基于凝血酶诱导自噬探讨化瘀解毒法对缺血性中风(瘀毒互结证)大鼠的影响

Effect of Blood Stasis Removing and Detoxifying Method on Ischemic Stroke(Entangled Stasis and Toxin) in Rats Based on Thrombin-Induced Autophagy

目的:观察化瘀解毒法对缺血性中风瘀毒互结证(IS-YD)大鼠不同缺血时相、不同脑区凝血酶表达以及自噬指标LC3Ⅱ/Ⅰ的影响,分析其发挥抗缺血性脑损伤作用的可能机制。方法:于缺血后12、24 h,将116只SD大鼠,随机分为假手术组(SHAM组)、模型组(IS-YD组)、化瘀解毒方组(HJF组)和阿加曲班组(ARG组),其中假手术组8只,其余每组36只,再分为12、24 h两个时间点,每组18只。除假手术组外,其余大鼠制备缺血性中风瘀毒互结证模型。化瘀解毒方组给予11.33 g/(kg·d)剂量灌胃,阿加曲班组予以11.16 mg/(kg·d)剂量腹腔注射,假手术组、模型组给予0.9%生理盐水灌胃。Western blot法检测大脑皮质、海马区的凝血酶、LC3Ⅱ/Ⅰ比值的表达;TTC染色法计算脑梗死体积;透射电镜观察法观察脑区自噬超微结构;HE染色法观察神经元形态学改变。结果:与假手术组比较,模型组大鼠12、24 h两个时间点的神经功能评分升高(P <0.01),脑梗死体积占比增加(P <0.05),大脑皮质、海马区凝血酶、LC3Ⅱ/Ⅰ比值表达均明显升高(P <0.01),皮质和海马区见自噬小体、自噬溶酶体数量明显增多,皮质区可见大片神经元坏死,细胞核皱缩或碎裂,海马区神经元细胞排列紊乱、细胞核固缩深染;与模型组比较,化瘀解毒方组和阿加曲班组大鼠12、24 h两个时间点的神经功能评分显著降低(P <0.01),脑梗死体积占比显著降低(P <0.05);化瘀解毒方组和阿加曲班组皮质和海马区的凝血酶、LC3Ⅱ/Ⅰ表达显著下降(P <0.05,P <0.01),自噬小体、自噬溶酶体数量减少,且病理形态损伤轻微,神经元损伤程度降低。结论:依据化瘀解毒法创立的化瘀解毒方能发挥抗脑缺血作用,可能机制是通过抑制凝血酶的过表达,下调自噬的发生,从而减轻缺血性脑损伤。

Objective: To observe the effect of stasis removing and detoxifying method on ischemic stroke, thrombin expression in different brain regions and autophagy-related index of LC3Ⅱ/Ⅰ in ischemic stroke rats with syndrome of stasis and toxin, and to analyze its possible mechanism in protecting brain function.Methods: 12 h and 24 h after ischemia, 116 SD rats were randomly divided into the sham operation group(n =8), the model group(n=36), the HJF(stasis removing and detoxifying) group(n=36) and the ARG(argatroban) group(n=36). The rat model of ischemic stroke with stasis-toxicity syndrome was established. HJF group was given intraperitoneal injection of Huayu Jiedu Formula at the dose of 11.33 g/(kg·d), the ARG group was given intraperitoneal injection of argatroban at the dose of 11. 16 mg/(kg·d), and the sham operation group and the model group were given 0. 9% normal saline by intragastric administration.Western blot method was used to detect the expression of thrombin and the ratio of LC3Ⅱ/Ⅰ in the cerebral cortex and hippocampus, TTC staining method was used to calculate the volume of cerebral infarction, the transmission electron microscopy was used to observe the ultrastructure of autophagy in the brain, and HE staining was used to observe the morphological changes of neurons. Results: Compared with those in the sham operation group, the neurological function scores increased(P<0.01), the proportion of cerebral infarction volume increased(P<0.05), the expression of thrombin and the ratio of LC3Ⅱ/Ⅰ in cerebral cortex and hippocampus increased at 12 h and 24 h time points in the model group(P<0.01);the number of autophagosomes and autolysosomes in the cortex and hippocampus was significantly increased;in the cortex, it showed large areas of neuron necrosis, shrunken or fragmented nuclei;in the hippocampus, it showed disordered arrangement of neuronal cells, as well as pyknosis and deep staining of nuclei. Compared with those in the model group, the neurological function scores significantly decreased at 12h and 24 h in the HJF group and the ARG group(P<0.01), and the proportion of cerebral infarction volume significantly decreased(P<0.05), the expression of thrombin and the ratio of LC3 Ⅱ/Ⅰ in the cortex and the hippocampus significantly decreased in the HJF group and the ARG group(P<0.05, P<0.01);the number of autophagosomes and autolysosomes in the cortex and hippocampus was significantly decreased, the pathological morphological damage was slight, and the degree of neuronal damage was reduced in the HJF group and the ARG group. Conclusion: Stasis removing and detoxifying method may reduce ischemic brain injury through inhibiting the over-expression of thrombin and down-regulating the occurrence of autophagy.