Cirbp在急性冷暴露致肝脏损伤中的作用及机制研究

Study on damage effect of cold exposure and its protection is an important aspect of preventive medicine research. Outdoor activities in high latitudes in winter, or working in low temperature environment such as cold storage will be facing with the threat of cold injury. Our previous study found that acute cold exposure may cause liver injury, increased serum levels of inflammatory cytokines. Cirbp is the first cold shock protein found in mammalian cells which participate in cold stress reaction by regulating RNA stability. Recent studies found that increased expression of Cirbp in hepatic ischemia-reperfusion and its entering the circulatory system, which mediated inflammatory reactions and may cause liver injury. The aim of our study is to establishing acute cold exposure models in vivo and in vitro, observing the injury level of hepatocytes and expression level of Cirbp by using immuno-histochemistry and molecular biology method. And to detecting the liver inflammatory injury effects of Cirbp, which may caused by its possible regulative effects on inflammatory cytokines such as NF-κB, by blocking its expression using overexpression and/or underexpression cell culture and animal model. This study may shed further light on the revealing of key influence factors and damage feature of acute cold exposure induced liver injury. And clarifying the injury effects, as well as its relationship with injury level, of Cirbp during acute cold exposure. It will provide further preventive and therapeutic clues and theory for treating cold exposure-related organ damage.

冷暴露致机体损伤与防护研究是预防医学研究的重要内容。在高纬度地区冬季进行户外活动或在冷库等低温环境下作业的人员将面临冷暴露损伤威胁。我们前期研究发现急性冷暴露可以引起肝损伤，血清炎性因子水平增加。Cirbp是首先在哺乳动物细胞中发现的冷应激蛋白，通过调节RNA稳定性而参与冷应激反应。新近研究发现，Cirbp在肝缺血再灌注后表达增加并进入循环系统，介导炎症反应而引起肝脏损伤。本研究拟通过建立体内和体外冷暴露模型，利用组织化学和分子生物学方法，观察冷暴露后肝细胞损伤和Cirbp表达变化情况，并通过构建Cirbp高表达/低表达细胞与转基因动物探讨Cirbp对NF-κB等炎性因子的调控与冷暴露致肝脏炎症损伤的关系。本研究将揭示急性冷暴露致肝脏损伤的关键因素及损伤特点，阐明Cirbp在急性冷暴露引起肝脏损伤中的作用及其与肝脏损伤程度的关系，为进一步预防和治疗冷暴露相关脏器损伤提供新的线索和理论依据。

广泛人群如冷库等低温环境作业人员和高纬度地区冬季的户外活动者均面临冷暴露损伤的威胁。肝脏作为人体产热与能量代谢的重要器官，参与机体寒冷应答，在冷暴露过程中可能发生损伤。Cirbp是首先在哺乳动物细胞中发现的冷应激蛋白，在肝脏缺血再灌注后表达增加并进入循环系统，介导炎症反应从而引起肝脏损伤，因此我们推测Cirbp可能在冷暴露后表达上调并参与冷暴露诱导的肝脏损伤。主要结果：（1）体外研究发现低温暴露可引起细胞周期阻滞于S期，细胞活力下降，但低温暴露并未引起细胞凋亡水平显著升高；（2）体内研究发现4 h急性冷暴露及复温4 h可以引起大鼠肝组织损伤，并以复温4 h后损伤更为显著。表现为细胞ATP水平下降、凋亡细胞增加和炎性损伤相关因子增加，分子生物学、血清学和形态学检测结果进一步验证了以上现象，表现为①肝组织Caspase-3蛋白剪切水平和线粒体Bax转位增加，肝组织TNF-α和NF-κB表达水平增加；②血清ALT/AST等肝脏损伤相关指标增加；③TUNEL染色阳性细胞增多和细胞、线粒体超微结构损伤；（3）在这一过程中，Cirbp蛋白水平上调，虽然未发生细胞内的转位，但可以在血清中检测到Cirbp含量显著升高；（4）通过腺相关病毒构建肝脏Cirbp低表达动物模型，证实Cirbp参与急性冷暴露及复温后肝组织的损伤过程，抑制Cirbp能够减轻急性冷暴露复温后肝组织Caspase-3蛋白剪切水平，同时降低肝组织TNF-α和NF-κB的水平，血清学指标同样验证了以上现象，表现为血清ALT/AST水平的降低和IL-1β及IL-6水平的降低。 以上结果为揭示急性冷暴露诱导肝组织损伤的分子机制提供了理论和实验依据，为进一步研究急性冷暴露诱导机体损伤的防治措施提供了新思路。