麻黄连翘赤小豆汤对5-HT所致瘙痒模型大鼠与不同细胞系中5-HT1A及GRPR表达的影响

Effects of Mahuang Lianqiao Chixiaodou Decoction on Expression of 5-HT1a and GRPR in Rat Pruritus Models and in Vitro Cell

目的通过建立大鼠瘙痒模型联合体外细胞培养,探讨麻黄连翘赤小豆汤的止痒效果和对5-HT1a受体(5-羟色胺1a受体,Serotonin 1a receptor)、GRPR(胃泌素释放肽受体,Gastrin-releasing peptide receptor)表达的影响。方法将40只雄性Sprague Dawley(SD)大鼠作为研究对象,随机分成空白组、模型组、麻黄连翘赤小豆汤(MLC)低、中、高3个剂量组,每组8只。除正常组外,其余各组均采取颈背部皮下注射2%5-羟色胺(5-hydroxytryptamine,5-HT)溶液建立大鼠瘙痒模型。造模完成1天以后,药物干预组大鼠采用麻黄连翘赤小豆汤溶液灌胃治疗14天,浓度分别为4.3 g·kg^(-1)、8.6 g·kg^(-1)、17.2 g·kg^(-1),正常组和模型组灌胃等量等体积的生理盐水,观察并记录大鼠造模当天、治疗第7天、治疗第14天各组大鼠搔抓次数。治疗14天后取大鼠脊髓,采用蛋白免疫印迹法(Western Blot,WB)和免疫组化法检测5-HT1a受体和GRPR的表达水平及组织内分布情况。同时选用2种不同类型细胞系,分别分为空白组,对照组与干预组,对照组与干预组采用生理盐水与麻黄连翘赤小豆汤处理,48h后采用实时荧光定量PCR技术(Quantitative real-time PCR,qPCR)检测5-HT1a受体与GRPR mRNA的表达情况。结果麻黄连翘赤小豆汤对瘙痒模型大鼠有止痒作用(P<0.05),其抑制5-HT1a受体及GRPR的表达水平(P<0.05),在细胞层面也下调5-HT1a受体及GRPR的表达。结论麻黄连翘赤小豆汤可通过抑制5-HT1a与GPRP的表达进而达到止痒作用。

Objective Through the establishment of rat pruritus model combined with cell culture in vitro,the antipruritic effect of Mahuang Lianqiao Chixiaodou Decoction and its influence on the expression of 5-HT1 a receptor and GRPR were discussed.Methods Forty male Sprague Dawley(SD) rats were randomly divided into three groups:blank group,model group,low,medium and high dose groups of Mahuanglianqiao Chixiaodou Decoction,with 8 rats in each group.Except for the normal group,all the other groups were injected with 2% serotonin solution subcutaneously on the back of the neck to establish a rat model of itching.One day after the completion of model building,rats in the drug intervention group were treated with Mahuang Lianqiao Chixiaodou Decoction for 14 days at concentrations of 4.3 g·kg^(-1),8.6 g·kg^(-1),and 17.2 g·kg^(-1).The normal group Gave the same volume of normal saline as the model group.Observe and record the number of scratching times of rats in each group on the day of modeling,the 7 th day of treatment,and the14 th day of treatment.After 14 days of treatment,the rats’ spinal cords were taken,and the expression levels of 5-HTla receptor and GRPR and the distribution in the tissue were detected by Western blotting and immunohistochemistry.At the same time,2 different cell lines were selected and divided into the blank group,control group,and intervention group.The control group and intervention group were treated with saline and Mahuang Lianqiao Chixiaodou Decoction.After 48 hours,real-time fluorescent quantitative PCR was used to detect 5-HT1 a receptors and GRPR mRNA expression.Results Mahuang Lianqiao Chixiaodou Decoction had an antipruritic effect on itching rats(P<0.05).It inhibited the expression levels of 5-HT1 a receptor and GRPR(P<0.05),and down-regulated 5-HT1 a receptor and GRPR at the cell level.Conclusion Mahuang Lianqiao Chixiaodou Decoction can achieve an antipruritic effect by inhibiting the expression of 5-HT1 a and GPRP.