Role Of Mitogen-Activated Protein Kinases In Human Skin Fibroblast Cells Based On A Lipopolysaccharide Stimulated Model

Hypertrophic Scar(HTS)is common in burn patients,according to researches,30%?91%patients develop hypertrophic scar after burn injuries.Huamn skin fibroblast cell(HSF)is the effect cell of developing HTS,thus plays an important role in HTS,therefore,researches on HSF in becoming a hot spot in treating scars.Researchers believe that the local infection and inflammation of wound area is the key factor that leads HSF to change on the phenotype level.Reseachers in China had established an in vitro model with certain concentration level of lipopolysaccharide(LPS)that could stimulate normal HSF to have the statistically sameness on the phenotype level with HTS patitens' HSF.This study focuses on the signaling pathway based on this model.Objective:Using in vitro model of HSF to reveal the activation or inhibition of Mitogen-Activated Protein Kinases(MAPK)pathway,experiments on three major sub-pathway of Erk,JNK and p38 could show us the changes of each pathway and how they react with different stimulation time and intensity.Next step we are using inhibitors of the activated pathway in the model,the performing experiments on the HSF include proliferation,TGF-?1 and IFN-? level,expression,synthesis and secretion of type ?and ? collagen and gene chip analysis.Methods:(1)Using Western Blotting to analyze the expression level of Erk,p-Erk,JNK,p-JNK,p38,p-p38,different conditions include time and concentration level.Concentration level of LPS 0.1ug/mLwith stimulation duration 0h,24 hours,48 hours and 72 hours;Stimulation duration 48 hours but different concentration level of LPS 0ug/mL,0.005ug/mL,O.Olug/mL,0.05ug/mL,0.1ug/mL,0.5ug/mL and lug/mL.(2)Using inhibitors of SP600125 and PD98059 to inhibit the activated sub-pathway of MAPK,then give a stimulation of 0.1ug/mL LPS with 48 hours to establish the model.We performed following experiments to analyse the changes of HSF.(2-1)Experiments on proliferation:Form of change with inverted microscope,curve of proliferation with MTT test,count of proliferation with trypanblau test and cycle of proliferation with flow cytometry.(2-2)Experiments on secretion level of TGF-? 1 and IFN-y:Phenotype change with HE immunohistochemistry,detailed cell change with transmission electron microscope and secretion level of TGF-?1 and IFN-? with ELISA.(2-3)Experiments on expression,synthesis and secretion of type ? and ? collagen:Pepsin digestion after incorporation of 3H-proline into stable,single-layered,confluent HSF,expression level of mRNA of type ? and ? collagen with RT-QPCR,expression level of type ? and ?collagen in cells with immunofluorescence staining and expression level of type ? and ?collagen and hydroxyproline in the supernate with ELISA.Results:(1)Results of same LPS concentration level and different stimulation duration:Compared with control group,inhibited groups show the increased level of p-JNK and p-Erk with time.Results of same LPS stimulation duration and different concentration level:The activation of MAPK/JNK and MAPK/Erk were increased with LPS concentration level,and reached highest point at 0.1ug/mL.(2-1)Results on the proliferation of HSF:Compared with control group,the curve of proliferation,counts of proliferation and S stage of cell cycle in single inhibition groups are lower,double inhibition gourp is lowest.(2-1)Results on the secretion of TGF-?1 and IFN-? level:Compared with control group,the a-SMA and al(?)level are lower,cell damage are worse,with lower level of TGF-?1 and higher level of IFN-y,trending of double inhibition group is more evident.(2-3)Results on expression,synthesis and secretion of type ? and ? collagen:Compared with control group,single inhibition group have less collagen DNA synthesis,less mRNA of type ? and ? collagen,less type ? and ?collagen in supernatant,expression,synthesis and secretion of type ? and ? collagen.Conclusion:(1)LPS stimulated the activation of MAPK/Erk and MAPK/JNK pathwaysin the in vitro model of hypertrophic scar,this could be an important mechanisem in the formation of hypertrophic scar.(2)Inhibiting the activated pathway(s)could interfere the phenotype change and the synthesis of collagencollagen,this provides a new perspective and treating strategy for treating hypertrophic scar.