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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 expressi