Mangiferin Inhibits High-Fat Diet Induced Vascular Endothelial Injury Via Regulation Of PTEN/AKT/eNOS Pathway

Part one Mangiferin improvement of aorta injury induced by high-fat diet in miceObjective: Long-term high-fat diet not only causes obesity,but also leads to the rise of blood lipids,and may cause hyperlipidemia.High blood lipids can lead to the occurrence of vascular endothelial injury,and then affect the cardiovascular function and cause cardiovascular disease.Vascular endothelial injury is one of the important factors that lead to the occurrence of cardiovascular disease.Mangiferin(Mangiferin,MAN)is bis pyridine ketone flavonoids,dried rhizome mainly from Liliaceae perennial herb Zhimu.Mangiferin has antitussive expectorant,anti-inflammatory analgesic,immune choleretic and anti-lipid peroxidation,anticancer,antidiabetic,antibacterial,antiviral and other pharmacological activities.But the effect in high-fat diet induced endothelial injury of Mangiferin has not been reported.Methods: High-fat diet induced C57/BJ6 male mice model was used to investigate the effects of Mangiferin on blood lipids and aorta in mice,Blood lipid in serum and HE staining of blood vessels were investigated and to clarify the protective effects of Mangiferin on blood lipid regulation and aorta.Results:1.Compared with the normal group,the level of cholesterol and triglyceride in high fat model group was significantly higher than the normal group.At the same time,high-density lipoprotein cholesterol decreased significantly,low density lipoprotein cholesterol increased significantly,serum inflammatory factors TNF-? and IL-6 increased significantly in model group,while mangiferin significantly improved blood lipids and inflammatory factors in mice.2.Compared with the normal group,the level of serum NO in the high-fat model group was significantly lower than the control group.The level of NO increased significantly after low dose and high dose of mangiferin.Positive control drugs can improve the level of blood lipid in mice,but have no significant effect on the level of IL-10 and NO.3.Histopathological results showed that mice in control group,vascular endothelial integrity,membrane visible fusiform smooth muscle cells,elastic fibers are arranged annularly,clear and complete,the outer membrane is a thin connective tissue;while in the high-fat diet mice,the aortic intimal became thickening,elastic fibers were distributed discontinuously arranged disorder,vascular smooth muscle cells hyperplasia,arranged in disorder.The thickening of the inner wall of the blood vessel was relieved and the integrity of the endothelium was recovered after the dosage of mangiferin.There was no thickening of the inner wall of the vessel in the high dose group,and the endometrium was smooth.No obvious intimal thickening was found in the positive control group.Part two Mangiferin improves ox-LDL induced endothelial cells injury by upregulated NOObjective: Oxidized low density lipoprotein(ox-LDL)is an important factor in vascular endothelial injury.Ox-LDL can increase the permeability of endothelial cells to LDL,resulting in vacuolar degeneration of cytoplasm,shrinkage of serosa and endothelial cell apoptosis and necrosis.Ox-LDL also could inhibit the synthesis of NO.NO is an important regulator for maintaining endothelial function,NO can promote endothelial relaxation,can regulate platelet aggregation,prevent the occurrence of atherosclerosis.NO is mainly composed of endothelial type eNOS.PI3K/Akt/eNOS is the main signal pathway to promote NO synthesis.Mangiferin has a protective effect on vascular injury caused by high fat,but its mechanism is unknown.Methods: ox-LDL induced HUVEC injury model was established,and invisgated the effects of Mangiferin on model of cell survival,apoptosis,NO level,ROS level of influence,and the impact on the synthesis of NO related proteins.Results:1.Compared with the normal group,the viability of ox-LDL induced cell decreased significantly,lactate dehydrogenase elevated significantly.After treatment with Mangiferin,the viability of endothelial cell was increased significantly and the activity of lactate dehydrogenase was decreased,and the effect of Mangiferin was inhibited by PI3 K inhibitor and eNOS inhibitor respectively.2.Compared with the normal group,the activity of eNOS and NO level induced by ox-LDL in HUVEC cells decreased significantly,ROS level increased significantly.Mangiferin could significantly increase the activity of LDH and eNOS in HUVEC cells,the level of NO was also increased,and reduced the level of ROS cells.When the mangiferin combined with PI3 K inhibitor or eNOS inhibitor,the eNOS activity,NO,ROS has significant difference with the single mangiferin group.3.Compared with the normal group,ox-LDL in HUVEC cells induced the phosphorylation levels of Akt and eNOS decreased,mangiferin could significantly increase Akt,eNOS phosphorylation and inhibition of PTEN levels in model cell,but no significant effect on PI3 K regulation;when the mangiferin combination with PI3 K inhibitor,Akt and eNOS decreased significantly,the regulation of Akt still exists when combined with eNOS inhibitor,but the regulation of eNOS disappeared.Part three Mangiferin regulates NO in endothelial cells through PTEN/PI3K/Akt/eNOS pathwayObjective: eNOS/NO are an important protective mechanism for vascular endothelium,and PI3K/Akt is an important regulatory approach to eNOS.Phosphorylated Akt can phosphorylate eNOS,which produces ser1177-eNOS to promote the activation of eNOS,and the phosphorylated Akt also participates in the regulation of eNOS activity.PTEN is a negative regulatory protein of PI3K/Akt,and PTEN blocks the PI3K/ Akt pathway,and PTEN and PI3 K regulate the Akt pathway together.The second part found that mangiferin has protective effect on ox-LDL induced HUVEC cells injury,and is involved in regulating the level of eNOS and NO in vascular endothelial cells,and could regulated the level of Akt and eNOS phosphorylation.However,its molecular regulation mechanism needs to be elucidated.In this part,PTEN gene silencing cells were used to investigate the regulatory mechanism of mangiferin on PI3 K / Akt pathwayMethods: PTEN gene silencing cells were used to investigate the regulatory effects of Mangiferin on NO,eNOS activity and molecular pathway related proteins in the ox-LDL model.The aim is to elucidate the relationship between the molecular mechanism of NO regulation by Mangiferin and PTEN/Akt/eNOS pathway.Results:1.Compared with the normal group,there was no significant difference in cell viability and LDH in siRNA negative control and transfection of siRNA-PTEN cell,and when treated with ox-LDL,the cell viability was significantly decreased,LDH increased significantly,NO and eNOS activity were significantly decreased significantly with siRNA-PTEN cells,when treated with mangiferin.On cell viability,LDH,NO and eNOS activity had no significant changes.2.Compared with normal group,siRNA negative control and PTEN cells transfected with siRNA-Akt,there was no significant difference in the phosphorylation level of eNOS,the phosphorylation of Akt and eNOS was decreased after pretreatment with ox-LDL.While the administration of Mangiferin on Akt,phosphorylation of eNOS compared with the model group had no significant effect.Conclusions:1.Mangiferin can effectively improve aortic injury,reduce blood lipid levels and alleviate inflammation in high-fat mice.2.Mangiferin can improve the ox-LDL induced injury of HUVEC by up-regulating NO level,enhancing endothelial cell eNOS activity and decreasing LDH activity.3.Mangiferin can up-regulate NO level in endothelial cells by inhibiting PTEN,activating PI3K/Akt/eNOS signaling pathway,and then protect ox-LDL induced endothelial injury.