Differential Proteome Analysis Of Reverted And Activated Hepatic Stellate Cells

Activated hepatic stellate cell (HSC) is the main myofibroblast cell type in the liverfibrosis(LF). In normal liver, hepatic stellate cells play a key role in the storage andtransport of retinoids (vitamin A compounds). Once liver injured, hepatic stellate cell“activation” refers to the conversion of a resting vitamin A-rich cell to one that isproliferating, brogenic, and contractile. The fibrotic liver could revert to be lessfibrotic or even normal architecture after the elimination of pathological factors. Animportant characteristic of the recovery of LF is reversal of myofibroblast-likephenotype to a quiescent-like phenotype in addition to undergoing apoptosis andsenescense. Thus, understanding the changes of cellular and secreted proteins in thereversion of activated HSCs may provide the broader view of cellular regulatorynetworks and discover candidate markers or targets for therapeutic strategies of LF.Mass spectrometric-based SILAC (Stable isotope labeling by amino acids in cellculture) technology was adopted to study differential expressed celluarproteome/secreatome of LX-2(hepatic stellate cell line) between reversion andactivation. In total celluar proteome,2,293proteins were confidently identified (falsediscovery rate <0.05), of which1,347proteins were quantified. Compared withactivated HSCs,273proteins showed significant differences in reverted HSCs (≥1.5fold). GeneGO/MetaCore software analysis showed that up-regulated proteinsassociated with reversion of HSCs mainly related to oxidation-reduction and lipidmetabolism, while the top of down-regulated proteins was found in regulatedcytoskeleton formation. In total secreatome,330proteins showed significant differencesin reverted HSCs. Among these,109up-regulated proteins were mainly involved inamino acid metabolism pathway and glucose metabolism pathway usingGeneGO/MetaCore software, while221down-regulated proteins are closely associatedwith HSCs activation, such as cytoskeleton remodeling, chemokines and cell adhesion.Changes in the expression levels of cellular and selected proteins were verified byWestern blot analysis, especially Signal transducer and activator of transcription1(STAT1), Filamin A (FLNA), LIM and SH3domain protein (LASP1), nicotinamidephosphoribosyltransferase (NAMPT), Vitronectin, laminin beta1(LAMB1) andUbiquitin conjugation factor E4B (UBE4B) proteins. The distinct roles of STAT1were further analyzed between activated and reverted of HSCs，and STAT1can be viewed asa key regulator for HSCs reversion.Taken together, the proteomic analysis could provide insights into the differencebetween reverted and activated of HSCs and may accelerate our understanding themechanisms of HSC reversion on cessation of fibrogenic stimuli, identified somepotential biomarkers of LF in clinical studies and provide new targets for antifibroticliver therapy.