基于Notch/Snail信号的艳山姜挥发油对内皮间质转分化的保护作用研究

目的：探讨艳山姜挥发油对转化生长因子β₁（TGF-β₁）诱导的内皮间质转分化（EndMT）的干预作用及信号机制。方法：以10 ng·mL^-1 TGF-β₁诱导人脐静脉内皮细胞（HUVECs）建立EndMT模型。实验共分为两个部分：（1）分组：正常组，模型组（10 ng·mL^-1 TGF-β₁），EOFAZ低剂量组（10 ng·mL^-1 TGF-β₁+1 μg·L^-1 EOFAZ），EOFAZ高剂量组（10 ng·mL^-1 TGF-β₁+4 μg·L^-1 EOFAZ）。EOFAZ干预作用2 h后加入TGF-β₁共同孵育72 h。采用Transwell小室实验检测细胞迁移能力；波形蛋白（Vimentin）和血小板-内皮细胞粘附分子（CD31）以及Notch-1的蛋白表达情况通过蛋白免疫印迹法检测。（2）分组：正常组，模型组（10 ng·mL^-1 TGF-β₁），γ-分泌酶抑制剂组（10 ng·mL^-1 TGF-β₁+15 μmol·L^-1 DAPT），EOFAZ高剂量组（10 ng·mL^-1 TGF-β₁+4 μg·L^-1 EOFAZ）。DAPT及EOFAZ干预作用2 h后加入TGF-β₁共同孵育72 h。通过蛋白免疫印迹法检测Notch-1，Snail和Slug的表达。结果：内皮细胞经TGF-β₁处理72 h后，细胞迁移能力明显增强（P<0.01），CD31蛋白表达水平显著降低（P<0.01），Vimentin，Notch-1，Snail和Slug蛋白表达水平显著提高（P<0.01，P<0.05），给予EOFAZ作用后能逆转上述改变。同时在给予DAPT阻断Notch信号后，Notch-1，Snail和Slug蛋白水平下降（P<0.05）。结论：EOFAZ干预TGF-β₁诱导EndMT的作用与Notch信号相关，其可以进一步调控Snail和Slug的表达。

Protective effect of Essential Oil from Fructus Alpinia Zerumbeton on Endothelial-to-mesenchymal transition based on Notch/Snail signal

OBJECTIVE To investigate the intervention effect and mechanism of EOFAZ on Endothelial-to-mesenchymal transition (EndMT) induced by transforming growth factor β₁ (TGF-β₁).METHODS Human umbilical vein endothelial cells (HUVECs) was induced by 10 ng·mL^-1 TGF-β₁ to establish EndMT model. The experiment was divided into two parts: (1)Grouping: normal group and model group (10 ng·mL^-1 TGF-β₁), EOFAZ low dose group (10 ng·mL^-1 TGF-β₁ +1 μg·L^-1 EOFAZ), EOFAZ high dose group (10 ng·mL^-1 TGF-β₁ + 4 μg·L^-1 EOFAZ). After EOFAZ intervention for 2 hours, TGF-β₁ was added to incubate for 72 hours. Transwell experiment was used to detect the ability of cell migration. Western blot was used to detect the protein expression of mesenchymal cell marker Vimentin and endothelial cell marker Platelet endothelial cell adhesion molecule-1 (CD31), and the protein expression of Notch-1. (2)Grouping: normal group and model group (10 ng·mL^-1 TGF-β₁), γ-secretase inhibitor group (10 ng·mL^-1 TGF-β₁+15 μmol·L^-1 DAPT), EOFAZ high dose group (10 ng·mL^-1 TGF-β₁ + 4 μg·L^-1 EOFAZ). After EOFAZ and DAPT intervention for 2 hours, TGF-β₁ was added to incubate for 72 hours. Western blot was used to detect the protein expression of Notch-1, Snail and Slug.RESULTS After treated with TGF-β₁ for 72 h, the migration ability of HUVECs cells was significantly increased (P<0.01), the expression of CD31 protein was significantly decreased (P<0.01). The expression of Vimentin, Notch-1, Snail and Slug protein was significantly increased (P<0.01)(P<0.05), which could be reversed by EOFAZ. At the same time, after blocking Notch signal with DAPT, the protein levels of Notch-1, Snail and Slug decreased(P<0.05).CONCLUSION The effect of EOFAZ on EndMT induced by TGF-β₁ was related to Notch signal, and could further regulate the expression of Snail and Slug.