香芍颗粒对脑卒中后抑郁小鼠行为和内侧前额叶皮质OPCs、OLs的影响

Effects of Xiangshao granules on behavior and OPCs and OLs in the medial prefrontal cortex of mice with post-stroke depression

目的:探讨香芍颗粒对脑卒中后抑郁(PSD)小鼠行为和内侧前额叶皮质(mPFC)少突胶质细胞前体细胞(OPCs)、少突胶质细胞(OLs)的影响。方法:80只C57BL/6小鼠按照随机数字表法分为假手术组、大脑中动脉闭塞(MCAO)组、PSD+PBS组和PSD+香芍组,每组20只。采用MCAO后轻度慢性不可预见性应激(CUMS)和孤独饲养的方法构建PSD模型。采用激光散斑成像、改良神经严重程度评分(mNSS)和TTC染色评估MCAO模型,采用体质量、糖水偏好实验和悬尾实验评估PSD模型。PSD建模后4组小鼠均灌胃28 d,假手术组、MCAO组、PSD+PBS组给予溶剂(PBS)0.2 mL灌胃,1次/d;PSD+香芍组按照60 mg/kg剂量将香芍颗粒溶于0.2 mL PBS中灌胃,1次/d。采用免疫荧光染色法检测mPFC中OPCs和OLs的数量,采用Western blotting实验检测mPFC中髓鞘碱性蛋白(MBP)和磷脂酰肌醇3-激酶/丝氨酸苏氨酸蛋白激酶/雷帕霉素靶蛋白(PI3K/AKT/mTOR)通路蛋白的表达。结果:(1)模型验证结果:激光散斑成像实验观察到小鼠在MCAO术前、术中及再灌注后手术侧大脑中动脉供血区脑血流的明显改变;MCAO组、PSD+PBS组、PSD+香芍组小鼠中TTC染色后均观察到典型的红色非梗死区和白色梗死区;MCAO组、PSD+PBS组、PSD+香芍组小鼠mNSS评分均>4分且差异无统计学意义(P>0.05);提示MCAO模型构建成功。PSD建模后治疗前,4组小鼠行为学评估结果显示,与假手术组和MCAO组相比,PSD+PBS组和PSD+香芍组小鼠体质量明显下降,糖水偏好度明显降低,悬尾不动时间明显延长,差异均有统计学意义(P<0.05),提示PSD模型构建成功。(2)治疗后小鼠行为学实验结果:治疗28 d后,4组小鼠体质量、糖水偏好度和悬尾不动时间差异均有统计学意义(P<0.05)。与PSD+PBS组相比,PSD+香芍组小鼠体质量和糖水偏好度明显上升,悬尾不动时间明显缩短,差异均有统计学意义(P<0.05)。(3)免疫荧光染色及Western blotting实验结果:4组小鼠mPFC中OPCs(Olig2+PDGFRa+)数量、增殖性OPCs(Ki-67+PDGFRa+、EdU+PDGFRa+)数量、OLs(Olig2+CC1+)数量,以及MBP蛋白相对表达量、p-PI3K、p-AKT、p-mTOR蛋白相对表达量差异均有统计学意义(P<0.05)。与PSD+PBS组比较,PSD+香芍组小鼠上述细胞数量以及蛋白相对表达量均明显升高,差异均有统计学意义(P<0.05)。结论:香芍颗粒可通过激活mPFC区的PI3K/AKT/mTOR信号通路促进OPCs增殖和OLs成熟,进而发挥治疗PSD的作用。

Objective To investigate the effects of Xiangshao granules on behavior and oligodendrocyte precursor cells(OPCs)and oligodendrocytes(OLs)in the medial prefrontal cortex(mPFC)of post-stroke depression(PSD)mice.Methods Eighty C57BL/6 mice were divided into sham-operated group,middle cerebral artery occlusion(MCAO)group,PSD+PBS group,and PSD+Xiangshao group(n=20).PSD models were constructed using mild chronic unforeseeable stress(CUMS)and solitary feeding after MCAO.MCAO models were evaluated by laser speckle contrast imaging(LSCI),modified neurological severity score(mNSS)and TTC staining.PSD models were evaluated by body mass,sugar and water preference test and tail suspension test.After PSD modeling,mice in the sham-operated group,MCAO group,and PSD+PBS group were given 0.2 mL PBS,while mice in the PSD+Xiangshao group was given Xiangshao granules at dosage of 60 mg/kg(dissolved in 0.2 mL PBS);all were given via intragastric administration once a d for 28 d.Number of OPCs and OLs in mPFC was detected by immunofluorescence.Expressions of myelin basic protein(MBP)and phosphatidylinositol 3-kinase/serine/threonine kinase/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway proteins in mPFC were detected by Western blotting.Results(1)Model verification results:LSCI showed obvious changes of cerebral blood flow in the middle cerebral artery supply area before,during and after MCAO;TTC staining showed typical red non-infarct area and white infarct area in MCAO group,PSD+PBS group and PSD+Xiangshao group;mNSS scores in MCAO group,PSD+PBS group and PSD+Xiangshao group were all>4,without significant differences(P>0.05);the MCAO model was successfully constructed.After PSD and before treatment,the PSD+PBS group and PSD+Xiangshao group had significantly decreased body weight and sugar-water preference,and statistically prolonged tail suspension immobilization time compared with sham-operated group and MCAO group(P<0.05);the PSD model was successfully constructed.(2)Results of mouse behavior experiment after treatment:significant differences in body weight,sugar-water preference and tail suspension time were noted in mice of the 4 groups 28 d after treatment(P<0.05);PSD+Xiangshao group had significantly increased body weight and sugar-water preference and decreased tail suspension immobilization time compared with PSD+PBS group(P<0.05).(3)Number of OPCs(Olig2+PDGFRa+),proliferative OPCs(Ki-67+PDGFRa+,EdU+PDGFRa+)and OLs(Olig2+CC1+),and relative MBP,p-PI3K,p-AKT and p-mTOR protein expressions in mPFC of the 4 groups were significantly different(P<0.05);compared with those in PSD+PBS group,the above cell number and relative protein expressions in PSD+Xiangshao group were significantly increased(P<0.05).Conclusion Xiangshao granules can promote the OPCs proliferation and OLs maturation by activating PI3K/AKT/mTOR signaling pathway in mPFC,thus playing a role in PSD.