ICAM-1expression of fibroblast, and determine the level ofphosphor-p38MAPK and AP-1protein in the lung fibroblasts. The aim of thepresent study is to investigate the roles of p38MAPK in adhesion moleculesexpression, which could be a theoretical base of pathogenesis and treatmentpulmonary fibrosis.Methods: Explant cultures of embryo lung fibroblasts: Anaesthetized thewistar parturient pregnant rats immediately by ethylether, remove the fetal ratand take its lung tissue. Discard bronchial, connective tissue, and then cut thelung tissue into1mm×1mm×1mm size small, put it into DMEM medium,percuss it evenly applied to the culture flasks with a sterile pipette, until thecells covered the bottom of the bottle, add the appropriate culture medium andcontinued to cultivate, in this process to discard red blood cells, epithelial cells,small tissue block. That is fifth or sixth generations of lung fibroblast withconsistent in morphology and structure. Use fibroblast in fifth or sixthgenerations for testing.The cultured lung fibroblasts were divided into three groups (n=6wells):(1)Control group: cultured lung fibroblasts in with dimethylsulfoxide(DMEM) for6hours;(2)Intervention group with IL-1β: use the culturemedium which was contains IL-1β（15ng/ml） to stimulate lung fibroblasts6hours;(3)The intervention group with p38MAPK inhibitor (SB203580) +IL-1β: Deal with lung fibroblasts for20minutes in advance, then use theculture medium which was contains IL-1β（15ng/ml）to stimulate lungfibroblasts6hours. RT-PCR method was used to determination the expressionlevels of ICAM-1mRNA. Western Blot protein immune imprinting was usedto determination the expression level of phosphor-p38MAPK and AP-1/Junprotein.Results:1Successfully primary cultured Wister pregnant rat lung fibroblastsThis experiment adopts the tissue culture technology in Wistar rat fetuseslung fibroblasts, some round cell can be found around the tissue block under amicroscope after5-10hours. After about one week later, the round cellbecome oval-shaped and rhombus with the fusion cells after2-3times oftrypsin digestion, the legacy organization, red blood cells, epithelial cells weregradually cleared. The rest whose form, structure, growth state are consistentwith each other(Consistent Cell morphology, fullness cell body, uniformcytoplasm, nucleolus clear, visible mitotic, good growth condition) is the ideallung fibroblast cells in experimental models.2Using RT-PCR methods to determination the changes of ICAM-1mRNAexpression level in lung fibroblast cellICAM-1mRNA electrophoresis bands of light density scanning display,in lung fibroblasts has a certain amount of basal expression of ICAM-1mRNA,control group was0.303±0.011; intervention group with IL-1βwas0.887±0.051, significantly higher than that the control group (P<0.05); Theintervention group with p38MAPK inhibitor (SB203580)+IL-1β was0.719±0.014, significantly lower than intervention group with IL-1β(P<0.05), higher than that in control group (P <0.05).3Using Western blot method to determination the phosphor-p38MAPK andAP-1/Jun protein expression level changes in the lung fibroblasts:In lung fibroblasts has basic expression of phosphor-p38MAPK, thecontrol group was0.295±0.033; IL-1β intervention group was0.735±0.025,significantly higher than that in the control group (P<0.05); The intervention group with SB203580was0.492±0.010, significantly lower than interventiongroup with IL-1β (P<0.05), higher than that in control group (P <0.05).In lung fibroblasts has basic expression of AP-1/Jun, the control groupwas0.188±0.002; IL-1β intervention group was0.712±0.033, significantlyhigher than that in the control group (P<0.05); The intervention group withSB203580was0.321±0.006, significantly lower than intervention group withIL-1β (P<0.05), higher than that in control group (P <0.05).Conclusion:1ICAM-1expression level is very low in the normal lung fibroblast, butafter IL-1β stimulated lung fibroblast, the expression of ICAM-1wassignificantly increased;2IL-1β may up-regulate expression of ICAM-1in lung fibroblaststhrough p38MAPK signaling pathway;3P38MAPK signaling pathway activated by IL-1β, then subsequentlyactivated the AP-1, Ap-1is able to bind site in promoter of ICAM-1.