基于疏肝健脾法探讨Zn^(2+)对HPRL大鼠DAT的调控机制

Regulatory mechanism of Zn^(2+)on DAT in HPRL rats based on method of dredging liver and strengthening spleen

目的以疏肝健脾法为核心,探讨逍遥散加减方对高催乳激素血症(HPRL)模型大鼠Zn^(^(2+))的表达影响,进一步讨论Zn^(2+)对其中枢多巴胺转运体(DAT)的调控机制。方法将96只大鼠随机分6组:正常组、模型组、西药组(溴隐亭,0.001g·kg^(-1)·d^(-1))、逍遥散加减方高、中、低剂量组(逍遥散加减方60,30,15 g·kg^(-1)·d^(-1))。制作HPRL动物模型的方法采用ip甲氧氯普胺联合慢性复合应激。给药14d后,通过酶联免疫法(ELISA)检测各组大鼠血清催乳素(PRL)水平;锌离子荧光探针检测各组大鼠下丘脑多巴胺能神经元Zn^(2+)表达的情况;蛋白免疫印迹法(WB)检测各组大鼠下丘脑多巴胺转运体(DAT)蛋白表达的情况。结果模型组PRL、DAT表达与正常组相比,显著升高(P<0.01),模型组Zn^(2+)表达与正常组相比,显著降低(P<0.01);中药中剂量组、西药组的PRL、DAT表达与模型组相比,均显著降低(P<0.01),中药高、低剂量组的DAT表达与模型组相比,均显著降低(P<0.01),中药低剂量组、西药组的Zn^(2+)表达与模型组相比,明显升高(P<0.01,P<0.05)。结论逍遥散加减方能升高Zn^(2+)的表达,通过Zn^(2+)调控DAT的蛋白表达,从而调节PRL水平。

Objective Using the method of dredging the liver and strengthening the spleen as the core,we investigated the expression of Zn^(2+)in rats in the model of hyperprolactinemia(HPRL)by modified Xiaoyaosan,and further discussed the regulation mechanism of Zn^(2+)on their central dopamine transporter(DAT).Methods Ninety-six rats were randomly divided into six groups,the Control group,model group,western medicine(bromocriptine,0.001g·kg^(-1)·d^(-1)),and the high,medium,and low dose groups(modified xiaoyaosan60,30,and 15 g·kg^(-1)·d^(-1)).The method used to produce the HPRL animal model was intraperitoneal injection of metoclopramide combined with chronic stress.After 14 days of administration,the prolactin level(PRL)in the serum of the rats of each group was measured by enzyme-linked immunosorbent assay(ELISA);the Zn^(2+)expression of the hypothalamic dopaminergic neurons of each group was detected by zinc ion fluorescence probe;the protein expression of the dopamine transporter(DAT)in the hypothalamus of the rats of each group was detected by protein immunoblotting(WB).Results Compared with the Control group,the model group exhibited significantly increased PRL and DAT(P<0.01),and the model group exhibited significantly decreased Zn^(2+)(P<0.01);compared with the model group,bromocriptine and modified Xiaoyaosan at the medium dose significantly lowered the content of PRL and DAT(P<0.01),the expression levels of DAT in the high-and low-dose modified Xiaoyaosan group were significantly decreased(P<0.01),the modified Xiaoyaosan at the low doses and bromocriptine elevated the expression of Zn^(2+)(P<0.01,P<0.05).Conclusion Modified xiaoyaosan can increase the expression of Zn^(2+)and regulate the protein expression of DAT by Zn^(2+),thereby regulating the PRL level.