DARPP-32磷酸化参与天芪平颤颗粒缓解帕金森病运动并发症的发生

Phosphorylated DARPP-32 involved in the remission of levodopa-induced dyskinesia by Tianqi anti-tremor granules in Parkinson's disease

目的研究天芪平颤颗粒对帕金森病(PD)运动并发症大鼠异常不自主行为学及纹状体DARPP-32(Thr75)磷酸化表达的影响,探讨长期左旋多巴使用后天芪平颤颗粒对异动症(LID)的作用机制。方法 6-羟基多巴胺(6-OHDA)制备PD大鼠模型并给予左旋多巴治疗4周,制备LID大鼠模型,将LID大鼠随机分为5组,分别给予大鼠生理盐水(LID模型组)、左旋多巴(西药组)以及小、中、大剂量天芪平颤颗粒组,另设假手术组(n=5)为对照组,并用免疫组化和Western blotting法观察各组大鼠纹状体内磷酸化DARPP-32(Thr75)的表达情况。结果长期使用左旋多巴后,PD大鼠出现刻板运动和进行性增加的对侧旋转等行为学改变。天芪平颤颗粒可明显缓解LID大鼠的不自主运动行为。免疫组化结果显示,西药组磷酸化DARPP-32(THr75)表达较LID模型组降低〔(3.53±0.20)×104 vs.(3.85±0.30)×104,P<0.05〕,大、中、小剂量中药组磷酸化DARPP-32(THr75)表达量分别为(8.54±0.17)×104、(8.10±0.31)×104、(7.06±0.69)×104,较西药组升高(P<0.05)。Western blotting结果与免疫组化基本一致,西药组磷酸化DARPP-32(THr75)灰度值与LID模型组比较无统计学差异〔(0.97±0.24)×106 vs.(1.08±0.12)×106,P>0.05〕,大、中、小剂量中药组大鼠纹状体磷酸化DARPP-32(THr75)灰度值分别为(2.40±0.09)×106、(2.37±0.16)×106、(1.44±0.14)×106,较西药组升高(P<0.05)。结论天芪平颤颗粒可降低LID大鼠的异常不自主运动评分,其机制可能是通过逆转磷酸化DARPP-32(THr75)表达的进一步下降,从而抑制了PKA通路的过度活化;DARPP-32磷酸化参与了治疗PD运动并发症的机制。

Objective To study effects of Tianqi anti-tremor granules on behavior and expression of phosphorylated DARPP-32（Thr75）in striatum of Parkinsonian rats with abnormal involuntary movements（AIM）,and to explore the effects of Tianqi anti-tremor granules on mechanism of levodopa-induced dyskinesia.Methods Six unilateral hydroxydopamine-lesioned rat,the model of Parkinson disease（PD）,were treated with 1evodopa/benserazide twice daily for 4 weeks in order to construct the model of LID.In contiguous 4 weeks,LID rats were randomly divided into 5 groups and treated with saline,levodopa,traditional Chinese medicine（TCM）-low,TCM-medium,and TCM-high,respectively.Other 5 rats with sham-operation were served as controls.Rats were then sacrificed,and immunohistochemical and Western blotting techniques were used to measure the expression of phosphorylated DARPP-32（Thr75）in striatum.Results Pulsatile treatment with levodopa gradually induced AIM,including stereotypy（limb dyskinesia,axial dystonia and masticatory dyskinesia）towards the side contralateral to the dopamine-denervated striatum,and increased contralateral rotation.Treatment with Tianqi anti-tremor granules can relieve AIMs of LID rats apparently.Immunohistochemical study showed that the expression of phosphorylated DARPP-32（Thr75）in striatum of levodopa group diminished to（3.53±0.20）×10^4（P〈0.05）,while it was increased to（8.54±0.17）×10^4,（8.10±0.31）×10^4,（7.06±0.69）×10^4（P〈0.05,respectively）in the 3 groups treated with Levodopa plus 3 doses of Tianqi anti-tremor granules.Western blotting showed that the gray value of phosphorylated DARPP-32（Thr75）was decreased in the levodopa group（0.97±0.24）×10^6（P〉0.05）,while it was increased to（2.40±0.09）×10^6,（2.37±0.16）×10^6,（1.44±0.14）×10^6,respectively,in the high,medium and low dose groups（P〈0.05）.Conclusions Tianqi anti-tremor granules can decrease AIM scores of LID rats,inhibit the overactive of PKA pathway through reversing the decreased expression of phosphorylated DARPP-32（Thr75）;DARPP-32 takes part in healing levodopa-induced dyskinesia.