Xiaoyao Powder Modulates the Myelin Function of mPFC-BLA Neural Circuit through PI3K/AKT/mTOR Pathway to Alleviates the Anxiety and Depression Phenotype in VaD Mice
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摘要:
目的 研究逍遥散(XYP)对血管性痴呆(VaD)小鼠焦虑抑郁行为表型的作用及可能机制。 方法 3月龄雄性C57BL/6小鼠, 采用双侧颈总动脉狭窄术(BCAS)并给予慢性束缚应激(CRS), 构建VaD伴焦虑抑郁小鼠模型。将造模小鼠分为模型组、氟西汀(阳性对照)组以及逍遥散低、中、高剂量组, 对照组为假手术无束缚组。逍遥散低、中、高剂量组予逍遥散水煎剂(5、10、20 g·kg-1·d-1)灌胃, 氟西汀组给予氟西汀(10 mg·kg-1·d-1)灌胃, 对照组、模型组给予等体积生理盐水灌胃, 共计4周, 给药期间给予束缚应激, 每日维持6 h; 旷场实验、强迫游泳实验、高架十字迷宫实验、糖水偏好实验检测小鼠焦虑抑郁行为表型, 免疫荧光法检测小鼠前额叶皮层(mPFC)、基底外侧杏仁核(BLA)和胼胝体(CC)髓鞘碱性蛋白(MBP)荧光表达水平; Western blot分别检测小鼠mPFC和BLA神经核团中磷脂酰肌醇-3-激酶(PI3K)、磷酸化磷脂酰肌醇-3-激酶(p-PI3K)、蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)、雷帕霉素靶蛋白(mTOR)、磷酸化雷帕霉素靶蛋白(p-mTOR)以及MBP、少突胶质细胞糖蛋白(MOG)、髓鞘相关糖蛋白(MAG)的蛋白表达水平; LFB髓鞘染色观察小鼠CC的髓鞘形态。 结果 行为学检测结果显示: 与对照组相比, 模型组小鼠在高架十字迷宫和糖水偏好实验中进入开放臂时间百分比、开放臂次数百分比、糖水偏好率均下降(P < 0.01), 在强迫游泳实验中不动时间明显增加(P < 0.01), 旷场实验中总运动距离、中心区域运动时间明显减少(P < 0.01);与模型组相比, 逍遥散中、高剂量组小鼠不动时间缩短(P < 0.05，P < 0.01), 糖水偏好率增加(P < 0.01), 开放臂时间百分比、开放臂次数百分比均增加(P < 0.05, P < 0.01), 逍遥散高剂量组总运动距离、中心区域运动时间明显增加(P < 0.01)。免疫荧光结果显示: 与对照组相比, 模型组小鼠在CC、mPFC、BLA脑区的荧光强度显著降低(P < 0.01), 中、高剂量逍遥散, 氟西汀均可不同程度增加MBP荧光强度(P < 0.05, P < 0.01), 其中逍遥散高剂量组最为明显(P < 0.01)。在LFB染色中, 与对照组相比, 模型组小鼠CC髓鞘纤维排列疏松, 髓鞘染色变浅, 存在脱髓鞘改变, 逍遥散可以改善模型组小鼠CC髓鞘结构损伤, 效应在中、高剂量组显著(P < 0.05, P < 0.01)。Western blot结果显示: 与对照组相比, 模型组小鼠mPFC、BLA脑区中MBP、MOG、MAG表达下降(P < 0.01);逍遥散中、高剂量组显著逆转上述趋势(P < 0.05, P < 0.01), 氟西汀对MAG蛋白表达下降有一定的逆转作用(P < 0.05), 对MBP、MOG表达无明显影响。与对照组相比, 模型组小鼠p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR水平明显降低(P < 0.01), 而在给予中、高剂量逍遥散，氟西汀干预后, p-PI3K/PI3K、p-AKT/AKT、p-mTOR/mTOR蛋白表达水平明显增加(P < 0.05, P < 0.01)。 结论 逍遥散可能通过激活PI3K/AKT/mTOR通路促进mPFC-BLA神经环路髓鞘再生并增加髓鞘结构完整性, 改善VaD小鼠焦虑抑郁表型。 Abstract:OBJECTIVE To study the effect of Xiaoyao Powder (XYP) on anxiety and depression behavior phenotype in vascular dementia (VaD) mice and its possible mechanism. METHODS Three-month-old male C57BL/6 mice were treated with bilateral carotid artey stenosis (BCAS) and chronic restraint stress (CRS) to construct the VaD mice model with anxiety and depression. The model mice were divided into model group, fluoxetine positive drug group and XYP low-dose, medium-dose and high-dose groups. The control group were treated with sham operation and unbound. XYP low, medium and high groups were given XYP water Decoction (5, 10, 20 g·kg-1·d-1) by intragastric administration, fluoxetine positive control group was given fluoxetine (10 mg·kg-1·d-1) by intragastric administration, control group and model group were given equal volume of normal saline for 4 weeks. And restraint stress was maintained for 6 h·d-1 during drug intervention. The open field test, forced swimming test, elevated cross maze test and sucrose preference test were used to detect the behavioral phenotypes of anxiety and depression in mice. The fluorescence expression levels of myelin basic protein (MBP) in the prefrontal cortex (mPFC), basolateral amygdala (BLA) and corpus callosum (CC) were detected by immunofluorescence assay. Western blot was used to test the protein expression levels in mPFC and BLA of phosphatidylinositol-3 kinase (PI3K), phosphorylated phosphatidylinositol-3 kinase (p-PI3K), protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), target of rapamycin (mTOR), phosphorylated target of rapamycin (p-mTOR), as well as oligodendrocyte glycoprotein (MOG), myelin associated glycoprotein (MAG) and MBP. The myelin forms of CC in mice were observed by LFB staining. RESULTS Compared with the control group, the percentages of open arm time and open arm entries