| Cardiovascular diseases?CVDs?are the diseases that cause the most deaths worldwide.Among them,atherosclerosis?AS?caused by lipid metabolism disorders is the main cause of CVDs.The initial stage of AS is closely related to vascular endothelial damage.Oxidized low-density lipoprotein?Ox-LDL?plays an important role in vascular endothelial cell inflammation and lipid damage.Quercetin is one of the most abundant antioxidants in the diet.It has anti-lipid peroxidation and anti-inflammatory effects,and can effectively prevent and interfere with AS.However,the target and molecular mechanism of quercetin in AS intervention have not yet been elucidated.In addition,quercetin is limited in its application in the food field due to poor water solubility,easy decomposition when exposed to light,and short reaction time.Therefore,based on the previous experimental research,we constructed an Ox-LDL-induced HUVECs injury model,and analyzed the therapeutic targets and signaling pathways of quercetin to alleviate vascular endothelial injury from the perspective of non-coding RNA?ncNRA?,providing a new theoretical basis for elucidating the molecular mechanism of quercetin's intervention in AS;a Pickering emulsion delivery system was constructed,and the cardiovascular protection effect of quercetin-containing emulsion was evaluated through animal experiments,which provided a technical reference for the further development of related functional foods.The specific results are as follows:?1?A protective model of quercetin against Ox-LDL-induced HUVECs injury was established.HUVECs were treated with Ox-LDL?25-200?g/mL?and quercetin?10-300?M?.The results showed that the IC50 value of Ox-LDL to reduce the survival rate of HUVECs cells was 128.2?g/mL.According to IC50 combined with actual experiments In the case,the final Ox-LDL concentration of 125?g/mL was selected as the damage concentration for subsequent experiments.In addition,the results also show that quercetin is not toxic to HUVECs cells in the experimental concentration range of 0-30?M,so a concentration gradient of 5,10,15,20,25?M was selected for subsequent experiments.?2?Protective effect of quercetin on endothelial lipid damage.Quercetin pretreatment can increase the survival rate of HUVECs induced by ox-LDL,and reduce the production and release of intracellular reactive oxygen species?ROS?.Further analysis showed that quercetin can significantly induce the content/enzyme activity of superoxide dismutase?SOD?and glutathione peroxidase?GSH-Px?in cells,reduce LDH activity and reduce the accumulation of malondialdehyde?MDA?,effectively restored the balance of the state of intracellular oxidative stress.In addition,quercetin treatment can also rescue the loss of mitochondrial membrane potential caused by Ox-LDL injury,and then reduce the occurrence of apoptosis.?3?High-throughput sequencing of whole transcriptome analyzed the protective effect of quercetin in Ox-LDL-induced HUVECs cell injury model.Transcriptome analysis was used to enrich GO functions,KEGG pathways,and hierarchical clustering analysis.The results indicate that PTEN/PI3K/AKT/Caspase9 may be a potential downstream pathway of quercetin.Verification by qRT-PCR and Western-blot confirmed that quercetin may regulate cells to resist Ox-LDL damage through PTEN/PI3K/AKT/Caspase9 cascade signaling pathway.Based on the results of bioinformatics analysis such as transcriptomics and software prediction?Tatgetscan7.1 and Starbase2.0?,miR-494-3p related to downstream PTEN and its competitive inhibitory lncRNA?MALAT1?may be selected as upstream targets.The ceRNA mode of action directly mediates the regulation of quercetin on downstream signaling pathways.Further verified by qRT-PCR,Western-blot,inhibitor/overexpression vector transfection,double luciferase experiments,and apparent effects?survival,ROS,mitochondrial membrane potential detection?,we proved miR-494-3p and MALAT1 may be upstream targets of quercetin and function through ceRNA regulation.The protective effect of quercetin on endothelial cells may be achieved through miR-494-3p/MALAT1/PTEN/PI3K/AKT/Caspase9 axis.?4?Zein/hawthorn pectin composite colloidal particles?ZHPs?were constructed.The particle size,polydispersity coefficient?PDI?,zeta-potential,and three-phase contact angle of colloidal particles with different composite ratios were analyzed,and it was found that the 1:1composite ratio exhibited proper neutral