Protective effect of alizarin on vascular endothelial dysfunction via inhibiting the type 2 diabetes-induced synthesis of THBS1 and activating the AMPK signaling pathway

In this study, we investigated the protective effects of alizarin (AZ) on endothelial dysfunction (ED). AZ has inhibition of the type 2 diabetes-induced synthesis of THBS1. AMP-activated protein kinase (AMPK), particularly AMPKα2 isoform, plays a critical role in maintaining cardiac homeostasis. The aim of this study was to investigate the ameliorative effect of AZ on vascular injury caused by diabetes and to reveal the potential mechanism of AZ in high glucose (HG)-stimulated HUVECs and diabetic model rats. HUVECs cells, rats and AMPK−/− transgenic mice were used to investigate the mitigating effects of AZ on vascular endothelial dysfunction caused by T2DM and its ex vivo and in vivo molecular mechanisms. In type 2 diabetes mellitus (T2DM) and HUVECs, the inhibitory effect of alizarin on THBS1 synthesis was verified by immunohistochemistry (IHC), immunofluorescence (IF) and Western blot (WB) so that increase endothelial nitric oxide synthase (eNOS) content in vitro and in vivo. In addition, we verified protein interactions with immunoprecipitation (IP). To probe the mechanism, we also performed AMPKα2 transfection. AMPK's pivotal role in AZ-mediated prevention against T2DM-induced vascular endothelial dysfunction was tested using AMPKα2 gene (AMPKα2-KO) mice. We first demonstrated that THBS1 and AMPK are targets of AZ. In T2DM, THBS1 was robustly induced by high glucose and inhibited by AZ. Furthermore, AZ activates the AMPK signaling pathway, and recoupled endothelial nitric oxide synthase in stressed endothelial cells which plays a protective role in vascular endothelial dysfunction. The main finding of this study is that chymotrypsin can play a role in different pathways of vascular injury due to type 2 diabetes. Mechanistically, alizarin inhibits the increase in THBS1 protein synthesis after high glucose induction and activates AMPKα2, which increases NO release from eNOS, which is essential in the prevention of vascular endothelial dysfunction caused by T2DM.