ERK通路参与可持续释放左旋多巴微球减少帕金森病运动并发症的实验研究

摘要

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/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 利用PLGA包裹左旋多巴或苄丝肼制成微球,采用高效液相法测定微球在大鼠体外释放左旋多巴/苄丝肼的浓度.6-羟基多巴胺(6-OHDA)腹腔注射制备PD大鼠模型,将造模成功的PD大鼠分为PD组、左旋多巴处理组和微球处理组,并设假手术组,各组12只.给予左旋多巴处理组大鼠皮下注射左旋多巴(12 mg/kg)和苄丝肼(15 mg/kg),给予微球处理组大鼠皮下注射含同等剂量左旋多巴和苄丝肼的微球.于治疗的第1、7、14天计数大鼠经阿扑吗啡诱导的旋转圈数.2周后行大鼠异常不自主评分(AIM)并利用Western blot检测大鼠纹状体区细胞外信号调节激酶(ERK1/2)的磷酸化水平.结果 体外释放试验结果显示微球内的左旋多巴/苄丝肼均匀缓慢释放,第7天时左旋多巴和苄丝肼释放量分别达到91.2％％和97.1％.治疗2周后,微球处理组大鼠和左旋多巴处理组大鼠阿扑吗啡诱导的旋转圈数均明显下降(均P＜0.05),但在治疗的第1、7、14天两组比较无明显统计学差异.微球处理组大鼠于治疗后的第1、2、4、6、8、10、12、14天的AIM评分(轴性+上肢+口面)与左旋多巴处理组大鼠有统计学差异(均P＜0.05).Western blot结果显示左旋多巴处理组大鼠纹状体ERK1/2水平较PD组和假手术组明显升高(均P＜0.05).微球处理组大鼠纹状体ERK1/2磷酸化水平较左旋多巴处理组大鼠明显降低(P＜0.01).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,这可能与微球释放的左旋多巴持续性刺激PD大鼠从而减少ERK1/2的磷酸化水平有关.%Objective To investigate the effects of levodopa/benserazide-loaded poly-lactide-co-glycolide (PLGA) microspheres on motor deficits and levodopa-induced dyskinesia in 6-hydroxydopamine (6-OHDA) -lesioned parkinsonian rats and to explore the mechanisms underlying these effects. Methods Levodopa and benserazide were loaded by PLGA. The content of levodopa/benserazide released from the microspheres in vitro was determined by high-performance liquid chromatography. The rat model of Parkinson' s disease (PD) was induced by intraperitoneal injection of 6-OHDA. Then the PD rats were divided into the PD group, the levodopa-treated group, the microsphere-treated group and the sham operation group. There were 12 rats in each group. Rats in the levodopa-treated group and the microsphere-treated group were treated with levodopa (12 mg/kg) / benserazide (15 mg/kg) or microspheres. Apomorphine-induced rotations in rats were measured on 1, 7 and 14 days after treatments. 2 weeks after treatments, the abnormal involuntary movements (AIM) were measured. Moreover, phosphorylated extracellular signal-regulated kinases 1/2 (ERK1/2) levels were determined by Western blot. Results Vitro release test showed that 91. 2% of levodopa and 97. 1% benserazide released slowly from the microspheres on day 7. Decreased apomorphine-induced rotations were observed in rats treated with levodopa or microspheres 2 weeks after treatments (P<0. 05, respectively). However, there was no difference in rotations between levodopa-treated and microsphere-treated groups on day 1, 7 and 14 of treatments. The AIM scores (axial + limb + orolingual) of microspheres-treated rats decreased significantly compared with levodopa-treated rats on day 1, 2, 4, 6, 8, 10, 12 and 14 of treatments (all P<0.05). Western blot indicated that phosphorylated levels of ERK1/2 in levodopa-treated rats increased significantly comparing to sham-operated rats and PD rats (P<0. 05, respectively). Moreover, phosphorylated levels of ERK1/2 in microspheres-treated rats decreased significantly comparing to levodopa-treated rats (P<0. 01). Conclusions Microspheres can be used to improve the motor deficits and attenuate 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 ERK1/2 in these rats.