wettability(?O/W=93o).Scanning electron microscope and transmission electron microscope were used to observe the microstructure of ZHPs.It was found that the encapsulation of zein colloidal particles?ZPs?by pectin macromolecules.Combining Fourier transform infrared spectroscopy?FTIR?scanning with previous zeta-potential results,it is proved that the encapsulation of pectin macromolecules on ZPs in composite particles is the result of hydrogen bonding and electrostatic adsorption.The synthesized ZHPs with a composite ratio of 1:1 have good amphiphilic interface properties and are used in the subsequent preparation of Pickering emulsions.?5?Analysis of Pickering Emulsions?ZHPEs?Prepared by ZHPs Composite Colloidal Particles.The effects of different oil phase fractions on the properties of the stable Pickering emulsions of ZHPs were studied by means of stability experiments,microscopic observations,emulsion particle size,low-field nuclear magnetic resonance?LF-NMR?,and rheology.The results showed that after 1 month of storage at room temperature,ZHPEs with oil phase fractions of 0.1 to 0.4 began to show water phase separation;ZHPEs with oil phase fractions of0.5 to 0.7 remained stable during storage;ZHPEs with oil phase fractions of 0.6 and 0.7 were stored in 1 After a few days,it turned into a semi-solid emulsion gel,which improved the storage stability of Pickering emulsion.The surface microstructure of the droplets of ZHPEs emulsion was observed by laser confocal microscope?CLSM?and cold field scanning electron microscope?cryo-SEM?.Cryo-SEM results showed that spherical particles?ZHPs?were densely packed on the surface of the droplets,proving that ZHPEs belong to Pickering emulsion.More interestingly,the complex network of interactions observed between droplets may limit the relative motion between the droplets.CLSM also clearly shows the entangled connections between the composite particles on the surface of the droplets and the local aggregation of particles,which is why the ZHPEs show significant colloidal characteristics.These results show that by changing the oil phase fraction of ZHPEs,the rheological behavior and interfacial microstructure of the emulsion carrier system can be adjusted to meet the diverse needs of the physical properties of the emulsion carrier system for different use cases.?6?Inhibitory effect of hawthorn pectin on lipid oxidation of Pickering emulsions?ZHPEs?.The hydroxyl radical scavenging ability and total antioxidant capacity?T-AOC?of hawthorn pectin and the effect on the content of MDA in ZHPEs were studied.Compared with commercial pectin,hawthorn pectin shows stronger hydroxyl radical scavenging ability and total antioxidant capacity,which is mainly due to the molecular weight of hawthorn pectin is lower than that of ordinary pectin,and at the same time,some small molecules have antioxidant activity during purification.Ingredients?active components of hawthorn source?residues/graft are related in hawthorn pectin.MDA results showed that the MDA content of corn germ oil without emulsification was 25.46±1.03?mol/kg,which was significantly higher than the MDA content of the emulsion.Compared with Pickering emulsions?ZCPEs and ZAPEs?prepared by commercial pectin,ZHPEs have a significant inhibitory effect on the MDA content?8.32±0.70?mol/kg?in the oil phase.This proves that the antioxidant activity of hawthorn pectin itself also provides additional protection for the emulsion?oil phase?.?7?Animal experiments evaluated the anti-cholesterol damage effect of quercetin in vivo and the bioavailability of Pickering emulsion delivery system.Paigen diet for 10 weeks caused significant cholesterol damage in mice,marking a successful injury model.Serum anti-oxidative enzymes,blood lipids,liver function?related enzymes?were detected in mice,while the mice were sacrificed and their tissues were analyzed by means of sectioning,H&E or oil red O staining.The results show that direct administration of quercetin can significantly reduce cholesterol damage and restore homeostasis of lipid metabolism,especially alleviate fatty liver damage and aortic lipid damage.In addition,compared to direct administration,the same dose of quercetin delivered by the Pickering emulsion system showed a stronger protective effect.This indicates that the delivery system can increase the effect of quercetin in vivo without increasing the dose. |
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