Background

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.

Methods

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.

Results

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.

Conclusions

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.

中文翻译：

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酒精性肝纤维化中肝星状细胞的蛋白质组学分析揭示了参与胶原蛋白生产的蛋白质。

背景

肝星状细胞（HSC）激活在酒精诱导的肝纤维化中具有重要作用。酒精性肝纤维化（ALF）中HSC的蛋白质仍未完全了解。在这里，我们进行了蛋白质组学研究，使用从大鼠模型中分离出的HSCs发现与ALF相关的蛋白质。

方法

Sprague–Dawley大鼠用乙醇喂养2或6周。使用Sirius红和油红O染色评估肝组织学。使用Percoll密度梯度离心法富集HSC，并使用流式细胞仪进行分析。使用二维电泳（2DE）分离从HSC中提取的蛋白质。使用液相色谱-质谱（LC-MS）鉴定差异表达的蛋白质。使用UniProtKB数据库和STRING软件分析差异表达蛋白的特征。使用实时RT-PCR分析了两种差异表达蛋白的mRNA水平，其中在ALF模型和人类中使用Western blot（WB）和免疫组化分析进一步研究了NADH脱氢酶（泛醌）黄素蛋白2，线粒体（Ndufv2）。肝组织。使用WB评估了Ndufv2与酒精刺激之间的关系。接下来，Ndufv2在HSC-T6细胞系中被shRNA敲除。检测到与HSC激活相关的三个基因（编码胶原蛋白，金属蛋白酶抑制剂1 [TIMP-1]和α-平滑肌肌动蛋白[a-SMA]）。

结果

成功建立了ALF模型，肝纤维化评分为1-2（S1-2），并且出现了一些较大的脂肪液泡。使用质谱法鉴定了21种差异超过2倍的非丰富蛋白质，包括7种上调的蛋白质和14种下调的蛋白质。这些差异蛋白是对抗原呈递，线粒体代谢，乙醇和胶原蛋白降解的反应。其中，两种上调的蛋白（Ndufv2和ATP合酶亚基α，线粒体[ATP5a1]）参与了ALF的线粒体代谢，并显示出mRNA和蛋白水平的同时变化。如WB所示，Ndufv2在HSC中在大鼠模型和人肝组织的非实质细胞（NPC）中上调，并使用免疫组织化学检测。酒精刺激后，Ndufv2也被上调。

结论

进行了蛋白质组学研究，以发现从大鼠模型中分离的HSC中与ALF相关的蛋白质。鉴定出二十一种差异表达的蛋白质，包括参与线粒体代谢和抗原呈递的蛋白质。Ndufv2是ALF中的一种上调蛋白，可能通过调节纤维化因子的产生而参与ALF。