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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 possible mechanism of Tribulus terrestris in the treatment of hypertension. Methods Hypothalamic neurons were cultured in vitro in primary Wistar rats and induced by angiotensin Ⅱ (Ang Ⅱ). Pharmacodynamics cells were divided into five groups: model group (2 × 10-6mol / L AngⅡ), Tribulus terrestris low dose group (3μg / ml Tribulus terrestris 2 h after adding 2 × 10-6mol / L AngⅡ), Tribulus terrestris High dose group (2μg / ml Tribulus terrestris 2h after adding 2 × 10-6mol / L Ang Ⅱ), valsartan group (10-5mol / L valsartan preincubation 2h after adding 2 × 10-6mol / L Ang Ⅱ group and normal group without any intervention. The absolute number of cells, length of neuron axon, cell body area, survival rate and apoptosis rate were detected at 24 h, 48 h, 72 h, respectively. At 72 h The mRNA expression of IKK-α, IKK-β, NF-κB p65, IκBα, JAK2, STAT3, AT1, GnRH, PKC and PI3K were detected. 6mmol / L AngⅡ), Tribulus terrestris group (2 × 10-6mol / L AngⅡafter 30μg / ml Tribulus terrestris preincubation for 2h), 2 hours after pretreatment with Valsartan (10-5mol / L valsartan) 10-6mol / L AngⅡ), antagonist group (2 × 10-6mol / L AngⅡ after 20μmol / L TPCA-1 preincubation for 2 hours), Tribulus Terrestris + antagonist group (30μg / ml Tribulus terrestris + 20μmol / 1 preincubation 2 h after adding 2 × 10-6mol / L Ang Ⅱ), normal group (without any intervention), according to pharmacodynamics AngⅡ action time observation result of the determination and related cytokines, and detecting the expression of cell-associated group factor mRNA and protein. Results Compared with the normal group, the absolute number of cells, cell body area, survival rate and apoptosis rate of model group increased significantly at 24 h after Ang Ⅱ intervention, and the length of neuron axon decreased significantly. Compared with model group, The absolute number of cells, cell body area, survival rate and apoptosis rate of high dose group and valsartan group decreased significantly at each time point, and neurite axon length increased (P <0.05). Compared with the normal group, the expression of IKK-β, NF-κB p65, JAK2, STAT3, AT1, GnRH, PKC and PI3K were significantly up-regulated and the expressions of IKK-α and IKBα were significantly down-regulated in the model group (P <0.05). Compared with the model group, the expression of IKK-β, NF-κB p65, JAK2, AT1, GnRH and PKC in high-dose Tribulus Terrestris group decreased and the expression of IKK- The expression of IKK-αand IκBα were increased in AT1 and PKC groups. The expressions of IKK-β, NF-κB p65, AT1, GnRH and PKC were decreased and the expressions of IKK-αand IκBα were up-regulated in valsartan group (P <0.05). According to the above results, the intervention time of AngⅡ was 24 h and the relevant cytokines were IκBα, IKK-β, NF-κB p65 and NF-κB p50 in pharmacological observation. Compared with the normal group, the expressions of IKK-β, NF-κB p50, NF-κB p65mRNA and protein in the hypothalamus neurons were up-regulated and the expression of IκBα was down-regulated in the model group. Compared with the model group, the expressions of NF- , While the expression of IκBα was up-regulated. The expression of NF-κB p65 and IKK-β was down-regulated and the expression of IκBα was up-regulated in Tribulus Terrestris group compared with valsartan group (P <0.05). Conclusion Tribulus terrestris can effectively maintain the central function of blood pressure regulation, and its antihypertensive mechanism may be related to regulating the activity of IKK-β / NF-κB signaling pathway in the hypothalamus.