Apigenin Interferes With The Development Of Atherosclerosis By Regulating Autophagy

ObjectiveApigenin is a candidate natural compound for intervention in development of atherosclerosis.Its intervention effect is related to two factors—hyperlipidemia and inflammation,that regulate the development of the disease.But the molecular mechanism is not yet clear.Therefore,this paper intends to explore the molecular mechanism of apigenin intervention in the development of atherosclerosis from the perspective of regulation of lipid metabolism inflammation through the LPS-induced Raw264.7 cellular inflammation model,the acute hyperlipidemia model and the high-fat diet induced ApoE-/-atherosclerosis model.Methods1.Effect of apigenin on the inflammatory response of Raw264.7 cells induced by LPS:Raw264.7 cells experiments were designed according to different detection requirements.LPS(500ng/mL)was used to stimulate the inflammatory response,and cck8 kit was used to detect the cell activity of apigenin at different concentrations.ROS and mitochondrial autophagy kit were used to detect ROS secretion and mitochondrial autophagy activity.ELISA kit was used to detect the secretion of downstream inflammatory factors of ROS.2.Study on the mechanism of apigenin in the treatment of acute hyperlipidemia:Experiment 1:60 C57BL/6 mice were randomly divided into six groups,the normal group,model group,the fenofibrate group(26 mg/kg),apigenin high-dose group(25 mg/kg),apigenin middle-dose group(12.5 mg/kg)and apigenin low-dose group(6.25 mg/kg).Triton WR-1339(0.12g/mL)was used for intramuscular injection to induce acute hyperlipidemia in mice.Blood lipid-related indexes were detected by biochemical analyzer.RT-PCR was used to detect the expression of lipid-related and autophagy-related mRNA and autophagy-related microRNA in mouse liver.Pathological changes of liver were observed by microscope.The co-localization of LC3B/ADRP and UVRAG/ADRP was observed by laser confocal observation.The formation of autophagosomes was observed by electron microscopy.Experiment 2:60 C57BL/6 mice were randomly divided into 6 groups,the normal group,model group,the fenofibrate group(26 mg/kg),apigenin group(25 mg/kg),inhibitor group(LY294002,1.5 mg/kg),apigenin+inhibitor groups.Blood lipid-related indexes were detected by biochemical analyzer.The mRNA expression of autophagy related genes was detected by RT-PCR.3.Study on the mechanism of apigenin in the development of atherosclerosis:5 male and 5 female of C57BL/6 mice as control group,25 male and 25 female ApoE-/-mice were randomly divided into five groups,model group,rosuvastatin group,high dose apigenin group(25 mg/kg),middle dose apigenin group(12.5 mg/kg)and low dose apigenin group(6.25 mg/kg).Atherosclerosis model caused by high fat diet.Blood lipid-related indexes were detected by biochemical analyzer.The mRNAs and proteins expression of autophagy and lipid metabolism-related genes in mouse liver were detected by RT-PCR and Western Blot.Pathological changes of liver were observed by microscope.The thoracic aorta and heart were stained with oil red o reagent.Results1.In the inflammatory response test of Raw264.7 cells by LPS-induced,Apigenin dose concentration had no effect on cell activity at about 1μM.apigenin significantly decreased the production of ROS and enhanced the activity of mitophagy induced by LPS.at the same time,reduced the secretion of downstream inflammatory cytokines of ROS.2.In the experiment of acute hyperlipidemia of C57BL/6 mice induced by triton WR-1339,apigenin significantly reduced the blood lipid level of mice and improved liver function,Apigenin increased the content of TC and TG in the liver of mice.Apigenin up-regulates the expression of liver autophagy related genes and other genes related to lipid metabolism and down-regulates the expression of microRNA negatively regulated of autophagy.Through laser confocal observation,it was found that apigenin increased the co-localization of LC3B/ADRP and UVRAG/ADRP.The results showed that apigenin increased the formation of autophagosomes.Under the intervention of PI3K inhibitor LY294002,apigenin could not reduce the lipid level of mice and could not up-regulate the mRNA expression of autophagy related genes.3.In the atherosclerosis experiment of ApoE-/-mice induced by high-fat diet,the oil red 0 experiment showed that apigenin significantly reduced the formation of thoracic aorta and heart plaque in mice,suggesting that apigenin can interfere the development of atherosclerosis;Apigenin significantly reduced the level of blood lipid in atherosclerosis mice,reduced the content of TC and TG in the liver,and reduced the degree of liver lesions and improved liver function.Apigenin up-regulated the expression of autophagy related genes,and increased the protein expressions of autophagy and lipid metabolism related proteins.ConclusionThis topic under the research of anti-inflammatory,lipid-lowering and intervention AS role of the apigenin,preliminarily clarified that the efficacy of apigenin in intervention of the development of atherosclerosis is correlated with the regulation of apigenin on macrophage mitophagy to inhibit the secretion of inflammatory factors and the regulation of autophagy-dependented lipid metabolism—lipophagy.Results of the topic Provides a new idea and pharmacological experimental basis for the research of antiatherogenic drugs in prevention and treatment of apigenin.