Background & Aims

Smoking, an independent risk factor for pancreatitis, accelerates the development of alcoholic pancreatitis. Alcohol feeding of mice induces up-regulation of spliced X-box binding protein 1 (XBP1s), which regulates the endoplasmic reticulum (ER) unfolded protein response and promotes cell survival upon ER stress. We examined whether smoking affects the adaptive mechanisms induced by alcohol and accelerates disorders of the ER in pancreatic acinar cells.

Methods

We studied the combined effects of ethanol (EtOH) and cigarette smoke extract (CSE) on ER stress and cell death responses in mouse and human primary acini and the acinar cell line AR42J. Cells were incubated with EtOH (50 mmol/L), CSE (20–40 μg/mL), or both (CSE+EtOH), and analyzed by immunoblotting, quantitative reverse-transcription polymerase chain reaction, and cell death assays. Some cells were incubated with MKC-3946, an inhibitor of endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1) that blocks XBP1s formation. Male Sprague-Dawley rats were fed isocaloric amounts of an EtOH-containing (Lieber-DeCarli) or control diet for 11 weeks and exposed to cigarette smoke or room air in an exposure chamber for 2 hours each day. During the last 3 weeks, a subset of rats received intravenous injections of lipopolysaccharide (LPS, 3 mg/kg per week) to induce pancreatitis or saline (control). Pancreatic tissues were collected and analyzed by histology and immunostaining techniques.

Results

In AR42J and primary acini, CSE+EtOH induced cell death (necrosis and apoptosis), but neither agent alone had this effect. Cell death was associated with a significant decrease in expression of XBP1s. CSE+EtOH, but neither agent alone, slightly decreased adenosine triphosphate levels in AR42J cells, but induced oxidative stress and sustained activation (phosphorylation) of eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3, also called PERK) and increased protein levels of DNA damage inducible transcript 3 (DDIT3, also called CHOP). CHOP regulates transcription to promote apoptosis. Incubation of AR42J or primary mouse or human acinar cells with MKC-3946 reduced expression of XBP1s, increased levels of CHOP, and induced cell death. In rats fed an EtOH diet, exposure to cigarette smoke increased ER stress in acinar cells and sensitized the pancreas to LPS-induced pathology.

Conclusions

Cigarette smoke promotes cell death and features of pancreatitis in EtOH-sensitized acinar cells by suppressing the adaptive unfolded protein response signaling pathway. It also activates ER stress pathways that promote acinar cell death.

中文翻译：

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酒精和香烟烟雾的结合诱导胰腺腺泡细胞内质网应激和细胞死亡

背景与目标

吸烟是胰腺炎的独立危险因素，可加速酒精性胰腺炎的发展。小鼠饮酒会诱导剪接的X-box结合蛋白1（XBP1s）上调，从而调节内质网（ER）展开的蛋白反应并在ER应激时促进细胞存活。我们检查了吸烟是否会影响酒精诱导的适应性机制并加速胰腺腺泡细胞内质网的失调。

方法

我们研究了乙醇（EtOH）和香烟烟雾提取物（CSE）对小鼠和人类原发性腺泡和腺泡细胞系AR42J的内质网应激和细胞死亡反应的联合作用。将细胞与EtOH（50 mmol / L），CSE（20–40μg/ mL）或两者（CSE + EtOH）一起孵育，并通过免疫印迹，定量逆转录聚合酶链反应和细胞死亡分析进行分析。一些细胞与MKC-3946一起孵育，MKC-3946是内质网对细胞核信号传导1（ERN1，也称为IRE1）的抑制剂，可阻止XBP1s的形成。给雄性Sprague-Dawley大鼠喂食等热量的含EtOH（Lieber-DeCarli）或对照饮食的食物，持续11周，每天在暴露室内暴露于香烟烟雾或室内空气中2小时。在最近3周内，部分大鼠接受了静脉注射脂多糖（LPS，每周3 mg / kg），以诱发胰腺炎或生理盐水（对照组）。收集胰腺组织，并通过组织学和免疫染色技术进行分析。

结果

在AR42J和原发性腺泡中，CSE + EtOH诱导细胞死亡（坏死和凋亡），但没有一种药物单独具有这种作用。细胞死亡与XBP1s表达的显着降低有关。CSE + EtOH，但没有单独使用，只能使AR42J细胞中的三磷酸腺苷水平略有降低，但会诱导氧化应激和真核翻译起始因子2α激酶3（EIF2AK3，也称为PERK）的持续活化（磷酸化），并增加DNA的蛋白质水平损伤诱导转录本3（DDIT3，也称为CHOP）。CHOP调节转录以促进细胞凋亡。用MKC-3946将AR42J或原代小鼠或人腺泡细胞孵育后，XBP1s的表达减少，CHOP水平升高，并诱导细胞死亡。在喂了EtOH饮食的大鼠中，

结论

香烟烟雾通过抑制适应性未折叠的蛋白质反应信号通路，促进了EtOH敏感的腺泡细胞的细胞死亡和胰腺炎的特征。它还激活ER应力通路，从而促进腺泡细胞死亡。