针刺对宫内窘迫HIBD大鼠BNIP3介导的线粒体自噬保护机制研究

Hypoxic-ischemic encephalopathy (HIE) is the direct factor of severe nervous system sequelae like cerebral palsy. As one of the very treating way of hypoxic-ischemic encephalopathy (HIE), Jin's 3-needle therapy has been proved to be effective clinically, and the State Administration of Traditional Chinese Medicine has recognized the Jin's 3-needle therapy as the clinical appropriate treatment, and has been promoted all around the country. Animal studies have confirmed that acupuncture can inhibit neuron apoptosis and help repair damaged neurons through the mitochondrial pathway. In recent years, mitochondrial autophagy as a self-protection mechanism was found that can improve the HIE pathological changes. Our previous studies found that acupuncture treatment can repair neurons, and at the same time, can increase the expression of cells autophagy. This may be the key mechanisms of acupuncture treatment to HIE, which has not been proven yet. In this study, we intend to start from the mechanism of BNIP3-mediated mitochondrial autophagy, establish ameliorated HIBD rat model by intrauterine asphyxia, use electron microscopy, flow cytometry techniques (FCM), Western Blot techniques etc. in order to reveal the self-protection mechanism of acupuncture to BNIP3-mediated mitochondrial autophagy on morphology, proteomics and function, and prove our hypothesis that Jin's 3-needle therapy can repair damaged neurons by promoting BNIP3-mediated mitochondrial autophagy as one of the self-protection mechanism, providing new laboratory evidence for acupuncture in the clinical use of HIE.

新生儿缺氧缺血性脑病（HIE）是导致儿童脑瘫等神经系统严重伤残疾病的直接因素。临床上以"靳三针"头部穴组（脑三针、颞三针、智三针）为主针刺治疗HIE疗效肯定，；我们前期研究已证实针刺可抑制细胞凋亡，同时提高线粒体自噬的表达，最终修复受损神经元。近年来，线粒体自噬作为一种自我保护机制被发现可以改善HIE脑组织病理变化。因此，我们推断，针刺对自噬表达的增强可能是其治疗HIE的关键机制。本研究拟从BNIP3介导的线粒体自噬机制入手，制备改良的宫内窘迫缺氧缺血性脑损伤（HIBD）大鼠模型，针刺头部相应穴位，应用电镜、流式细胞术、Western Blot、免疫组化等方法，从形态、蛋白、功能多方面观察BNIP3、Beclin-1、LC3的表达，揭示针刺对BNIP3介导的线粒体自噬自我保护机制的影响。证实"针刺'靳三针'头部穴组促进线粒体自噬的自我保护机制是其修复受损神经元的重要作用机制之一"的假说。

新生儿缺氧缺血性脑损伤（HIBD）严重危害新生儿的健康，是导致脑瘫，智力低下，自闭症等小儿脑病的直接因素。临床上“靳三针头穴”治疗小儿脑病二十余年，疗效肯定，国家中医药管理局已将“靳三针治疗儿童精神发育迟滞和儿童自闭症临床规范化研究”两项成果作为临床适宜疗法向全国推广。目前对“靳三针疗法”治疗小儿脑病的临床研究很多，却缺乏对其作用机理的基础研究。近年来如何实现受损神经元的修复成为研究热点。.本课题组前期研究中（国家自然科学基金课题81072878）从抑制线粒体通路介导神经干细胞凋亡入手，对宫内窘迫诱导HIBD模型新生大鼠针刺“靳三针头穴”后缺血灶脑组织切片，采用免疫组化等多种方法检测脑组织，如BrdU、Nesting、Bcl-2、Bax、Beclin-1、LC3等，证实了针刺可抑制线粒体通路介导的神经干细胞死亡，证实了“针刺‘靳三针’头穴抑制新生神经干细胞的凋亡是其促进神经干细胞修复受损神经元的中药作用机制之一”的科学家说。同时课题组在研究中发现针刺可促进自噬相关蛋白Beclin-1和LC3的表达，提示了针刺修复受损神经元的作用机制与自噬的内在关系。.本期课题组从Bnip3通路介导的线粒体自噬入手，对改良宫内窘迫诱导HIBD模型新生大鼠针刺“靳三针头穴”后行为学，形态学及分子生物学等进行检测观察。多次预实验改良宫内窘迫HIBD新生大鼠模型使成模率显著提高，死亡率显著下降。行为学测试项目中在翻身实验，悬吊试验，斜坡实验，旷场试验基础上添加姿态观察，肌张力，不自主动作，拒俘反应实验及水迷宫实验，更加接近临床新生儿行为学指标观测。采用透射电镜，HPLC，TTC染色，TUNEL检测，免疫印迹检测及qPCR等检测观察不同时间节点24h，14d，21d及28d新生鼠脑组织。如Bnip3，Beclin-1，p62，LC3，mTOR，NIX，Bcl-2等。证实了针刺可促进神经干细胞中由Bnip3介导的线粒体自噬修复受损神经元，且通过添加自噬激活剂Rapamycin及自噬抑制剂3-MA解释针刺“靳三针头穴”促进神经元修复的通路上下游作用机制。为临床应用“靳三针头穴”治疗HIBD导致的各种新生儿脑损伤疾病提供可靠的理论依据。