目的 基于Nrf2/D1R-ERK信号通路探讨甘松Nardostachys jatamansi对左旋多巴（levodopa，L-DOPA）诱导的异动症（levodopa-induced dyskinesia，LID）模型大鼠的作用机制。方法 将SD大鼠随机分为假手术组、帕金森（Parkinson’s disease，PD）组、L-DOPA组和甘松＋L-DOPA组。PD组、L-DOPA组和甘松＋L-DOPA组颈背部sc鱼藤酮葵花籽油（1.5 mg/kg）14 d制备PD模型，假手术组sc等体积葵花籽油，之后进行14 d的药物干预：L-DOPA组ig L-DOPA（100 mg/kg）和苄丝肼（25 mg/kg），甘松＋L-DOPA组ig甘松（1240 mg/kg）、L-DOPA（100 mg/kg）和苄丝肼（25 mg/kg），假手术组和PD组ig等体积0.9%氯化钠溶液。通过前肢功能检测实验评估L-DOPA对PD大鼠运动能力的影响；通过异常不自主运动（abnormal involuntary movement，AIM）评分评估L-DOPA导致LID大鼠的AIM程度；采用免疫组化法考察大鼠黑质中酪氨酸羟化酶（tyrosine hydroxylase，TH）的表达；Western blotting法检测大鼠纹状体内转录因子E2相关因子2（nuclear factor erythroid-2 related factor 2，Nrf2）、多巴胺D1受体（dopamine D1 receptor，D1R）、细胞外调节蛋白激酶（extracellular regulated protein kinases，ERK）、磷酸化ERK（（phosphorylated ERK，p-ERK）和∆；FosB蛋白的表达；ELISA法检测大鼠脑纹状体中活性氧自由基（reactive oxygen species，ROS）、血红素加氧酶-1（heme oxygenase-1，HO-1）、超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）、多巴胺和cAMP调节的磷蛋白-32（dopamine and adenosine 3'，5'-monophosphate-regulated phospho-protein，DARPP-32）和p-DARPP-32的含量。结果 前肢功能检测结果显示，L-DOPA单用和甘松与L-DOPA联用均能显著增加PD大鼠的前肢跨步次数（P＜0.001）。AIM评分结果显示，与L-DOPA单用相比，甘松与L-DOPA联用可显著降低LID大鼠的AIM症状（P＜0.01）。免疫组化结果显示，PD组和L-DOPA组的TH表达没有显著性差异，而甘松与L-DOPA联用可明显增强PD大鼠的TH表达（P＜0.05），同时显著增强L-DOPA单用导致的LID大鼠的TH表达（P＜0.01）。Nrf2信号通路结果显示，PD组和L-DOPA组没有显著性差异，而甘松与L-DOPA联用可明显降低PD大鼠的ROS含量（P＜0.05），显著促进Nrf2表达（P＜0.01），并显著提高HO-1、SOD、GSH-Px的含量（P＜0.001）；同时还能显著降低LID大鼠的ROS含量（P＜0.001），明显提高Nrf2、HO-1、SOD的含量（P＜0.05），并显著提高GSH-Px含量（P＜0.01）。D1R-ERK信号通路结果显示，甘松与L-DOPA联用能显著降低LID大鼠的D1R表达（P＜0.001），同时明显减低∆；FosB、p-DARPP-32/DARPP-32、p-ERK/ERK的表达（P＜0.05）。结论 L-DOPA可以改善PD大鼠的运动障碍，但不能降低TH的损伤，而且还会引起氧化应激反应和D1R-ERK通路的过表达。甘松联合L-DOPA给药可明显改善PD大鼠的运动损伤，抑制L-DOPA引起的AIM现象，同时提高TH的表达。其机制可能是甘松通过激活Nrf2通路相关蛋白的表达，并抑制D1R-ERK通路相关蛋白的过表达发挥抗LID作用。

ObjectiveTo explore the mechanism of Nardostachys jatamansi (Nar) on levodopa (L-DOPA)-induced dyskinesia (LID) model rat based on Nrf2/D1R-ERK signaling pathway. Methods SD rats were randomly divided into sham group, Parkinson’s disease (PD) group, L-DOPA group and Nar + L-DOPA group. Rats in PD group, L-DOPA group, and Nar + L-DOPA group were sc rotenone sunflower oil (1.5 mg/kg) on the back of neck for 14 d to prepare PD model. Rats in sham group was sc same amount of sunflower oil. Then 14 d of drug intervention was carried out: rats in L-DOPA group were ig L-DOPA (100 mg/kg) and benserazide (25 mg/kg), rats in Nar + L-DOPA group were ig Nar (1240 mg/kg), L-DOPA (100mg/kg) and benserazide (25 mg/kg), rats in sham group and PD group were ig same amount of normal saline. Effect of L-DOPA on motor ability of PD rats was evaluated by forelimb function test. Abnormal involuntary movement (AIM) score was used to assess the AIM degree of LID induced by L-DOPA in rats. Expression of tyrosine hydroxylase (TH) in substantia nigra of rats was investigated by immunohistochemical staining. Expressions of nuclear factor E2-related factor 2 (Nrf2), dopamine D1 receptor (D1R), extracellular regulated protein kinases (ERK), p-ERK and ∆FosB protein in rats striatum was detected by Western blotting. ELISA method was used to detected the contents of reactive oxygen species (ROS), heme oxygenase-1 (HO-1), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), dopamine and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32), p-DARPP-32 in the striatum of rats. ResultsThe results of the forelimb function test showed that L-DOPA alone and Nar + L-DOPA combined significantly increased the number of forelimb strides in PD rats (P< 0.001). The AIM score results showed that compared with L-DOPA alone, the combination of Nar + L-DOPA significantly reduced the AIM symptoms of LID rats (P< 0.01). Immunohistochemical results showed that there was no significant difference in TH expression between PD group and L-DOPA group. The combination of Nar + L-DOPA significantly enhanced TH expression in PD rats (P< 0.05), and significantly enhanced TH expression in LID rats induced by L-DOPA alone (P< 0.01). Nrf2 signaling pathway showed no significant difference between PD group and L-DOPA group. The combination of Nar + L-DOPA significantly decreased ROS content (P< 0.05), significantly promoted Nrf2 expression (P＜0.01), significantly increased the contents of HO-1, SOD and GSH-Px of PD rats (P< 0.001). The combination of Nar + L-DOPA significantly decreased ROS content (P< 0.001), significantly increased Nrf2, HO-1, SOD content (P< 0.05), and increased GSH-Px content in LID rats (P< 0.01). D1R-ERK signaling pathway results showed that the combination of Nar + L-DOPA significantly decreased the expression of D1R in LID rats (P< 0.001), significantly reduced the expressions of ∆FosB, p-DARPP-32/DARPP-32 and p-ERK/ERK (P< 0.05). Conclusion L-DOPA can alleviate dyskinesia in PD rats, but it can not reduce the damage of TH, and can induce an oxidative stress response and overexpression of D1R-ERK pathway. Nar combined with L-DOPA significantly alleviated the motor injury of PD rats, inhibited AIM phenomenon caused by L-DOPA and increased TH expression. The mechanism may be that Nar plays an anti-LID role by activating the expression of related proteins in Nrf2 pathway and inhibiting the overexpression of related proteins in D1R-ERK pathway.

R285.5

国家自然科学基金面上项目（82073971）