目的 考察甘松80%乙醇提取物对鱼藤酮诱导的帕金森(Parkinson's disease,PD)大鼠运动障碍的改善作用,探究甘松通过"肠-脑轴"改善PD大鼠运动障碍的作用机制。方法 采用鱼藤酮致大鼠PD模型,将大鼠分为对照组、模型组、左旋多巴(50 mg/kg)组及甘松低、中、高剂量(0.41、0.62、0.93 g/kg)组,通过旷场实验、斜板实验研究PD大鼠的运动障碍,ELISA测定PD大鼠纹状体多巴胺(dopamine,DA)含量,筛选出甘松80%乙醇提取物抗PD运动障碍的最优剂量。选择最优剂量组进行机制探究,通过免疫组化实验检测大鼠中脑黑质部位多巴胺DA能神经元特征标记物酪氨酸羟化酶(tyrosine hydroxylase,TH)的表达;采用ELISA测定大鼠结肠及纹状体中DA、5-羟色胺(5-hydroxytryptamine,5-HT)及其代谢产物3,4-二羟基苯乙酸(3,4-dihydroxyphenylaceticacid,DOPAC)、高香草酸(homovanillic acid,HVA)和5-羟吲哚乙酸(5-hydroxyindoleacetic acid,5-HIAA)含量;采用Western blotting测定大鼠结肠和纹状体中神经胶质细胞生物标志物胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和小胶质细胞的表面标记物离子钙结合蛋白-1(ionized calcium binding adaptor molecule-1,Iba-1)的表达。结果 与模型组比较,甘松均能在不同程度上改善PD大鼠的运动功能障碍,其中甘松中剂量组效果最优,能显著提升大鼠旷场运动总路程和斜板停留角度(P<0.001),显著提高PD大鼠纹状体DA和黑质TH阳性表达含量(P<0.01、0.001)。给予甘松干预后,PD大鼠结肠及纹状体中DA、5-HT及其代谢产物DOPAC、HVA和5-HIAA的含量下降趋势均得到不同程度的逆转(P<0.05、0.01、0.001),且DA的代谢速率得到了抑制(P<0.05);结肠和纹状体中GFAP和Iba-1的表达显著降低(P<0.001),抑制肠神经胶质细胞、神经胶质细胞和小胶质细胞的激活。结论 甘松能明显改善PD大鼠的运动障碍,其机制可能与甘松抑制肠道胶质细胞和神经胶质细胞的过度激活,从而阻止神经炎症的发展,并抑制结肠与纹状体中DA及5-HT等神经递质的代谢过程从而提高其含量有关。验证了甘松通过"肠-脑轴"发挥抗PD作用的理论,可为入脾胃经中药的抗PD的研究提供新思路。

Objective To investigate the effect of 80% ethanol extract of Nardostachys jatamansi (NJ) on dyskinesia in Parkinson's (PD) rats, and explore the mechanism of NJ against Parkinson's disease through the "gut-brain axis". Methods The rotenone was used to induce PD rats model, rats were set up as blank control group, model group, levodopa (50 mg/kg) group and NJ low-, medium- and high-dose (0.41, 0.62, 0.93 g/kg) groups. To screen optimal dose of 80% ethanol extract of NJ for anti-PD movement disorders, the movement disorders of PD rats were investigated by open field test and inclined plate test, the striatal DA content was measured by ELISA. Immunohistochemical staining was used to investigate the expression of tyrosine hydroxylase (TH), a characteristic marker of dopamine (DA) neurons in the substantia nigra of rat midbrain; ELISA was used to investigate the contents of DA, 5-hydroxytryptamine (5-HT) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyphenylacetic acid (5-HIAA) in colon and striatum of rats; Expressions of enteric glial cell biomarker glial fibrillary acidic protein (GFAP) and microglia surface marker ionized calcium binding adapter molecule 1 (Iba-1) from colon and striatum were detected by Western blotting. ResultsCompared with model group, NJ could improve the motor dysfunction of PD rats to varying degrees, and NJ medium-dose group had the best effect, which significantly improved the total distance of open field test and angle of stay of inclined plate test (P ＜ 0.001), significantly increased DA content in striatum and TH positive expression in substantia nigra of PD rats (P ＜ 0.01, 0.001). After administration of NJ, the decreasing trend of DA, 5-HT and its metabolites DOPAC, HVA and 5-HIAA in colon and striatum of PD rats were reversed to varying degrees (P ＜ 0.05, 0.01, 0.001), and DA metabolic rate was inhibited (P ＜ 0.05); NJ significantly reduced the expressions of GFAP and Iba-1 in colon and striatum (P ＜ 0.001), inhibited the activation of enteric glial cells, glial cells and microglia. Conclusion NJ can significantly improve the motor dysfunction of PD rats, and its mechanism may be related to that inhibiting the excessive activation of enteric glial cells and glial cells to prevent the development of neuroinflammation, and inhibiting the metabolic process of neurotransmitters such as DA and 5-HT in colon and striatum to increase their content. This study verifies the theory that NJ exerts its anti-PD effect through the "gut-brain axis", and can provide new ideas for the research on anti-PD of traditional Chinese medicine that attributed to the spleen and stomach meridians.

R285.5

国家自然科学基金面上项目（82073971）