Inhibitory Effect of Essential Oil from Fructus Alpinia zerumbet on Endothelial-to-mesenchymal Transition Induced by High Glucose via Nrf2/Notch1 Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

Objective:To investigate the inhibitory effect and the possible mechanism of essential oil from fructus Alpinia zerumbet （EOFAZ） on endothelial-to-mesenchymal transition （EndMT） induced by high glucose （HG）. Method:Human umbilical vein endothelial cells （HUVECs） was cultured in vitro to analyze the pharmacodynamic effects of EOFAZ on EndMT and oxidative stress damage induced by HG. The experiment was set the blank group， HG group （35 mmol·L-1）， EOFAZ low dose group （1 μg·L-1） and EOFAZ high dose group （4 μg·L-1）. After EOFAZ intervention for 2 h， HG was added to incubate for 72 h in order to establish EndMT cell model. Western blot was used to detect the protein expression of vimentin and platelet endothelial cell adhesion molecule （CD31）. Angiogenesis experiment was used to detect the ability of cell migration ability in order to analyze the effect of EOFAZ on EndMT. The changes of reactive oxygen species （ROS） levels were detected by 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence probe and the contents of malondialdehyde （MDA）， superoxide dismutase （SOD） and catalase （CAT） in cells were detected by the kit method to analyze the effect of EOFAZ on oxidative stress. Western blot was used to detect the protein expression levels of nuclear transcription factor E2 related factor 2 （Nrf2） and Notch1. The overexpression of Nrf2 was achieved by adenovirus （AD） transfection and the mechanism of EOFAZ inhibiting EndMT was further analyzed. The experiment was set the blank group， HG group （35 mmol·L-1）， AD-Nrf2 group， EOFAZ group （4 μg·L-1）， AD-Nrf2+EOFAZ group （4 μg·L-1）. The cells were infected with recombinant adenovirus overexpression plasmid of Nrf2 gene for 6 h， then replaced with normal medium for 24 h. After EOFAZ intervention for 2 h， HG was added to co-incubate for 72 h to induce EndMT. Western blot was used to detect the protein expressions of Nrf2， CD31， vimentin， Notch1 and Snail. Result:Compared with the HG group， after treatment with EOFAZ， the protein expression of CD31 was significantly up-regulated （P<0.05）， the protein expression of vimentin was significantly down-regulated （P<0.01）， the ability of cell migration was decreased （P<0.01）， and the contents of ROS and MDA were decreased （P<0.05， P<0.01）， the levels of CAT and SOD were increased （P<0.01）. In addition， EOFAZ could significantly up-regulate the protein expression of antioxidant signal Nrf2 （P<0.01） and down-regulate the protein expression of Notch1 （P<0.01）. High expression of Nrf2 was achieved by stable AD transfection into HUVECs. The results of Western blot showed that， compared with the HG group， the protein expression levels of Nrf2 and CD31 in each treatment group were significantly increased （P<0.01）， while the protein expression levels of vimentin， Notch1 and Snail were down-regulated （P<0.01）. At the same time， compared with the AD-Nrf2 group， the AD-Nrf2+EOFAZ group could further up-regulate the protein expressions of Nrf2 and CD31 （P<0.05， P<0.01）， while decrease the protein expression levels of vimentin， Notch1 and Snail （P<0.01）. Conclusion:EOFAZ ameliorates oxidative stress injury of vascular endothelial cells induced by HG and inhibits EndMT， which is related to Nrf2/Notch1 signaling pathway.