摘要
目的 从多巴胺稳态探讨补肾活血颗粒治疗帕金森病的可能机制。方法 将72只小鼠随机分为空白组、模型组、美多芭组和补肾活血颗粒低、中、高剂量组,每组12只。除空白组外其余各组小鼠采用腹腔注射1-甲基-4-苯基-1'2'3'6-四氢吡啶(MPTP)连续7天诱导构建帕金森病小鼠模型。腹腔注射结束后次日,补肾活血颗粒低、中、高剂量组小鼠分别给予补肾活血颗粒1.25、2.5、5.0 mg/(g·d)灌胃,美多芭组小鼠给予美多芭片0.093 8 mg/(g·d)灌胃,空白组与模型组小鼠给予蒸馏水0.2 ml/10 g灌胃,均每日1次,持续14天。采用旷场实验、爬杆实验与抓力实验进行行为学评价;免疫组织化学法检测脑纹状体酪氨酸羟化酶(TH)表达;尼氏染色检测纹状体神经元数量;超高效液相色谱串联质谱法检测纹状体多巴胺(DA)与3'4-二羟基苯基乙酸(DOPAC)含量;透射电镜观察纹状体神经元突触小泡;免疫荧光检测纹状体囊泡单胺转运蛋白2(VMAT2)表达;蛋白质印迹法检测纹状体细胞外调节蛋白激酶(ERK)、磷酸化细胞外调节蛋白激酶(p-ERK)、环磷腺苷效应元件结合蛋白(CREB)、磷酸化环磷腺苷效应元件结合蛋白(p-CREB)及VMAT2蛋白水平。结果 与空白组比较,模型组小鼠旷场实验总路程与平均速度下降、总静止时间增加,爬杆实验中小鼠从开始运动到头完全朝下所需的时间(T-turn)与小鼠从开始运动到木杆底部的总时间(T-total)均增加,小鼠前肢抓力下降,脑纹状体TH平均光密度值、VMAT2蛋白平均荧光强度、DA含量降低,纹状体神经元数量减少,DOPAC/DA升高,纹状体p-ERK/ERK、p-CREB/CREB与VMAT2水平降低(P<0.01),透射电镜结果显示纹状体神经元突触小泡数量与体积明显降低。与模型组比较,美多芭组和补肾活血低、中、高剂量组小鼠上述各指标均显著改善(P<0.05或P<0.01)。与美多芭组比较,补肾活血高剂量组纹状体DA含量、p-CREB/CREB升高(P<0.05)。与补肾活血低剂量组比较,补肾活血高剂量组总路程、平均速度增加,总静止时间、T-turn与T-total均降低,小鼠前肢抓力增加,脑纹状体VMAT2蛋白平均荧光强度、DA含量、p-ERK/ERK、p-CREB/CREB、VMAT2水平升高(P<0.05或P<0.01)。结论 补肾活血颗粒可能通过调节ERK/CREB/VMAT2信号通路促进多巴胺稳态平衡,缓解帕金森病多巴胺能神经元损伤。
Objective To investigate the possible mechanism of Bushen Huoxue Granule(补肾活血颗粒,BHG)in treating Parkinson's disease(PD)from the perspecitve of dopamine(DA)homeostasis.Methods Seventy-two mice were randomly divided into blank group,model group,madopar group and BHG low-,medium-and high-dose groups,with 12 mice in each group.Except for the blank group,all mice were administered intraperitoneal injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)for 7 consecutive days to induce a PD model.On the day following the injection,BHG low-,medium-and high-dose groups were administered BHG at doses of 1.25,2.5,and 5.0 mg/(g·d)by oral gavage,respectively,while the madopar group received madopar tablets at dose of 0.0938 mg/(g·d)by oral gavage.The blank group and the model group were given 0.2 ml/10 g of distilled water by gavage.All treatments were given once daily for 14 days.Open field test,pole climbing test and grip test were used to evaluate the behavior of mice in each group.Immunohistochemistry was used to detect the expression of tyrosine hydroxylase(TH)in striatum.Nissl staining was used to detect the activity of striatal neurons.The contents of DA and 3,4-dihydroxyphenylacetic acid(DOPAC)in striatum were detected by ultra performance liquid chromatography tandem mass spectrometry.The number and volume of synaptic vesicles were observed by transmission electron microscope.The expression of vesicular monoamine transporter 2(VMAT2)in striatum was detected by immunofluorescence.Western Blot was used to detect the expression of extracellular signal-regulated kinase(ERK),phosphorylated ERK(p-ERK),cAMP response element-binding protein(CREB),phosphorylated CREB(p-CREB)and VMAT2 in striatum.Results Compared to the blank group,mice in the model group showed a significant decline in total distance and average speed in the open field test,along with an increase in total resting time;in the pole test,both the time required for the mice to turn completely downward(T-turn)and the total time taken to reach the bottom of the pole(T-total)were prolonged;forelimb grip strength was reduced;in the striatum,the mean optical density of TH,the average fluorescence intensity of VMAT2 protein,and DA content all decreased,while the number of striatal neurons was reduced,and the DOPAC/DA ratio was elevated;the levels of p-ERK/ERK,p-CREB/CREB,and VMAT2 in the striatum significantly decreased(P<0.01);transmission electron microscopy revealed that both the number and volume of synaptic vesicles in striatal neurons were markedly reduced.Compared to the model group,mice in the madopar group and BHG low-,medium-and high-dose groups showed significant improvements in all the above indicators(P<0.05 or P<0.01).Compared to madopar group,the BHG high-dose group exhibited increased DA content and elevated p-CREB/CREB ratio in the striatum(P<0.05).Compared to the BHG low-dose group,the BHG high-dose group showed increased total distance and mean velocity,decreased total resting time,T-turn,and T-total,as well as enhanced forelimb grip strength;moreover,the average fluorescence intensity of VMAT2 protein,DA content,p-ERK/ERK,p-CREB/CREB,and VMAT2 levels in the striatum were all significantly elevated(P<0.05 or P<0.01).Conclusion BHG may restore DA homeostasis and alleviate the damage of dopaminergic neurons by regulating ERK/CREB/VMAT2 signaling pathway.
作者
孙和昊
谌盈帆
王鹏
耿小涵
张煜梽
张倩
李敏
李绍旦
杨明会
SUN Hehao;CHEN Yingfan;WANG Peng;GENG Xiaohan;ZHANG Yuzhi;ZHANG Qian;LI Min;LI Shaodan;YANG Minghui(Department of Traditional Chinese medicine,The sixth medical center,The People's Liberation Army General Hospital,Beijing,100048;Chinese People's Liberation Army Medical School)
出处
《中医杂志》
北大核心
2025年第23期2484-2493,共10页
Journal of Traditional Chinese Medicine
基金
国家自然科学基金(82130117)
中医药传承与创新“百千万”人才工程(岐黄工程)岐黄学者(国中医药人教函[2018]284号)。
关键词
帕金森病
多巴胺稳态
补肾活血颗粒
纹状体
囊泡单胺转运蛋白2
细胞外调节蛋白激酶
环磷腺苷效应元件结合蛋白
Parkinson's disease
dopamine homeostasis
Bushen Huoxue Granules(补肾活血颗粒)
striatum
vesi-cular monoamine transporter 2
extracellular regulated protein kinase
cAMP response element-binding protein
