Ethanol and fish oil induce NFκB transactivation of the collagen α2(I) promoter through lipid peroxidation -: Driven activation of the PKC-PI3H-Akt pathway
Ethanol and fish oil induce NFκB transactivation of the collagen α2(I) promoter through lipid peroxidation -: Driven activation of the PKC-PI3H-Akt pathway
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DOI：
10.1002/hep.21659
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发表时间：
2007-06-01
期刊：
影响因子：
13.5
通讯作者：
Nieto, Natalia
中科院分区：
文献类型：
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作者：
Nieto, Natalia
为了分析鱼油作为n-3系列多不饱和脂肪酸的来源是否能在体内与乙醇协同促进胶原蛋白I的上调，我们将胶原蛋白α 2(I)启动子- β Gal （col1a2 - β Gal）转基因小鼠喂食在乙醇（乙醇组）或葡萄糖（对照组）存在下富含鱼油的饲料。乙醇喂养小鼠表现出轻度脂肪变性，谷丙转氨酶（ALT）、天冬氨酸转氨酶（AST）、非甾体脂肪酸和血浆酒精水平升高，细胞色素P450 20活性升高，脂质过氧化终产物升高，谷胱甘肽（GSH）水平降低，提示氧化应激和肝损伤增强。通过β - Gal活性评估的COL1A2启动子在体内和在体外转染胶原蛋白(1)启动子（COL1A1）和COL1A2启动子的缺失构建物，显示了COL1A2启动子的反活化增加。通过核体外转录运行、northern blot分析和定量聚合酶链反应验证COL1A1和COL1A2启动子的转录调控，随后胶原I蛋白上调，基质金属蛋白酶13 （MMP 13）未发生变化。为了进一步分析I型胶原蛋白上调的可能机制，我们设计了体外共培养模型，将原代星状细胞接种于Boyden室底板上，其余肝细胞接种于细胞培养插入物上，并添加鱼油或鱼油加乙醇。鱼油和乙醇的组合在体内和共培养中增加了核因子kappa B与COL1A2启动子的结合，也导致蛋白激酶C的磷酸化，PI3激酶的活化和Akt的磷酸化增加。体外添加维生素E阻止了这种激活和胶原蛋白I的增加。此外，所有3种激酶的抑制剂阻断了胶原I和NF κ B与COL1A2启动子结合的增加；后者也可以通过维生素e来预防。这些结果表明，鱼油（主要是n-3多不饱和脂肪酸[PUFAs]）可以与乙醇协同诱导胶原1，在没有明显脂肪变性和炎症的情况下，通过脂质过氧化- pkc - pi3k - akt - nf κ b驱动机制激活COL1A2启动子。
To analyze whether fish oil, as a source of polyunsaturated fatty acids from the n-3 series, could synergize with ethanol to promote collagen I upregulation in vivo, collagen alpha 2(I) promoter-beta Gal (COL1A2-beta Gal transgenic mice were fed a diet enriched in fish oil in the presence of ethanol (ethanol group) or dextrose (control group). Ethanol-fed mice showed mild steatosis, increased alanine aminotransferase (ALT, aspartate aminotransferase (AST), nonsterified fatty acids, and plasma alcohol levels along with elevated cytochrome P450 20 activity, lipid peroxidation end products, and low glutathione (GSH) levels, which suggested enhanced oxidant stress and liver injury. Increased transactivation of the COL1A2 promoter assessed by beta Gal activity was shown in vivo and by transfection with deletion constructs for the collagen al (1) promoter (COL1A1) and COL1A2 promoters in vitro. Transcriptional regulation of both COL1A1 and COL1A2 promoters was validated by nuclear in vitro transcription run-on, northern blot analysis, and quantitative polymerase chain reaction, which was followed by the subsequent upregulation of collagen I protein with no changes in matrix metalloproteinase 13 (MMP 13). To further analyze the potential mechanism for collagen I upregulation, an in vitro coculture model was designed with primary stellate cells seeded on the bottom plate of a Boyden chamber and the rest of the liver cells plated on a cell culture insert, and fish oil or fish oil plus ethanol were added. The combination of fish oil plus ethanol increased nuclear factor kappa B binding to the COL1A2 promoter both in vivo and in the cocultures and also resulted in increased phosphorylation of protein kinase C, activation of PI3 kinase, and phosphorylation of Akt. The in vitro addition of vitamin E prevented such activation and collagen I increase. Furthermore, inhibitors of all 3 kinases blocked the increase in collagen I and NF kappa B binding to the COL1A2 promoter; the latter was also prevented by vitamin E. Conclusion. These results suggest that fish oil (mainly n-3 polyunsaturated fatty acids [PUFAs]) can synergize with ethanol to induce collagen 1, transactivating the COL1A2 promoter through a lipid peroxidation-PKC-PI3K-Akt-NF kappa B-driven mechanism in the absence of overt steatosis and inflammation.