【摘要】 目的 基于小窝蛋白1（caveolin-1,Cav1）/音猬因子（sonic hedgehog,Shh）信号通路探讨补阳还五汤对脑缺血后星形胶质细胞转分化的作用机制。方法 雄性C57BL/6小鼠随机分为假手术组、模型组及补阳还五汤低、中、高剂量（9.25、18.50、39.00 g/kg）组和丁苯酞（54 mg/kg）组，除假手术组外，其余各组采用大脑中动脉栓塞（middle cerebral artery occlusion,MCAO）法复制脑缺血模型，给予药物干预21 d后，采用神经行为学评分、苏木素-伊红染色及免疫组化评估补阳还五汤疗效。随后将雄性野生（WT）小鼠及Cav1^-/-（KO）小鼠分别随机分为假手术组、模型组和补阳还五汤（18.5 g/kg）组，造模前7 d予以脑内注射GFAP-EGFP腺相关病毒，采用MCAO法制备脑缺血模型，给予药物干预21 d。采用免疫荧光检测缺血侧皮质区星形胶质细胞转分化情况，Western blotting法检测缺血侧皮质区Shh、平滑同源物（smootened,Smo）及神经胶质瘤关联癌基因同源物1（glioma associated oncogene homolog 1,Gli1）的蛋白表达，qRT-PCR法检测神经分化因子acoete-scute同系物1（achaete-scute complex homolog 1,Ascl1）、神经源性分化因子1（neurogenic differentiation 1,NeuroD1）、神经源性分化因子2（neurogenic differentiation 2,NeuroD2）、神经元素1（neurogenin-1,Ngn1）、神经元素2（neurogenin-2,Ngn2）及配对盒基因2（paired box gene 2,Pax2）的表达。结果 与模型组比较，补阳还五汤各剂量组及丁苯酞组的神经行为学评分显著降低（P<0.05、0.01），缺血侧皮质区病理损伤明显改善，NeuN表达明显上升（P<0.05、0.01）。此外，补阳还五汤低剂量组和补阳还五汤中剂量组的神经行为学评分及NeuN的表达有显著差异（P<0.01），而补阳还五汤中、高剂量组及丁苯酞组之间无明显差异。与同基因型模型组比较，WT、KO补阳还五汤组小鼠缺血侧皮质区EGFP-NeuN共定位明显增加（P<0.01）,Shh、Smo及Gli1蛋白表达明显上升（P<0.05、0.01），神经分化因子Ascl1、NeuroD1、NeuroD2、Ngn1、Ngn2及Pax2表达明显上升（P<0.05、0.01）。与WT模型组及WT补阳还五汤组比较，KO模型组及KO补阳还五汤组小鼠缺血侧皮质EGFP-NeuN共定位明显下降（P<0.01）,Shh、Smo及Gli1蛋白表达明显下降（P<0.05），神经分化因子Ascl1、NeuroD1、NeuroD2、Ngn1、Ngn2及Pax2表达明显下降（P<0.05、0.01）。结论 补阳还五汤能够促进脑缺血后星形胶质细胞向神经元转分化，其作用机制可能与通过Cav1调控Shh信号通路，上调各神经分化因子的表达有关。更多还原

【Abstract】 Objective To examine the mechanism of Buyang Huanwu Decoction（补阳还五汤, BHD） on astrocyte transdifferentiation after cerebral ischemia based on caveolin-1（Cav1）/sonic hedgehog（Shh） signaling pathway. Methods Male C57BL/6 mice were randomly divided into sham group, model group, BHD low-, medium-, high-dose（9.25, 18.50, 39.00 g/kg） groups and butylphthalide（54 mg/kg） group. Except the sham group, the other groups were treated with middle cerebral artery occlusion（MCAO）to replicate the cerebral ischemia model. Following 21 d of drug intervention, the efficacy of BHD was evaluated using neurological behavior scoring, hematoxylin-eosin（HE） staining and immunohistochemistry. Subsequently, male wild type（WT） mice and Cav1^-/-（KO） mice were randomly divided into sham group, model group and BHD（18.5 g/kg） group. The brain was injected with GFAPEGFP adeno-associated virus 7 d prior to the operation, and the MCAO method was employed to establish a model of cerebral ischemia.Drug intervention was administered for a duration of 21 d. Immunofluorescence was utilized to examine the trans-differentiation of astrocytes in the ischemic cortex, Western blotting was employed to assess the protein expressions of Shh, smooth homolog（Smo）,and glioma associated oncogene homolog 1（Gli1） in the ischemic cortex. The expression of nerve differentiation factors, including achaete-scute complex homolog 1（Ascl1）, neurogenic differentiation 1（NeuroD1）, neurogenic differentiation 2（NeuroD2）,neurogenin-1（Ngn1）, neurogenin-2（Ngn2）, and paired box gene 2（PAX2） were detected using qRT-PCR. Results Compared with model group, the neurobehavioral scores were significantly decreased in BHD each dose group and butylphthalide group（P < 0.05,0.01）. Additionally, the pathological injury of the ischemic cortex showed significant improvement, and the expression of NeuN was significantly increased（P < 0.05, 0.01）. Furthermore, there were significant differences in neurobehavioral scores and NeuN expression between the low-dose group and medium-dose group of BHD（P < 0.01）, while no significant differences were observed among the medium-, high-dose groups of BHD and butylphthalide groups. Compared with model group of the same genotype, the co-localization of EGFP-NeuN in the ischemic cortex of mice in WT and KO BHD groups were significantly increased（P < 0.01）, the protein expressions of Shh, Smo and Gli1 were significantly increased（P < 0.05, 0.01）, and the expressions of neural differentiation factors Ascl1, NeuroD1, NeuroD2, Ngn1, Ngn2 and Pax2 were significantly increased（P < 0.05, 0.01）. Compared with WT model group and WT BHD group, the co-localization of EGFP-NeuN in the ischemic cortex of the KO model group and KO BHD group exhibited a significant decrease（P < 0.01）. Additionally, there was a significant decrease in the expressions of Shh, Smo, and Gli1 proteins（P <0.05）, while the expressions of neural differentiation factors Ascl1, NeuroD1, NeuroD2, Ngn1, Ngn2 and Pax2 showed a significant decrease（P < 0.05, 0.01）. Conclusion BHD has the potential to induce the trans-differentiation of astrocytes into neurons following cerebral ischemia. Its mechanism is likely associated with the regulation of the Shh signaling pathway through Cav1, as well as the upregulation of nerve differentiation factors.更多还原