桃叶珊瑚苷对注意缺陷多动障碍模型小鼠行为及星形胶质细胞过度活化的作用

Improvement effects of aucubin on ADHD-like behaviors in mice via the inhibition of excessive astrocytic activation

目的:探讨桃叶珊瑚苷对注意缺陷多动障碍(attention deficit/hyperactivity disorder,ADHD)模型小鼠行为及星形胶质细胞过度活化的作用。方法:清洁级C57BL/6雌性野生型孕鼠12只,采用孕期腹腔注射艾司氯胺酮(15 mg/kg)法建立子代小鼠ADHD模型。子代小鼠按照窝别匹配原则分为对照+生理盐水组、对照+桃叶珊瑚苷组、艾司氯胺酮+生理盐水组和艾司氯胺酮+桃叶珊瑚苷组(每组n=15)。子代小鼠出生后14 d,对照+桃叶珊瑚苷组和艾司氯胺酮+桃叶珊瑚苷组小鼠给予桃叶珊瑚苷(5 mg/kg,1次/d)灌胃,对照+生理盐水组和艾司氯胺酮+生理盐水组小鼠给予等体积0.9%氯化钠溶液,连续给灌胃5 d,子代小鼠与母鼠同笼饲养至出生后21 d。子代小鼠出生后21 d,通过旷场实验、高架十字迷宫实验评估子代小鼠行为学指标;免疫荧光法检测小鼠杏仁核区谷氨酸脱羧酶2(glutamic acid decarboxylase 2,GAD2)、γ-氨基丁酸(γ-aminobutyric acid,GABA)及胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达,Sholl analysis定量分析星形胶质细胞形态变化。采用GraphPad Prism 9.0.1软件进行统计分析,多组间比较采用单因素方差分析或Kruskal-Wallis检验。结果:(1)行为学实验结果显示,4组小鼠旷场实验中总路程以及高架十字迷宫实验中开放臂停留时间均差异有统计学意义(F=236.90,H=39.92,均P<0.001)。艾司氯胺酮+生理盐水组小鼠的运动总路程[(7044±249)mm,(22891±2175)mm,P<0.05]和开放臂停留时间[12.69(9.86,17.24)s,2.72(0.57,3.87)s,P<0.05]均高于对照+生理盐水组。艾司氯胺酮+桃叶珊瑚苷组小鼠的运动总路程[(22891±2175)mm,(8252±839)mm,P<0.05]和开放臂停留时间[12.69(9.86,17.24)s,5.45(1.13,10.99)s,P<0.05]均低于艾司氯胺酮+生理盐水组。(2)免疫荧光结果显示,4组小鼠杏仁核区GAD2、GABA、GFAP荧光强度均差异有统计学意义(F=145.50,50.08,53.83,均P<0.05)。与对照+生理盐水组比较,艾司氯胺酮+生理盐水组小鼠杏仁核区GAD2[(100.00±9.60)%,(24.86±4.14)%,P<0.05]、GABA[(100.00±16.84)%,(25.48±5.70)%,P<0.05]荧光强度均下调,GFAP[(100.00±18.02)%,(223.80±25.85)%,P<0.05]荧光强度上调;与艾司氯胺酮+生理盐水组比较,艾司氯胺酮+桃叶珊瑚苷组小鼠杏仁核区GAD2[(24.86±4.14)%,(56.08±6.55)%,P<0.05]、GABA[(25.48±5.70)%,(52.59±15.74)%,P<0.05]荧光强度上调,GFAP[(223.80±25.85)%,(157.10±22.10)%,P<0.05]荧光强度下调。(3)Sholl analysis结果显示,4组小鼠星形胶质细胞突起与同心圆的交点数目差异有统计学意义(F=12.47,P<0.05)。与对照+生理盐水组比较,艾司氯胺酮+生理盐水组星形胶质细胞突起及突起分支与同心圆交点数目增加[(2.07±0.48)个,(1.67±0.72)个,P<0.05];与艾司氯胺酮+生理盐水组小鼠比较,艾司氯胺酮+桃叶珊瑚苷组星形胶质细胞突起及突起分支与同心圆交点数目减少[(1.20±0.78)个,(2.07±0.48)个,P<0.05]。结论:桃叶珊瑚苷能够改善ADHD模型小鼠多动行为,其机制可能与桃叶珊瑚苷能够抑制小鼠脑杏仁核区星形胶质细胞过度活化相关。

