摘要
近年來，隨著美國各州擴大大麻使用的合法化，除了醫用大麻以外，於美國的市面上可以買到各種娛樂用大麻產品。在如此開放的環境下，大麻在年輕人中使用的比例增加，也引發其對健康的疑慮。根據美國疾病管制暨預防中心（CDC）統計來自各州的通報，截至2020年總計有2,807例的肺損傷及68例死亡，是與病患使用含有大麻主成份— delta-9-四氫大麻酚（Δ9-THC）的電子煙有關，這項統計顯示大麻對使用者肺部的健康可能具有危害，然而Δ9-THC 引起肺損傷的機制尚不清楚。本論文主要研究目的是根據先前流行病學統計看到Δ9-THC有造成電子煙肺病(EVALI)的風險，利用動物及細胞實驗評估Δ9-THC對肺部健康的影響，並進一步探討其中的大麻藥理機轉。 於動物實驗中，我們使用 8 周大的C57BL/6J 雄性小鼠，隨機分為三組：Δ9-THC 吸入組、Δ9-THC 腹腔注射組（i.p.）以及對照組。特別的是，吸入組我們使用mesh nebulizer霧化Δ9-THC，讓Δ9-THC保持原型狀態。經過 2 個月的慢性給藥實驗，我們利用H&E stain、Masson's trichrome stain、IF stain觀察肺部切片的免疫細胞浸潤，並利用快速多重蛋白質與核酸標的偵測（Multiplex Immunoassays）檢測小鼠血清中發炎的程度。我們用兩種方式量測小鼠的肺功能：非侵入性的微型電腦斷層掃描( Micro- CT )及侵入性的呼吸參數測定儀（flexiVent ; SCIREQ）。於細胞實驗中，我們採用人類支氣管上皮細胞BEAS-2B以及人類肺泡上皮type2- like細胞A549來評估Δ9-THC對於肺部細胞發炎以及細胞存活率之影響。 動物實驗中，H&E stain結果顯示，在Δ9-THC處理的小鼠肺臟組織有較多的免疫細胞浸潤，而Masson's trichrome stain顯示在發炎部位有collagen堆積之情形。IF stain顯示CD68、F4/80等巨噬細胞標誌物及TNF-α、NFκB等發炎介質在Δ9-THC處理的小鼠肺部表現增加。Multiplex Cytokine Assay結果顯示，發炎因子如 IL-1β、IL-6、IFN-γ、TNF-α，在 Δ9-THC 處理的小鼠血清中增加。 Micro- CT 及flexiVent 結果顯示，吸入及腹腔注射Δ9-THC 會損害小鼠的肺功能。 而在細胞實驗中，我們發現Δ9-THC以劑量依賴性方式造成BEAS-2B 和 A549 中發炎相關因子的表現上升，包含NFκB、TNF-α、IL-1β、IL-6的表現上升。細胞存活率分析(MTT assay)結果顯示，Δ9-THC會促進 BEAS-2B 的增生，反觀在A549則是造成細胞毒性。流式細胞儀結果驗證了Δ9-THC造成BEAS-2B 的增生以及A549呈現細胞週期停滯之結果。 綜合上述結果顯示，大麻活性成分Δ9-THC會導致小鼠肺部以及全身循環發炎，並進一步造成肺功能失調；亦會造成人類支氣管上皮細胞以及肺泡上皮細胞的發炎及對細胞存活率造成影響。這些現象皆可能導致Δ9-THC會對肺部健康造成不良影響，後續值得我們更深入探討其中的分子機轉。
並列摘要
As the expanding legalization of marijuana use in U.S. states, all sorts of recreational marijuana products are currently available on the market followed by increased marijuana use among the general public. A multistate outbreak of e-cigarette, or vaping, product use–associated lung injury (EVALI) including 2,807 hospitalized cases and 68 deaths has raised concerns about the impact of marijuana on public health. Most of these cases reported using delta-9-tetrahydrocannabinol (Δ9-THC)-containing products, indicating that marijuana has a negative effect on users’ lung health, while the mechanism underlying Δ9-THC-induced lung injury remains unclear. The purpose of this study is to investigate whether chronic exposure to Δ9-THC causes lung damage and to figure out underlying mechanisms. A chronic Δ9-THC treatment to 8-week-old C57BL/6J male mice was carried out for 2 months. Mice were randomly allocated into three groups, Δ9-THC inhalation, Δ9-THC intraperitoneal (i.p.) injection, and a control group treated with vehicle (n=5 mice in each group). A mesh nebulizer was used in Δ9-THC inhalation group. Immunofluorescence (IF) staining was used to detect immune cell infiltration and inflammation in mouse lung sections. Multiplex Cytokine Assay was used to examine the level of inflammatory cytokines in mice serum. A computer-controlled piston ventilator (flexiVent system) and Micro-CT were used to measure lung function in mice. Human bronchial epithelial cells (BEAS- 2B) and human lung epithelial cells (A549) were used to investigate the effect of Δ9-THC on the lung in vitro. H&E staining suggested more immune cell infiltration in Δ9-THC- treated mice lungs compared to the control. IF staining showed that expressions of macrophage CD68 and F4/80 as well as proinflammatory markers such as TNF-α, NF-κB were increased in mice lungs after Δ9-THC treatment. Multiplex Cytokine Assay showed inflammatory cytokines such as IL-1β, IL-6, IFN-γ, and TNF-α increased in Δ9-THC-treated mice serum. Using flexiVent system and Micro-CT, we found that both inhaled delivery and i.p. injection of Δ9-THC may impair lung function in mice. In vitro cell-based assays showed that Δ9-THC induced inflammation in BEAS-2B and A549 in a dose-dependent manner showed as the expression of NFκB、TNF-α、IL-1β、IL-6 elevation, and further had an effect on cell viability showed as proliferation in BEAS-2B and cytotoxicity in A549. Cell cycle analysis confirmed proliferation in BEAS-2B and G1 phase cell cycle arrest in A549 with Δ9-THC treatment. Our study showed the primary psychoactive component of smoked cannabis, Δ9-THC can cause lung injury. Both inhalation and injection of Δ9-THC caused lung and systemic inflammation and lung function impairment. Δ9-THC also induced inflammation and cause proliferation in bronchial epithelial cells BEAS- 2B while cytotoxicity in human lung epithelial cells A549.
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