Proteomic profiling of hepatic stellate cells in alcohol liver fibrosis reveals proteins involved in collagen production.

Abstract

Hepatic stellate cell (HSC) activation has central functions in alcohol-induced liver fibrosis. Proteins of HSCs in alcoholic liver fibrosis (ALF) are still not completely understood. Here, we performed a proteomic study to discover proteins related to ALF using HSCs isolated from a rat model. Sprague-Dawley rats were fed with ethanol for 2 or 6 weeks. Liver histology was assessed using Sirius red and Oil red O staining. HSCs were enriched by using Percoll density gradient centrifugation, and analyzed using flow cytometry. Proteins extracted from HSCs were separated using two-dimensional electrophoresis (2DE). Differentially expressed proteins were identified using liquid chromatography-mass spectrometry (LC-MS). The characteristics of the differentially expressed proteins were analyzed using the UniProtKB database and STRING software. The mRNA levels of two differentially expressed proteins were analyzed using real-time RT-PCR, of which NADH dehydrogenase (ubiquinone) flavoprotein 2, mitochondrial (Ndufv2) was further investigated using Western blot (WB) and immunohistochemical analysis in the ALF model and human liver tissues. The relationship between Ndufv2 and alcohol stimulation was evaluated using WB. Next, Ndufv2 was knocked-down by shRNA in the HSC-T6 cell line. Three genes (encoding collagen, metalloproteinase inhibitor 1 [TIMP-1], and α-smooth muscle actin [a-SMA]) related to HSC activation were detected. An ALF model was successfully established, with a liver fibrosis score of 1-2 (S1-2), and some big fat vacuoles development. Twenty-one non-abundant proteins with more than a 2-fold difference were identified using mass spectrometry, including 7 upregulated and 14 downregulated proteins. These differential proteins are a response to antigen presentation, mitochondrial metabolism, ethanol, and collagen degradation. Among them, two upregulated proteins (Ndufv2 and ATP synthase subunit alpha, mitochondrial [ATP5a1]) were involved in mitochondrial metabolism in ALF, and showed concurrent changes in mRNA and protein levels. Ndufv2 was upregulated in HSCs, as shown by WB, in non-parenchymal cells (NPCs) in the rat model and human liver tissues, and detected using immunohistochemistry. Ndufv2 was also upregulated after alcohol stimulation. Following Ndufv2 knockdown, collagen, TIMP-1, and α-SMA were downregulated compared with that in the controls. A proteomic study was performed to discover proteins related to ALF in HSCs isolated from a rat model. Twenty-one differentially expressed proteins were identified, including proteins involved in mitochondrial metabolism and antigen presentation. Ndufv2, an upregulated protein in ALF, might be involved in ALF through regulating the production of fibrosis factors.