针刺对TBI脑功能重塑的影响及其对细胞自噬的双向良性调节作用研究

The main pathology after traumatic brain injury (TBI) is the micro-environment changes caused by cerebral ischemia and hypoxia. However, a stable internal environment is the key to brain function remodeling. And the autophagy plays a key role in the pathology and brain function remodeling after TBI as it mainly maintains the stability of the internal environment. Meanwhile, it also presents a bidirectional effect on the brain function remodeling after TBI. The preliminary studies have found that acupuncture can bidirectionally regulate autophagy . Our previous studies showed that acupuncture can benignly bidirectionally regulate glial cells to differentiation and promote nerve repair after TBI. Therefore, we put forward a hypothesis: Benignly bidirectionally regulate autophagy by regulating related signaling pathways to maintaining brain environment stability and promoting brain functional remodeling is possible the key mechanism of acupuncture treatment for TBI. This study intends to adopt TBI model rat that conducting acupuncture treatment, and proceed multi-period dynamically observations: using the UHF-MRI, TEM, flow cytometery and tissue patch-clamp technique to detect brain environment and brain function remodeling. Moreover, the RT-qPCR and Western blot are used to detect the autophagy markers, the expressions of mRNA and protein of related gens.Our objective is to explore the effects and mechanism of acupuncture on brain function remodeling and benign bidirectional regulation to autophagy after TBI. And also look for new therapeutic targets for acupuncture treatment in TBI and central nervous system diseases, and provide a new experimental basis for the benign bidirectional regulative effect of acupuncture at the nerve central level.

颅脑损伤（TBI）后主要病理是脑缺血缺氧造成的微环境改变，内环境稳定是损伤后修复（脑功能重塑）的关键。细胞自噬的主要功能是维持内环境稳定，在损伤后病理及脑功能重塑中起关键作用，呈双向性。初步研究发现针刺可双向调节自噬，我们前期研究也发现针刺可双向良性调节TBI后胶质细胞分化、促进神经修复。故提出假说：针刺治疗TBI的关键机制可能是通过调控相关信号通路来双向良性调节细胞自噬，维持脑内环境稳定，促进脑功能重塑。本研究拟采用针刺治疗TBI大鼠模型，多时段动态观测：UHF-MRI、电镜、流式细胞仪、组织膜片钳技术分别检测脑内环境、脑功能重塑；RT-qPCR、Western blot检测自噬标记物及相关调控基因表达，探讨针刺对TBI后脑功能重塑的影响及其对细胞自噬的双向良性调节作用与机制，为TBI及中枢神经系统疾病寻找新的治疗靶点，在中枢层面为针刺双向良性调节作用提供新的实验依据并扩大其内涵。

项目背景：创伤性颅脑损伤（TBI）是指由外力引起的脑部功能改变或病理性损害，给医疗保健系统及社会经济发展带来了严峻的挑战。细胞自噬的主要功能是维持内环境稳定，在损伤后病理及脑功能重塑中起关键作用，良性调控自噬将为TBI治疗提供新的思路和方向。.主要研究内容：采用CCI法建立TBI大鼠模型，进行针刺治疗，采用病理染色、透射电镜、小动物磁共振、激光散斑血流成像、流式细胞术、膜片钳等方法观察针刺对TBI大鼠损伤区病理形态、细胞自噬、组织结构和代谢、血流灌注、脑功能修复以及对自噬标记物和相关信号通路的调控。.重要结果、关键数据及意义：.1. 建立TBI大鼠模型，mNSS评分法评估造模情况，结果符合中度颅脑损伤（见附件1图2）。HE和Nissl染色结果表明：针刺可明显减轻TBI大鼠脑组织病理损伤程度（见附件1图3和图4）。.2.流式和WB结果表明，针刺可抑制早期脑细胞凋亡，促进PI3K、AKT、Bcl-2蛋白表达，抑制Bax和Caspase-3蛋白表达。针刺可通过调控凋亡基因减少脑细胞凋亡。（见附件1图5-9）.3.透射电镜结果显示，在3d，针刺组自噬小体较模型组多；7d和14d，模型组可见神经元核溶解，自噬体仍较多，针刺组神经元形态结构改善，自噬体减少。免疫荧光结果显示，在3d，针刺组神经元的LC3表达增强；7d和14d，模型组神经元的LC3表达仍然增高，针刺组神经元的LC3表达较模型组降低。AMPK/mTOR/ULK1、p-p38/p38-MAPK、p-p53/p53通路及LC3Ⅱ/Ⅰ和p62等自噬相关基因蛋白表达情况与电镜和免疫荧光趋势基本一致。（见附件1图10-23）.4.小动物磁共振、激光散斑血流成像和流式结果表明，针刺可减少损伤病灶体积，减轻脑组织水肿和组织结构损伤，改善血流灌注，诱导神经干细胞增殖分化，促进神经元再生修复和脑组织结构重构（见附件1图24-34）。.结论：针刺具有双向良性调节作用，可通过多信号相互协同的治疗效应起到神经保护作用，早期促进自噬，有助于清理异常细胞结构，维持细胞稳态，减轻神经损伤；后期抑制细胞过度自噬，促进神经元再生修复和脑组织结构重构，从而促进脑功能重塑。