Hepatic fibrosis is an inevitable process in the transformation of chronic HBV infection to hepatic cirrhosis, but its detailed mechanism is still unknown. Fontana et al. retrospectively analyzed the clinical progression of serum hepatic fibrosis markers obtained from currently laboratory methods and discovered that these clinical test methods can only reliably recognize advanced fibrosis, and cannot accurately identify the degree of fibrosis present during the early and middle portions of its progression . Therefore, identification of HBV hepatic fibrosis biomarkers is of great significance for early diagnosis and prevention of fibrosis.
Serum proteomics studies serum proteins which are readily available. However, because of proteome analysis restrictions for sample size, highly abundant proteins often make it difficult to separation and identify less abundant proteins, an issue which is particularly evident in the proteomic analyses of serum . Thus, it is necessary to first remove the interfering high abundance proteins prior to serum proteome analysis. In this study, we removed the albumin which improves the detection rate of low abundance proteins with good reproducibility. A total of 27 differentially expressed genes were found in patients with HBV hepatic fibrosis compared to the plasma of HBV carriers, of which 19 were up-regulated and 8 were down-regulated in the serum of patients with HBV hepatic fibrosis.
As one of the key enzymes of glycolysis, enolase-1 (α-enolase) widely exists in many tissues and its expression varies with cellular pathological physiology, metabolism, inflammation, and the state of cell development [11, 12]. Enolase-1 also plays an important role in cell energy metabolism . Enolase-1 is expressed at the cell surface where it promotes cancer invasion, and is subjected to a specific array of post-translational modifications, namely acetylation, methylation and phosphorylation. Enolase-1 binds plasminogen at the cell surface, enhancing local plasmin production and monocyte migration through epithelial monolayers, and promoting matrix degradation. These data suggest an important mechanism of inflammatory cell invasion is mediated by increased cell-surface expression of enolase-1 . Both enolase-1 over expression and its post-translational modifications could be of diagnostic and prognostic value in cancer . Takashima et al. analyzed the hepatic tissue of patients with hepatitis b virus-related hepatocellular carcinoma (HCC) by proteomics analysis and found that expression of enolase-1 was enhanced, which is particularly apparent in poorly-differentiated HCC . Enolase-1 acts as a central element in colon cancer susceptibility and protein biosynthesis . However there are no reports about enolase-1 and hepatic fibrosis. Our experimental results indicated that the expression level of enolase-1 in the serum of patients with HBV hepatic fibrosis was significantly higher than that in HBV carriers. Its change of concentration in the blood may reflect the degree of hepatic fibrosis suggesting that enolase-1 can be used as a serum marker for the prediction of hepatic fibrosis.
Thrombospondin-1 (TSP-1) is a glycoprotein with a molecular weight of 450 kDa, and is a major component of platelets . It is involved in angiogenesis and inflammation and the effects of TSP-1 have been studied in numerous preclinical tumor models. Many normal cells, including endothelial cells, fibroblasts, adipocytes, smooth muscle cells, monocytes, and macrophages were all founded to secrete TSP-1 [19, 20]. TSP-1 binds to the protein components of the extracellular matrix such as fibronectin. TSP-1 is also expressed in glomerulopathies and is considered an early marker of inflammation and fibrosis .
TSP-1 can bind to various receptors and functions in regulating cell proliferation, proteases and the activity of transforming growth factor beta-1 (TGF-β1) [22, 23]. As an activator of TGF-β1, TSP-1 modulates the functions of TGF-β1 in cardiovascular diseases, atherosclerosis, and obesity. In addition, TSP-1 is selectively expressed in the infracted border suggesting that TSP-1 might inhibit the expansion of inflammation by activating TGF-β1 . TSP1 is also a major endogenous activator of TGF-β1 in experimental inflammatory glomerular disease. Regulation of TSP-1 with regards to its influence on TGF-β1 activity may be one of the causes of fibrosis, as TGF-β1 positively regulates HCV RNA replication which is likely manifested in the liver fibrosis associated with hepatitis C infection . It has been reported that TSP-1 is closely related to renal fibrosis in which TSP-1 is highly expressed . In studies of hepatic fibrosis, TSP-1 is found to be highly expressed. TSP-1 might play an additional role in liver fibrogenesis by stimulating angiogenesis and could be a potential target to prevent fibrogenesis in chronic inflammatory diseases of the liver .
NF-κB is a transcription factor present in almost all animal cell types. It is involved in many biological processes such as inflammation, immunity, cell differentiation, cell growth, tumorigenesis and apoptosis . Studies indicate that NF-κB and TSP-1 together, modulated by the expression of the androgen receptor, exert antitumor effects in prostate cancer . In the tumor microenvironment, TSP-1 has been shown to suppress tumor growth by inhibiting angiogenesis and activating TGF-β1. NF-κB mRNA expression and activity were significantly enhanced by proteinuria-loading and were synchronized with high expression of TSP-1, and TGF-β1 mRNA in the kidney . We found that expression of TSP-1, TGF-β1, and NF-κB in patients with HBV hepatic fibrosis were all significantly higher than that in the serum of HBV carriers, suggesting that TSP-1, TGF-β1, and NF-κB play important roles in the process of hepatic fibrosis. The detailed mechanisms involved in the interplay between TSP-1, TGF-β1, NF-κB and hepatic fibrosis require further study.
In conclusion, using a proteomics method, we obtained 27 differentially expressed proteins by comparing the serum of HBV carriers and hepatic fibrosis patients. The 27 proteins identified by proteomic methods are involved in cell growth, receptor binding, metastasis, blood coagulation, calcium ion binding, DNA binding, as well as being biomarkers and enzymes. Additional work is required to understand the relationship between these proteins and hepatic fibrosis. The results indicated that TSP-1, TGF-β1, NF-κB, and enolase-1 likely play important roles in the process of hepatic fibrosis. Although the levels of enolase-1 and TSP-1 in the serum of HBV carriers and hepatic fibrosis patients can be detected, the relationship between the degree of liver fibrosis and their expression needs further study.