| Objective: The aim of this study was to clarify the inhibitory effects of ATRA on fibrotic proteomics induced by TGF-β1 in rat PSCs,and to provide experimental basis for the treatment of pancreatic fibrosis in chronic pancreatitis.Methods: Rat PSC line(RP-2 cell)was used for proteomics study.RP-2 cells were placed on 10 cm dishes and cultured in 2.5% FBS DMEM for 24 h.The cells were cultured in 1% FBS DMEM for another 12 h.The cells were then divided into 3groups,(1)Ctrl(vehicle buffer),(2)TGF-β1(10 ng/ml)and(3)TGF-β1(10 ng/ml)plus ATRA(5 μM),and incubated for 48 h.The cultured cells were collected individually.Protein extraction and trypsin hydrolysis were carried out.LC-MS/MS was used for protein separation and determination.The mass spectrum data were analyzed by bioinformatics analysis,GO analysis and cluster analysis.The differential screening was carried out according to the quantitative results,and the 1.5-fold change was taken as the threshold of differential protein expression,and the statistical diagram of differential analysis was drawn.Results: 644 differential expressed proteins were obtained by proteomic analysis.253 proteins were up-regulated and 267 proteins were down-regulated in TGF-β1 vs Ctrl group.69 proteins were up-regulated and 114 proteins were down-regulated in TGF-β1+ATRA vs TGF-β1 group.24 proteins were up-regulated in TGF-β1 vs Ctrl group and down-regulated in TGF-β1+ATRA vs TGF-β1 group,which were mainly involved in the synthesis and degradation of ECM,the formation of cytoskeleton proteins,energy metabolism,immune response,cell proliferation,autophagy and other processes.16 proteins were down-regulated in TGF-β 1 vs Ctrl group and up-regulated in TGF-β1+ATRA vs TGF-β1 group.Most of them are related to DNA replication,transcription,inhibition of cell proliferation and induction of apoptosis.Through comparative proteomic analysis and Gene Cards database search,23 differential proteins related to fibrosis were obtained,including 8 extracellular matrix components,2 cytoskeleton proteins,12 fibrosis related factors and 1 enzyme mediating TGF-β1-induced fibrogenesis.These 23 proteins was up-regulated in TGF-β1 vs Ctrl group and down-regulated in TGF-β1+ATRA vs TGF-β1 group.Through the construction of protein-protein interaction network map,the first ten important node proteins were obtained in each group,in which TGF-β1 vs Ctrl group was EGFR,BUB1,CCNA2,PIK3 CD,RAP1A,RAF1,PRKACB,CAV1,COL4A2,ITGA1;TGF-β1+ATRA vs TGF-β1 group was FBN1,SPARC,COL5A2,COL3A1,COL5A1,P4HA3,FASN,CAV1,BECN1,EGFR.These proteins are mainly involved in ECM synthesis and degradation,immune response,cell proliferation,apoptosis,autophagy,energy metabolism and so on.Conclusion: In this study,we complete a comparative proteomic analysis using rat PSC line(RP-2 cell),confirming that ATRA can inhibit pancreatic fibrosis induced by TGF-β1 in the cells.This study provides experimental data and theoretical basis for the administration of ATRA in treating fibrosis in chronic pancreatitis. |
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