蒺藜超微粉对血管紧张素Ⅱ诱导的下丘脑神经元细胞损伤IKK-β/NF-κB信号通路的影响

Effects of Jili [Tribulus terrestris L. 蒺藜] on IKK-β/NF-κB Signaling Pathway in Hypothalamic Neuron Injury Induced by Angiotensin Ⅱ

目的探讨蒺藜治疗高血压病的可能作用机制。方法体外原代培养Wistar乳鼠下丘脑神经元细胞,并以血管紧张素Ⅱ(AngⅡ)诱导细胞损伤即高血压细胞模型。药效学观察时细胞分为5组,模型组(2×10-6mol/L AngⅡ)、蒺藜低剂量组(3μg/ml蒺藜预孵育2 h后加入2×10-6mol/L AngⅡ)、蒺藜高剂量组(30μg/ml蒺藜预孵育2 h后加入2×10-6mol/L AngⅡ)、缬沙坦组(10-5mol/L缬沙坦预孵育2 h后加入2×10-6mol/L AngⅡ)、正常组(无任何干预),分别于AngⅡ作用24、48、72 h时检测各组细胞绝对数量、神经元轴突长度、胞体面积、存活率、凋亡率,并于72 h时检测细胞因子IKK-α、IKK-β、NF-κB p65、IκBα、JAK2、STAT3、AT1、GnRH、PKC、PI3K的mRNA表达;药理学观察时细胞分为6组,模型组(2×10-6mol/L AngⅡ)、蒺藜组(30μg/ml蒺藜预孵育2 h后加入2×10-6mol/L AngⅡ)、缬沙坦组(10-5mol/L缬沙坦预孵育2 h后加入2×10-6mol/L AngⅡ)、拮抗剂组(20μmol/L TPCA-1预孵育2 h后加入2×10-6mol/L AngⅡ)、蒺藜+拮抗剂组(30μg/ml蒺藜+20μmol/L TPCA-1预孵育2 h后加入2×10-6mol/L AngⅡ)、正常组(无任何干预),根据药效学观察结果确定AngⅡ作用时间及相关细胞因子,并检测各组细胞相关因子mRNA及蛋白的表达。结果与正常组比较,模型组从AngⅡ干预24 h开始的各时间点细胞绝对数量、胞体面积、存活率、凋亡率均显著升高,神经元轴突长度显著降低;与模型组比较,蒺藜高剂量组、缬沙坦组各时间点细胞绝对数量、胞体面积、存活率、凋亡率均显著下降,神经元轴突长度增加(P<0.05)。与正常组比较,模型组IKK-β、NF-κB p65、JAK2、STAT3、AT1、GnRH、PKC、PI3K表达显著上调,IKK-α、IKBα表达显著下调(P<0.05)。与模型组比较,蒺藜高剂量组IKK-β、NF-κB p65、JAK2、AT1、GnRH、PKC表达下降,IKK-α、IκBα表达升高;蒺藜低剂量组IKK-β、NF-κB p65、AT1、PKC表达下降,IKK-α、IκBα表达升高;缬沙坦组IKK-β、NF-κB p65、AT1、GnRH、PKC表达下降,IKK-α、IκBα表达上调(P<0.05)。根据以上结果确定药理学观察时AngⅡ干预时间为24 h,相关细胞因子为IκBα、IKK-β、NF-κB p65、NF-κB p50。与正常组比较,模型组下丘脑神经元细胞IKK-β、NF-κB p50、NF-κB p65mRNA和蛋白表达上调,IκBα表达下调;与模型组比较,缬沙坦组和蒺藜组NF-κB p50、NF-κB p65、IKK-β的表达下调,IκBα表达上调;与缬沙坦组比较,蒺藜组NF-κB p65、IKK-β表达下调,IκBα表达上调(P<0.05)。结论蒺藜可有效维持血压调节中枢功能,其降压机制可能与调节下丘脑IKK-β/NF-κB信号通路活性有关。

