A Study On The Inhibitory Roles Of Dihydromyricetin In High Glucose-induced Vascular Endothelial Cell Injury Through Activating AMPK

According to the latest research,adult diabetes mellitus(DM)prevalence rate in our country is 11.6%,which has become ‘the third killer’ threat to people’s health,next only to cardiovascular disease and cancer.Not only is diabetic cardiovascular disease is one of the most common complications of diabetes,but also it is the main cause of disabilities and death in diabetic patients.In addition,high glucose-induced endothelial cell damage is the start of diabetic vascular complications.Based on the above description,the strict control of blood glucose is the primary therapeutic method,but the efficiency of glucose control in diabetic patients is still low.Therefore,the research on the mechanisms of high glucose-induced vascular endothelial cells damage and its protective measures have important theory value and application prospect.As a large number of researches show,AMP-activated protein kinase(AMPK)is an important target for the prevention of glucolipid metabolic disorder-related diseases.In recent years,some studies have found that AMPK signaling pathway plays an important regulatory role in vascular endothelial cells function.AMPK is heterotrimeric complex composed of a catalytic subunit α and regulatory subunit β and γ.Upstream kinase activates AMPK by phosphorylating the Thr172 loci in α subunit.The research has found that glycogen cause allosteric inhibition of AMPK activity through combining the glycogen-binding domain(GBD)in β subunit.Glycogen synthetase(GS)is a key enzyme of glycogen synthesis regulation,and protein kinase B(Akt)as well as glycogen synthase kinase-3β(GSK3β)also play an important regulatory role in the activity of GS.γ subunit cause allosteric regulation of AMPK through perceiving the AMP/ATP ratio changes in cells.The research has proved that the high glucose-induced myocardial as well as endothelial progenitor cells damage are closely related to the inhibition of cellular AMPK activity,but the impact of AMPK in high glucose-induced endothelial cell injury is still unclear.Ampelopsis grossedentata is a wild liane of the vitaceae ampelopsis michx,which mainly distributes in the southern mountain areas in China such as Hunan,Hubei,Guanxi and Fujian.In China many minority groups have the habit of drinking ampelopsis grossedentata,and view it as a traditional medicine for cold fever,sore throat and icteric model hepatitis,etc.Dihydromyricetin is a kind of flavonoids chemical with an extremely rich content in ampelopsis grossedentata(about 25%).It is widely accepted that dihydromyricetin has a comprehensive range of benefits such as antioxidant,bacteriostasis,anti-hyperlipidemia and anti-tumor,thus it is considered as the main active ingredients in ampelopsis grossedentata.Our previous study has demonstrated that myricetin plays a protective role in high glucose-induced vascular endothelial cells oxidative stress injury.Based on the similar molecular structure between dihydromyricetin and myricetin,we hypothesize that dihydromyricetin may also have a protective effect on high glucose-induced endothelial cell damage.In vitro,high glucose-induced vascular endothelial cell damage model is established through stimulating the primary human umbilical vein endothelial cells(HUVECs)with high glucose.Cell activity assay,flow cytometry analysis,protein immunoblot,anthranone-sulphuric acid colorimetric,real-time fluorescent quantitative PCR,chemiluminescence,ELISA and other experimental methods are used to explore the role of AMPK activity alterations in high glucose-induced vascular endothelial cell injury and its underlying mechanisms,and find the protective roles and mechanisms of dihydromyricetin intervention in high glucose-induced vascular endothelial cell injury,which aims to provide experiment evidence for dihydromyricetin intervention in vascular complications of diabetes.The main research results and conclusions are as follows:1.With the stimulation of high glucose,HUVECs has a significant decrease in cell viability and increase in apoptosis rate,and dihydromyricetin intervention significantly inhibited the above alterations,which indicates that dihydromyricetin has a protective role in high glucose-induced HUVECs injury.2.In high glucose-induced HUVECs,the phosphorylation of AMPK and its downstream target protein ACC are significantly decreased,which indicates that high glucose inhibited AMPK activity in HUVECs.The pretreatment of AMPK activator-AICAR inhibits the decreased cell viability and the increased apoptosis rate induced by high glucose.By contrast,AMPK inhibitor-compound C significantly counteracted the protective effect of AICAR on the decreased cell viability and the increased apoptosis induced by high glocose,indicating that the inhibition of AMPK activity contributes to high glucose-induced cell injury.3.Compared with the control group,dihydromyricetin significantly increases the expression levels of p-AMPK and p-ACC.In addition,dihydromyricetin significantly reverses the decreased expression levels of p-AMPK and p-ACC induced by high glucose,which indicates that dihydromyricetin can reverse the inhibitory role of high glucose in AMPK activity.By contrast,AMPK inhibitor-compound C alleviates the protective role of dihydromyricetin,further indicating that dihydromyricetin inhibits high glucose-induced HUVECs injury through activation AMPK.4.Compared with the control group,intracellular glycogen content,GS m RNA and protein expression,Akt and GSK3β protein expression and phosphorylation increases significantly in high glucose-induced HUVECs,indicating that high glucose promotes glycogen synthesis and further inhibits AMPK activity through Akt-GSK3β-GS pathways.5.Compared with the control group,in dihydromyricetin-induced HUVECs the levels of ATP and the ATP synthase complex(also called Complex V or F1F0-ATPase)declined obviously,which indicates that dihydromyricetin activate AMPK by inhibiting the activity of ATP synthetase and further reducing the content of ATP.To sum up,high glucose increased glycogen content in HUVECs and further decreased AMPK activity through Akt-GSK3β-GS pathway,thus causing the injury of HUVECs.But dihydromyricetin pretreatment can reverse the effects of high glucose on HUVECs through inhibiting the activity of mitochondrial enzyme complex F1F0-ATPase,reducing the synthesis of ATP and increasing AMPK activity.