濫用愷他命對非神經性細胞危害機制探討

The damaging effect of ketamine on non-neural cells

徐心儀(Hsin-Yi Shyu)

指導教授 ： 李明學

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.11189

摘要

非法濫用藥物嚴重危害社會大眾健康與安全。愷他命屬於解離型的麻醉藥劑，但自2006年起躍升為臺灣緝獲量第一的濫用藥物。濫用愷他命除了導致神經性毒害外，在腎臟損傷及抑制免疫上亦時有所聞，然而其分子作用機制目前尚不清楚。因此，本論文的研究目的是在探討愷他命如何致使腎臟功能受損及抑制免疫反應的分子作用機制。
第一部份：愷他命藉由調控GSK-3β訊息傳遞路徑增加遠端腎小管管柱細胞通透性及誘使上皮細胞間質轉化。
臨床上，濫用愷他命亦會引起腎臟方面不良反應，然而其分子作用機制目前尚不清楚。在腎臟疾病中，上皮細胞間質轉化（Epithelial phenotypic changes, EPCs）會造成細胞黏附特性降低、失去細胞極性和增加細胞移動侵襲能力；我們推論愷他命可能藉由誘發上皮細胞間質轉化而損傷腎臟功能。運用遠端腎小管MDCK細胞株作為實驗模式細胞。當處理愷他命時，發現愷他命會降低此分化細胞的跨上皮電阻值（transepithelial electric resistance）、增加物質通透性並使細胞間連結瓦解，且伴隨著上皮細胞標誌（ZO-1, occludin, E-cadherin）表現減低，間質細胞標誌（N-cadherin, fibronectin, vimentin）表現增加的情形。同時，上皮細胞標誌E-cadherin的轉錄抑制分子Snail, Slug, Twist, ZEB1均同時增加表現。再者，愷他命處理會促進MDCK細胞移動侵襲能力。愷他命活化PI3K/Akt並進而抑制下游GSK-3β，用以增加細胞通透性及上皮細胞間質轉化。當預先投予PI3K/Akt抑制劑（LY294002）可阻斷愷他命所引起的遠端腎小管管柱細胞通透性、上皮細胞間質轉化及移動侵襲能力；另一方面，利用GSK-3β抑制劑（3F8）則可再現愷他命所造成的通透性、上皮細胞間質轉化及移動侵襲能力。綜合以上，我們的實驗結果顯示愷他命可以透過活化Akt和抑制GSK-3β以調控上皮及間質細胞標誌表現，造成遠端腎小管管柱細胞發生上皮細胞間質的轉化。
第二部分：愷他命經由TGF-β/Smad訊息傳遞路徑致使腎臟損傷。
臨床研究發現濫用愷他命會影響腎臟及下泌尿道系統功能，然而，愷他命影響腎臟損傷的病理作用機制迄今尚不清楚。因此，本論文進一步探討研究愷他命對腎臟及近端腎小管NRK-52E細胞的影響並尋找其參與之訊息傳遞路徑。
