Naringin Inhibits Autophagy Mediated By PI3K-Akt-mTOR Pathway To Ameliorate Endothelial Cell Dysfunction Induced By High Glucose/High Fat Stress

Objective:Sugar and fat are necessary for our daily diet,but excessive intake will burden people's bodies,which has caused many "health killers" including obesity and cardiovascular diseases to take advantage of the opportunity.Autophagy is a process that engulfs self-damaged and useless proteins or organelles,and coats them into vesicles to form autophagolysosomes with lysosomes,and then degrades the enclosed contents to produce amino acids and fatty acids.This achieves the homeostasis of the cell.Autophagy can occur in both physiological and pathological processes,but the positive and negative effects of autophagy have not been elucidated.Naringin(Nar)as a flavonoid compound has various pharmacological effects such as lowering plasma cholesterol,reducing thrombosis and improving local microcirculation and nutrition supply.However,effects of Nar on function and autophagy of vascular endothelial cells under high glucose and high fat(HG/HF)stress are largely unclear.The purpose of this syudy is to observe the effect of Nar on the function of human umbilical vein endothelial cells(HUVECs)under HG/HF stress and to explore its possible mechanism.Methods:In this experiment,HUVECs were used as experimental objects.HUVECs were treated with different concentrations of glucose/palmitic acid to establish a model of HG/HF-induced endothelial cell injury.The cell function and autophagy levels of the normal group and the damaged group were measured first to judge HG Of HF stress on cell function and autophagy.Then add low(43 ?M),medium(86 ?M),and high(172 ?M)concentrations of Nar on the basis of the impaired group to determine the effect of Nar on cell function and autophagy.Next,cells were treated with the autophagy inducer rapamycin(RAPA)and the autophagy inhibitor 3-methyladenine(3-MA)to compare the functional levels of different groups to judge the relationship between cell function and autophagy.Finally,the molecular action mechanism of Narwas explored with PI3 K,Akt,and mTOR inhibitors.The experiment was divided into Control group,Vehicle group,HG/HF group,Nar group,autophagy inhibitor group and pathway inhibitor group.HE staining was used to observe changes in cell morphology and quantity;Western blot analysis was performed to detect the expression of protein levels such as p-eNOS/eNOS,LC3-II/I,p62,Beclin 1,p-PI3K/PI3 K,p-Akt/Akt,p-mTOR/mTOR;Nitric oxide(NO),endothelin(ET-1)and lactate dehydrogenase(LDH)of each group were measured by spectrophotometer;the reactive oxygen species(ROS)levels were assessed using flow cytometry;MDC staining was used to detect autophagosome;cell scratch test was used to detect cell proliferation and migration function;transwell method was used to detect cell infiltration function.Results:1.HUVECs under HG/HF stress,with the increase of HG/HF treatment concentration and time,HE staining results show that the cell morphology is gradually damaged and the number of cells is gradually reduced;MTT results also show that the cell viability is gradually reduced;the results of flow cytometry showed that the increase of HG/HF concentration will also increase the content of ROS in the cell;the results of microplate reader also showed a decrease in the level of NO content,suggesting that HG/HF stress will cause damage to cells or even death.Western blot results showed that under the influence of HG/HF stress,the expression of p-eNOS/eNOS was significantly down-regulated,the expression of autophagy key proteins LC3-II/I and Beclin1 was significantly up-regulated,and the expression of p62 was significantly down-regulated.The number of autophagosomes also increased under the microscope,indicating that HG/HF stress will promote autophagy.In this experiment,cells treated with 44/1.0 mM HG/HF for 24 h were regarded as the best function-impaired group.The results of MDC staining showed that the level of cell autophagy also increased significantly at this time,so it was also called the best autophagy(OA)group.2.HUVECs under HG/HF stress,under the effect of adding different concentrations of Nar,cell scratch experiments showed that the cell proliferation and migration ability was significantly improved;the results of flow cytometry showedthat the intracellular ROS content decreased.The spectrophotometric method measured the increase of NO content and the decrease of ET-1 content,and the above experiments all had the most obvious effect with the addition of medium concentration of Nar,indicating that HUVECs under HG/HF stress have improved cell function under the effect of adding Nar,and the most significant improvement was achieved with the medium concentration of Nar.Western blot results showed that the expression of p-eNOS/eNOS was significantly up-regulated,the expression of LC3-II/I and Beclin1 were significantly down-regulated,and the expression of p62 was significantly up-regulated,which together with the decrease in the number of autophagosomes shown by MDC staining results indicated that Nar would inhibit HG/HF Autophagy of HUVECs under stress.Therefore,in this experiment,cells with medium concentration of Nar for 24 h were used as the optimal Nar group,namely the ON group.3.Transwell experiment showed that adding autophagy regulator on the basis of OA group and OA+ON group,the autophagy inducer RAPA will reduce the infiltration function of cells,while the autophagy inhibitor 3-MA will increase the infiltration function of cells;LDH experiments show that the addition of the autophagy inducer RAPA will increase the LDH content in the cell,while the autophagy inhibitor 3-MA will reduce the LDH content in the cell.The above shows that the autophagy inducer will partially cancel the improvement effect of Nar on the function of HUVECs under HG/HF stress,while the autophagy inhibitor will increase the protective effect of Nar,indicating that autophagy negatively regulates cell function.4.Western blot results showed that the expressions of p-PI3K/PI3 K,p-Akt/Akt,and p-mTOR/mTOR in the OA+ON group were significantly increased compared to the OA group,indicating that the addition of Nar activated the PI3K-Akt-mTOR pathway.Moreover,under the action of PI3 K,Akt and mTOR pathway inhibitors,the expression of autophagy-related proteins LC3-II/I and Beclin1 were significantly up-regulated,and the expression of p62 was significantly down-regulated,indicating that inhibition of this pathway will weaken Nar's down-regulation of autophagy,further confirmed that Nar exerts the effect of inhibiting autophagy by activatingPI3K-Akt-mTOR pathway.In addition,the addition of pathway inhibitors significantly down-regulated p-eNOS/eNOS expression and significantly increased ROS content,indicating that Nar can improve cell function by activating PI3K-Akt-mTOR pathway.Conclusion: Nar improves the function of HUVECs under HG/HF stress through activating the PI3K-Akt-mTOR pathway to inhibit autophagy.