摘要目的:探索华佗再造丸（ Huatuo Zaizao pills, HT）对慢性脑低灌注模型小鼠脑白质损伤及认知损害的保护作用及相关机制。 方法:使用成年雄性C57BL/6J小鼠40只，随机分为假手术组、双侧颈动脉狭窄（bilateral carotid artery stenosis, BCAS）模型组和HT组。应用弹簧圈圈套双侧颈总动脉法制作BCAS模型。采用5 g/kg剂量HT（或等量纯净水）连续处理小鼠4周后，使用新物体识别实验评估认知功能。通过LFB髓鞘染色评估神经髓鞘形态结构变化，通过免疫荧光染色检测胼胝体髓鞘相关糖蛋白（myelin-associated glycoprotein, MAG）、胼胝体和海马离子钙结合衔接分子1（ionized calcium-binding adapter molecule 1, IBA-1）和胶质细胞原纤维酸性蛋白（glial fibrillary acidic protein, GFAP）评估白质损伤及胶质细胞表达。利用实时荧光定量聚合酶链反应（quantitative polymerase chain reaction, qPCR）检测胼胝体髓鞘相关蛋白、Janus激酶2（Janus kinase 2, JAK2）和信号转导及转录激活因子3（signal transducer and activator of the transcription 3, STAT3）以及海马脑源性神经营养因子（brain-derived neurotrophic factor, BDNF）、谷胱甘肽过氧化物酶-1（glutathione peroxidase-1, GPx-1）和各种炎性因子mRNA表达。结果:新物体识别实验显示，小鼠在BCAS后4周出现显著工作记忆障碍（ P<0.01），而HT组工作记忆障碍较BCAS组显著改善（ P<0.01）。LFB髓鞘染色显示，BCAS组显著髓鞘损伤（ P<0.001），而HT组髓鞘损伤程度较BCAS组显著改善。免疫荧光染色显示，BCAS组和HT组小鼠胼胝体和海马均出现小胶质细胞增生，两组差异无统计学意义；相比之下，HT组胼胝体星形胶质细胞活化较BCAS组显著改善（ P<0.05）。qPCR显示，BCAS组髓鞘相关蛋白以及JAK2和STAT3 mRNA表达上调；与BCAS组比较，HT组JAK2和STAT3 mRNA表达下降（ P均<0.05），而髓鞘相关蛋白表达上调（ P均<0.05）。此外，BCAS组和HT组海马组织炎性因子、BDNF和GPX1 mRNA表达差异无统计学意义。 结论:HT可改善慢性脑低灌注小鼠的认知损害及脑白质损伤，JAK2-STAT3通路可能为其效应通路之一。

abstractsObjective:To investigate the protective effect and related mechanisms of Huatuo Zaizao pills (HT) on white matter injury and cognitive impairment induced by chronic cerebral hypoperfusion in mice. Methods:Forty adult male C57BL/6J mice were randomly divided into sham-operation group, bilateral carotid artery stenosis (BCAS) model group, and HT group. An animal model of BCAS was constructed using the spring loop into the bilateral common carotid arteries. After continuous treatment with 5 g/kg HT (or an equal amount of purified water) for 4 weeks, cognitive function was evaluated using the novel object recognition test. Morphological and structural changes in myelin sheath were evaluated by LFB myelin staining. White matter damage and glial cell expression were detected by myelin associated glycoprotein (MAG) in the corpus callosum, ionized calcium-binding adapter molecule 1 (IBA-1), and glial fibrillar acidic protein (GFAP) in corpus callosum and hippocampus through immunofluorescence staining. Real time quantitative polymerase chain reaction (qPCR) was used to detect mRNA expressions of myelin-associated proteins, Janus kinase 2 (JAK2), signal transducer and activator of the transcription 3 (STAT3) in corpus callosum, as well as brain-derived neurotrophic factor (BDNF), glutathione peroxidase 1 (GPx-1), and various inflammatory factors in hippocampus.Results:The novel object recognition test showed that mice had significant working memory impairment at 4 weeks after BCAS ( P<0.01), while the HT group showed significant improvement in working memory impairment compared to the BCAS group ( P<0.01). LFB myelin staining showed significant myelin damage in the BCAS group ( P<0.001), while the degree of myelin damage in the HT group was significantly improved compared to the BCAS group. Immunofluorescence staining showed that both the BCAS and HT groups had proliferation of microglia in the corpus callosum and hippocampus, and there was no significant difference between the two groups. In contrast, the activation of astrocytes in the corpus callosum was significantly improved in the HT group compared to the BCAS group ( P<0.05). qPCR showed upregulation of myelin-associated proteins as well as JAK2 and STAT3 mRNA expression in the BCAS group. Compared with the BCAS group, the expressions of JAK2 and STAT3 mRNA were decreased in the HT group (all P<0.05), while the expression of myelin-associated proteins were upregulated (all P<0.05). There were no significant difference in the expressions of inflammatory factors, BDNF, and GPX1 mRNA in the hippocampal tissue between the BCAS group and the HT group. Conclusion:HT may improve cognitive impairment and white matter damage in mice with chronic cerebral hypoperfusion, and the JAK2-STAT3 pathway may be one of its effect pathways.