Effect Of LPS On Expression Of Src Suppressed C Kinase Substrate In Rat Pulmonary Microvascular Endothelial Cells

Background Vascular hyperpermeability is one of the most important pathological features and pathogenesis of acute lung injury(ALI),when it occours,fluid and protein transfer from vascular vessel to interstitial and alveolar,that cause fatal pulmonary edema and respiratory failure.Acute respiratory distress syndrome(ARDS) is a serious phase of ALI.Therefore,study on pulmonary vascular permeability is a key subject in ALI and ARDS fields,investigation of the mechanism of vascular permeability increasing is very important.Inflammatory mediators cause vasopenneability increasing by changing the cytoskeletal protein's contents,distribution or functions of vascular endothelial cell(EC),and result in diversity of EC cytoskeleton,that lead to EC recovery,larger gap juctions and EC monolayer hyperpermeability,src suppressed C kinase substrate(SSeCKS) is a new-found cytoskeletal protein,people did a lot of work to illustrate the role it took part in tumorigenesis,but payed little attention to how it worked in regulation of vasopermeability.SSeCKS is also considered to be an inflammation response protein.In this study,we first cultured rat pulmonary microvascular endothelial cells(RPMVEC) in vitro,then used bacterial endotoxin-LPS to induce inflammation,and observed changes of SSeCKS's expression.Objective To study the expression of src suppressed C kinase substrate(SSeCKS) mRNA and protein in rat pulmonary microvascular endothelial cells(RPMVEC) induced by lipopolysaccharide,and the interfering effect of methylprednisolone sodium succinate.Obersve the effect of methylprednisolone sodium succinate on LPS induced RPMVEC monolayer permeability injury.Methods RPMVEC were isolated and cultured in vitro,then groped randomly according to different culture time and different dosage of LPS,and the expression of SSeCKS mRNA in RPMVEC of different groups were determined by reverse transcription polymerase chain reaction(RT—PCR),the expression of SSeCKS protein in RPMVEC of different groups were determined by Western blot.Micro infiltrato rwas used to measure the changes of RPMVEC monolayer permeability coefficient(Kf) after exposure to LPS or LPS with Rotterin together.Results1.RPMVEC were isolated and cultured in vitro successfully,and was proved by morphology and FITC-BSI binding experiment.2.Amplificated SSeCKS specific band from RPMVEC total RNA using RT-PCR,detected SSeCKS protein blot band from RPMVEC cell protein extract.3.Normally,the expression of SSeCKS in RPMVEC was maintained on a low level,and it could be elevated by stimulation of LPS,and the changes in the expression exhibited in dosage and time dependent manne.After being stimulated with LPS for 1 hour,the extent of increase in SSeCKS mRNA in RPMVEC coincided with the increased doseage of LPS,SSeCKS protein had the same increase tendency;When RPMVEC were stimulated with 10mg/L of LPS,the cxpression of SSeCKS mRNA began to increase at 0.5 hour,peaked at 1 hour,then decreased gradually,and it remained high wven at 12 hours,after methylprednisolone sodium succinate had been given,the increase of SSeCKS mRNA induced by LPS could be inhibited;the expression of SSeCKS protein began to increase at 0.5 hour,peaked at 3 hour,then decreased gradually,and it remained high even at 12 hours.4. Methylprednisolone sodium succinate could release LPS induced RPMVEC Kf increase. Conclusion 1.We have successfully cultured and identified the primary RPMVEC in vitro.2.LPS could induce up-regulation of SSeCKS mRNA and protein,the elevation was in time and doseage dependent manner.Methylprednisolone sodium succinate was involved in inhibition of increase of SSeCKS mRNA induced by LPS.3. Methylprednisolone sodium succinate could release LPS induced RPMVEC Kf increase,this indicated SSeCKS was related with LPS induced injury of RPMVEC.