“嗅三针”干预对帕金森病模型小鼠嗅球超微结构及胶质纤维酸性蛋白表达的影响

Effects of Xiusanzhen treatment on ultrastructure of olfactory bulb and GFAP expression in mice with Parkinson's disease

目的:观察"嗅三针"干预对脂多糖(LPS)经鼻诱导的帕金森病小鼠嗅球超微结构和中脑黑质胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达的影响,为帕金森病的早期诊治提供思路及证据。方法:选用C57BL/6雄性小鼠40只,随机分为空白组、模型组、电针组、药物组,每组10只。除空白组外,其余各组均用经鼻灌注LPS 30 d建立帕金森模型。电针组自造模结束第1天起取双侧"迎香""印堂"穴进行电针干预,留针20 min,每日1次,5 d为一疗程,疗程间休息2 d,共进行4个疗程。药物组腹腔注射左旋多巴注射液10 mg/m L,每日1次,5 d为一疗程,疗程间休息2 d,共进行4个疗程。空白组与模型组不进行干预。各组小鼠于干预结束后第1天采用足迹分析及游泳试验评分法进行行为学测试,并采用透射电镜观察嗅球超微结构变化,免疫印迹法测定中脑黑质GFAP蛋白表达的变化。结果:(1)造模后,模型组、电针组、药物组小鼠震颤、怕冷等症状显著,且体质量较空白组显著下降(均P<0.01);行为学测试结果显示,模型组、电针组、药物组小鼠步长及游泳时间均较空白组小鼠显著减少(均P<0.01),确认造模成功。(2)第1个疗程后,电针组未见震颤、怕冷、立毛等症状。经4个疗程干预后,电针组小鼠体质量较模型组显著升高(P<0.05),药物组未见震颤、怕冷、立毛等症状,但体质量较模型组未发生明显变化(P>0.05)。(3)干预后,与空白组比较,模型组小鼠步长及游泳时间均显著减少(均P<0.01);与模型组比较,电针组和药物组小鼠步长和游泳时间明显增加(均P<0.01)。(4)干预后,与空白组比较,模型组嗅球组织细胞器及超微结构病理改变明显,与模型组比较,电针组改善了嗅球超微结构。(5)干预后,与空白组比较,模型组小鼠黑质中GFAP蛋白的表达显著升高(P<0.05);与模型组比较,电针组与药物组黑质中GFAP表达均下降(均P<0.05)。结论:"嗅三针"早期干预可以改善LPS经鼻诱导的帕金森病模型小鼠行为学障碍,其机制可能与"嗅三针"改善嗅球超微结构及降底黑质GFAP的表达有关。

Objective To observe the effects of Xiusanzhen treatment on the ultrastructure of olfactory bulb and the expression of substantia nigra glial fibrillary acidic protein（GFAP） in mice with Parkinson ＇s disease（PD） induced by lipopolysaccharide（LPS）, and to provide methods and evidence for early prevention and treatment of PD. Methods Forty C57 BL/6 male mice were randomly divided into a blank group, a model group, an electroacupuncture（EA） group and a medication group, 10 mice in each one. The mice in the model group, EA group and medication group were treated with 30-day nasal perfusion of LPS to establish PD model. From the first day of model establishment, the mice in the EA group were treated with electroacupuncture at bilateral ＂Yingxiang＂（LI 20） and ＂Yintang＂（GV 29） for 20 min, once a day; 5-day treatment was taken as one session, and 4 sessions were given with an interval of 2 days between sessions. The mice in the medication group were treated with intraperitoneal injection of L-DOPA, 10 mg/m L, once a day; 5-day treatmentwas taken as one session, and 4 sessions were given with an interval of 2 days between sessions. After treatment, the behavioristics changes were observed by using footprint analysis and swimming te st score; the ultrastructure of olfactory bulb was observed by using transmission electron microscopy; the expression of GFAP in substantia nigra was measured by using western blot method. Results（1） After model establishment, the mice in the model group, the EA group and medication group showed significant symptoms of quiver and fear of chill, and the BMI was significantly lower than that in the blank group（all P〈0.05）. The results of behavioristics test indicated footprint length and swimming time in the model group, the EA group and medication group was significantly lower than those in the blank group（all P〈0.01）, indicating the successful establishment of PD model.（2） After one-session treatment, the symptoms of quiver and fear of chill were not observed in the EA group. After 4-session treatment, the BMI in the EA group was significantly higher than that in the model group（P〈0.05）; the symptoms of quiver and fear of chill were not observed in the medication group, but the BMI was similar with the model group（P〉0.05）.（3） After treatment, the footprint and swimming time in the model group were significantly lower than that in the blank group（both P〈0.01）; the footprint and swimming time in the EA group and medication group were significantly higher than those in the model group（all P〈0.01）.（4） After treatment, compared with the blank group, the organelles and ultrastructure of olfactory bulb in the model group were significantly improved; the ultrastructure of olfactory bulb in the EA group was improved compared with that in the model group.（5） After treatment, the expression of substantia nigra GFAP in the model group was significantly higher than that in the blank group（P〈0.01）; the expression of substantia nigra GFAP in the EA group and medication group was significantly lower than that in the model group（both P〈0.05）. Conclusion The early treatment of Xiusanzhen can improve behavioral disorders in LPS-induced early PD mice, and the mechanism may be related to the regulation of olfactory disorders and the expression of GFAP in substantia nigra.