我們觀察到當在小鼠中連續注射愷他命後，尿液白蛋白量、血清肌酸酐指數、乙型轉型生長因子TGF-β、發炎趨化因子MCP-1及KC均有上升情形；在腎臟組織的病理特徵則有腎小管間質病兆和纖維蛋白沈積等情形，且伴隨著上皮細胞標誌（occludin and E-cadherin）表現減低，間質細胞標誌（fibronectin）表現增加的現象。愷他命可能藉由乙型轉型生長因子(TGF-β)訊息傳遞路徑誘導上皮細胞間質轉化進而導致腎臟損傷。運用近端腎小管NRK-52E細胞株作為實驗模式細胞。當處理愷他命時，發現愷他命會降低此分化細胞的跨上皮電阻值（transepithelial electric resistance）、增加物質通透性並使細胞間連結瓦解。此外，愷他命會在近端腎小管NRK-52E細胞中產生顯著的型態改變，例如：F-actin細胞骨架分佈及纖維粘連蛋白fibronectin增加；同時，愷他命誘導近端腎小管管柱細胞進入上皮細胞間質轉化，致使上皮細胞標誌（ZO-1, occludin, E-cadherin ) 表現下降，而間質細胞標記（N-cadherin, fibronectin, vimentin）表現上升；並有提升細胞移動侵襲及不貼附生長的能力。愷他命活化乙型轉型生長因子TGF-β訊息傳遞路徑與其誘使上皮細胞間質轉化有關，因為預先投予第一型TGF-β受器抑制劑（SB431542）可阻斷愷他命所引起的前述細胞生理現象。綜合以上，我們的實驗結果顯示乙型轉型生長因子TGF-β訊息傳遞路徑參與在愷他命毒害腎臟的分子作用機制中。阻斷該訊息傳遞路徑未來也許可以作為減緩或治療愷他命濫用者腎臟功能傷害的方法。
第三部分：愷他命抑制單核球細胞分化成巨噬細胞。
愷他命會抑制促發炎反應細胞激素的產生，防止嗜中性顆粒球的成熟、阻礙樹狀細胞分化及延緩清除入侵細菌等免疫反應。單核球細胞分化成巨噬細胞（monocyte-macrophage differentiation）在免疫反應中扮演重要角色，因此本研究的目的是在探討愷他命對巨噬細胞分化的影響。運用可自行分裂增生的人類單核球THP-1細胞株，並以巴豆醇酯（phorbol 12-myristate 13-acetate, PMA）促分化訊號刺激此單核球THP-1細胞朝巨噬細胞方向演變，用以探討愷他命對巨噬細胞分化過程中可能的影響及其分子作用機制。我們的實驗結果顯示預先投予的愷他命，可抑制由PMA誘發人類單核球THP-1細胞分化成巨噬細胞的作用。再者，愷他命亦能抑制促發炎反應細胞激素（TNF-α、IL-6及IL-8）的生成，並降低MMP-9的表現和活性，進而導致細胞侵襲能力下降。研究結果亦發現預先投予的愷他命可減緩由PMA誘發活性氧化物質的量及細胞生長的抑制作用。重要的是愷他命可以抑制PMA處理引起的MAP kinases (ERK1/2, p38, JNK）蛋白磷酸化，顯示出愷他命可經由阻斷MAP kinases訊息傳遞路徑，進而抑制單核球細胞分化成巨噬細胞。綜合以上，我們的實驗結果顯示愷他命對單核球細胞分化成巨噬細胞的過程有拮抗作用，其可能藉由阻礙單核球細胞分化而導致免疫缺陷。
愷他命在臺灣屬第三級管制藥品，由於取得容易、價格低廉、效果快速，且施用遭查獲僅需罰緩及接受講習，故為傳播極快的軟性濫用藥物。本篇研究論文說明愷他命濫用會造成腎臟機能及免疫功能的損害，並對其作用機制提供了新的見解。期望此新的研究發現能讓愷他命濫用者知此藥物對身體的嚴重危害、降低其濫用程度並使政府加以重視國人愷他命濫用問題，用以改善管理政策及防制策略，以減少國人濫用愷他命及其所造成的毒害。

