Effect Of Resistin On Endothelial Cell Function And Autophagy Based On AMPK And Comparison With High Glucose

Arteriosclerotic cardiovascular disease(ASCVD)is the main content of cardiovascular disease.Vascular endothelial dysfunction is the initiation of atherosclerosis(AS),and it runs through the development of atherosclerosis.Resistin is a cytokine that is specifically secreted by an adipocyte and macrophage,named for its role in the development of insulin resistance.Studies have shown that resistin is an independent risk factor of cardiovascular,cerebrovascular events and coronary atherosclerotic heart disease.Resistin can enhance the adhesion of endothelial cells to monocytes,induces inflammatory reaction and promote insulin resistance,which causes endothelial cell damage and promotes the development of atherosclerosis.Therefore,it is crucial to explore the effect of resistin on endothelial cell function and its mechanism for the prevention of AS.AMP-activated protein kinase(AMPK)as a sensor of cellular energy metabolism,plays an important role in cellular insulin resistance,energy metabolism regulation,and autophagy.AMPK is considered to be a potential target of anti-atherosclerotic treatment.Studies have shown that resistin can inhibit AMPK phosphorylation in endothelial cells,suggesting that AMPK may be a target of resistin in endothelial cells.Autophagy is a process of transporting abnormal or aging proteins and organelles into lysosomes for digestion and degradation.It plays an important role in maintaining cell homeostasis and regulating cell energy metabolism.Studies have shown that endothelial cells autophagy is contacting with the development of AS,and AMPK is a key molecule in the regulation of autophagy.The absence of autophagy in endothelial cells leads to dysfunction of endothelial cells and promotes the occurrence of AS.Resistin may effect the autophagy in endothelial cells through inhibiting AMPK phosphorylation.Insulin resistance caused by resistin contributes to elevate blood glucose.Studies also have shown that high glucose increaseed the expression of resistin gene in endothelial cells,suggesting a mutual promotion between high glucose and resistin in AS.In addition,there is a similar effect of high glucose and resistin on enhancing inflammation reaction and inducing the insulin resistance in endothelial cells.The above suggests that high glucose is close connect to resistin.Comparing and exploring the effects of high glucose,resistin and cobined action of them in endothelial cells can provide new target of the treatment for atherosclerosis in diabetic patients.Part One: Resistin affects endothelial cellfunction and autophagy via AMPK Objective: Human umbilical vascular endothelial cells(HUVECs)were used as model to investigate the effects of resistin on endothelial cell function and autophagy based on AMPK.Methods:The experimental group was set as: blank control group(Con group),resistin group(R group),resistin combined with AMPK inhibitor Compound C group(Compound C10?M pretreatment 1h+resistin 100ng/ml,R+C group),and resistin combined with AMPK agonists AICAR(AICAR 100 ?M pretreatment 30 min + resistin 100 ng/ml,R+A group).After starvation for 12 h in low serum medium,endothelial cells were treated for 24 h according to the groups.Endothelial proliferation and apoptosis were detected by scratch test and Annexin V-FITC/PI staining respectively.The basal and insulin simulated glucose uptak in HUVECs was detected by 2-NBDG flow cytometry.Nile Red staining and H2 DCFDA staining with flow cytometry were used to detect intracellular lipid content and ROS content respectively.Finally,the the protein expression of p-IRS-1,cell adhesion factors VCMA-1 and ICAM-1,AMPK and p-AMPK,and autophagy-related proteins LC3 B,beclin-1 and p62 were decteced by Western blot.Results:1.Compared to control cells,HUVECs exposed to resistin increased the migration and expression of VCAM-1 and ICAM-1,and decreased the insulin-stimulated glucose uptake and the expression of p-IRS in HUVECs,inhibited the AMPK phosphorylation.However,there was no significant effect of resistin on ROS content,lipids content,and apoptosis in HUVECs.2.Compared to control cells,HUVECs exposed to resistin with AICAR decreased cell apoptosis,reduced ROS and lipid contents,and stimulated AMPK phosphorylation.AICAR inhibitd the HUVECs migration and decreased the expression of VCAM-1 and ICAM-1 againsted resistin.AICAR also attenuated the effect of resistin on glucose uptake and the expression of p-IRS-1 in HUVECs3.Compared to control cells,HUVECs exposed to resistin with Compound C increased the content of ROS and reduced the basal glucose uptake.Compound C enhanced the effect of resistin on AMPK phosphorylation and expression of VCAM-1,but there was no significant difference in endothelial cell migration,apoptosis,and lipid content between R+C group and R group.4.Resistin decreased the expression of LC3? and beclin-1 in HUVECs,and increased the expression of P62.Compound C enhanced the role of resistin on expression of beclin-1 and p62 in HUVECs.AICAR increased the expression of LC3? and beclin-1,elevated the ratio of LC3?/?,and decreased the expression of p62 against to resistin in HUVECs.Conclusion:Resistin induces HUVECs dysfunction and inhibits autophagy through AMPK,AICAR can activate AMPK to against the effect of resistin in HUVECs.Part Two:Comparison of the effects of resistin and high glucose on endothelial cells Objective: To investigate and compare the effects of high glucose and resistin on HUVECs' functions,and the collective effects of them.Methods: The HUVECs were divided into four groups: blank control group(Con group),high glucose group(glucose 30 m M,HG group),resistin group(resistin 100 ng/ml,R group),high glucose + resistin group(glucose 30 m M + resistin 100 ng/ml),R+HG group).After starvation for 12 h in low serum medium,endothelial cells were treated for 24 h according to the groups to detect endothelial cell migration,apoptosis,glucose uptake,lipid and ROS contents,the protein expression of p-IRS–1,VCAM-1 and ICAM-1.Results: 1.On the one hand,different from resistin,HUVECs exposed to high glucose decreased the migration,and increased the ROS content and apoptosis rate compared to control cells.On the other hand,similar to resistin,HUVECs exposed to high glucose increased the expression of VCAM-1 and ICAM-1,decreased insulin-stimulated glucose uptake.High glucose also inhibited p-IRS-1 as well as resistin.2.Compared to cells cultured in high glucose,HUVECs co-cultured in high glucose and resistin decreased the insulin-stimulated glucose uptake inHUVECs and increased ICAM-1 expression.And compared to cells cultured in resistin,HUVECs co-cultured in high glucose and resistin increased apoptosis and ROS contents,and decreased the migration of HUVECs.Compared to cell cultured in high glucose or resistin alone,HUVECs cocultured in high glucose and resistin increased the expression of VCAM-1 in HUVECs,but no significant difference on p-IRS-1 expression and lipid contents.Conclusion: There were similar effects of resistin and high glucose on cell adhesion molecule expression and insulin resistance in HUVECs.The interaction between them may aggravated the endothelial cell damage.