包裹左旋多巴/苄丝肼PLGA微球通过Tau蛋白/△FosB信号通路治疗异动症大鼠的实验研究

Controlled-release levodopa methyl ester/benserazide-loaded PLGA microspheres ameliorated levodopa-induced dyskinesia by inhibiting Tau/△FosB singal pathway in dyskinetic rats

目的探讨可缓释包裹左旋多巴/苄丝肼微球对异动症大鼠的治疗作用及其机制。方法通过注射6-羟基多巴胺(6-OHDA)制作帕金森病(Parkinson disease,PD)大鼠模型,制模成功的PD大鼠模型接受左旋多巴(25mg/kg)/苄丝肼(12.5mg/kg)腹腔注射21d制作异动症大鼠模型。将异动症大鼠分成普通剂型组(n=13)和微球组(n=13)两组。普通剂型组给予普通剂型左旋多巴/苄丝肼皮下注射(按体质量左旋多巴12mg/kg、苄丝肼15mg/kg),微球组给予等剂量包裹左旋多巴/苄丝肼微球皮下注射。分别于治疗第1、5、10、15和第20天,在大鼠腹腔注射阿扑吗啡后计数其旋转圈数,并于治疗3周后对大鼠进行异常不自主运动(AIM)评分(包括上肢、口面部和轴性3部分)。另设PD组和假手术组大鼠各13只。以Western Blot检测大鼠纹状体区△FosB、Tau蛋白和磷酸化Tau蛋白水平,免疫组织化学法测定大鼠纹状体区磷酸化Tau蛋白阳性细胞水平。结果普通剂型组大鼠在治疗后的第1、5、10、15和20天阿扑吗啡诱导的旋转圈数分别为221±34.4、180±25.4、123±17.3、91±13.1、84±10.7,微球组大鼠的旋转圈数分别为223±35.1、172±26.8、131±18.7、97±14.9、82±10.5,两组各时点分别比较差异均无统计学意义(P>0.05)。微球组大鼠轴性AIM、上肢AIM、口面AIM评分分别为6.5±0.9、4.1±0.5、5.8±0.7,均低于普通剂型组大鼠(分别为10.3±1.9、6.3±0.9、8.2±1.2)(均P<0.05)。Western blot结果显示,普通剂型组大鼠纹状体△FosB水平〔(620.7±48.3)%〕高于PD组〔(290.2±31.5%)〕(t=2.11,P<0.05),但低于微球组〔(320.5±32.8)%〕(t=4.56,P<0.01)。普通剂型组纹状体区磷酸化Tau蛋白水平〔(340.4±27.1)%〕高于PD组〔(130.4±21.5)%〕(t=2.67,P<0.05),微球组〔(134.6±14.1)%〕低于普通剂型组(t=4.13,P<0.01)。免疫组化结果示,普通剂型组大鼠纹状体区磷酸化Tau蛋白阳性细胞指数为(14.6±2.3)×104,高于PD组〔(6.9±1.1)×104〕(t=3.98,P<0.01),微球组〔(7.2±1.1)×104〕明显低于普通剂型组(t=3.76,P<0.01)。结论微球治疗减轻了异动症大鼠的症状,其原因可能是由于可缓释包裹左旋多巴/苄丝肼微球通过影响Tau蛋白/△FosB信号通路的活性,进而改善了异动症大鼠的症状。

Objeetive To observe the effect of levodopa/benserazide-loaded poly-lactide-coglycolide （PLGA） microspheres on dyskinetie rats and to explore the mechnisms underlying this effect. Methods A rat model of Parkinson disease was induced by 6-OHDA injections. Then the valid PD rats was treated with levodopa （25 mg/kg, i. p. ） /benserazide （12.5 mg/kg, i. p. ） for 21 days to induce a rat model of levodopa-induced dyskinesia. Then the dyskinetic rats were divided into two groups （levodap group and microsphere group） and treated with levodopa （12 mg/kg, s. c. ） /benserazide （15 mg/kg, s. c） or mierospheres. Apomorphine- induced rotations were measured on the treatment day of 1, 5, 10, 15 and 20. Abnormal involuntary movements （AIM） was determined after 3 week of the treatment. Western blot was used to determine the levels of AFosB, tau and phosphorylated tau in the striata of rats. Immunohischemistry （IHC） was employed to measure phosphorylated tau-positive cells in the striata. Results The degree o{ reduction of apomorphine induced rotations was comparable in dyskinetic rats treated with levodopa plus benserazide or levodopa /benserazide- loaded microspheres. The subtypes of AIMs in dyskinetic rats treated with levodopa/benserazide-loaded microspheres decreased compared to dyskinetic rats treated with levodopa plus benserazide. Western blot showed that the levels of A FosB and phosphorylated tau in dyskinetic rats treated with microspheres were （320.5 ± 32.8） % and （134.6±14.1）% respectively, which were significantly reduced compared to dyskinetic rats treated with levodopa plus benserazide [ （620.7 ± 48.3）% and （340.4±27.1） %]. IHC indicated that the level of phosphorylated tau were （7.2±1.1） X 104 in dyskinetic rats treated with microspheres. However, the level of which was only （14.6±2.3））〈 104 in levodopa plus benserazide-treated dyskinetic rats. There was a significant difference between the two group （t= 3.76, P〈0.01）. There was significant difference between the dyskinetic rats and the PD rats （6.9±1.1）×10^4 （ t=3.98, P〈0.01）. Conclusions The microspheres treatment ameliorate the expression of levodopa-induced dyskinesia in dyskinetic rats. It may be due to reduced activity of Tau/AFosB signal induced by microspheres.