| Objective: Type 2 diabetes mellitus(T2DM)is a metabolic disease with a high prevalence rate in the world and a major public health problem in China.Vascular complications are the most common and serious complications of T2 DM,and the prevention and treatment of vascular complications is an important goal of the tertiary prevention of T2 DM.Endothelial dysfunction is the initiating link and key factor of vascular complications in T2 DM.However,the biomarkers and pathogenesis of endothelial dysfunction remain unclear.VAC is a kind of polyphenolic plant chemical.Previous studies have found that VAC can protect endothelial cells and their functions,but its protective mechanism has not been fully clarified.This study aims to explore biomarkers and pathogenesis of endothelial dysfunction in T2 DM,as well as VAC’s mechanism of action,which is of great significance for further understanding of vascular complications in T2 DM,and provide evidence for the application of polyphenolic phytochemicals in vascular protection of T2 DM.Methods: This study is divided into four parts:Part 1: To investigate the physiological status of T2 DM mice,the condition of aorta disease,and the biological effects of VAC.T2 DM mice model was established by high fat diet(high fat diet,HFD)combined with streptozotocin(STZ)intraperitoneal injection.After successful establishment of the model,the mice in the administration group were given VAC intragastric administration(1 mg/kg)for six weeks.Mice were sacrificed to obtain materials after oral glucose tolerance test(OGTT)and insulin tolerance test(ITT).Serum was collected to detect biochemical indexes.The thoracic aorta vessels were taken for histopathological staining,and the expression of signals related to inflammation,oxidative stress and mitochondrial damage was detected by q RT-PCR and Western blot.Part 2: Human umbilical vein endothelial cells(HUVEC)were stimulated with 35 m M high glucose(HG)solution for 24 h to simulate the vascular endothelium in T2 DM with hyperglycemia,and then treated with 5 μM VAC for 16 h.q RT-PCR,Western blot and fluorescent probe were used to detect endothelial inflammation,oxidative stress and mitochondrial damage caused by HG.By overexpression or knockdown of histone deacetylase1(HDAC1)and BCL2 interacting protein 3(BNIP3),the mechanism of HG-induced endothelial injury and the mechanism of VAC action are explored.Part 3: mi RNAs that may regulate HDAC1 and BNIP3 were screened using mi RNA databases such as Targetscan,and mi RNA-570-3p(mi R-570-3p)was preliminary identified as the target.The binding of mi R-570-3p to HDAC1 and BNIP3 was verified by luciferase reporter assay.The expression of mi R-570-3p was increased and inhibited using mi R-570-3p mimic and inhibitor to explore the role of mi R-570-3p in endothelial dysfunction caused by HG and whether it is related to VAC’s action.Part 4: Because VAC is a water-soluble substance with a large molecular weight,it cannot directly enter cells to play its role,so the mechanism of action of key membrane proteins in VAC’s relief of endothelial dysfunction caused by HG is explored.The disturbed membrane proteins in HG-induced HUVEC were screened by liquid chromatography and mass spectrometry(LC-MSMS),and the key targets were quantitatively analyzed by q RT-PCR.The target was identified as G protein subunit beta 1(GNB1)by molecular docking technique.The role of GNB1 in HG-induced endothelial dysfunction and the mechanism of VAC’s action were explored through overexpression and knockdown of GNB1.Results: Part 1: T2 DM mice showed decreased body weight,increased blood glucose,decreased glucose tolerance and insulin sensitivity,lipid metabolism disorder,liver function injury,and abnormal morphology of thoracic aorta vessels,which were improved after VAC administration.At the same time,thoracic aorta inflammation,oxidative stress and mitochondrial damage were increased in T2 DM mice,VAC could alleviate endothelial dysfunction.Part 2: The expression of inflammatory factors TNF-α and IL-1β,the production of oxidative stress indicators ROS and MDA increased,and the activities of GSH-Px and SOD decreased in HG-stimulated HUVEC.VAC or HDAC1 si RNA treatments significantly inhibited inflammatory and oxidative stress;VAC was unable to exert its anti-inflammatory and antioxidant effects while HDAC1 overexpression.Besides,the mitochondrial membrane potential(Δψm)decreased,the production of autolysosome and mitochondrial superoxide increased,and the PINK1/Parkin pathway was activated in HG-stimulated HUVEC.VAC significantly inhibited mitochondrial damage.VAC was unable to mitigate mitochondrial damage while BNIP3 overexpression.It has been shown that VAC exerts anti-inflammatory and antioxidant effects by inhibiting HDAC1 and anti-mitochondrial damage by inhibiting BNIP3.Part 3: VAC increased the expression of mi R-570-3p in HG-induced HUVEC and T2 DM thoracic aorta vessels.mi R-570-3p targets HDAC1 and BNIP3 3’-UTR.VAC or mi R-570-3p mimic could significantly inhibit the expression of HDAC1 and BNIP3,and relieve inflammation,oxidative stress and mitochondrial damage.When mi R-570-3p was inhibited,VAC was not effective in alleviating endothelial dysfunction.It is suggested that VAC can alleviate endothelial dysfunction by increasing mi R-570-3p.Part 4: GNB1 was significantly decreased in HG-stimulated HUVEC.The expression of mi R-570-3p was significantly increased after VAC treatment or GNB1 overexpression,thus alleviating endothelial dysfunction.The effect of VAC on endothelial dysfunction was also suppressed when GNB1 was inhibited.It is indicated that VAC regulates the expression of mi R-570-3p partly through GNB1,and thus plays a role in alleviating endothelial dysfunction.Conclusion: VAC can relieve physiological disorder,abnormal morphology and function of thoracic aorta in T2 DM mice.Through in vitro experiment,it was found that VAC exerts its vascular protective effect,which may be related to the regulation of HDAC1 and BNIP3 expression by GNB1/mi R-570-3p signaling axis.This study explored biomarkers of T2 DM endothelial dysfunction and the mechanism by which VAC alleviates T2 DM endothelial dysfunction.It provides the research basis and theoretical basis for mi R-570-3p as a biomarker of endothelial dysfunction in T2 DM,further understanding of vascular complications in T2 DM and application of polyphenol plant chemicals similar to VAC. |
