羟基红花黄色素A调控重症中暑急性肺损伤中RIP3介导的程序性坏死的研究

This research is focus on the acute lung injury which have an important role on the prognosis of severe heat stroke, and also based on the important pathophysiology of pulmonary microvascular endothelial cells in the induction of acute lung injury. We studied the role of RIP1 and RIP3 complex (necrosome)-induced pulmonary microvascular endothelial cells necroptosis on the pathogenesis of acute lung injury caused by severe heat stroke in vivo and in vitro studies, and to verify whether Hydroxysafflor yellow A (HSYA ) have the inhibition of necroptosis to play the role of lungs protection. This study is helpful to improve the understanding of the pathophysiological mechanism of acute lung injury in severe heat stroke, and further clarify the role of necroptosis on the development of severe heat stroke. Whether HSYA can prevent the occurrence of necroptosis and improve the acute Lung injury, which help to reveal the role and mechanism of HYSA in the treatment of severe acute lung injury induced by severe heat stroke. Furthermore, it is helpful to reveal the effect and mechanism of HSYA in treatment of severe heat-stroke. Moreover, it provide theoretical basis and evidence for HSYA treatment in heat-stroke. The characteristics and advantages of the traditional Chinese Medicine is functioning adequately and explicated by modern medical methods. The eventual objective of this research is increasing the success rate and decreasing mortality and disability in the clinical treatment for severe heat-stroke.

本研究从对重症中暑预后有重要影响的急性肺损伤入手，同时基于肺微血管内皮细胞在介导急性肺损伤中的重要病理生作用，我们在前期研究的基础上通过体内及体外实验研究RIP1及RIP3形成复合体（Necrosome小体）介导的肺微血管内皮细胞程序性坏死在重症中暑急性肺损伤中的作用机制，并给予羟基红花黄色素A（Hydroxysafflor yellow A，HSYA）干预，验证其是否通过抑制程序性坏死发挥肺保护作用，并对其具体机制进行深入研究。本研究有利于提高对重症中暑急性肺损伤病理生理机制的认识，进一步阐明程序性坏死在重症中暑发生发展中的作用，HSYA药物治疗是否可阻断程序性坏死从而改善急性肺损伤，为临床开展HSYA治疗重症中暑奠定理论基础并充实循证依据，充分发挥祖国中医药学的特色和优势，利用现代医学手段进行阐释和分析，研究成果可在短期内迅速转化成临床治疗，最终达到提高临床救治重症中暑成功率的目的。

在重症中暑研究领域，程序性坏死的相关机制类研究较少，在本课题的研究中，我们实验结果发现HSYA可增加重症中暑小鼠热耐受，降低循环中炎症因子及HMGB1水平，抑制RIP1-RIP3复合物的形成，可缓解重症中暑导致的急性肺损伤，降低肺泡灌洗液中炎症因子、HMGB1及RIP3水平，延长重症中暑小鼠生存时间。我们同时通过鼠尾静脉注射RIP3敲除腺病毒（AD-RIP3-/-）成功 建立AD-RIP3-/-小鼠模型，与鼠尾静脉注射空载腺病毒（AD-control）组相比可明显改善小鼠热耐受能力，AD-RIP3-/-处理可明显延长重症中暑小鼠生存时间，改善生存率。我们成功体外分离小鼠肺微血管内皮细胞（PMVECs），热打击使肺微血管内皮细胞中RIP1及MLKL蛋白表达水平显著升高，而RIP3水平在热打击后无明显增加，热打击使肺微血管内皮细胞中RIP1-RIP3复合物（Necrosome小体）形成，从而导致细胞发生程序性坏死，细胞给予RIP1活化抑制剂Nec-1及使用RIP1siRNA及RIP3siRNA预处理可抑制肺微血管内皮细胞发生程序性坏死，以上结果表明热打击使PMVECs发生了程序性坏死，且为RIP1及RIP3所介导。HSYA可降低热打击导致的PMVECs程序性坏死, 给予HSYA预处理后可抑制HMGB1从细胞核向细胞浆转移, 热打击可导致肺微血管内皮细胞中TLR4蛋白表达增加，而TNFR、TLR2及Fas无明显改变，使用TLR4封闭抗体预处理细胞可抑制MLKL磷酸化，结果表明热打击可能通过TLR4受体介导肺微血管内皮细胞Necrosome小体激活，从而使下游MLKL发生磷酸化，从而导致细胞发生程序性坏死，给予HSYA预处理细胞可抑制TLR4表达，HSYA通过抑制TLR4受体抑制下游MLKL磷酸化，从而降低程序性坏死发生。.本研究填补重症中暑小鼠模型中程序性坏死在急性肺损伤研究领域的空白，证明重症中暑肺损伤中出现程序性坏死现象，早期出现Necrosome小体激活，由以肺微血管内皮细胞坏死释放HMGB1，介导及加重肺部炎症及水肿，促进急性肺损伤发生及发展，推动重症中暑肺损伤向致命性ARDs发展，阐明了HSYA药物预处理可通过抑制TLR4受体表达抑制MLKL激活及HMGB1释放从而发挥肺保护作用，为选择重症中暑急性肺损伤的治疗提供可能有效的用药方案及理论依据。