關鍵字

愷他命 ； 腎小管管柱細胞 ； 上皮細胞間質轉化 ； 腎臟功能失調 ； 巨噬細胞分化 ； 免疫缺陷

並列摘要

Illicit drug abuse has caused tremendous harm to public health and safety. Since 2006, the annual apprehended amount of ketamine has become the highest one among all illegal drugs in Taiwan. In addition to its neuropathic toxicity, ketamine abuse has numerous effects, including renal failure and immune suppression; however, the underlying mechanisms are elusive. In this study, the molecular mechanisms in ketamine-induced renal dysfunction and immunosuppression were further explored.
Part I: Ketamine increases permeability and alters epithelial phenotype of renal distal tubular cells via a GSK-3β-dependent mechanism
Ketamine abuse has been reported with renal damages; however, the underlying mechanism is poorly understood. The process called epithelial phenotypic changes (EPCs) causes the loss of cell-cell adhesion and cell polarity in renal diseases, and the acquisition of migratory and invasive properties. Madin-Darby canine kidney (MDCK) cells, an in vitro renal distal epithelial cell model, were subjected to experimental manipulation to investigate whether ketamine could promote EPCs. Our data showed that ketamine dramatically decreased transepithelial electrical resistance and increased paracellular permeability and junction disruption, which were coupled to decreased levels of apical junctional proteins (ZO-1, occludin and E-cadherin), and to elevated levels of the mesenchymal markers (N-cadherin, fibronectin and vimentin) and the E-cadherin repressors (Snail, Slug, Twist, and ZEB1). Moreover, ketamine significantly enhanced the cell motility. Ketamine could cause an inhibition of GSK-3β activity through Ser-9 phosphorylation by the PI3K/Akt pathway. Inhibition of PI3K/Akt with LY294002 reactivated GSK-3β and suppressed ketamine-enhanced permeability, EPCs and motility, while the inactivation of GSK-3β using the inhibitor 3F8 recapitulated ketamine action on the cells. The results provide evidence that ketamine induces renal distal tubular EPCs through the activation of PI3K/Akt and the inactivation of GSK-3β.
Part II: Ketamine modulates TGF-β/Smad signaling to induce kidney injury
Ketamine abuse has been shown to affect kidney and the lower urinary tract; however, the etiology how ketamine affects kidney remains unclear. To explore the effect of ketamine on kidney injury, animal model and renal proximal tubular epithelial NRK-52E cells were used in this study. Ketamine treatment could significantly increase the levels of urinary albumin, serum creatinine, TGF-β, MCP-1 and KC in mice, and cause tubulointerstitial injury including tubular atrophy and collagen deposition coupled with downregulation in occludin and E-cadherin and upregulation in fibronectin. Ketamine also could induce the EPCs of polarized NRK-52E cells by induction of tight junction disruption, elevated paracellular permeability, TGF-β signaling and F-actin stress fiber formation, that were associated with decreased epithelial markers (ZO-1, occludin and E-cadherin) and increased mesenchymal markers (N-cadherin, fibronectin and vimentin,). Consistently, ketamine also promoted cell migration, invasion and anchorage-independent cell growth of NRK-52E cells. In addition, type I TGF-βR kinase inhibitor SB431542 attenuated ketamine-induced EPCs of renal epithelial cells, indicating that the ketamine induced EPCs are TGF-β dependent. The data together indicate that TGF-β signaling is involved in ketamine-induced tubulointerstitial injury, and imply that inhibitors of this pathway may have potentials for ketamine-caused renal dysfunction.
Part III: Ketamine affects the differentiation of monocytes into macrophages
Ketamine has been shown to interfere some pro-inflammatory cytokine production, maturation of neutrophils, differentiation of dendritic cells or bacterial clearance. In this study, we further investigated the influence of ketamine on monocyte-to-macrophage differentiation, which is a key step in the immune response. We then used a well-established monocyte model of human THP-1 leukemia cells which are ably induced by PMA (phorbol 12-myristate 13-acetate) to undergo a differentiation process along to the macrophage lineage. Our results showed that ketamine could antagonize the PMA-induced THP-1 monocyte differentiation to macrophages. Moreover, PMA-induced pro-inflammatory cytokine production (TNF-α, IL-6 and IL-8) was suppressed by ketamine in a dose-dependent manner. Our data further showed that PMA-induced MMP-9 expression, ROS generation, or growth inhibition was significantly attenuated by ketamine. In addition, ketamine suppressed PMA-induced phosphorylation of ERK1/2, p38 and JNK, suggesting that ketamine-inhibited monocyte-macrophage differentiation may be via suppressing MAPK signaling. These data together indicate that ketamine exhibits an antagonist effect on monocyte differentiation to macrophage through the suppression of MAPKs. Ketamine abuse may contribute to immune defect via interrupting monocyte maturation.
Ketamine is cataloged as a third-class controlled drug in Taiwan. Due to its accessibility, low cost and quickness to induce hallucination with less fines and penalty if caught, ketamine abuse robustly explores and recently emerges as the first recreational drug among illicit drugs in Taiwan. The present findings from our studies provide a new spectrum to explain how ketamine injures renal and immune systems as well as provide updated information for the public to keep ketamine or other drug abuse away. To reduce the harm from ketamine abuse, we heartily expect that our findings can encourage our government to pay attention to the serious problem of ketamine abuse and readjust the current national policy and strategies on controlling ketamine abuse in order to reduce the damages of ketamine abuse on people health.

並列關鍵字

Ketamine ； Renal tubular cells ； Epithelial phenotypic changes ； Renal failure ； Macrophage differentiation ； Immune defect

參考文獻

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