Department of Pathology and Laboratory Medicine, Lewis Katz School of Medicine, Temple University, Philadelphia, PA, 19140, USA.
Cell Commun Signal. 2024 Jan 15;22(1):39. doi: 10.1186/s12964-023-01461-1.
Use of nicotine containing products like electronic cigarettes (e-Cig) and alcohol are associated with mitochondrial membrane depolarization, resulting in the extracellular release of ATP, and mitochondrial DNA (mtDNA), mediating inflammatory responses. While nicotine effects on lungs is well-known, chronic alcohol (ETH) exposure also weakens lung immune responses and cause inflammation. Extracellular ATP (eATP) released by inflammatory/stressed cells stimulate purinergic P2X7 receptors (P2X7r) activation in adjacent cells. We hypothesized that injury caused by alcohol and e-Cig to pulmonary alveolar epithelial cells (hPAEpiC) promote the release of eATP, mtDNA and P2X7r in circulation. This induces a paracrine signaling communication either directly or via EVs to affect brain cells (human brain endothelial cells - hBMVEC).
We used a model of primary human pulmonary alveolar epithelial cells (hPAEpiC) and exposed the cells to 100 mM ethanol (ETH), 100 µM acetaldehyde (ALD), or e-Cig (1.75 µg/mL of 1.8% or 0% nicotine) conditioned media, and measured the mitochondrial efficiency using Agilent Seahorse machine. Gene expression was measured by Taqman RT-qPCR and digital PCR. hPAEpiC-EVs were extracted from culture supernatant and characterized by flow cytometric analysis. Calcium (Ca) and eATP levels were quantified using commercial kits. To study intercellular communication via paracrine signaling or by EVs, we stimulated hBMVECs with hPAEpiC cell culture medium conditioned with ETH, ALD or e-cig or hPAEpiC-EVs and measured Ca levels.
ETH, ALD, or e-Cig (1.8% nicotine) stimulation depleted the mitochondrial spare respiration capacity in hPAEpiC. We observed increased expression of P2X7r and TRPV1 genes (3-6-fold) and increased intracellular Ca accumulation (20-30-fold increase) in hPAEpiC, resulting in greater expression of endoplasmic reticulum (ER) stress markers. hPAEpiC stimulated by ETH, ALD, and e-Cig conditioned media shed more EVs with larger particle sizes, carrying higher amounts of eATP and mtDNA. ETH, ALD and e-Cig (1.8% nicotine) exposure also increased the P2X7r shedding in media and via EVs. hPAEpiC-EVs carrying P2X7r and eATP cargo triggered paracrine signaling in human brain microvascular endothelial cells (BMVECs) and increased Ca levels. P2X7r inhibition by A804598 compound normalized mitochondrial spare respiration, reduced ER stress and diminished EV release, thus protecting the BBB function.
Abusive drugs like ETH and e-Cig promote mitochondrial and endoplasmic reticulum stress in hPAEpiC and disrupts the cell functions via P2X7 receptor signaling. EVs released by lung epithelial cells against ETH/e-cig insults, carry a cargo of secondary messengers that stimulate brain cells via paracrine signals.
尼古丁类产品（如烟碱电子烟）和酒精的使用会导致线粒体膜去极化，导致细胞外 ATP 和线粒体 DNA（mtDNA）的释放，从而介导炎症反应。虽然尼古丁对肺部的影响众所周知，但慢性酒精（ETH）暴露也会削弱肺部的免疫反应并引起炎症。炎症/应激细胞释放的细胞外 ATP（eATP）会刺激邻近细胞中嘌呤能 P2X7 受体（P2X7r）的激活。我们假设酒精和电子烟对肺泡上皮细胞（hPAEpiC）造成的损伤会促进 eATP、mtDNA 和 P2X7r 在循环中的释放。这会通过旁分泌信号转导直接或通过 EV 影响脑细胞（人脑微血管内皮细胞 - hBMVEC）。
我们使用原代人肺泡上皮细胞（hPAEpiC）模型，用 100 mM 乙醇（ETH）、100 μM 乙醛（ALD）或电子烟（1.75 µg/mL 1.8%或 0%尼古丁）条件培养基处理细胞，并使用安捷伦 Seahorse 机器测量线粒体效率。通过 Taqman RT-qPCR 和数字 PCR 测量基因表达。从培养上清液中提取 hPAEpiC-EVs，并通过流式细胞分析进行表征。使用商业试剂盒定量测定 Ca 和 eATP 水平。为了研究通过旁分泌信号或 EV 进行的细胞间通讯，我们用 ETH、ALD 或电子烟处理的 hPAEpiC 细胞培养基或 hPAEpiC-EVs 刺激 hBMVECs，并测量 Ca 水平。
ETH、ALD 或电子烟（1.8%尼古丁）刺激耗尽了 hPAEpiC 的线粒体备用呼吸能力。我们观察到 hPAEpiC 中 P2X7r 和 TRPV1 基因的表达增加了 3-6 倍，细胞内 Ca 积累增加了 20-30 倍，导致内质网（ER）应激标志物的表达增加。用 ETH、ALD 和电子烟条件培养基刺激的 hPAEpiC 释放出更多携带更高 mtDNA 和 eATP 的更大粒径的 EVs。ETH、ALD 和电子烟（1.8%尼古丁）暴露还增加了细胞培养基和 EV 中 P2X7r 的脱落。携带 P2X7r 和 eATP 货物的 hPAEpiC-EVs 触发了人脑微血管内皮细胞（BMVECs）的旁分泌信号，并增加了 Ca 水平。通过 A804598 化合物抑制 P2X7r 可使线粒体备用呼吸正常化，减少 ER 应激并减少 EV 释放，从而保护 BBB 功能。
像 ETH 和电子烟这样的滥用药物会促进 hPAEpiC 中的线粒体和内质网应激，并通过 P2X7 受体信号转导破坏细胞功能。肺上皮细胞在 ETH/e-cig 攻击下释放的 EVs 携带第二信使货物，通过旁分泌信号刺激脑细胞。
