复方地黄颗粒和毛蕊花糖苷治疗帕金森病模型大鼠的作用靶标研究

Study on the target of compound Rehmannia granule and verbascoside in the rat model of Parkinson's disease

目的探讨复方地黄颗粒和毛蕊花糖苷治疗帕金森病(P D)的作用靶标。方法采用6-羟基多巴胺(6-OHDA)偏侧损毁黑质制备PD大鼠模型,分层随机分为模型组、美多芭组、毛蕊花糖苷组、复方地黄颗粒(低、中、高剂量)组,另设不作处理的正常对照组和只注射抗坏血酸的假手术组,每组12只。复方地黄颗粒(3.5、7、14g/kg)、毛蕊花糖苷(60mg/kg)和美多芭(150mg/kg)用蒸馏水溶解后分别给予复方地黄颗粒低、中、高剂量组,毛蕊花糖苷组和美多芭组大鼠灌胃,1次/d。正常对照组、假手术组和模型组以等体积蒸馏水(2ml/只)灌胃。灌胃6周后,取大鼠患侧黑质、纹状体,应用RT-PCR法检测各组大鼠N-甲基-D-天冬氨酸受体1(NMDA-R1)、NMDA-R2、γ-氨基丁酸B1受体(GABA-RB1)m RNA水平,HPLC法检测天门冬氨酸(ASP)、谷氨酸(GLU)、甘氨酸(GLY)、γ-氨基丁酸(GABA)等含量变化,免疫组化法检测酪氨酸羟化酶(TH)阳性细胞数。结果复方地黄颗粒和毛蕊花糖苷可使PD模型大鼠右侧纹状体NMDA-R1、NMDA-R2 m RNA水平明显降低,GABA-RB1 m RNA表达升高(P<0.01);ASP、GLU含量升高,GLY、GABA含量降低(P<0.01);TH阳性表达细胞数明显增多(P<0.01)。其中以复方地黄颗粒中剂量组改善最为明显,差异有统计学意义(P<0.01)。镜下观察可见TH阳性信号定位于细胞质,在正常对照组、假手术组的右侧黑质均可见密集的TH阳性细胞,呈棕褐色,细胞形状主要为圆形或卵圆形,少数呈多边形;模型组右侧黑质可见少量、稀疏分布的TH阳性细胞;给药干预后TH阳性细胞数明显增加。结论复方地黄颗粒和毛蕊花糖苷的作用靶标可能包括TH以及氨基酸类神经递质及其受体基因。

Objective To explore the target of compound Rehmannia granule and verbascoside in the rat model of Parkinson＇s disease. Methods Single-side substantia nigra was lesioned by using 6-hydroxydopamine for preparation of PD rat model. The rats were layer randomly divided into model group, Madopar group, verbascoside group, compound Rehmannia granule group （low, medium, high doses）. Another 12 rats were not treated as normal control group, and only the rats of injected ascorbic acid as sham operation group. Compound Rehmannia granule （3.5, 7, 14g/kg）, verbascoside （60mg/kg） and Madopar （150mg/kg） were dissolved in distilled water, and given separately to the compound Rehmannia granule （low, medium and high doses） groups, verbascoside group and Madopar group rats for gastric gavage, one time a day. The equal volume distilled water （2ml/only） was administered to the stomach in the normal control group, the sham operation group and the model group. After 6 weeks of the gavage, 3% pentobarbital sodium （50mg/kg） was used for anesthesia and the rats were sacrificed. The substantia nigra and striatum at the sick side were taken for determining the gene expression changes in NMDA-R1, NMDA-R2, GABA-RB1 by RT-PCR method and the content changes in ASP, GLU, GLY, GABA by HPLC method and the number of tyrosine hydroxylase （TH）-positive cells by immunohistochemical method in these rats. Results Compound Rehmannia granule and verbascoside significantly decreased the expressions of NMDA-R1 and NMDA-R2 mRNA, and increased the expression of GABA-RB 1 mRNA in the right striatum of model rats with Parkinson＇s disease （P〈0.01）; decreased contents of ASP and GLU and increased the contents of GLY and GABA （P〈0.01）; and also increased the number of TH-positive cells （P〈0.01）. The improvement effect was most obvious in medium dose group of compound Rehmannia granule, and the difference was statistically significant （P〈0.01）. Microscopic observation showed that TH- positive signal was localized in cytoplasm. There were dense TH-positive ceils in the right substantia nigra of the normal control group and the sham operation group, which were brown, and the cells were mainly round or oval, with a few polygons. In the right substantia nigra of the model group, a few and sparse distribution of TH-positive cells were found. With the intervention of the drug,the expression of TH-positive ceils increased gradually. Conclusion The target of compound Rehmannia granule and verbascoside are neurotransmitter and its receptor gene of amino acids and TH.