基于“线粒体-肠道菌群交互作用”探讨益气化痰通络法抗脑缺血/再灌注损伤作用及机制研究

Intestinal microecology is closely related to neuronal damage after stroke, and the destruction of mitochondrial structure and function is a key link of cerebral Ischemia/Reperfusion injury. Recent studies have found that there is crosstalk between microbiota-mitochondria, the mechanism is mainly related to immune and inflammatory mechanisms. Therefore, "microbiota-mitochondria crosstalk" has become a new hot spot in the research of anti-cerebral Ischemia/Reperfusion injury. Yiqi Huatan Tongluo method is based on the development and enrichment of previous research by the applicant team. The clinical curative effect of Yiqi Huatan Tongluo Recipe on cerebral Ischemia/Reperfusion injury is exact. Therefore, this study focuses on the "microbiota-mitochondria crosstalk", combined with intestinal flora transplantation method, the Yiqi Huatan Tongluo method of brain protection. Through the establishment of rat model of MCAO, preparation of Yiqi Huatan Tongluo extract dry powder water solution to observe changes of microbiota-mitochondria after cerebral Ischemia/Reperfusion injury and the intervention of Yiqi Huatan Tongluo medicine; Preparation of Yiqi Huatan Tongluo medicine gut extract, through the flora method to observe the MCAO model rat of microbiota-mitochondria, and then explore the Yiqi Huatan Tongluo method whether through the impact of "microbiota-mitochondria crosstalk" play anti-cerebral Ischemia/Reperfusion injury.

肠道微生态与脑卒中后神经元损伤密切相关，线粒体结构和功能的破坏是脑缺血/再灌注损伤的关键环节。新近研究发现，线粒体与肠道菌群存在交互作用，其机制主要与免疫炎症机制相关。因此，干预“线粒体-肠道菌群交互作用”成为抗脑缺血/再灌注损伤研究的新热点。益气化痰通络法是基于申请者团队前期研究的发展和丰富，益气化痰通络方抗脑缺血/再灌注损伤临床疗效确切。因此，本研究着眼于“线粒体-肠道菌群交互作用”，结合肠道菌群移植方法，对益气化痰通络法脑保护作用机制进行探讨。通过建立大鼠MCAO模型，制备益气化痰通络方浸膏干粉水溶液，观察大鼠脑缺血/再灌注损伤后线粒体-肠道菌群的变化及益气化痰通络方的干预作用；制备耐益气化痰通络方肠道菌群提取物，通过菌群移植的方法观察其对MCAO模型大鼠模型线粒体-肠道菌群的影响，进而探究益气化痰通络方是否通过影响“线粒体-肠道菌群交互作用”起到抗脑缺血/再灌注损伤的作用。

线粒体结构完整性及其功能稳态平衡的破坏是脑缺血/再灌注损伤的关键环节，保护线粒体结构和功能是脑缺血/再灌注损伤后神经元保护的重点；调节肠道菌群是启动神经保护的重要切入点，这种保护机制与免疫炎症反应密切相关。“线粒体-肠道菌群的交互作用”与免疫炎症机制密切相关，干预“线粒体-肠道菌群交互作用”可起到脑保护的作用。本研究建立大鼠MCAO模型，以益气化痰通络方干预，证实其无急性肝肾毒性，从行为学、形态学方面，运用透射电镜观察大鼠缺血侧脑皮质神经元及线粒体超微结构，TUNEL染色检测大鼠缺血侧神经元细胞凋亡指数，采用ELISA法检测脑组织线粒体中ROS、Ca2+浓度，采用羟胺法检测SOD活力、硫代巴比妥酸（TBA）法检测MDA含量、硝酸还原酶法检测NO含量，采用Western blot法检测缺血侧脑组织Bcl-2、Bax、Cyt-C及caspase-3蛋白表达水平，并取大鼠粪便进行16SrDNA肠道微生物多样性信息分析，取血清进行LC-MS代谢产物分析，采用ELISA法检测IL-10、IL-17含量，RT-PCR法检测缺血侧脑组织、脾脏、回肠黏膜CXCL1、CXCL2及Foxp3 mRNA表达情况，并采用流式细胞术检测外周血、脾脏、脑膜Th-17、Treg细胞比例，结果发现益气化痰通络方可改善tMCAO模型大鼠线粒体结构及功能稳态，抑制线粒体凋亡，且能减轻机体免疫炎症反应，从而调控肠道微生态环境，并且通过耐益气化痰通络方菌群移植干预，发现其亦可以通过保护线粒体而抑制线粒体凋亡，并抑制免疫炎症反应，从而进一步证实，益气化痰通络方可能是通过“线粒体-肠道菌群交互作用”抗脑缺血/再灌注损伤，为指导和推广其临床应用提供参考。