| Objective:To investigate the effect of Rhizoma Paridis total saponin(RPTS) on the protein expression profile of HepG2 cells,and to screen the RPTS anti-tumor related proteins.Methods:①RPTS was prepared according to the mothod of saponin extraction.②The effect of RPTS on the proliferation of HepG2 cells was performed by MTT assay.The apoptosis assay and the cell cycle distribution were analyzed by Flow cytometry(FCM).③HepG2 cells were treated with or without 10μg/ml RPTS for 48h,and then the total proteins extracted from HepG2 cells were separated and visualized by immobilized pH gradient(IPG)two-dimensional gel electrophoresis(2-DE).The differential expression proteins between RPTS-treated group and control group were analyzed using ImageMaster software and identified by peptide mass fingerprinting(PMF)based on matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS).Mascot software was used to identify the differential protein spots.The proteins were searched in NCBI protein databases.Results:①RPTS was successfully obtained and identified by TLC and HPLC.②RPTS could markedly inhibit the proliferation of HepG2,and the inhibiting effect was concentration and time dependent.The IC50of RPTS in HepG2 cells for 48h treatment is 10.0±1.2μg/ml.③After incubation with 10μg/ml RPTS for 48h,apoptosis rate of HepG2 cells were 20.3%,compared with 1.7%in HepG2 cells without RPTS treatment.HepG2 cells with 10μg/ml RPTS treatment for 48h resulted in a higher number of cells in the S phase(52.88%)compared to the control(23.58%).④The 2-DE patterns with high resolution and reproducibility were obtained.For RPTS-treated group,average spots of the gels were 1690±25,1394±17 spots were matched with an average matching rate of 82.5%.For control group,average spots of the gels were 1700±39,and 1421±13 spots were matched with an average matching rate of 83.6%.The average position deviation of matched spots was 1.232±0.218 mm in isoelectric focusing (IEF)direction,and 1.126±0.142mm in sodium dodecylsulfatepolyacrylamide gel electrophoresis(SDS-PAGE)direction.Fifty-one differentially expressed protein spots were found between control group and RPTS-treated group,of which 31 spots down-regulated and 20 spots up-regulated in RPTS-treated group compared with control group.Fifteen PMF maps were obtained from 15 extremely differentially expressed protein spots (increased or decreased density more than with＞2 fold between control group and RPTS-treated group)by MALDI-TOF-MS,and twelve proteins including dUTPase,hnRNP K,GMP synthase,DNase gamma,Nucleoside diphosphate kinase A,were primarily identified after database searching,some of which were associated with tumor initiation,promotion,and progression.Conclusions:①RPTS could inhibit the proliferation of HepG2 cells.②RPTS not only induced apoptosis of HepG2 cells,but also affected the cell cycle distribution.The cytotoxicity of RPTS might be related to the induction of apoptosis and cell cycle S phase arrest.③RPTS might exert its inhibit effects on HepG2 cells through regulating multiple proteins expression directly or indirectly,such as such as dUTPase,hnRNP K,GMP synthase,DNase gamma and NDKA.These findings would offer valuable insights into the mechanism of anti-tumor effect affected by RPTS treatment in HepG2 cells.
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