基于人脐静脉内皮细胞的OGD/R模型研究当归-川芎药对中7个活性成分对VEGF-PI3K-AKT/NF-κB信号通路的影响

HUVEC-Based OGD/R Injury Model to Study the Effect of Danggui-Chuanxiong Herb Pair Medicine on the Main Pharmacological Components on VEGF-PI3K-AKT/NF-κB Signaling Pathway

目的探讨当归-川芎药对中7个活性成分(绿原酸、阿魏酸、咖啡酸、丁烯基苯酞、洋川芎内酯H、洋川芎内酯A、藁本内酯)对VEGF-PI3K-AKT/NF-κB信号通路关键蛋白及其上下游血管活性物质、黏附因子和炎症因子的调控作用。方法构建人脐静脉内皮细胞(HUVEC)的氧糖剥夺/复氧(OGD/R)模型,采用细胞增殖试剂盒(CCK-8法)检测细胞活力,探索7个成分的最佳造模时间;通过检测乳酸脱氢酶(LDH)释放探索最佳给药浓度;采用酶联免疫吸附法(ELISA)检测7个成分对VEGF、VCAM-1、PAI-1、NF-κB、IL-1和IL-6表达的影响;采用逆转录-聚合酶链式反应(RT-PCR)法检测7个成分对VEGF-PI3K-AKT/NF-κB信号通路关键蛋白mRNA表达的影响。结果HUVEC氧糖剥夺6 h再复氧为最佳造模时间。高剂量绿原酸组、阿魏酸组、洋川芎内酯H组,低、中剂量丁烯基苯酞组,中、高剂量洋川芎内酯A、藁本内酯组显著降低LDH漏出率(P<0.05,P<0.01);绿原酸、阿魏酸、洋川芎内酯H部分剂量组细胞中VEGF、ICAM-1、VCAM-1的表达显著降低,绿原酸、阿魏酸、洋川芎内酯H、藁本内酯部分剂量组细胞NF-κB的表达显著下降,绿原酸、咖啡酸、丁烯基苯酞、洋川芎内酯H、洋川芎内酯A部分剂量组细胞中IL-6的表达显著增加,绿原酸、阿魏酸、洋川芎内酯A部分剂量组细胞IL-1表达显著减少,阿魏酸、洋川芎内酯H部分剂量组细胞中PAI-1的表达量显著下降(P<0.05,P<0.01);绿原酸、阿魏酸、咖啡酸、丁烯基苯酞和洋川芎内酯A部分剂量组细胞中ERK、VEGF、NF-κB、VEGFR2和MMP9的mRNA相对表达量显著下调,洋川芎内酯H和洋川芎内酯A部分剂量组细胞中AKT的mRNA相对表达量均显著上调(P<0.05,P<0.01)。结论当归-川芎中的药效成分可能通过抑制黏附因子、炎症因子和VEGF-PI3K-AKT/NF-κB信号通路关键蛋白mRNA的表达,从而发挥抗缺血性中风的作用。

Objective To study the effects of Danggui-Chuanxiong herb pair medicine on vasoactive substances,adhesion factors,inflammatory factors,and VEGF-PI3K-AKT/NF-κB signaling pathways,in order to elucidate the mechanism of Danggui-Chuanxiong herb pair on the treatment of ischemic stroke(IS).Methods The oxygen glucose deprivation/reoxygenation(OGD/R)model of human umbilical vein endothelial cells(HUVEC)was constructed,and the cell viability was detected by cell proliferation kit(CCK-8 method)to explore the optimal modeling time of seven components;The release of lactate dehydrogenase(LDH)was detected by cytotoxicity kit;The expression of related cytokines was detected by enzyme-linked immunosorbent assay(ELISA);The mRNA expression of key proteins in the signaling pathway was detected by reverse transcription-polymerase chain reaction(RT-PCR).Results Reoxygenation after 6 h of oxygen-glucose deprivation of HUVEC is the best modeling time.High-dose chlorogenic acid group,ferulic acid group,senkyunolide H,low-dose and medium-dose butylidenephthalide group,medium-dose and high-dose senkyunolide A and ligustilide groups significantly decreased LDH leakage rate(P<0.05,P<0.01);The expression of IL-6 in the cells of the partial dose group of chlorogenic acid,caffeic acid,butenylphthalide,senkyunolide H and senkyunolide A was significantly increased,the expression of IL-1 in the cells of the partial dose group of chlorogenic acid,ferulic acid and senkyunolide A was significantly decreased,the expression of VEGF,ICAM-1 and VCAM-1 in the cells of the partial dose group of chlorogenic acid,ferulic acid and senkyunolide H was significantly decreased,the expression of NF-κB in the cells of the partial dose group of chlorogenic acid,ferulic acid,senkyunolide H and ligustilide was significantly decreased,the expression of PAI-1 in the cells of ferulic acid and senkyunolide H partial dose group decreased significantly(P<0.05,P<0.01);The mRNA relative expression levels of ERK,VEGF,NF-κB,VEGFR2 and MMP9 were significantly down-regulated in the cells of chlorogenic acid,ferulic acid,caffeic acid,butylidenephthalide and senkyunolide A partial dose group,while the mRNA relative expression levels of AKT were significantly up-regulated in the cells of senkyunolide H and senkyunolide A partial dose groups(P<0.05,P<0.01).Conclusion The medicinal components of Danggui-Chuanxiong herb pair may play a role in IS by inhibiting the mRNA expression of adhesion factor,inflammatory factor and key protein of VEGF-PI3K-AKT/NF-κB signaling pathway in HUVEC.