TRPV1介导的线粒体自噬在氮芥诱导角质形成细胞损伤中的作用及机制研究

Keratinocytes (KC) are the targets of Mechlorethamine (NM), and their dysfunction plays a key role in NM-induced skin injury, however, the underlying mechanism has not been thoroughly elucidated. Recently, it has been found that transient receptor potential channel-vanillin subtype 1 (TRPV1) is highly expressed in KC and plays an important role in KC injury caused by various physical and chemical stimuli. Meanwhile, it has also been demonstrated that mitophagy is important for the biological regulation of TRPV1. Our previous studies found that NM can significantly increase TRPV1 expression and induce mitophagy in KC; in contrast, inhibition of TRPV1 significantly inhibits NM-induced mitophagy and cell death. Accordingly, we hypothesized that NM might cause KC injury by regulating mitophagy via the TRPV1-Ca2+-AMPK signaling pathway. To confirm this hypothesis, we investigate the role of mitophagy in NM-induced KC injury in vitro and in vivo. Furthermore, the potential involvement of the TRPV1-Ca2+-AMPK signaling pathway is also investigated. Our results will provide new insights into the mechanism of NM-induced KC injury as well as providing an important experimental evidence for targeting TRPV1 to prevent and treat NM-induced skin damage.

角质形成细胞（KC）是氮芥（NM）的作用靶点，其功能紊乱是NM诱导皮肤损伤的关键，但作用机制尚未完全阐明。最新研究发现，瞬时受体电位通道-香草醛亚型1（TRPV1）高表达于KC，在多种理化刺激引起的KC损伤中有重要作用；同时发现线粒体自噬与TRPV1发挥其生物调控作用关系密切。申请者前期研究发现，NM可显著增加KC中TRPV1表达，诱导线粒体自噬；反之，抑制TRPV1可明显抑制NM引起的线粒体自噬和细胞死亡。据此，本项目提出“NM可能通过激活TRPV1-Ca2+-AMPK信号通路调控线粒体自噬，从而引起KC损伤”的研究假说。拟通过基于KC、TRPV1-/-小鼠和无毛小鼠的研究，明确线粒体自噬在NM诱导KC损伤中的作用，并阐明“TRPV1-Ca2+-AMPK”信号通路在其中的可能作用，以期从全新视角揭示NM诱导KC损伤的分子机制，并为靶向TRPV1防治NM诱导的皮肤损伤提供实验依据。

氮芥（NM）导致的难愈性皮肤损伤，尚缺乏靶向治疗方法。NM诱导皮肤损伤机制尚未完全阐明，是限制其特效防治药物研发的瓶颈所在。最新研究表明，自噬在物理和化学暴露引起的皮肤损伤中起重要作用。然而，自噬在NM诱导皮肤损伤中的作用尚不清楚。本项目通过建立体内外NM染毒皮肤损伤模型，深入研究了瞬时受体电位通道-香草醛亚型1（TRPV1）介导的自噬在NM诱导皮肤损伤中的作用及具体机制。我们发现NM可剂量依赖性地引起细胞死亡并诱导角质形成细胞自噬。利用3-MA，氯喹和bafilomycin A1或ATG5 siRNA抑制自噬后，减少了NM诱导的角质形成细胞死亡。同时发现，NM可增加TRPV1的表达、细胞内Ca2+含量以及Ca2+ /钙调蛋白依赖性激酶激酶β（CaMKKβ）、AMP激活的蛋白激酶（AMPK）、unc-51-like激酶1（ULK1）和雷帕霉素哺乳动物靶标（mTOR）活性。通过使用各种抑制剂TRPV1（capsazepine）、CaMKKβ（STO-609）、AMPK（compoud C）和ULK1（SBI-0206965）以及TRPV1、CaMKKβ、AMPK siRNA转染，可以消除NM诱导的角质形成细胞自噬。此外，mTOR抑制剂（雷帕霉素）对NM诱导的自噬或角质形成细胞死亡没有明显影响。最后，在NM处理的皮肤组织中取得了一致的结果。以上结果表明，NM通过激活TRPV1-Ca2+-CaMKKβ-AMPK-ULK1信号通路来诱导过度自噬而引起皮肤毒性，提示阻断TRPV1依赖性自噬可能是NM引起皮肤损伤的潜在治疗策略。我们的研究结果为NM诱导难愈性皮肤损伤的作用机制研究提供了全新的视角，同时也为靶向TRPV1防治NM诱导皮肤损伤药物的研发提供了重要的实验证据支持，具有重要的军事意义和民用价值。