Inhibitory Effect of Essential Oil from Fructus Alpiniae zerumbet on Endothelial-to-Mesenchymal Transformation Induced by TGF-β1 and down-regulating KLF4

Background: Endothelial Mesenchymal Transformation (EndMT) contributes to the development of cardiovascular disease. Krüpple factor 4 (KLF4) is a zinc finger transcription factor whose N-terminus can recruit acetyltransferase to promote histone acetylation, thereby affecting the transcription activation of downstream genes. Our previous studies have shown that EOFAZ has protective effects on HUVECs oxidative stress induced by TGF-β1. However, whether EOFAZ has a protective effect on EndMT induced by TGF-β1 and whether it is related to the regulation of downstream signals by KLF4 has not yet been elucidated.Methods: The protective effects of EOFAZ were evaluated in TGF-β1-treated EndMT in Human umbilical vein endothelial cells (HUVECs). Cell mobility was evaluated by wound-healing, transwell assays and angiogenesis experiment. Western blot analysis, Quantitative real-time PCR analysis (qRT-PCR) and immunofluorescence staining were utilized to determine the expression of endothelial and mesenchymal markers , KLF4, Histone 3 acetylation and Notch/Snail signaling axis. Small interfering RNA (siRNA) and adenovirus infection were used to determine the effciency of KLF4 inhibition and overexpression. Immunoprecipitation experiments were performed to analyze protein interactions.Results: We reported that EOFAZ has a protective effect on EndMT induced by TGF-β1. Deletion of KLF4 inhibited EndMT induced by TGF-β1 in HUVECs. EOFAZ pretreatment and KLF4 knockout reduced the migration ability of HUVECs , and increased endothelial markers accompanied by decreased mesenchymal markers, meanwhile caused the change of Notch/Snail signal axis. In addition, TGF-β1 upregulated the expression of KLF4, while the high expression of KLF4 promoted the acetylation of histone H3, and there was a protein interaction between the acetylated histone H3 and KLF4. Conclusions: These results suggest that TGF-β1 may promote the acetylation of histone H3 and activate the transduction of Notch/Snail signal axis by up-regulating the expression of KLF4, which may induce EndMT and this effect may be reversed by EOFAZ. Therefore, EOFAZ may inhibit EndMT induced by TGF-β1 by down-regulating KLF4 expression.