加减薯蓣丸抑制EphA4/RhoA/Rock信号通路改善血管性痴呆伴抑郁小鼠髓鞘再生及抑郁行为

目的 观察加减薯蓣丸对血管性痴呆（vascular dementia，VD）伴抑郁小鼠的疗效并探究其作用机制。方法 采用双侧颈总动脉狭窄法结合慢性不可预测温和刺激复制VD伴抑郁小鼠模型，将小鼠分为假手术组、模型组、加减薯蓣丸组以及氟西汀组。采用行为学实验检测小鼠抑郁行为，免疫荧光法检测小鼠胼胝体血小板衍生生长因子受体α（platelet-derived growth factor receptor alpha，PDGFRα）、髓鞘碱性蛋白（myelin basic protein，MBP）表达水平，Western blot法检测MBP、少突胶质细胞糖蛋白（myelin oligodendrocyte glycoprotein，MOG）、髓鞘相关糖蛋白（myelin associated glycoprotein，MAG）、酪氨酸蛋白激酶EPH受体A4（Ephrin receptor A4，EphA4）、Ras同源基因家族成员-A（Ras homolog gene family,member A，RhoA）、轴突生长抑制因子-A（neurite outgrowth inhibitor-A，Nogo-A）、Rho相关卷曲螺旋蛋白激酶（Rho associated coiled-coil forming protein kinase，Rock）、Nogo受体（Nogo receptor,NgR）的蛋白表达水平，劳克坚牢蓝(Luxol fast bue,LFB)染色法观察胼胝体髓鞘结构，超微透射电子显微镜观察髓鞘超微结构。结果 行为学检测结果显示，加减薯蓣丸组及氟西汀组小鼠在旷场实验中活动总路程显著增加（P＜0.05），糖水偏好率显著升高（P＜0.05），强迫游泳实验中不动时间明显增加（P＜0.05）；免疫荧光染色结果显示，加减薯蓣丸、氟西汀均可增加PDGFRα、MBP荧光强度；LFB染色结果显示，与假手术组比较，模型组小鼠LFB髓鞘染色缺失，存在脱髓鞘改变，加减薯蓣丸能改善VD抑郁小鼠髓鞘着色程度；Western blot法检测结果显示，与假手术组比较，模型组小鼠胼胝体中MBP、MOG、MAG表达水平显著下降（P＜0.05），加减薯蓣丸组小鼠MBP、MOG、MAG表达水平显著升高（P＜0.05）；加减薯蓣丸组小鼠胼胝体中EphA4、RhoA、Rock、Nogo-A、NgR蛋白表达水平显著降低（P＜0.05）。结论 加减薯蓣丸可能通过抑制EphA4/RhoA/Rock通路促进髓鞘再生，改善VD小鼠的抑郁状态。

Modified Dioscorea opposita Thunb. Pill Improves Myelin Regeneration and Depressive Behavior in Mice with Vascular Dementia and Depression by Inhibiting the EphA4/RhoA/Rock Signaling Pathway

Objective To investigate the clinical effect and possible mechanism of Modified Dioscorea opposita Thunb. Pill in mice with vascular dementia (VD) and depression.Methods Bilateral common carotid artery stenosis (BCAS) combined with chronic unpredictable stimulation (CUMS) was used to establish a mouse model of VD and depression, and the mice were divided into sham-operation group, model group,Modified Dioscorea opposita Thunb.Pill group,and fluoxetine group.The behavioral test was used to observe the depressive behavior of mice;immunofluorescence assay was used to measure the fluorescence expression levels of platelet-derived growth factor receptor α (PDGFR-α) and myelin basic protein (MBP) in the corpus callosum of mice;Western blot was used to measure the protein expression levels of MBP,myelin oligodendrocyte glycoprotein (MOG), myelin-associated glycoprotein (MAG),Ephrin receptor A4 (EphA4),Ras homolog gene family, member A (RhoA),neurite outgrowth inhibitor-A (Nogo-A),Rho-associated coiled-coil forming protein kinase (Rock), and Nogo receptor (NgR);Luxol Fast Blue (LFB) staining was used to observe the myelin structure of the corpus callosum,and a transmission electron microscope was used to observe myelin ultrastructure.Results The behavioral test showed that the Modified Dioscorea opposita Thunb.Pill group and the fluoxetine group had significant increases in the total distance of activity in the open field test (P<0.05),the percentage of sugar and water preference (P<0.05),and the immobility time in the forced swimming test (P<0.05). Immunofluorescence assay showed that both Modified Dioscorea opposita Thunb.Pill and fluoxetine increased the fluorescence intensity of PDGFRα and MBP.LFB staining showed that compared with the control group,the model group had the absence of LFB myelin staining and the presence of demyelination changes,and Modified Dioscorea opposita Thunb.Pill improved the degree of myelin staining in the mice with VD and depression.Western blot showed that compared with the control group,the model group had significant reductions in the expression of MBP,MOG,and MAG in the corpus callosum (P<0.05),and Modified Dioscorea opposita Thunb.Pill significantly upregulated the expression levels of MBP,MOG,and MAG (P<0.05).The Modified Dioscorea opposita Thunb.Pill group showed significant reductions in the relative protein expression levels of EphA4,RhoA,Rock,Nogo-A,and NgR in the corpus callosum (P<0.05).Conclusion Modified Dioscorea opposita Thunb.Pill may promote myelin regeneration and improve the depressive state of VD mice by inhibiting the EphA4/RhoA/Rock pathway.