ObjectiveTo investigate the effect of aucubin on behaviors and excessive activation of astrocytic in attention deficit/hyperactivity disorder(ADHD)model mice.MethodsTwelve wild-type C57BL/6 pregnant mice(female,clean grade)were intraperitoneally administered with esketamine(15 mg/kg)to establish an ADHD model in offspring mice.The offspring mice were divided into control+saline group,control+aucubin group,Ketamine+saline group and Ketamine+aucubin group according to the nest matching principle with 15 in each group.At 14 days after birth,mice in the control+aucubin group and Ketamine+aucubin group were administered with aucubin(5 mg/kg,once a day)by gavage for 5 days.Mice in control+saline group and Ketamine+saline group were administered with equal volume of 0.9%sodium chloride solution.The offspring mice were housed with their mothers in the same cage until 21 days after birth.Twenty-one days after birth,the offspring mice were evaluated by open field test and elevated plus maze tests.Immunofluorescence assay was used to detect the expression of glutamate decarboxylase 2(GAD2),γ-aminobutyric acid(GABA)and glial fibrillary acidic protein(GFAP)in the amygdala.The morphological changes of astrocytes were quantitatively analyzed by Sholl analysis.GraphPad Prim 9.0.1 software was used for statistical analysis.The comparison of multiple groups was conducted by one-way ANOVA or Kruskal-Wallis test.Results(1)The results of behavioral experiments showed that the total distance traveled in the open field test and the residence time in open arm of the elevated plus maze were statistically significant(F=236.90,H=39.92,both P<0.001).The total distance((7044±249)mm,(22891±2175)mm,P<0.05)and the residence time in open arm(12.69(9.86,17.24)s,2.72(0.57,3.87)s,P<0.05)of mice in Ketamine+saline group were both higher than those in control+saline group.The total distance((22891±2175)mm,(8252±839)mm,P<0.05)and the the residence time in open arm(5.45(1.13,10.99)s,12.69(9.86,17.24)s,P<0.05)of Ketamine+aucubin group were both lower than those of Ketamine+saline group.(2)The immunofluorescence results showed that the levels of GAD2,GABA and GFAP intensity in amygdala of mice in the four groups were statistically significant(F=145.50,50.08,53.83,all P<0.05).Compared with control+saline group,the fluorescence intensities of GAD2((100.00±9.60)%,(24.86±4.14)%,P<0.05)and GABA((100.00±16.84))%,(25.48±5.70)%,P<0.05)of Ketamine+saline group were down-regulated,and the GFAP((100.00±18.02)%,(223.80±25.85)%,P<0.05)was up-regulated.Compared with Ketamine+saline group,the fluorescence intensities of GAD2((24.86±4.14)%,(56.08±6.55)%,P<0.05)and GABA((25.48±5.70)%,(52.59±15.74)%,P<0.05)in Ketamine+aucubin group were up-regulated,but the fluorescence intensity of GFAP((223.80±25.85)%,(157.10±22.10)%,P<0.05)was down-regulated.(3)Sholl analysis indicated that the number of the intersections between the astrocyte processes or the branches of astrocyte processes was statistically significant in the 4 groups(F=12.47,P<0.05).Compared with control+saline group,the number of the intersections in Ketamine+saline group((2.07±0.48),(1.67±0.72),P<0.05)increased.While the number of the intersections in Ketamine+aucubin group was lower than that of Ketamine+saline group((1.20±0.78),(2.07±0.48),P<0.05).ConclusionAucubin administration can alleviate ADHD-like behaviors in offspring mice,and the mechanism may be associated with the inhibition of excessive astrocytic activation.