Betulinic Acid Induces eNOS Expression via the AMPK-Dependent KLF2 Signaling Pathway.

Betulinic acid (BA) is a natural pentacyclic triterpenoid with protective effects against inflammation, metabolic diseases, and cardiovascular diseases. We have previously shown that BA prevents endothelial dysfunction by increasing nitric oxide (NO) synthesis through activating endothelial nitric oxide synthase (eNOS) in human endothelial cells. However, the effect of BA on eNOS expression remains unclear. Thus, the aim of our study was to investigate the intracellular pathways associated with the effect of BA to regulate eNOS expression in human endothelial cells. BA significantly increased eNOS expression in a time- and concentration-dependent manner. Additionally, BA upregulated the expression of the transcription factor KLF2, which is known to regulate eNOS expression. KLF2 silencing in human endothelial cells attenuated the ability of BA to upregulate eNOS. BA also increased levels of intracellular Ca2+, activating CaMKK beta, CaMKII alpha, and AMPK. Inhibition of the TRPC calcium channel abolished BA-mediated effects on intracellular Ca2+ levels. Moreover, BA increased the phosphorylation levels of ERKS, HDACS, and MEF2C. Pretreatment of cells with compound C (AMPK inhibitor), LMK235 (HDACS inhibitor), and XMD8-92 (ERKS inhibitor) attenuated the BA-induced eNOS expression. Collectively, these findings suggest that BA induces eNOS expression by activating the HDACS/ERKS/KLF2 pathway in endothelial cells. The data presented here provide strong evidence supporting the use of BA to prevent endothelial dysfunction and treat vascular diseases, such as atherosclerosis.