基于Nrf2/ARE信号通路研究姜叶三七挥发油对氧化低密度脂蛋白诱导人脐静脉内皮细胞损伤的保护作用

The protective effect of essential oil of rhizome from Stahlianthus involucratus on ox-LDL-induced HUVECs damage based on Nrf2/ARE signaling pathway

目的研究姜叶三七挥发油(essential oil from Stahlianthus involucratus rhizomes,EOSIR)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的人脐静脉内皮细胞损伤的保护作用及机制。方法实验采用ox-LDL诱导人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)建立细胞损伤模型,分为空白对照组、模型组、EOSIR低、中、高剂量组(0.01、0.05、0.10μg/mL)以及阿司匹林组。通过水蒸气蒸馏法提取EOSIR,采用GC-MS联用技术对EOSIR的成分进行分析。采用MTT法确定ox-LDL诱导HUVECs细胞损伤的最佳浓度与时间,分析EOSIR对ox-LDL诱导HUVECs细胞存活率的影响。Hoechst 33342染色实验观察EOSIR对ox-LDL诱导HUVECs细胞损伤形态的变化。JC-1染色实验观察EOSIR对ox-LDL诱导HUVECs细胞的线粒体膜电位变化的影响。ELISA法分析EOSIR对ox-LDL诱导HUVECs细胞内乳酸脱氢酶(lactate dehydrogenase,LDH)、丙二醛(malondialdehyde,MDA)、一氧化氮(nitric oxide,NO)、超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)的含量影响。流式细胞术检测EOSIR对ox-LDL诱导HUVECs细胞内活性氧(reactive oxygen species,ROS)含量以及细胞凋亡的影响。实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)检测EOSIR对ox-LDL诱导HUVECs细胞的核因子E2相关因子2(nuclear factory erythroid-2 related,Nrf2)、醌氧化还原酶1(NADPH quinone oxidoreductase 1,NQO1)、血红素加氧酶-1(heme oxygenase-1,HO-1)、Bcl-2、Bax mRNA表达水平的影响。蛋白免疫印迹法(Western blot)检测EOSIR对ox-LDL诱导HUVECs细胞的Nrf2、NQO1、HO-1、Bcl-2、Bax蛋白表达水平的影响。结果(1)水蒸气蒸馏法提取得到的挥发油出油率为3.78%,GC-MS技术初步分析鉴定了EOSIR中54种化合物,占总挥发油含量的98.86%,其中含量最高的是莰烯,占22.38%;其次是α-蒎烯,占16.11%;居第三位的是D-樟脑,占9.74%;第四位的是3,3,6,6-四甲基-3,6,7,8-四氢化-并茚-1(2H)-酮,占8.41%。(2)MTT结果显示:与空白对照组相比,EOSIR在0.01~60μg/mL时对正常的HUVECs细胞没有毒性,但大于80μg/mL时有明显的细胞毒性(P<0.01);在200μg/mL的ox-LDL诱导24小时条件下,HUVECs存活率为54.32%(P<0.01),以此作为造模的最佳条件;与模型组比较,EOSIR能显著抑制ox-LDL诱导的HUVECs细胞存活率下降(P<0.01)。(3)Hoechst 33342染色结果显示:与空白对照组相比,模型组出现较强的蓝色荧光,给药EOSIR能明显减弱荧光强度并显著改善细胞损伤的形态。(4)JC-1染色结果显示,与空白对照组相比,模型组损伤的细胞表现出绿色荧光强度增强,红色荧光强度下降,给药EOSIR能减弱绿色荧光强度并增强红色荧光强度。(5)ELISA结果显示,与空白对照组相比,模型组的LDH、MDA含量升高,SOD、CAT、GSH-Px、NO的含量减少(P<0.05,P<0.01)。给药EOSIR能降低LDH、MDA含量,升高SOD、CAT、GSH-Px、NO的含量(P<0.05,P<0.01)。(6)流式细胞术结果发现,与空白对照组相比,模型组的ROS含量显著升高,凋亡率显著增加(P<0.05)。给药EOSIR能显著下调ROS含量并降低细胞的凋亡率(P<0.05,P<0.01)。(7)RT-qPCR分析表明,ox-LDL能诱导激活促凋亡基因Bax mRNA的表达,降低抗凋亡基因Bcl-2 mRNA的表达,并且降低Nrf2、NQO1、HO-1 mRNA的表达(P<0.01)。EOSIR能显著下调Bax mRNA表达水平,上调Nrf2、NQO1、HO-1、Bcl-2 mRNA表达水平(P<0.05,P<0.01)。(8)蛋白免疫印迹法结果表明,ox-LDL能诱导激活促凋亡蛋白Bax的表达水平,降低抗凋亡蛋白Bcl-2的表达水平,并且降低Nrf2、NQO1、HO-1蛋白表达水平(P<0.01)。EOSIR能显著下调Bax蛋白表达水平,上调Nrf2、NQO1、HO-1、Bcl-2蛋白表达水平(P<0.05,P<0.01)。EOSIR抑制了ox-LDL诱导的HUVEC细胞毒性和凋亡。同时,EOSIR显著抑制了ox-LDL处理的细胞中ROS、LDH和MDA水平的增加,并增加了NO、SOD、CAT和GSH-Px水平。EOSIR上调了Nrf2、HO-1和NQO1的mRNA和蛋白表达水平。此外,EOSIR能够显著降低了与细胞凋亡相关的生化变化,例如MMP的丢失、Bax mRNA和蛋白质水平的下调以及ox-LDL处理的细胞中Bcl-2 mRNA和蛋白质水平的上调。结论EOSIR能够通过调节线粒体依赖性细胞凋亡和激活Nrf2/ARE信号通路来预防ox-LDL诱导的内皮损伤。

