香菸與加熱菸誘發發炎反應與細胞毒性之研究

Abstract

研究背景 吸菸被視為造成許多疾病的來源之一，如慢性阻塞性肺病(chronic obstructive pulmonary disease, COPD)，以及神經退化性疾病。加熱菸被希望成為傳統香煙的替代品，然而在菸品成分方面，加熱菸與香菸的組成結構相似，所以仍可能造成損傷。至目前為止尚未有明確實驗說明加熱菸與香菸的危害不同之處及發炎反應的相關機制。高遷移率族蛋白1(high-mobility group box 1)為一廣泛存之蛋白，在細胞受損傷時可作為損傷相關分子模式(damage-associated molecular pattern molecules, DAMPs)誘導發炎相關反應，可能作為菸品對人體造成的損傷途徑之一。 研究目的 本研究探討加熱菸氣霧對於肺上皮細胞和神經母細胞之細胞毒性，以及是否能引響細胞之HMGB1與自噬作用的表現。並比較單支香菸與加熱菸的尼古丁含量以及對細胞造成的傷害。另外比較具調味之加熱菸對細胞產生細胞毒性相較於無調味之加熱菸之程度是否較大。 研究方法 本研究利用MTT assay檢測加熱菸氣霧提取物(heated tobacco product extract, HTE)、香菸煙霧提取物(cigarette smoke extract, CSE)對肺上皮細胞A549和神經母細胞N2a之細胞毒性，再利用西方墨點法與ELISA偵測HMGB1與自噬作用標記蛋白之表現，以及利用HPLC檢測各提取物中尼古丁之含量。 研究結果 研究結果顯示HTE與CSE對A549上皮細胞及N2a神經細胞皆具有細胞毒性。在實驗濃度與時間上升時皆能影響此二細胞釋出HMGB1蛋白；在N2a神經細胞中HMGB1蛋白釋出受HTE與CSE的濃度影響；細胞內自噬作用標記蛋白LC3B-II的量也受HTE與CSE的影響。而HPLC分析結果顯示HTE與CSE中尼古丁之含量存在差異，單支香菸中尼古丁的含量較加熱菸與調味加熱菸高。另外分別測定尼古丁、HTE、CSE及調味加熱菸對A549上皮細胞及N2a神經細胞之細胞毒性，顯示在相當於單支菸尼古丁含量的HTE或CSE，造成細胞毒性皆大於等量純尼古丁的作用。 結論 本研究結果顯示HTE也會對細胞造成毒性，雖然比CSE所造成之細胞毒性低。同時HTE與CSE皆可促使A549上皮細胞釋放HMGB1，可能參與發炎反應之發生。HTE與CSE也可促使N2a神經細胞釋放HMGB1增加，導致自噬作用活化，說明加熱菸與香菸皆可導致細胞受損。，由於相對於純尼古丁，HTE及CSE對細胞造成的毒性較大，因此尼古丁可能非造成細胞毒性之主要成分。而調味加熱菸，相較於原味加熱菸，會產生較大之細胞毒性，亦暗示加熱菸氣霧中的其他添加成份可能會造成之潛在危害，未來需要更進一步探討。

Background Smoking is considered as one of the causes of many diseases, such as chronic obstructive pulmonary disease (COPD) and neurodegenerative diseases. Heated tobacco products are thought to be a harmless alternative to traditional cigarettes. Although heated at lower temperature, heated tobacco products may be still harmful with regard to the similar composition to traditional cigarettes. However, the biohazards and the mechanisms of inflammatory effect caused by the heated tobacco products remain unclear. High-mobility group box 1 (HMGB1) is a widely present protein that can act as a damage-associated molecular pattern molecule (DAMP) to induce inflammatory responses when cells are damaged, potentially serving as one of the pathways through which tobacco products cause harm to the human body. Objective This study investigates the cytotoxicity of heated tobacco aerosols to lung epithelial cells and neuroblastoma cells, as well as the effect on the expression of HMGB1 and autophagy. The effects are compared between heated tobacco product extract (HTE) and cigarette smoke extract (CSE). The cytotoxicity of CSE, HTE with different flavors and the equivalent nicotine is compared in neuroblastoma cells to elucidate the potential toxic components in the extracts in addition to nicotine. Methods The cytotoxicity of HTE and CSE on lung epithelial cells and neuroblastoma cells was measured by MTT assay. The expression and release of HMGB1 and autophagy marker proteins were measured by western blot and ELISA assay. The nicotine contents of the extracts were measured by HPLC. Results Both HTE and CSE caused cytotoxicity on A549 epithelial cells and N2a neuroblastoma cells. As the experimental concentration and time increased, both types of cells released HMGB1 protein. In N2a neuroblastoma cells, HMGB1 protein release was caused by the increased concentration of HTE and CSE. The autophagy marker protein LC3B-II is also affected by the exposure to HTE and CSE. HPLC analysis results reveal differences in nicotine content between HTE and CSE, with a single cigarette containing higher nicotine levels than heated tobacco product and flavored heated tobacco product. Additionally, the cytotoxicity of nicotine, HTE, CSE, and flavored heated tobacco product on A549 epithelial cells and N2a neuroblastoma cells was measured, revealing that HTE or CSE at nicotine levels equivalent to a single cigarette induced greater cytotoxicity than an equivalent amount of pure nicotine. Conclusion The results of this study show that HTE also causes cytotoxicity, although it is lower than the cytotoxicity caused by CSE. Both HTE and CSE can promote the release of HMGB1 from A549 epithelial cells, which may be involved in the inflammatory response. HTE and CSE can also increase the release of HMGB1 from N2a neuroblastoma cells, which may be related to the activation of autophagy, leading to cell damage. Furthermore, nicotine is not the primary cause of cytotoxicity, as HTE and CSE have a greater toxic impact on cells compared to pure nicotine. Additionally, flavored heated tobacco product shows greater cytotoxicity compared to original heated tobacco product. Future research is needed to further investigate the potential hazards caused by other toxic substances in heated tobacco aerosols.