王不留行黄酮苷通过NLRP3/Caspase-1信号通路抑制ox-LDL诱导的细胞焦亡

Vaccarin Attenuated Ox-LDL-induced HUVECs Pyroptosis by Suppressing NLRP3/Caspase-1 Signaling Pathway

[目的]探讨王不留行黄酮苷对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤的保护作用及作用机制。[方法]以ox-LDL(100μg·mL-1)刺激HUVECs建立模型。采用细胞增殖活性检测(cell counting kit-8,CCK-8)法检测1~10μmol·L-1王不留行黄酮苷对ox-LDL诱导的HUVECs的保护作用;采用乳酸脱氢酶(lactate dehydrogenase,LDH)法检测LDH的释放;采用AnnexinⅤ-FITC/碘化丙啶(propidium iodide,PI)凋亡试剂盒检测细胞凋亡;使用2’,7’-二氯荧光素二乙酸酯(2’,7’-dichlorodihydrofluorescein diacetate,DCFD-A)荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;采用实时荧光定量聚合酶链式反应(Real-time polymerase chain reaction,Real-time PCR)检测白细胞介素-6(interleukin-6,IL-6)、单核细胞趋化蛋白-1(monocyte chemoattracctant protein-1,MCP-1)、人血管内皮细胞黏附分子-1(human vascular cell adhesion molecule-1,VCAM-1)的mRNA表达;采用免疫印迹法检测B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关x蛋白(Bcl-2 associated X protein,Bax)、NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3,NLRP3)、胱天蛋白酶-1(cysteinyl aspartate specific proteinase-1,caspase-1)、裂解的胱天蛋白酶-1(cleaved caspase-1)、IL-1β和IL-18的蛋白表达。[结果]1~10μg·mL-1王不留行黄酮苷减轻100μg·mL-1的ox-LDL诱导的HUVECs损伤(P<0.05,P<0.01)。王不留行黄酮苷减轻ox-LDL诱导的LDH释放,减少细胞凋亡,同时促进Bcl-2蛋白表达,抑制Bax蛋白表达(P<0.01)。王不留行黄酮苷显著减少了ox-LDL诱导的ROS生成,抑制ox-LDL诱导的炎症因子IL-6、MCP-1和VCAM-1的水平(P<0.01)。王不留行黄酮苷抑制了ox-LDL诱导的NLRP3炎症小体介导的焦亡相关蛋白表达(P<0.05,P<0.01)。[结论]王不留行黄酮苷能够减轻ox-LDL诱导的HUVECs凋亡,缓解炎症反应,其机制可能与NLRP3介导的焦亡有关。

[Objective]To explore the protective effect and mechanism of Vaccarin on oxidized low-density lipoprotein(ox-LDL)-induced damage of human umbilical vein endothelial cells(HUVECs).[Methods]The protective effect of 1~10μmol·L-1 Vaccarin on ox-LDLinduced HUVECs was detected by cell counting kit-8(CCK-8);the release of lactate dehydrogenase(LDH)was detected by LDH method;the cells apoptosis was detected using AnnexinⅤ-FITC/propidium iodide(PI)apoptosis kit;2',7'-dichlorofluorescein diacetate(DCFD-A)fluorescent probe was used to detect intracellular reactive oxygen species(ROS)levels;Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of interleukin-6(IL-6),monocyte chemoattractant protein-1(MCP-1)and vascular cell adhesion molecule-1(VCAM-1);Western blot was used to detect the protein expression of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),NOD-like receptor thermal protein domain associated protein 3(NLRP3),cysteinyl aspartate specific proteinase-1(caspase-1),cleaved caspase-1,IL-1βand IL-18.[Results]Vaccarin 1~10μg·mL-1 ameliorated the damage of HUVECs induced by ox-LDL at 100μg·mL-1(P<0.05,P<0.01).Vaccarin attenuated ox-LDL-induced LDH release and reduced apoptosis,while promoting Bcl-2 protein expression and inhibiting Bax protein expression(P<0.01).Vaccarin significantly reduced ox-LDL-induced ROS generation and inhibited ox-LDL-induced inflammatory factors IL-6,MCP-1 and VCAM-1 levels(P<0.01).Vaccarin inhibited ox-LDLinduced expression of NLRP3 inflammasome-mediated pyroptosis-related proteins(P<0.05,P<0.01).[Conclusion]Vaccarin could alleviate the apoptosis of HUVECs induced by ox-LDL and reduce the inflammatory response,and its mechanism may be tightly related to NLRP3 inflammasome activation-mediated pyroptosis.