Abstract: Background　Neuronal apoptosis caused by cellular autophagy is an important link of nerve injury after cerebral ischemia.Tongluoqingnao powder injection，a representative therapy of TCM treatment for clearing heat and detoxifying and clearing collaterals，has been proved effective for cerebral ischemia in rats even without syndrome differentiation，indicating that the mechanism of cerebral protective effect of this therapy may be correlated with inhibition of cellular autophagy.Objective　To investigate the effect of tongluoqingnao powder injection on protecting the brain and expression of neuronic autophagy-related proteins in a rat model of cerebral ischemia-reperfusion injury.Methods　This experiment was conduct from January 2019 to March 2020.We selected 36 adult male specific pathogen free SD rats，and equally randomized them into sham-operated group，model group and tongluoqingnao group after coding.Suture-occluded method，namely middle cerebral artery occlusion，was used to prepare rat models of cerebral ischemia-reperfusion injury：rats in sham-operated group only received angiodiastasis，and those in tongluoqingnao group received tongluoqingnao solution 3.2 ml/kg（with a concentration of 18.75 mg/ml）injected slowly via the caudal vein，while those in other two groups received isopyknic 0.9% sodium chloride solution.Neurological functional score and proportion of cerebral infarction volume 24 hours after modeling，as well as neuron injury in hippocampus，formation of autophagosomes in hippocampus and cortex and neuronic autophagy-related proteins（including p-NF-κB，Beclin1 and LC3）in hippocampus 24 hours after reperfusion were compared among the three groups.Results　The neurological functional score and proportion of cerebral infarction volume 24 hours after modeling in sham-operated group，tongluoqingnao group，and model group increased successively（P<0.05）.Twenty-four hours after reperfusion，the sham-operated group showed no obvious neuronal necrosis in hippocampus，but with occasional autophagosomes in hippocampus and cortex，the model group demonstrated serious structural damage and a lot of loss of neurons in the ischemic region of hippocampus，with bilayer autophagosomes in hippocampus and cortex.In contrast to the model group，tongluoqingnao group had obviously shrinked range of neuronal necrosis，obviously relieved neuron damage，with obviously reduced number of autophagosomes.At 24 hours after reperfusion，the sham-operated group had less relative expression quantities of p-NF-κB，and LC3 in hippocampus than tongluoqingnao group（P<0.01），and this group also showed less relative expression quantities of p-NF-κB，Beclin1 and LC3 in hippocampus than the model group（P<0.01）.The relative expression quantities of p-NF-κB，Beclin1 and LC3 in hippocampus in tongluoqingnao group were significantly lower than those in model group（P<0.01）.Conclusion　In rat models of cerebral ischemia-reperfusion injury，tongluoqingnao powder injection may effectively improve the neurological function，relieve the degree of cerebral infarction and neuron injury，reduce the formation of autophagosomes and neuronal apoptosis，inhibit the expression of neuronic autophagy-related proteins such as p-NF-κB，Beclin1 and LC3，and its mechanism of cerebral protective effect may be correlated with the inhibition of neuronic excessive autophagy.

Key words: Brain ischemia, Reperfusion injury, Tongluoqingnao powder injection, Neurons, Autophagy

摘要： 背景　细胞自噬所致神经元凋亡是脑缺血后神经损伤的重要环节，而以通络清脑注射粉针为代表的清热解毒通络疗法在只辨病、不辨证的情况下治疗大鼠脑缺血亦能取得良好疗效，因此推测通络清脑注射粉针发挥脑保护作用的机制很可能与抑制细胞自噬有关。目的　探讨通络清脑注射粉针对脑缺血-再灌注损伤大鼠的脑保护作用及神经元自噬相关蛋白表达的影响。方法　本实验于2019年1月—2020年3月实施，共选取SPF级成年雄性SD大鼠36只，编号后采用随机数字表法分为假手术组、模型组、通络清脑组，每组12只。采用线栓法制备大鼠脑缺血-再灌注损伤模型（假手术组大鼠只进行血管分离、不进行其他操作），通络清脑组大鼠经尾静脉缓慢推注浓度为18.75 mg/ml的通络清脑溶液3.2 ml/kg，假手术组、模型组大鼠经尾静脉注射等体积0.9%氯化钠溶液。比较三组大鼠建模24 h后神经功能学评分、脑梗死体积占比及再灌注24 h后海马区神经元损伤情况、海马区和皮质区自噬体形成情况、海马区神经元自噬相关蛋白〔包括磷酸化核因子-κB（p-NF-κB）、Beclin1、自噬微管相关蛋白1轻链3（LC3）〕表达情况。结果　模型组、通络清脑组大鼠建模24 h后神经功能学评分、脑梗死体积占比高于假手术组，而通络清脑组大鼠建模24 h后神经功能学评分、脑梗死体积占比低于模型组（P<0.05）。再灌注24 h后假手术组大鼠海马区神经元未见明显坏死、海马区和皮质区均偶见自噬体，模型组大鼠海马区缺血区神经元结构破坏严重、大量神经元丢失且海马区和皮质区均可见双层膜结构自噬体，通络清脑组大鼠海马区缺血区神经元坏死范围较模型组缩小、神经元损伤明显缓解且海马区和皮质区自噬体数量均减少。再灌注24 h后模型组、通络清脑组大鼠海马区p-NF-κB、LC3相对表达量及模型组Beclin1相对表达量高于假手术组，而通络清脑组大鼠海马区p-NF-κB、Beclin1、LC3相对表达量低于模型组（P<0.01）。结论　通络清脑注射粉针可有效改善脑缺血-再灌注损伤大鼠神经功能，减轻脑梗死程度、神经元损伤，减少自噬体形成及神经元凋亡，抑制神经元自噬相关蛋白p-NF-κB、Beclin1、LC3的表达，其发挥脑保护作用的机制可能与抑制神经元过度自噬有关。

关键词: 脑缺血, 再灌注损伤, 通络清脑注射粉针, 神经元, 自噬