Protective Effect And Mechanism Of Apigenin And Its Esters On Glycidol-induced Autophagy-dependent Apoptosis Of Human Umbilical Vein Endothelial Cells

Glycidol and glycidyl esters,a food contaminant formed during the deodorization step of refined vegetable oils,are also present in thermally processed foods containing salt and fat.Glycidyl esters can be hydrolyzed to glycidol in the gastrointestinal tract after ingestion.Glycidol can pass through the blood-brain barrier and is widely distributed in blood,liver,thyroid,spleen,kidney,skeletal muscle and adipose tissue.Therefore,vascular endothelial cells are constantly in contact with glycidol,which may have adverse effects on the human body.Apigenin(API),a natural flavonoid found in a variety of vegetables and fruits,has a variety of biological activities,including cancer,diabetes,inflammation,and cardiovascular disease.Apigenin-7,4’-O-dioctanoate(API-C8)is a derivative extracted from frying celery in soybean oil,and compared with API,it Has higher lipophilicity and cellular antioxidant activity.Therefore,in this paper,HUVECs were used as vascular endothelial cell model to study the cytotoxic effect of glycidol on HUVECs and its related mechanism.The protective effects of API and API-C8 against glycidol-induced cell injury in HUVECs and their associated mechanisms were also evaluated.The specific experimental methods and results are as follows:(1)In order to study the cytotoxic effect of glycidol on HUVECs,the effect of glycidol on the cell viability and cell cycle of HUVECs was first studied.The results showed that glycidol stimulated HUVECs cells for 24 hours,and glycidol significantly inhibited the proliferation of HUVECs and blocked the cell cycle in G2/M phase.Then,it was investigated whether glycidol induces apoptosis and autophagy in HUVECs.After24 hours of treatment with glycidol,the cells shrunken and chromatin agglutinated by Hoechst 33342 staining were observed under a fluorescence microscope;Western blot was used to detect the apoptosis marker protein Cleaved Caspase-3,Cleaved PARP,the protein content increased significantly with the increase of glycidol concentration;flow cytometry found that the percentage of cell apoptosis increased significantly with the increase of glycidol concentration,based on the above results that glycidol induced HUVECs apoptosis.We then further investigated whether glycidol induces autophagy in HUVECs.After 24 hours of glycidol treatment,MDC-stained autophagic vesicles were observed under a fluorescence microscope;Western blot was used to detect autophagy marker proteins.The protein contents of LC3-II and Beclin-1 increased significantly with the increase of glycidol concentration.The protein content of SQSTM1/P62 decreased significantly with the increase of LC3-II protein content;the autophagy inhibitor 3-MA inhibited autophagy and at the same time attenuated apoptosis,and 3-MA could significantly increase the glycidol treatment.cell viability.The results indicated that glycidol could induce autophagy-dependent apoptosis in HUVECs.(2)In order to deeply understand the mechanism of glycidol’s cytotoxicity on HUVECs,it was first studied that in the presence of ERK/JNK/p38 kinase inhibitor,compared with the treatment group without kinase inhibitor,the cell viability and survival rate were significantly improved;ERK,JNK,p38 phosphorylated protein increased significantly with the increase of glycidol concentration.It indicated that glycidol could activate the ERK/JNK/p38 signaling pathway in HUVECs.To further study whether the ERK/JNK/p38 signaling pathway regulates apoptosis and autophagy,in the presence of ERK/JNK/p38 signaling pathway kinase inhibitors,the contents of autophagy and apoptosis-related proteins were detected,and the study found that glycidol activates ERK/JNK/p38 signaling pathway induces apoptosis and autophagy in HUVECs(3)To evaluate the protective effect of API and API-C8 on glycidol-induced cytotoxicity of HUVECs and its mechanism.Firstly,the effects of API and API-C8 on the cell viability of HUVECs,and the cell viability of API and API-C8 in the presence of glycidol were respectively detected.The results showed that both API and API-C8 could significantly improve the cell viability,and the effect of API-C8 was found to be more significant.Then,Western blot was used to detect the levels of apoptosis and autophagy marker proteins.The results showed that both API and API-C8 could significantly attenuate apoptosis and autophagy.Finally,the results of the study found that API and API-C8 significantly activated the ERK/JNK/p38 signaling pathway in glycidol.