强志组方调控PI3K/Akt/TNF-α信号通路干预抽动障碍的实验研究

Experimental Study on the Intervention of Qiangzhi Formula in Tic Disorder by Regulating PI3K/Akt/TNF-αSignaling Pathway

目的:基于PI3K/Akt/TNF-α信号通路探究强志组方治疗抽动障碍(TD)大鼠的作用机制。方法:42只SD大鼠随机分为空白组、模型组、硫必利组与强志组方低、中、高剂量组,除空白组外其余大鼠腹腔注射亚氨基二丙腈(IDPN)制备抽动障碍大鼠模型。强志组方低、中、高剂量组分别予以4.455、8.91、17.82 g/(kg·d)强志组方水煎剂灌胃,硫必利组以0.027 g/(kg·d)硫必利混悬液灌胃,空白组与模型组以等剂量生理盐水灌胃,每天1次,连续28 d。采用刻板行为与运动行为评分评估大鼠行为学变化情况;干预结束后,采用蛋白免疫印迹法(Western blot)与实时荧光定量聚合酶链式反应(qPCR)检测纹状体PI3K、Akt、TNF-α的蛋白与mRNA表达;酶联反应吸附测定法(ELISA)检测血清IL-6、TNF-α含量。结果:与空白组比较,造模组大鼠出现异常运动行为与刻板行为。纹状体PI3K、TNF-α蛋白表达水平及p-Akt/Akt磷酸化水平升高(P<0.01,P<0.001,P<0.05),PI3K、Akt、TNF-αmRNA表达水平升高(P<0.001,P<0.01,P<0.05),血清IL-6、TNF-α含量升高(P<0.001,P<0.01)。药物干预28 d后,与模型组比较,硫必利组与强志组方各剂量组大鼠运动行为与刻板行为评分降低(P<0.001,P<0.05);强志组方低剂量组大鼠纹状体PI3K、TNF-α蛋白表达水平降低(P<0.05),强志组方中、高剂量组PI3K、Akt、TNF-α蛋白及p-Akt/Akt磷酸化表达水平降低(P<0.001,P<0.05);强志组方中、高剂量组纹状体PI3K、Akt mRNA表达水平降低(P<0.01,P<0.05);硫必利组与强志组方各剂量组血清IL-6水平降低(P<0.01,P<0.001),强志组方低、中、高剂量组血清TNF-α含量下降(P<0.01,P<0.05)。结论:强志组方能改善抽动障碍模型大鼠异常的运动行为与刻板行为,其作用机制可能与PI3K/Akt/TNF-α信号通路相关。

Objective:To explore the mechanism of Qiangzhi(QZ)Formula in the treatment of tic disorder(TD)based on PI3K/Akt/TNF-αsignaling pathway.Methods:Forty-two SD rats were randomly divided into blank group,model group,tiapride group,and low-dose,medium-dose and high-dose QZ formula groups.The tic disorder model was established by intraperitoneal injection of iminodipropionitrile(IDPN).The low-dose,medium-dose and high-dose QZ formula groups were given 4.455 g/(kg·d),8.91 g/(kg·d)and 17.82 g/(kg·d)water decoction by gavage respectively.Tiapride group was given tiapride suspension 0.027 g/(kg·d)by gavage.The rats in the blank group and the model group were given the same dose of normal saline by gavage once a day for 28 consecutive days.The behavioral changes of rats were evaluated by stereotyped behavior score and motor behavior score.After the intervention,Western blot and qPCR were used to detect the mRNA and protein expression of PI3K,AKT and TNF-αin the striatum.The serum levels of IL-6 and TNF-αwere detected by enzyme-linked reaction adsorption assay(ELISA).Results:After modeling,compared with the blank group,the rats in the model group showed abnormal motor behavior and stereotyped behavior.The protein expression levels of PI3K and TNF-αand the phosphorylation level of p-Akt/Akt increased in the striatum(P<0.01,P<0.01,P<0.05);PI3K,Aktand mRNA expression levels increased(P<0.001,P<0.01,P<0.05);and serum IL-6 and TNF-αlevels increased(P<0.001,P<0.01).After 28 days of drug intervention,compared with the model group,the motor behavior score and stereotype-behavior score of tiapride group and AZ formula groups decreased(P<0.001,P<0.05).The expression levels of PI3K and TNF-αprotein in striatum of QZ formula low-dose group decreased(P<0.05).The expression levels of PI3K,Akt,TNF-αand p-Akt/Akt phosphorylation in the middle and high dose of QZ formula group decreased(P<0.001,P<0.05,P<0.05).The expression levels of PI3K,Akt and mRNA in striatum of middle and high dose QZ formula group decreased(P<0.01,P<0.05).The level of serum IL-6 in tiapride group and QZ group decreased(P<0.01,P<0.001),and the serum TNF-αlevel decreased in each dose group of QZ formula(P<0.01,P<0.01,P<0.05).Conclusions:Qiangzhi Formula can alleviate the abnormal motor behavior and stereotype behavior of rats with tic disorders,which may be related to the PI3K/Akt/TNF-αsignaling pathway.