基于Wnt/Ca2+信号通路研究肢体缺血-再灌注损伤骨骼肌细胞凋亡机制及桃红四物汤的干预作用

The Study on Pathogenesis of Skeletal Muscle Apoptosis in LIRI and the Intervention Effect of Taohongsiwu Decoction Based on Wnt/Ca2+ Signal Pathway. Limb ischemia reperfusion injury (LIRI) is a common complex pathological process after severe trauma. The disease pathogenesis has not yet been fully grasped, the treatment lacks of specificity, and the treatment effect is uncertain, therefore it is of great significance to clarify the pathogenesis and the effective prevention and treatment of the disease. It is generally thought that the calcium overload and the body inflammation are important mechanisms of the ischemia-reperfusion injury of vital internal organs and Wnt/Ca2+ pathway plays a very important role in calcium ion metabolism and body inflammation. Based on the previous study, we found that Chinese herbal compound prescription for activating blood circulation and removing blood stasis reduced LIRI skeletal muscle apoptosis, changed the strength of expression of calmodulin, which preliminarily presented the strong correlation between Wnt/Ca2+ pathway and LIRI. This project is planed to further demonstrate the molecular mechanism of LIRI skeletal muscle apoptosis by establishing rats LIRI model and injury muscle cell model induced by hypoxia-reoxygenation, using techniques such as flow cytometry, laser confocal scanning microscopy system, and molecular biology, and observing the gene overexpression in Wnt/Ca2+ pathway and the impact intervention effect, and besides, to identify the targeting gene of Chinese herbal compound prescription for “activating blood circulation and removing blood stasis” on it by applying Taohongsiwu decoction as an intervention treatment. In this way, we expect to provide a new thought and theoretical basis for TCM treatment of LIRI...Key words: Wnt/Ca2+ Signal Pathway;Limb Ischemia Reperfusion Injury; Skeletal Muscle Cells Apoptosis; Taohongsiwu Decoction; Study of Pathological Mechanism

肢体缺血再灌注损伤（LIRI）为严重创伤后一种常见的复杂病理过程，其病理机制尚未完全阐明，治疗缺乏特异性且疗效不确定，因而阐明其病理机制及研究有效防治具有重要意义。目前钙超载及炎症反应被公认为内脏重要器官缺血再灌注损伤的重要病理机制，而Wnt/钙离子通路在钙离子代谢及炎症反应中起着至关重要的作用，我们前期研究发现：活血化瘀类中药可以减轻LIRI骨骼肌细胞凋亡，改变钙调蛋白表达的强弱，初步揭示Wnt/钙离子通路与LIRI应有高度相关性。本项目拟在前期研究基础上，建立大鼠LIRI模型及缺氧再给氧诱导损伤的肌母细胞模型，采用流式细胞仪、激光共聚焦扫描显微系统及分子生物学等技术，通过观察Wnt/钙离子通路中基因的过表达和干扰其表达对LIRI的影响，进一步阐明其骨骼肌细胞凋亡的分子机制，并以桃红四物汤干预治疗进行反证，明确桃红四物汤治疗该病的靶点基因，为中医药治疗LIRI提供新思路和理论依据。

肢体缺血-再灌注损伤（LIRI）病理机制尚未完全阐明，且治疗缺乏特异性疗效也不确定，因而阐明其病理机制及研究有效防治具有重要意义。目前钙超载及机体炎症反应被公认为内脏重要器官缺血-再灌注损伤的重要病理机制，而Wnt/Ca2+信号通路在钙离子代谢及机体炎症反应中起着至关重要的作用，我们前期临床工作中发现桃红四物汤在肢体缺血性疾病的治疗中有其独到的作用，实验研究也发现：活血化瘀类中药复方可以减轻LIRI骨骼肌细胞凋亡，使骨骼肌型钙调蛋白兰呢啶受体的表达减弱、钙泵的表达增强，初步揭示Wnt钙离子信号通路与LIRI应有高度相关性。基于以上背景，本项目主要从：LIRI中骨骼肌细胞Wnt/Ca2+信号通路蛋白表达特点、桃红四物汤治疗LIRI的效应及桃红四物汤治疗LIRI的分子机制（主要研究Wnt/Ca2+信号通路）三方面入手进行研究，我们采用流式细胞仪、激光共聚焦扫描显微镜及分子生物学等技术，使用Wnt/Ca2+信号通路中关键蛋白CaMKⅡ、IP3R、Wnt等阻断剂以及桃红四物汤进行干预发现：①在LIRI中骨骼肌细胞Wnt/Ca2+信号通路中关键蛋白Wnt5a、Wnt11、CaMKⅡ、IP3R表达均有所增强；②桃红四物汤可以降低SD大鼠LIRI模型及缺氧再给氧诱导损伤的大鼠肌母细胞模型中肌细胞的凋亡率、减轻LIRI损伤，其主要机制是使Wnt/Ca2+信号通路中关键蛋白Wnt5a、Wnt11、CaMKⅡ、IP3R表达下降，减轻钙超载而起到治疗LIRI的目的；③从我们的实验中看出：PKC虽为LIRI的靶点蛋白，但不是Wnt5a/Ca2+信号通路下游关键蛋白，所以桃红四物汤不能通过Wnt5a/Ca2+信号通路下调PKC。本项目的科学意义在于：通过实验发现Wnt钙离子信号通路所介导的钙超载是LIRI的主要病理机制，桃红四物汤可通过对该通路中多个基因靶点的作用而降低钙超载达到减轻LIRI的作用。