Subcellular proteome analysis unraveled annexin A2 related to immune liver fibrosis

Abstract

It is important to study the mechanism of liver fibrogenesis, and find new non-invasive biomarkers. In this study, we used subcellular proteomic technology to study the plasma membrane (PM) proteins related to immune liver fibrosis and search for new non-invasive biomarkers. A rat liver fibrosis model was induced by pig serum injection. The liver fibrogenesis from stage (S) S0-1, S2, S3-4, and S4 was detected by Masson staining and HE staining in this rat model after 2, 4, 6, and 8 weeks of treatment. The liver PM was enriched and analyzed using subcellular proteomic technology. The differentially expressed proteins were verified by Western blotting, immunohistochemistry, and ELISA. PM with 149-fold purification was obtained and 22 differentially expressed proteins were identified. Of which, annexin A2 (ANXA2) was detected to be increased obviously in S4 compared with S0-1, and verified by Western blotting of rat liver tissue and immunohistochemistry of rat and human liver tissue. The expression of ANXA2 in human plasma with S1-2 was also found to be up-regulated for 1.4-fold than that in S0. Furthermore, ANXA2 was detected to translocate from nuclear membrane and cytosol to PM as HBV stimulation through immunocytochemical analysis in vitro. This study identified 22 differentially expressed proteins related to liver fibrosis, and verified a potential biomarker (ANXA2) for non-invasive diagnosis of immune liver fibrosis. To our knowledge, it was the first time to dynamically study the proteins related to liver fibrosis and select biomarkers for liver fibrosis diagnosis through PM proteome research.

Proteome analysis of hepatic non-parenchymal cells of immune liver fibrosis rats

“…Animal studies were performed following the relevant national legislation and local guidelines. Establishment of the animal model was performed at the Center of Laboratory Animals according to a previously published procedure [21]. Each rat in pig serum and control groups was intraperitoneally injected with 0.5 mL pig serum (COSMO BIO, Japan) or 0.5 mL saline, respectively.…”

“…Five-micrometer paraffin sections were subjected to Masson's trichrome staining as described previously [21]. Hepatic parenchymal cell and NPC fractions purified by Percoll density centrifugation were fixed in 4% paraformaldehyde and then stained with Masson's trichome.…”

“…Pooled NPCs from 4-and 8-week time points were analyzed by 2DE using an IPGphor isoelectronic focusing system (GE Healthcare, USA) and Protein II electrophoresis apparatus (Bio-Rad, USA) as described previously [21,25]. A total of 1 mg of protein from each sample was applied to IPG dry strips (pH 3-10 NL, 180 mm×30 mm×0.5 mm).…”

“…After ImageMaster analysis, spots of differentially expressed proteins were cut out of the gels, digested, and then analyzed by nano flow liquid chromatograph (LC)-electrospray ionization tandem mass spectrometry (MS) according to our previous reports [21,26]. The tryptic peptide mixtures were injected into an Ultimate 3000 instrument (LC Packings, Dionex, USA) with a C18 µ-precolumn (300 µm id×5 mm, 5 µm, PepMap™) (LC Packings, Dionex, The Netherlands) at a flow rate of 20 µL min 1 .…”

“…We established the ILF model according to our previous study [21]. As shown in Figure S1 in Supporting Information, at 2 weeks after serum injection, inflammatory cell infiltration and hepatic fibrosis occurred around the portal area, and liver fibrosis was diagnosed as S0-1.…”