艳山姜挥发油对TGF-β_1诱导人脐静脉内皮细胞间质转分化的保护作用

Protective Effect of Essential Oil from Fructus Alpinia zerumbet Against Endothelialmesenchymal Transition in Human Umbilical Vein Endothelial Cells Induced by TGF-β_1

目的:研究艳山姜挥发油(EOFAZ)对转化生长因子-β_1(TGF-β_1)诱导人脐静脉内皮细胞(HUVECs)间质转分化的干预保护作用。方法:采用TGF-β_1(10μg·L^(-1),孵育48 h)诱导建立内皮-间质转化(End MT)细胞模型。实验分组为正常组,模型组(10μg·L^(-1)TGF-β_1),EOFAZ高剂量组(10μg·L^(-1)TGF-β_1+4μg·L^(-1)EOFAZ)及EOFAZ低剂量组(10μg·L^(-1)TGF-β_1+2μg·L^(-1)EOFAZ)。EOFAZ于造模前干预给药2 h,加入TGF-β_1共孵育复制End MT细胞模型。倒置显微镜下观察细胞形态,划痕实验分析细胞迁移能力,蛋白免疫印迹法(Western blot)检测EOFAZ对内皮细胞特异性标志物血管内皮细胞钙粘素(VE-cadherin),间充质细胞特异性标志物α-平滑肌肌动蛋白(α-SMA),锌指转录因子(Snail)及核转录因子-κB(NF-κB)p-p65蛋白表达的影响。结果:TGF-β_1诱导孵育后,细胞形态多呈长梭形,并可见明显的细胞丝状伪足,细胞间的紧密连接减少;划痕实验结果表明模型组细胞迁移能力增强(P<0.01);VE-cadherin蛋白表达显著减少(P<0.01),α-SMA,Snail,NF-κB p-p65蛋白表达量明显增多(P<0.01)。EOFAZ高、低剂量组干预给药后,能够明显抑制TGF-β_1诱导的细胞迁移能力(P<0.01),同时能增加VE-cadherin的表达,降低α-SMA,Snail及NF-κB p-p65蛋白的表达。结论:艳山姜挥发油对TGF-β_1诱导的HUVECs间质转分化具有干预保护作用,其机制与抑制NF-κB p65的磷酸化激活有关。

Objective： To explore the protective effect of essential oil from fructus Alpinia zerumbet（ EOFAZ） on endothelial-mesenchymal transition（ End MT） in human umbilical vein endothelial cells（ HUVECs）induced by transforming growth factor-β1（ TGF-β1）. Method： HUVECs were cultured in vitro and the End MT cell model was reproduced by 10 μg·L^-1TGF-β1incubated for 48 h. The experiment was divided into four groups,such as normal group,model group（ 10 μg·L^-1TGF-β1）,EOFAZ high dose group（ 10 μg·L^-1TGF-β1＋4 μg·L^-1EOFAZ） and EOFAZ low dose group（ 10 μg·L^-1TGF-β1＋ 2 μg·L^-1EOFAZ）. After pre-incubation with EOFAZ（ 4,2 μg·L^-1） for 2 h,the End MT cell model was reproduced. The cell morphology was observed under inverted microscope,and the cell migration ability was analyzed by scratch test. Western blot was used to detect the specific protein expression of vascular endothelial cadherin（ VE-cadherin）,α-smooth muscle actin（ α-SMA）, zinc finger transcription factor（ Snail） and nuclear transcription factor-κB（ NF-κB） p-p65. Result：After incubation with TGF-β1,the cell morphology of HUVECs was long spindle shape with visible cells of filamentous pseudopodia,the close connection between cells were reduced,and the cell migration ability of the model group was significantly increased（ P〈0. 01）, and the protein expression of VE-cadherin were downregulated significantly（ P〈0. 01）. On the contrary, the expression of α-SMA, Snail and NF-κB p-p65 was significantly increased（ P〈0. 01）. Pretreatment with high dose of EOFAZ, the cell migration ability was significantly inhibited; meanwhile,the expression of VE-cadherin was significantly increased（ P〈0. 01）,and expression of α-SMA,Snail and NF-κB p-p65 protein were significantly decreased（ P〈0. 01）. Conclusion：EOFAZ has protective effect on End MT in HUVECs induced by TGF-β1,and the mechanism is related to inhibition of phosphorylation of NF-κB p65.