基于Nrf2靶点及线粒体质量控制系统研究姜三七挥发油对内皮细胞损伤的保护作用

The protective effect of essential oil of rhizome from Stahlianthus involucratus on endothelial cell damage based on Nrf2 target and mitochondria quality control system

目的研究核因子E2相关因子2(nuclear E2-related factor 2,Nrf2)靶点及线粒体质量控制系统调控姜三七挥发油(essential oil from Stahlianthus involucratus rhizomes,EOSIR)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)损伤的保护作用机制。方法用氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导HUVECs建立细胞损伤模型,siRNA转染沉默Nrf2因子的表达,将培养好的细胞分为对照组、模型组、EOSIR组、转染组、转染阴性对照组,利用蛋白质印迹法(Western-blot)及定量即时聚合酶链锁反应(quantitative real time polymerase chain reaction,qRT-PCR)检测Nrf2及其下游因子的蛋白及mRNA的表达;Griess法检测一氧化氮含量;酶联免疫法(enzyme-linked immunosorbent assay,ELISA)分析人内皮素(endothelin 1,ET-1)、人前列环素(prostacyclin,PGI2)的含量;线粒体质量检测实验中将细胞分为空白组、ox-LDL诱导组、低剂量EOSIR组、中剂量EOSIR组、高剂量EOSIR组、阿司匹林阳性对照组,透射电镜观察HUVECs线粒体形态;Western-blot检测线粒体融合蛋白1(mitofusin 1,Mfn1)、线粒体融合蛋白2(mitofusin 1,Mfn2)、线粒体动力相关蛋白(dynamin-related protein 1,Drp1)、线粒体内膜融合蛋白(optic atrophy 1,Opa1)、线粒体转录因子A(mitochondrial transcription factor A,TFAM)、过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferatoractivated receptorγcoactivator-1α,PGC-1α)及轻链3A/B蛋白(LC3A/B)、泛素结合蛋白(p62)表达。结果EOSIR组的Nrf2及其下游因子的表达水平、NO、PGI2的含量较模型组均显著升高,ET-1水平较模型组相比显著降低,转染沉默Nrf2后,EOSIR对上述指标的改善作用被抑制;透射电镜观察线粒体形态,EOSIR干预组自噬小体数量较模型组明显减少,线粒体形态恢复正常,同时,EOSIR组显著改善了ox-LDL引起的线粒体相关蛋白表达的变化(P<0.05)。结论EOSIR可能通过激活Nrf2靶点及调控线粒体质量控制系统发挥对ox-LDL诱导的内皮细胞损伤的保护作用,延缓动脉粥样硬化的进程。

Objective To study the mechanism of Nuclear E2-related factor 2(Nrf2) mitochondria quality control system regulating the protective effect of essential oil from Stahlianthus involucratus rhizomes(EOSIR) on human umbilical vein endothelial cells(HUVECs) injury.Methods HUVECs were induced by oxidized low-density lipoprotein(ox-LDL) to establish a cell injury model,the expression of Nrf2 factor was silenced by siRNA transfection.The cultured cells were divided into control group,model group,EOSIR group,transfection group and transfection negative control group.The protein and mRNA expression of Nrf2 and its downstream factors were detected by Western blot and qRT-PCR;Griess method was used to detect the content of nitric oxide(NO);ELISA was used to analyze the content of human endothelin(ET-1) and human prostacyclin(PGI2);The cells were divided into control group,ox-LDL-induced group,low dose EOSIR group,medium dose EOSIR group,high dose EOSIR group and aspirin positive control group in the mitochondrial quality test.Transmission electron microscopy was used to observe the mitochondrial morphology of HUVECs,the expression levels of mitofusin 1(Mfn1),mitofusin 1(Mfn2),dynamin-related protein 1(Drp1),Optic atrophy 1(Opa1),mitochondrial transcription factor A(TFAM),peroxisome proliferator activated receptorγcoactivator-1α(PGC-1α)and autophagy related proteins LC3A/B,P62 was detected by Western blot.Results Compared with the model group,the expression levels of Nrf2 and its downstream factors,and the contents of NO and PGI2 in the EOSIR group were significantly increased,and the levels of ET-1 were significantly decrease than those of model group.After silencing Nrf2,the effect of EOSIR on the above indicators was inhibited;transmission electron microscopy observing the mitochondrial morphology,the number of autophagosomes in the EOSIR group was significantly reduced.compared with the model group,and the mitochondrial morphology returned to normal.At the same time,the EOSIR group significantly improved the changes in mitochondrial-related protein expression induced by ox-LDL(P<0.05).Conclusions EOSIR may play a protective role against ox-LDL-induced endothelial cell injury by activating the Keap1/Nrf2/ARE signaling pathway and regulating the mitochondrial quality control system.