Objective To explore the potential mechanism of Jili [Tribulus terrestris L. 蒺藜]in the treatment of hypertension. Methods The hypertension cell model was induced by angiotensin Ⅱ（ Ang Ⅱ） in the primary cultured hypothalamic neurons from neonatal Wistar rats. During the pharmacodynamics observation,the cells were divided into 5 groups： model group（ 2 × 10-6mol / L AngⅡ）,low dose Jili group（ preincubated with 3 μg / ml Jili for2 hours,then 2 × 10-6mol / L AngⅡ was added）,high dose Jili group（ preincubated with 30 μg / ml Jili for 2 hours,then 2 × 10-6mol / L AngⅡ was added）,Valsartan group（ preincubated with 10-5mol / L Valsartan for 2 hours,then2 × 10-6mol / L Ang Ⅱ was added） and control group（ with no intervention）. Then the cell number,the axonal length,the soma area,the survival rate and the apoptosis rate in each group were detected at the 24 th,48th and 72 nd hour after AngⅡ intervention. Also,the mRNA expression level of some cytokines,such as IκBα,IKK-α,IKK-β,NF-κB p65,JAK2,STAT3,AT1,GnRH,PKC and PI3 K,were detected at the 72 nd hour. During the pharmacological observation,the cells were divided into 6 groups： model group（ 2 × 10-6mol / L AngⅡ）,Jili group（ preincubated with 30 μg / ml Jili for 2 hours,then 2 × 10-6mol / L AngⅡwas added）,Valsartan group（ preincubated with 10-5mol / L Valsartan for 2 hours,then 2 × 10-6mol / L Ang Ⅱ was added）,antagonist group（ preincubated with 20 μmol / L TPCAK-1 for 2 hours,then 2 × 10-6mol / L AngⅡ was added）,Jili plus antagonist group（ preincubated with 30 μg /ml Jili and 20 μmol / L TPCA-1 for 2 hours,then 2 × 10-6mol / L AngⅡ was added） and control group（ with no intervention）. Then based on the results of pharmacodynamics observation,the action time of AngⅡ and the related cytokines were determined. Also,the expression level of mRNA and protein of related cytokines of each group were detected.Results Compared with those in the control group,the cell number,the soma area,the survival rate and the apoptosis rate in the model group increased significantly,while the axonal length deceased significantly at each time point after 24 hours after AngⅡ intervention; compared with those in the model group,the cell numbers,the soma areas,the survival rates and the apoptosis rates in the high dose Jili group and Valsartan group decreased significantly,while the axonal lengths increased significantly（ P 0. 05）. Compared with those in the control group,the expression levels of IKK-β,NF-κB p65,JAK2,STAT3,AT1,GnRH,PKC and PI3 K increased significantly,while the levels of IKK-αand IκBα decreased significantly in the model group（ P 0. 05）. Compared with those in the model group,the expression levels of IKK-β,NF-κB p65,JAK2,AT1,GnRH and PKC decreased,and the levels of IKK-α and IκBαincreased in the high does Jili group; the expression levels of IKK-β,NF-κB p65,AT1 and PKC decreased,and the levels of IKK-α and IκBα increased in the low does Jili group; the expression levels of IKK-β,NF-κB p65,AT1,GnRH and PKC decreased,and the levels of IKK-α and IκBα increased in Valsartan group（ P 0. 05）. Based on the results above,the intervention time of AngⅡ was determined as 24 h,and the related cytokines were determined as IκBα,IKK-β,NF-κB p65 and NF-κB p50. Compared with those in the control group,the expression levels of the mRNA and the protein of IKK-β,NF-κB p50 and NF-κB p65 increased,while those of IκBα decreased in the model group; compared with those in the model group,the expressions of IKK-β,NF-κB p50 and NF-κB p65 decreased,while the expression of IκBα increased in Valsartan group and Jili group; compared with those in Valsartan group,the expressions of NF-κB p65 and IKK-β decreased,while the expression of IκBα increased in Jili group（ P 0. 05）.Conclusion Jili could maintain the function of the blood pressure regulation center,and its potential mechanism of anti-hypertension might be related to regulating the IKK-β / NF-κB signaling pathway in hypothalamic neurons.