Ritzenthaler Jeffrey D, Ekuban Abigail, Horsman Benjamin, Roman Jesse, Watson Walter H
Division of Pulmonary, Allergy and Critical Care Medicine and the Jane & Leonard Korman Respiratory Institute, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Division of Gastroenterology, Hepatology and Nutrition, Department of Medicine, University of Louisville, Louisville, Kentucky, USA.
Alcohol Clin Exp Res (Hoboken). 2025 Mar;49(3):515-525. doi: 10.1111/acer.15533. Epub 2025 Jan 23.
Our previous study demonstrated that alcohol induced the expression of the α4 subunit of nicotinic acetylcholine receptors (nAChRs) in the livers of wild type mice (WT), and that whole-body α4 nAChR knockout mice (α4KO) showed protection against alcohol-induced steatosis, inflammation, and injury. Based on these findings, we hypothesized that hepatocyte-specific α4 nAChRs may directly contribute to the detrimental effects of alcohol on the liver.
Hepatocyte-specific α4 knockout mice (α4HepKO) were generated, and the absence of α4 nAChR was confirmed through PCR of genomic DNA. Female WT and α4HepKO mice were exposed to alcohol in the NIAAA chronic + binge model. After 10 days on the Lieber-DeCarli liquid diet containing 5% (vol/vol) alcohol or isocaloric maltose-dextrin, the mice were gavaged with a single dose of alcohol or isocaloric maltose-dextrin. The mice were euthanized 9 h later and their organs harvested. Additionally, hepatocytes were isolated from WT, α4HepKO, α4floxed, and α4KO mice and exposed to 80 mM alcohol in vitro for 24 h. Steatosis, inflammation, and cell injury were assessed in both liver and isolated hepatocytes.
In WT mice, alcohol exposure resulted in hepatic steatosis, inflammation, and injury as evidenced by increased liver triglycerides, neutrophil infiltration, and serum concentrations of liver enzymes. All of these responses were markedly lower in α4HepKO mice. mRNA expression of genes involved in lipogenesis (Srebf1, Fasn, and Dgat2) and inflammation (TNFα, Cxcl5, Cxcl1, and Serpine1) were increased in the livers of WT mice exposed to alcohol in vivo and in WT hepatocytes exposed to alcohol in vitro. These changes were not observed in liver or hepatocytes from mice lacking α4 nAChRs.
α4 nAChRs expressed in hepatocytes mediate alcohol-associated hepatoxicity. Therefore, the development of therapeutic strategies targeting hepatocyte α4-containing nAChRs could help reduce the burden of ALD.
我们之前的研究表明，酒精可诱导野生型小鼠（WT）肝脏中烟碱型乙酰胆碱受体（nAChRs）α4亚基的表达，且全身α4 nAChR基因敲除小鼠（α4KO）对酒精诱导的脂肪变性、炎症和损伤具有保护作用。基于这些发现，我们推测肝细胞特异性α4 nAChRs可能直接导致酒精对肝脏的有害影响。
构建肝细胞特异性α4基因敲除小鼠（α4HepKO），并通过基因组DNA的PCR确认α4 nAChR的缺失。将雌性WT和α4HepKO小鼠置于美国国立酒精滥用与酒精中毒研究所（NIAAA）的慢性+暴饮模型中接触酒精。在含有5%（体积/体积）酒精或等热量麦芽糖糊精的Lieber-DeCarli液体饮食喂养10天后，给小鼠单次灌胃酒精或等热量麦芽糖糊精。9小时后对小鼠实施安乐死并采集其器官。此外，从WT、α4HepKO、α4floxed和α4KO小鼠中分离肝细胞，并在体外使其暴露于80 mM酒精中24小时。对肝脏和分离出的肝细胞中的脂肪变性、炎症和细胞损伤进行评估。
在WT小鼠中，酒精暴露导致肝脏脂肪变性、炎症和损伤，表现为肝脏甘油三酯增加、中性粒细胞浸润以及肝酶血清浓度升高。所有这些反应在α4HepKO小鼠中均明显较低。在体内接触酒精的WT小鼠肝脏以及体外接触酒精的WT肝细胞中，参与脂肪生成（Srebf1、Fasn和Dgat2）和炎症（TNFα、Cxcl5、Cxcl1和Serpine1）的基因的mRNA表达增加。在缺乏α4 nAChRs的小鼠的肝脏或肝细胞中未观察到这些变化。
肝细胞中表达的α4 nAChRs介导酒精相关的肝毒性。因此，开发针对含肝细胞α4的nAChRs的治疗策略可能有助于减轻酒精性肝病（ALD）的负担。
