Dihydromyricetin Regulates Autophagy To Protect Human Umbilical Vein Endothelial Cells From High Glucose-induced Injury

Diabetes has been considered as the third-leading health threats after cardiovasculardiseases and cancer worldwide. Diabetic vascular complications including diabetic heartdiseases, diabetic retinopathy etc, are the leading cause of morbidity and mortality amongpeople suffering from diabetes, and high glucose-induced injury in vascular endothelial cellsis a major initiator of diabetic vascular complications. Thus, it is necessary to further clarifythe underlying mechanisms for diabetic vascular complications, and to explore thecorresponding strategies for prevention and treatment of diabetic vascular complications.Autophagy as a lysosomal degradation pathway of cytosolic components such as proteinsand organelles plays an important role in the maintenance of cellular homeostasis. Theoccurrence of autophagy in response to environmental stress by self-regulatory mechanisms isgenerally regarded as a cell survival mechanism. However, impaired or excessive autophagyinduced by certain stress factors may lead to cell death. Autophagy is considered as adouble-edged sword in cell survival and death. Therefore, it is very important to elucidate therole of autophagy in high glucose-induced injury in vascular endothelial cells, the resolutionof this problem has major implications in the design of autophagy-targeted strategies forcounteracting the pathophysiological processes of diabetic vascular complications.The roles of flavonoids in the prevention and treatment of cardiovascular diseases,diabetes and other chronic non-communicable diseases have attracted much attention in recentyears. It has been reported that certain flavonoids including genistein and apigenin couldinhibit high glucose-induced injury in vascular endothelial cells, suggesting that flavonoidshave potential applications in the prevention and treatment of diabetic vascular complications.Dihydromyricetin (DHM), also named ampelopsin, one of flavonids is the major bioactiveconstituent of Ampelopsis grossedentata, which is a kind of medicinal and edible plant. It hasbeen reported that DHM exerts a number of biological and pharmacological actions includinghypoglycemic, anti-oxidative, and hepato-protective effects etc. One study has demonstrated that DHM has a significant hypoglycemic effect in alloxan-induced diabetic mice, but it isunclear whether DHM has a protective effect on high glucose-induced injury in vascularendothelial cells.In the present study, we observed the effects of DHM on high glucose-induced injury incultured human umbilical vein endothelial cells (HUVECs) in vitro, and then explored the roleof autophagy in high glucose-induced injury in HUVECs as well as the intervention effects ofDHM on the altered autphagy induced by high glucose.The main results and conclusions were summarized as follows:1. High glucose treatment significantly decreased cell viability and activities of antioxidantenzymes SOD and CAT, increased apoptotic rate, intracellular levels of reactive oxygen species(ROS) and inflammatory cytokines TNF-α, ICAM-1, and MCP-1in primary cultured HUVECs,while DHM pretreatment could significantly inhibit the above changes induced by high glucose,indicating that DHM has a protective role in oxidative damage and inflammation in HUVECsinduced by high glucose.2. High glucose treatment increased autophagy activity in cultured HUVECs as evidencedby decreased Mcherry-GFP-LC3punta and decreased number of autophagosome withdouble-membrane structure visible by transmission electron microscopy (TEM), which wasaccompanied by down-regulated expression of autophagy-related genes including BECN1,ATG5, LC3-Ⅱand up-regulated expression of p62by western blotting, suggesting that highglucose inhibit autophagy in HUVECs.3. The pretreatment with autophagy inhibitor3-MA not only increased highglucose–induced inhibition of autophagy activity but also exacerbated high glucose–induceddamage in HUVECs, whereas the pretreatment with autophagy agonist rapamycin (RAPA) notonly attenuated high glucose–induced inhibition of autophagy activity but also decreased highglucose–induced damage in HUVECs. These results indicate that high glucose-inducedautophagy impairment is implicated in the HUVECs injury induced by high glucose.4. DHM treatment increased autophagy activity in cultured HUVECs as evidenced byincreased Mcherry-GFP-LC3punta and increased number of autophagosome withdouble-membrane structure visible by transmission electron microscopy (TEM), which wasaccompanied by up-regulated expression of autophagy-related genes including BECN1, ATG5,LC3-Ⅱand down-regulated expression of p62by western blotting. Meanwhile, the decreased Mcherry-GFP-LC3punta and the number of autophagosome as well as the expression ofBECN1, ATG5, p62and LC3-Ⅱ in high glucose-treated HUVECs were partly reversed bypretreatment with DHM. These data suggest that DHM could promote autophagy activity andeffectively inhibit high glucose-induced autophagy impairment in HUVECs.5. The pretreatment with autophagy inhibitor3-MA attenuated the protective effect ofDHM on high glucose-induced injury in HUVECs, whereas the pretreatment with autophagyagonist RAPA enhanced the protective effect of DHM on high glucose-induced injury inHUVECs, suggesting that the promotion of autophagy is one of the mechanisms for theprotective effect of DHM on high glucose-induced injury in HUVECs.In conclusion, the inhibition of autophagy induced by high glucose is closely correlatedwith high glucose-induced injury in HUVECs, while DHM could protect HUVECs from theinjury induced by high hlucose through promotion of autophagy. However, the underlyingmechanisms for the regulation of autophagic activity in HUVECs by high glucose and DHMare needed to further clairfy.