热敏灸调控线粒体凋亡信号通路减轻新生小鼠缺氧缺血性脑损伤的效应机制研究

Previous studies by our group suggested that acupoints at certain locations responded to disease. An acupoint has two states, the "resting" state and the" sensitized" state. Selecting the sensitized acupoints is the key to improving efficacy. There are a variety of types of sensitized acupoints, the heat-sensitive type is a new one among these. The theoretical basis for heat-sensitive moxibustion is that the acupoints which are activated from the resting state to the sensitized state by a pathological process become heat sensitive. Moxibustion placed above a heat-sensitive acupoint(ie, heat-sensitive moxibution)can achieve effect namely "less stimulation, greater effect", so the efficacy of moxibustion is significantly improved. . Neonatal hypoxic-ischemic encephalopathy (HIE) is a major cause of morbidity and mortality in infants and children, and it causes severe neurological disorders and lifelong disability in survivors. Currently, there are no effective pharmacological interventions available for protecting neonatal brains after HI injury. The animal experiments by our group discovered heat-sensitive moxibustion could significantly reduced neonatal hypoxic-ischemic brain injury in neonatal mice.So, we here put forward a new way of heat-sensitive moxibustion on neonatal hypoxic-ischemic encephalopathy and further explore its mechanism.. In this study, neonatal hypoxic-ischemic encephalopathy(NHIE) mice will be used as the pathological model, infrared imaging combined with tail temperature measurement will be used as the detecting technique of heat-sensitive acupoints, and the infarct volume, apoptosis,ROS/CytC/caspase 9/caspase 3 mitochondrial apoptotic signal pathway will be used as the observed indicators. Then we will compare the anti-apoptotic effects between heat-sensitive moxibustion and non heat-sensitive moxibustion and clarify the molecular mechanism for heat-sensitive moxibustion significantly attenuating neonatal hypoxic-ischemic brain injury. In addition, the study can also provide scientific clues to recognize the body's own regulatory systems that have greater potential function.

课题组以往的研究表明，穴位是敏化态的疾病在体表的反应部位，即穴位有“静息”与“敏化”的状态之别，选择敏化态穴位进行针灸是提高疗效的关键。穴位的敏化态有多种类型，热敏态是穴位敏化态的一种新类型。热敏态穴位对灸热刺激产生“小刺激大反应”，能够明显提高灸疗疗效。. 新生儿缺氧缺血性脑病（HIE）是导致患儿致死和致残的主要原因。目前对该病尚无有效的治疗方法。前期实验预试发现，热敏灸可减轻HIE模型小鼠脑损伤。为此，本课题提出热敏灸治疗HIE的新方法和新途径，并进一步探讨其提高疗效的机制。. 本研究以HIE小鼠为病理模型，红外成像结合尾温测定为穴位热敏态的检测技术，艾灸“大椎”穴为干预因素，线粒体凋亡信号通路等为观察指标，比较艾灸热敏态与非热敏态“大椎”穴在减轻缺氧缺血性脑损伤效应方面的差异，阐明热敏灸提高疗效的分子生物学基础，从而为认识自身调控系统功能具有较大潜力提供科学线索。

新生儿缺氧缺血性脑病（hypoxic-ischemic encephalopathy, HIE）具有几个显著的特点：一是涉及面广，目前仍是世界范围内足月儿普遍存在的问题；二是发病率高；三是致残率、致死率高。在治疗上，可供选择帮助患儿恢复的方法有限，幸存患儿常遗留有长期的神经后遗症。最新的研究报告也指出，尽管低温治疗已被证明可以减少足月和近期出生的中到重度缺氧缺血性脑病婴儿的死亡或残疾，但是仍然有许多幸存的患儿伴有残疾。可以看出，这种治疗手段达不到理想的治疗效果。因此，加强研究治疗新生儿HIE具有重要的意义。在医学界，中医药日益成为攻克神经系统疾病的有效工具。尤其是热敏灸在治疗多种疾病上表现出独特的优势，发挥了重要的作用。因此，本课题组将研究方向转向祖国医学的热敏灸疗法。实验研究发现，热敏灸具有减轻新生小鼠缺氧缺血性脑损伤的作用，然而其作用机制不清楚。本研究采用采用颈总动脉结扎术联合缺氧制备HIE新生小鼠模型，采用行为测试法、TTC（2,3,5-氯化三苯基四氮唑）染色法、DAPI染色法、HE染色法、TUNEL分析法、分光光度法、免疫组织化学法以及Western blot等观察热敏灸对小鼠模型行为学、形态学、生物化学、分子生物学等方面的影响，首次发现：.（1）热敏灸具有减轻新生小鼠缺氧缺血性脑损伤、改善行为学表现；.（2）热敏灸具有减少脑组织细胞凋亡的作用；.（3）热敏灸可增加脑组织SOD2蛋白表达、降低脑组织MDA含量；.（4）热敏灸可减少脑组织细胞色素c(Cyt c)蛋白表达、减少脑组织caspase-9和caspase-3阳性细胞数；.（5）热敏灸可增加脑组织Bcl-2 蛋白表达、降低Bax 蛋白表达。.上述结果提示，热敏灸具有减轻新生小鼠缺氧缺血性脑损伤、改善行为学表现的作用，其可能是通过调控线粒体凋亡信号通路（包括Bcl-2 家族和ROS/ cytochrome c/caspase-9/ caspase-3）实现的。