in open field test and the sucrose preference in sucrose preference test decreased in model group (P < 0.01), and the immobility time of model group increased significantly in forced swimming test (P < 0.01). In open field test, the total distance of movement and the time in the center area significantly decreased (P < 0.01). Compared with model group, the immobility time of mice in XYP high-dose and medium-dose groups decreased (P < 0.05), the percentage of sucrose preference increased (P < 0.01), the percentages of open arm time and open arm entries increased (P < 0.05, P < 0.01). The total distance of movement and the time of movement in the center area significantly increased (P < 0.01). Immunofluorescence results showed that, compared with the control group, the fluorescence intensity of model mice in the brain regions of CC, mPFC and BLA significantly decreased (P < 0.01), and the fluorescence intensity of MBP in XYP medium-dose and high-dose groups and fluoxetine increased to varying degrees(P < 0.05, P < 0.01), among which the XYP high-dose group was the most obvious (P < 0.01). In the LFB staining, compared with the control group, the myelin fibers in CC of the model group loosed arrangement, became lighter in colour and demyelinated. XYP could improve the damage of the myelin structure in the model group. Western blot results showed that, compared with the control group, the expressions of MBP, MOG and MAG in the brain regions of mPFC and BLA in model group decreased (P < 0.01). The above trends were significantly reversed in XYP high-dose and medium-dose groups (P < 0.05, P < 0.01). Fluoxetine had a certain effect on the decreasing trend of MAG (P < 0.05), but had no significant effect on the expressions of MBP and MOG. Compared with the control group, the levels of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR in model mice significantly decreased (P < 0.01), while the protein expression levels of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR in model mice significantly increased after XYP and fluoxetine intervention (P < 0.05, P < 0.01). CONCLUSION XYP may alleviate the anxiety and depression phenotype of VaD mice by activating PI3K/AKT/mTOR pathway to promote the regeneration of myelin in the mPFC-BLA neural circuit and increase the structural integrity of myelin. -
Key words:
- vascular dementia /
- Xiaoyao Powder /
- anxiety /
- depression /
- myelin sheath /
- mPFC /
- BLA /
- PI3K/AKT/mTOR pathway
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图 1 逍遥散对小鼠焦虑抑郁表型的影响
注: 与对照组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。x±s, n=10。
Figure 1. Effect of XYP on anxiety and depression phenotype in mice
图 2 逍遥散对小鼠mPFC-BLA髓鞘相关蛋白表达的影响
注: 与对照组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。x±s, n=6。
Figure 2. Effect of XYP on the expressions of myelin associated-proteins in mPFC and BLA of mice
图 3 逍遥散对mPFC中MBP荧光表达的影响
注: 与对照组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。x±s, n=4。bar=400 μm。
Figure 3. Effect of XYP on the fluorescence expression of MBP protein in mPFC
图 4 逍遥散对BLA中MBP荧光表达的影响
注: 与对照组比较, ##P < 0.01;与模型组比较, *P < 0.05, * *P < 0.01。x±s, n=4。bar=200 μm。
Figure 4. Effect of XYP on the fluorescence expression of MBP protein in BLA
图 5 各组小鼠CC中MBP免疫荧光图及相对荧光强度统计图
注: 与对照组比较, ##P < 0.01;与模型组比较, * *P < 0.01。x±s, n=4。bar=400 μm。
Figure 5. Immunofluorescence diagram and relative fluorescence intensity statistical diagram of MBP in CC in each group
图 6 各组小鼠CC LFB染色代表图
Figure 6. Representative diagram of LFB staining in CC in each group
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