可控释左旋多巴和苄丝肼微球减少帕金森病大鼠异动症的发生

An experiment on controlled release levodopa/benserazide-loaded microspheres in treatment of levodopa-induced dyskinesia of rats with Parkinson＇ s disease

目的观察聚乳酸-羟基乙酸共聚物（PLGA）包裹的可释放左旋多巴和苄丝肼的微球对帕金森病（PD）大鼠运动症状及异动症发生的影响并探讨其机制。方法PLGA包裹左旋多巴及苄丝肼制作微球，高效液相法测定微球在体内释放出的左旋多巴和苄丝肼的浓度，6-羟基多巴胺（6-OHDA）注射制作PD大鼠模型，制模成功的PD大鼠随机分成PD组、左旋多巴组、微球组（每组12只），另设溶剂注射假手术组（n=12）。左旋多巴组和微球组大鼠分别接受左旋多巴和苄丝肼（左旋多巴12mg／kg，苄丝肼15mg／kg）或等剂量微球皮下注射，在治疗的第1、4、7、10、14天行大鼠前肢功能测定，治疗2周后行大鼠异常不自主运动（AIM）评分，免疫组织化学及Westernblot法检测纹状体区磷酸化的多巴胺和环磷腺苷调节的磷酸化蛋白-32（DARPP-32）（Thr34）和AFosB水平。结果体内释放实验表明第7天时左旋多巴和苄丝肼释放量分别达76．2％和83．6％。微球处理组大鼠在治疗的第10天和第14天时前肢跨步数分别为5．8±1．6和5．2±1．5，比左旋多巴组（2．4±1．1、1．2±0．5）明显增加（t=4．12，5．43，均P〈0．01）。微球处理组大鼠第14天AIM评分[（16．0±2．1）分]较左旋多巴处理组[（26．0±3．2）分]显著下降，差异有统计学意义（t=6．59，P〈0．01）。免疫组织化学显示微球处理组大鼠纹状体磷酸化DARPP-32水平[（3．7±1．3）×10^4]较左旋多巴处理组[（7．9±2．2）×10^4]明显降低（t：2．95，P〈0．05）。Westernblot结果显示微球处理组大鼠磷酸化DARPP-32和AFosB水平分别为119．4％±11．3％和149．3％±12．3％，较左旋多巴组（184．8％±13．7％和300．4％±14．2％）显著下降（t=4．12、2．91，均P〈0．05）。结论微球皮下注射可以改善PD大鼠的运动症状，同时可以减少PD大鼠异动症的发生，这与微球释放的左旋多巴持续性刺激PD大鼠从而减少磷酸化DARPP-32和AFosB的水平有关。

Objective To investigate the effects of levodopa/benserazide-loaded poly-lactide-coglyeolide （PLGA） mierospheres on motor deficits and levodopa-induced dyskinesia in a rat model of Parkinson＇ s disease （PD） and to explore the mechanisms underlying this effects. Methods The content of levodopa/benserazide released from the microspheres was determined by high-performance liquid chromatography. The rat model of PD was induced by 6-OHDA injections. Then the valid PD rats were treated with levodopa （ 12 mg/kg, s.c. ）/benserazide （ 15 mg/kg, s.c. ） or microspheres. Forepaw adjusting steps were measured on 1, 4, 7, 10 and 14 days after treatment. After 2 weeks of treatment, the abnormal involuntary movements （AIM） were measured. Phosphorylated dopamine, cAMP-regulated phosphoprotein of 32 kDa （DARPP-32） at threonine 34 levels were determined by immunohistochemistry and Western blot respectively. In addition, the levels of AFosB were measured by Western blot. Results In vivo release test showed that 76. 2% of levodopa and 83.6% benserazide were released from the microspheres on day 7. Forepaw adjusting steps showed that the scores of forepaw adjusting steps in microspheres-treated PD rats were （5.8 ± 1.6 ） and （5.2 ± 1.5 ） respectively on 10 and 14 days of treatment, which were increased compared to levodopa-treaded PD rats （2. 4 ± 1.1 and 1.2 ± 0. 5; t = 4. 12, 5.43, all P 〈 0. 01 ）. The AIM scores of microspheres-treated rats （ 16.0 ± 2. 1 ） were decreased significantly compared to levodopa-treated rats （26. 0 ± 3.2） on day 14 （t = 6. 59, P 〈 0. 01 ）. Immunohistochemistry indicated that the phosphorylated levels of DARPP-32 in microspheres-treated rats （ （ 3.7 ± 1.3 ） ×10^4 ） were decreased significantly compared to levodopa-treated rats （ （7.9 ± 2. 2） ×10^4 ; t = 2. 95,P 〈 0. 05 ）. In addition, Western blot showed that the levels of phosphorylated DARPP-32 and AFosB were 119.4% ± 11.3% and 149. 3% ± 12. 3% respectively, which were decreased significantly compared to levodopatreated rats （184.8% ± 13.7% and 300.4% ± 14. 2%; t =4.12,2.91,all P 〈0.05）. Conclusions Microspheres can be used to improve the motor deficits and reduce the expression of dyskinesia in PD rats. This may be due to the continuous release of levodopa/beserazide from the microspheres, which leads to continuous stimulation of PD rats and reduces the levels of phosphorylated DARPP-32 and AFosB in the striata of these rats.