J Hepatol：喝酒伤肝的新机制：extracellular vesicles的新作用 - 外泌体之家 | 细胞外膜泡领域核心平台—exosomes & microvesicles—小膜泡大作用

在酒精性肝病中肝细胞在受到酒精的刺激后是通过什么机制激活包括巨噬细胞在内的炎症细胞目前尚不清楚。目前细胞外膜泡（EV）作为一种新的细胞与细胞间交流的方式已受到越来越多的关注。来自世界顶级医院的美国梅奥诊所（Mayo Clinic）的研究人员提出假设，酒精刺激肝细胞后可能增加细胞外膜泡的释放，从而激活巨噬细胞促进炎症反应的发生。

梅奥诊所的研究人员用乙醇处理原代肝细胞和过表达酒精代谢的酶-乙醇脱氢酶和细胞色素P450 的HepG2细胞系（HepG2ADH和HepG2Cyp2E1），然后检测细胞外膜泡分泌的情况。在体外巨噬细胞激活实验和体内小鼠酒精喂食实验中通过分析炎症因子和巨噬细胞相关的mRNA表达、免疫组化、生化分析血清中ALT和甘油三酯发现细胞外膜泡介导了巨噬细胞的激活。

在HepG2Cyp2E1细胞系中乙醇显著提高细胞外膜泡的分泌大3.3倍，并且这一过程与caspase-3的激活有关。利用药理工具药和基因操作阻断caspase的激活可消除乙醇诱导的细胞外膜泡的释放。细胞外膜泡可诱导巨噬细胞的激活和炎症因子的产生。通过芯片分析和抗体中和实验揭示CD40配体（CD40L）在细胞外膜泡介导巨噬细胞激活的过程中起着重要的作用。在小鼠体内实验中，利用Rho激酶抑制剂pan-caspase处理野生型小鼠、或敲除CD40 （CD40-/-）或caspase激活的TRAIL受体（TR-/-）均可保护肝脏免受酒精诱导的损伤和相关的巨噬细胞浸润。更重要的是，与正常人相比，酒精性肝炎的病人的血清中富含CD40L的细胞外膜泡的含量显著增加。

BACKGROUND/AIMS: The mechanisms bywhich hepatocyte exposure to alcohol activates inflammatory cells such asmacrophages in alcoholic liver disease (ALD) are unclear. The role of releasednano-sized membrane vesicles, termed extracellular vesicles (EV), incell-to-cell communication has become increasingly recognized. We tested thehypothesis that hepatocytes exposed to alcohol may increase EV release toelicit macrophage activation. METHODS:Primary hepatocytes or HepG2 hepatocyte cell lines overexpressingethanol-metabolizing enzymes Alcohol dehydrogenase (HepG2ADH) or cytochromeP450 2E1 (HepG2Cyp2E1) were treated with ethanol and EV release was quantifiedwith nanoparticle tracking analysis (NTA). EV mediated macrophage activationwas monitored by analyzing inflammatory cytokines and macrophage associatedmRNA expression, immunohistochemistry, biochemical serum ALT and triglyceridesanalysis in our in vitro macrophage activation and in vivo murine ethanolfeeding studies. RESULTS: Ethanolsignificantly increased EV release by 3.3 fold from HepG2Cyp2E1 cells and wasassociated with activation of caspase-3. Blockade of caspase activation withpharmacological or genetic approaches abrogated alcohol induced EV release. EVstimulated macrophage activation and inflammatory cytokine induction. Anunbiased microarray-based approach and antibody neutralization experimentsdemonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophageactivation. In vivo, wild-type (WT) mice receiving a pan-caspase, Rho kinaseinhibitor or with genetic deletion of CD40 (CD40-/-) or the caspase-activatingTRAIL receptor (TR-/-), were protected from alcohol-induced injury andassociated macrophage infiltration. Moreover, serum from patients withalcoholic hepatitis (AH) showed increased levels of CD40L enriched EV. CONCLUSION: In conclusion, hepatocytesrelease CD40L containing EV in a caspase dependent manner in response toalcohol exposure which promotes macrophage activation, contributing toinflammation in ALD.

外泌体之家 | 细胞外膜泡领域核心平台—exosomes &amp; microvesicles—小膜泡大作用

外泌体之家 | 细胞外膜泡领域核心平台—exosomes & microvesicles—小膜泡大作用