Elucidation of the molecular events underlying hepatic stellate cell (HSC) activation is an essential step toward understanding the biological properties of HSC and clarifying the potential roles of HSCs in liver fibrosis and other liver diseases, including hepatocellular carcinoma. High-throughput comparative proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) labeling combined with online two-dimensional nanoscale liquid chromatography and tandem mass spectrometry (2D nano-LC-MS/MS) were performed on an in vitro HSC activation model to obtain a comprehensive view of the protein ensembles associated with HSC activation. In total, 2,417 proteins were confidently identified (false discovery rate <1%), of which 2,322 proteins were quantified. Compared with quiescent HSCs, 519 proteins showed significant differences in activated HSCs (≥3.0-fold). Bioinformatics analyses using Ingenuity Pathway Analysis revealed that the 319 up-regulated proteins represented multiple cellular functions closely associated with HSC activation, such as extracellular matrix synthesis and proliferation. In addition to the well-known markers for HSC activation, such as α-smooth muscle actin and collagen types 1 and 3, some novel proteins potentially associated with HSC activation were identified, while the 200 down-regulated proteins were primarily related to immune response and lipid metabolism. Most intriguingly, the top biological function, top network, and top canonical pathway of down-regulated proteins were all involved in immune responses. The expression and/or biological function of a set of proteins were properly validated, especially Bcl2-associated athanogene 2, BAG3, and B7H3.
The present study provided the most comprehensive proteome profile of rat HSCs and some novel insights into HSC activation, especially the suppressed immune response. (HEPATOLOGY 2012;56:332–349)