世界中医药

目的：探讨益髓解毒方通过音猬因子(Shh)信号通路调控血管性痴呆(VD)大鼠海马突触可塑性的作用机制。方法：将无特定病原体(SPF)级雄性斯泼累格·多雷(SD)大鼠随机数字表法分为假手术组、模型组、阳性对照组、益髓解毒方组，每组10只。除假手术组外，其他3组采用2-VO法制备VD大鼠模型，假手术组双侧颈总动脉只置线不结扎。造模后第2天，阳性对照组大鼠给予5.2×10-4 g/kg盐酸多奈哌齐溶液灌胃，益髓解毒方组给予益髓解毒方11.11 g/kg灌胃，假手术组和模型组均给予等体积生理盐水灌胃，每日1次，连续给药4周。水迷宫测试大鼠行为学；苏木精-伊红(HE)染色及透射电镜(TEM)观察大鼠海马形态结构和CA1区突触超微结构；高尔基染色(Golgi)法检测大鼠海马CA1区锥体神经元树突及树突棘形态；实时荧光定量PCR（qPCR）检测海马Shh、细胞跨膜平滑蛋白(Smo)、胶质瘤相关癌基因1(Gli1)mRNA表达水平；蛋白质免疫印迹法检测海马突触素(SYN)、Shh、补缀同源物1(Ptch1)、Smo、Gli1蛋白表达。结果：与假手术组比较，模型组大鼠第4天逃避潜伏期长，穿越平台次数少(P<0.01)；与模型组比较，阳性对照组及益髓解毒方组大鼠第4天逃避潜伏期短，穿越平台次数多(P<0.01)。与模型组比较，阳性对照组及益髓解毒方组大鼠海马组织损伤减轻；海马突触结构较清晰，突触小泡数量增多，分布较密集，突触前、后膜连续性较好；海马CA1区神经元形态较规则，神经元树突分支及树突棘均有所增加。与假手术组比较，模型组大鼠海马组织中Shh、Smo、Gli1 mRNA表达低（P<0.01）；与模型组比较，阳性对照组和益髓解毒方组大鼠海马组织中Shh、Smo、Gli1 mRNA表达高（P<0.05）。与假手术组比较，模型组大鼠海马组织中SYN、Shh、Ptch1、Smo、Gli1蛋白表达低(P<0.05)。与模型组比较，阳性对照组大鼠海马组织中Ptch1、Smo、Gli1蛋白表达高(P<0.05)，SYN、Shh蛋白表达呈升高趋势，但差异无统计学意义(P>0.05)；益髓解毒方组大鼠海马SYN、Shh、Ptch1、Smo、Gli1蛋白表达高(P<0.05)。结论：益髓解毒方可明显提高VD大鼠学习记忆能力，其机制可能与激活Shh信号通路，修复海马神经元及突触超微结构损伤，促进并调节突触可塑性有关。

English Summary:

To explore the mechanism by which Yisui Jiedu Formula regulates synaptic plasticity in the hippocampus of vascular dementia(VD) rats through the Sonic Hedgehog(Shh) signaling pathway.Methods:Specific pathogen-free(SPF) male Sprague-Dawley(SD) rats were randomly assigned to the sham surgery group，model group，positive control group，and Yisui Jiedu Formula group according to the random number table method，with 10 rats in each group.Except for the sham surgery group，the other three groups were subjected to the 2-vessel occlusion(2-VO) method to establish the VD rat model.In the sham surgery group，only the common carotid arteries were cannulated without ligation.On the second day after modeling，rats in the positive control group were gavaged with 5.2 ×10-4 g/kg donepezil hydrochloride solution，rats in the Yisui Jiedu Formula group were gavaged with 11.11 g/kg of Yisui Jiedu Formula，and rats in the sham surgery and model groups were gavaged with an equal volume of physiological saline，once daily for 4 consecutive weeks.Behavioral tests were conducted using the Morris water maze.Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were used to observe the hippocampal morphology and synaptic ultrastructure in the CA1 region.Golgi staining was performed to assess the dendritic and dendritic spine morphology of pyramidal neurons in the hippocampal CA1 region.Real-time quantitative PCR(qPCR) was used to measure the mRNA expression levels of Shh，Smoothened(Smo)，and Gli1 in the hippocampus.Western blot was performed to detect the protein expression of synaptophysin(SYN)，Shh，Patched1(Ptch1)，Smo，and Gli1 in the hippocampus.Results:Compared to the sham surgery group，the model group showed longer escape latency and fewer platform crossings on day 4 of the Morris water maze test(P<0.01).Compared to the model group，the positive control and Yisui Jiedu Formula groups had shorter escape latency and more platform crossings on day 4(P<0.01).Compared to the model group，the positive control and Yisui Jiedu Formula groups showed less hippocampal tissue damage; the hippocampal synaptic structure was clearer，synaptic vesicle numbers were increased，and they were more densely distributed with better continuity of the pre-and post-synaptic membranes.Neurons in the hippocampal CA1 region showed more regular morphology，with increased dendritic branching and dendritic spines.Compared to the sham surgery group，the model group had lower mRNA expression levels of Shh，Smo，and Gli1 in the hippocampus(P<0.01).Compared to the model group，the positive control and Yisui Jiedu Formula groups had higher mRNA expression levels of Shh，Smo，and Gli1(P<0.05).Compared to the sham surgery group，the model group had lower protein expression of SYN，Shh，Ptch1，Smo，and Gli1 in the hippocampus(P<0.05).Compared to the model group，the positive control group had higher protein expression of Ptch1，Smo，and Gli1 in the hippocampus(P<0.05)，with SYN and Shh protein expression showing an increasing trend，but the difference was not statistically significant(P>0.05).The Yisui Jiedu Formula group showed higher protein expression of SYN，Shh，Ptch1，Smo，and Gli1 in the hippocampus(P<0.05).Conclusion:Yisui Jiedu Formula can significantly improve the learning and memory ability of VD rats.Its mechanism may be related to the activation of the Shh signaling pathway，repair of hippocampal neuron and synaptic ultrastructure damage，and promotion and regulation of synaptic plasticity.

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