Protective Effects And Mechanisms Of Vaccarin Against High Glucose-induced Vascular Endothelial Cell Injury

Cardiovascular complications are the most leading cause of morbidity and mortality in patients suffering from diabetes.Vascular endothelial dysfunction and apoptosis is a core pathophysiological event in the early stage of diabetes mellitus and leads to cardiovascular diseases’ complications.Vaccarin,an active flavonoid glycoside from vaccariae semen,exhibits extensive biological activities including vascular endothelial cell protection effects.In this study,we evaluated the effects of vaccarin on NO generation in response to high glucose(HG).Here,human microvascular endothelial cell-1(HMEC-1)cells were stimulated with 35 mM glucose for 48 h in the presence or absence of pre-incubation with vaccarin for 12 h.ELISA results showed that vaccarin dose-relatedly promoted NO production in HMEC-1 cells in response to HG,reaching its maximal effect at the concentration of 5 μM.Under HG condition,vaccarin increased NO generation and the phosphorylation of e NOS in HMEC-1 cells,which was abolished by pretreatment with AMPK inhibitor compound C(CC),miR-34 a mimic and eNOS inhibitor NG-Nitro-L-arginine Methyl Ester(L-NAME).Vaccarin downregulated the expression of miR-34 a,which was reversed by AMPK inhibitor CC and miR-34 a mimic.The phosphorylation of AMPK was activated by vaccarin,which was prevented by AMPK inhibitor CC.Reactive oxygen species(ROS)accumulation,the dephosphorylation of AMPK and eNOS,miR-34 a overexpression and the decrease of NO production in reponse to HG in HMEC-1 cells were all reversed using either vaccarin or ROS scavenger.These results reveal that vaccarin prevents endothelial dysfunction via ROS/AMPK/miR-34a/eNOS pathway.Further,we evaluated the protective effects of vaccarin against HG-induced endothelial cell apoptosis.CCK-8 assay showed that exposure of HMEC-1 cells to HG markedly decreased cell vitality,which was effectively attenuated by preconditioning with vaccarin in a dose-dependent manner.The maximum protective effect was observed with 5 μM vaccarin.Vaccarin rescued HG-evoked cell cycle arrest in HMEC-1 cells.The apoptosis of HMEC-1 was significantly inhibited by vaccarin,as evidenced by TUNEL assay and flow cytometry results.DHE staining showed that HG-induced ROS production was abolished by vaccarin.Pre-incubation with vaccarin partially rescued HG-triggered reductions of superoxide dismutase(SOD),catalase(CAT)and glutathion peroxidase(GSH-Px)activities.Inhibition of histone deacetylase1(HDAC1)was involved in the protective effects of vaccarin,as evidenced by western blot(WB)bands,TUNEL assay and flow cytometry results.Inhibition of ROS mediated the protective effects of vaccarin upon to HG.Taken together,these data suggested that vaccarin protected against HG-induced endothelial cell apoptosis via inhibition of ROS accumulation and HDAC1 over-expression.We investigated the effects of vaccarin on endothelial injury in vivo.In this study,we built the animal models for type 2 diabetes with a combination of high-dose streptozotocin(STZ)and high-fat diet(HFD).After four weeks’ operation,these mice were subjected to intraperitoneal injection of vaccarin or saline for four weeks.We found that vaccarin prevented the increase of blood glucose,reduced the level of glycosylated hemoglobin,increased glucose and insulin tolerance,relieved the disorder of lipid metabolism and oxidative stress in STZ/HFD-induced type 2 diabetes in mice.Vaccarin improved endothelium-dependent vasorelaxation in type 2 diabetes mice.Western blot showed that vaccarin promoted the phosphorylation of AMPK,eNOS and downregulated HDAC1 protein level in type 2 diabetes mice.MiR-34 a overexpression was prevented by vaccarin,as evidenced by qPCR assay.Vaccarin obviously suppressed caspase-3 activity detected by caspase-3 activity assay kit.Vaccarin may be a potential therapeutic agent for prevention and treatment of diabetes mellitus.