Objective To explore the protective effect and mechanism of the essential oil from Stahlianthus involucratus rhizomes(EOSIR)on vein endothelial cells damage induced by oxidized low-density lipoprotein(ox-LDL).Methods The experiment used ox-LDL to induce human umbilical vein endothelial cells(HUVECs)to establish a cell damage model,which was divided into blank control group,model group,low,middle and high dose group of EOSIR(0.01,0.05,0.10μg/mL)and aspirin group.EOSIR was extracted by steam distillation,and the components of EOSIR were analyzed by GC-MS.MTT assay was used to determine the optimal concentration and time of ox-LDL-induced HUVECs cell damage and to analyze the effect of EOSIR on the survival rate of HUVECs induced by ox-LDL.Hoechst 33342 staining experiment observed changes in the morphology of ox-LDL-induced HUVECs cell damage by EOSIR.JC-1 staining experiment observed the effect of EOSIR on the change of the mitochondrial membrane potential of ox-LDL-induced HUVECs.ELISA method was used to analyze the effect of EOSIR on the contents of lactate dehydrogenase(LDH),malondialdehyde(MDA)levels,increased nitric oxide(NO),superoxide dismutase(SOD),catalase(CAT),and glutathione peroxidase(GSH-Px)in HUVECs induced by ox-LDL.Flow cytometry was used to detect the effect of EOSIR on the content of reactive oxygen species(ROS)in ox-LDL-induced HUVECs and apoptosis.Real-time quantitative PCR(RT-qPCR)was used to detect the effect of EOSIR on the expression levels of nuclear factory erythroid-2 related(Nrf2),NADPH quinone oxidoreductase 1(NQO1),heme oxygenase-1(HO-1),Bcl-2 and Bax mRNA in ox-LDL-induced HUVECs.Western blot analysis was used to detect the effect of EOSIR on the protein expression levels of Nrf2,NQO1,HO-1,Bcl-2,Bax in HUVECs induced by ox-LDL.Results(1)The output rate of essential oil extracted by steam distillation was 3.78.The preliminary analysis of GC-MS technology identified 54 compounds in EOSIR,accounting for 98.86%of the total essential oil content,of which the highest content was camphene(22.38%),followed byα-pinene(16.11%),D-camphor(9.74%)and 3,6,7,8-tetrahydro-3,3,6,6-tetramethyl-as-indacen-1(2H)-one(8.41%).(2)MTT results showed that compared with the blank control group,EOSIR had no toxicity to normal HUVECs cells in the concentration range of 0.01-60μg/mL,but had significant cytotoxicity when the concentration was above 80μg/mL(P<0.01).Under the 24-hour ox-LDL induction condition of 200μg/mL,the survival rate of HUVECs was 54.32%(P<0.01),which was used as the optimal condition for modeling.Compared with the model group,EOSIR significantly inhibited the decline of survival rate of HUVECs induced by ox-LDL.(3)The Hoechst 33342 staining results showed that compared with the blank control group,the model group had stronger blue fluorescence.The treatment with EOSIR could significantly weaken the fluorescence intensity and improve the morphology of cell damage.(4)The JC-1 staining results showed that compared with the blank control group,the damaged cells in the model group showed an increase in green fluorescence intensity and a decrease in red fluorescence intensity and the treatment with EOSIR could weaken the green fluorescence intensity and enhance the red fluorescence intensity.(5)The ELISA results showed that compared with the blank control group,the contents of LDH and MDA in the model group increased,and the contents of SOD,CAT,GSH-Px and NO decreased(P<0.05,P<0.01).The treatment with EOSIR could decrease the contents of LDH,MDA and increase the contents of SOD,CAT,GSH-Px and NO(P<0.05,P<0.01).(6)The of flow cytometry results showed that compared with the blank control group,the ROS content of the model group and apoptosis rate significantly increased(P<0.01).The treatment with EOSIR could significantly down-regulate ROS content and decrease apoptosis rate(P<0.05,P<0.01).(7)RT-qPCR analysis showed that ox-LDL could activate the expression of pro-apoptotic gene Bax mRNA and reduce the expression of anti-apoptosis gene Bcl-2 mRNA,Nrf2,NQO1 and HO-1 mRNA(P<0.01).It was found that EOSIR could significantly down-regulate the expression of Bax mRNA and up-regulate the expression of Nrf2,NQO1,HO-1 and Bcl-2 mRNA(P<0.05,P<0.01).(8)Western blot results showed that ox-LDL could activate the expression of pro-apoptotic protein Bax and decrease the expression level of anti-apoptosis protein Bcl-2,Nrf2,NQO1 and HO-1 protein(P<0.01).EOSIR could significantly down-regulate the expression level of Bax protein and up-regulate the protein expression levels of Nrf2,NQO1,HO-1 and Bcl-2(P<0.05,P<0.01).Conclusion The EOSIR prevents ox-LDL-induced HUVECs damage by regulating the mitochondrial-dependent apoptosis and activating Nrf2/ARE signaling pathways.