Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.
Hepatology. 2010 Jun;51(6):1988-97. doi: 10.1002/hep.23572.
Acute alcoholic hepatitis is characterized by disproportionate macrophage inflammatory cytokine responses to bacterial lipopolysaccharide. Lack of knowledge of the underlying mechanism has limited progress toward effective therapy. We postulated a novel mechanism by which ethanol increases histone acetylation, increasing proinflammatory gene transcription and cytokine synthesis. Cytokine responses to lipopolysaccharide in a human macrophage cell line cultured in 86 mM ethanol, 1 mM acetate, and normal media were measured by multiplex immunoassay. Changes in histone acetylation were determined by immunofluorescence microscopy and chromatin immunoprecipitation on presentation. The effect of ethanol and acetate on acetyl-coenzyme A (acetyl-coA) synthetases, which convert acetate to acetyl-coA, the substrate for histone acetylation, was determined by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Knockdown of acetyl-coA synthetases by short hairpin RNA (shRNA) was used to determine their role in ethanol's enhancement of the inflammatory cytokine response. Ethanol-exposed macrophages developed enhanced interleukin 6 (IL6), IL8, and tumor necrosis factor alpha responses to lipopolysaccharide with time-dependent increases in histone acetylation that could be prevented by inhibition of ethanol metabolism. Chromatin immunoprecipitation confirmed increased histone acetylation at promoter regions of specific cytokine genes. The effect of ethanol was reproduced by incubation with acetate, the principal hepatic metabolite of ethanol, and both ethanol and acetate reduced histone deacetylase activity and up-regulated acetyl-coA synthetases. Knockdown of the acetyl-coA synthetases abrogated the effect of ethanol on cytokine production.
Synthesis of metabolically available acetyl-coA from acetate is critical to the increased acetylation of proinflammatory gene histones and consequent enhancement of the inflammatory response in ethanol-exposed macrophages. This mechanism is a potential therapeutic target in acute alcoholic hepatitis.
急性酒精性肝炎的特征是对细菌脂多糖的巨噬细胞炎症细胞因子反应不成比例。对潜在机制的了解有限，限制了有效治疗的进展。我们假设了一种新的机制，即乙醇增加组蛋白乙酰化，增加促炎基因转录和细胞因子合成。通过多重免疫测定法测量了在培养于 86mM 乙醇、1mM 乙酸盐和正常培养基中的人巨噬细胞系中对脂多糖的细胞因子反应。通过免疫荧光显微镜和呈现时的染色质免疫沉淀测定组蛋白乙酰化的变化。通过定量逆转录聚合酶链反应和免疫印迹测定乙醇和乙酸盐对乙酰辅酶 A（乙酰辅酶 A）合酶的影响，乙酰辅酶 A 合酶将乙酸盐转化为组蛋白乙酰化的底物。通过短发夹 RNA（shRNA）敲低乙酰辅酶 A 合酶来确定它们在乙醇增强炎症细胞因子反应中的作用。暴露于乙醇的巨噬细胞随着时间的推移发展出增强的白细胞介素 6（IL6）、IL8 和肿瘤坏死因子-α对脂多糖的反应，同时组蛋白乙酰化也呈时间依赖性增加，这可以通过抑制乙醇代谢来预防。染色质免疫沉淀证实了特定细胞因子基因启动子区域的组蛋白乙酰化增加。用乙酸盐孵育可重现乙醇的作用，乙酸盐是乙醇的主要肝代谢物，乙醇和乙酸盐均可降低组蛋白去乙酰化酶活性并上调乙酰辅酶 A 合酶。乙酰辅酶 A 合酶的敲低消除了乙醇对细胞因子产生的影响。
从乙酸盐合成代谢上可利用的乙酰辅酶 A 对于促炎基因组蛋白的乙酰化增加以及随后暴露于乙醇的巨噬细胞中炎症反应的增强至关重要。该机制是急性酒精性肝炎的潜在治疗靶点。
