Guantitative Proteomic Profiling The Effect Of Cocktail Therapy On Hepatic Stellate Cells

Background:Liver fibrosis is a tissue repair process,which caused by a variety of pathogenic factors, mainly for the excessive deposition of extracellular matrix in the liver, liver fibrosis is a common pathological basis of cirrhosis of the liver, even the liver cancer. Therefore, the search for effective treatment of liver fibrosis is necessary and urgent. In recent years, many studies have confirmed that liver fibrosis can be reversed, the research of drug against liver fibrosis has made some progress, but the complex mechanism of liver fibrosis, and drug therapy focus on a part of the blocking pathogenesis,it seems to little effect. Our research team used epigallocatechin gallate, taurine,and genistein three different mechanism of anti-fibrosis drug coalition cocktail and conducted a series of in vitro and in vivo experiments, we found it has anti-liver fibrosis effect.Objective:In this paper, we take the iTRAQ technology combined with high performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) method of guantitative proteomics,study changes of overall protein levels by the effect of HSC-T6cells with cocktail, analysize and identify the differentially expressed proteins of cells, to explore antifibrotic mechanism of cocktail therapy based on the protein level, and look for reliable biomarkers for the diagnosis of liver fibrosis, treatment and prognosis.Methods:HSC-T6as target cells,when it treated by cocktail, MTT assay was used to determine the impact of drugs on cell proliferation, to make sure the optimal concentration of Cocktail,which inhibit the proliferation of HSC-T6; Then we extract proteins from HSC-T6cells after treated by cocktail of the optimal concentration. The determination of protein concentration with bradford method; equal amount of protein digested by trypsin,and the iTRAQ labeled protein peptide, separated by the cation column (SCX), we use LC-ESI-MS/MS to detect differentially expressed proteins and then use Mascot software to search IPI rat protein database for protein identification, at last, we use bioinformatics to analysize the function of differentially expressed proteins.Results:(1) Cocktail therapy inhibit the proliferation of HSC-T6, the Cocktail of low,medium and high dose group have more significant difference than the control group in A value (0.122±0.009,0.154±0.016,0.187±0.052VS0.223±0.032,P＜0.05), there is no significant difference between the medium dose and low dose group (P>0.05), low-dose and middle dose group have better inhibitor of proliferation than high dose group (P<0.05);(2)Total727proteins were identified by MS. According to the difference of protein abundance reach1.2fold,and the p-value less than 0.05,we consider them as differentially expressed proteins.Compared with control group, three biological replicates identified the number of differentially expressed proteins respectively:a total of285differentially expressed proteins were found in the the first experiment,119proteins were up-regulated,166proteins were down-regulated; In second repeat experiment we found331differentially expressed proteins, of which126proteins were up-regulated,205proteins were down-regulated;330differentially expressed proteins were found in the third experiment,130proteins were up-regulated,200proteins were down-regulated. The analysis manifest36differentially expressed proteins were identified in three biological replicates simultaneously, of which11differentially expressed proteins were up-regulated,25differentially expressed proteins were down-regulated, we found that these differentially expressed proteins correlate with post-translational modification, transcription, recombination and signal transduction pathways.Conclusion:(1) Cocktail therapy inhibit proliferation of HSC-T6,the low-dose group (taurine15μg/ml+EGCG17.5μg/ml+genistein3.5μg/ml)has a good inhibitory effect;(2) serine/threonine protein kinase, matrix metalloproteinase inhibitors1, galectin-3binding protein, plasminogen activator inhibitorthe binding protein1RNA and laminin may be involved in the anti-fibrosis of cocktail on HSC-T6;(3) galectin-3binding protein, plasminogen activator inhibitor1RNA-binding protein can be used as the candidate markers during early diagnosis of liver fibrosis;(4) Cocktail therapy has the effect of inhibiting the proliferation of HSC-T6and anti-fibrosis, may be accomplished by regulation of protein expression.