The Study On The Anti-inflammatory Effect Of Glycyrrhizic Acid In The Intervention Of Macrophage Exosomes

Inflammatory factors mainly refer to various cytokines that are produced by monocytes/macrophages or lymphocytes and participate in inflammatory reactions,such as IL-1β、IL-6、IL-8、TNF-α and so on.These cytokines are involved in a variety of physiological and pathological processes such as inflammation,infection and immunity of various diseases of the body,and can cause sepsis,multiple organ dysfunction syndrome,systemic inflammatory cascade syndrome and so on.Especially in the new coronary pneumonia,the cytokine cascade storm caused by these inflammatory factors is widely recognized because of the high fatality rate.However,the inflammatory response is a complex cascade network formed by the interaction of multiple factors and multiple targets.The inflammatory regulatory network is a complex system involving multiple factors and multiple targets.Drugs also exert anti-inflammatory effects through multiple targets and multiple systems,targeting the inflammatory network.The overall regulation mechanism of the disease is still unclear.With the deepening of research,it has been discovered that exosomes derived from macrophages can participate in the regulation of the inflammatory network and play an important role in the regulation of inflammation.Exosomes are extracellular vesicles secreted by cells,which play an important role in cell-to-cell communication,immune response,antigen presentation,and tumor invasion.It also has important biological significance in the fields of disease diagnosis and prognosis markers,tissue repair and drug delivery.Macrophage-derived exosomes carry a variety of proteins and genetic information,can play a role in cell-to-cell communication,and play an important role in information transmission and regulation of the occurrence and development of inflammatory processes.For example,recent studies have shown that the microRNA carried by exosomes participates in the inflammatory factor regulation network,which can regulate the secretion of various cytokines such as IL-1β,IL-6,IL-8,TNF-α,and can also be affected and regulated by these inflammatory factors.At present,as a new mechanism of cell-to-cell signal transmission in inflammatory response,exosomes are receiving great attention from researchers.Research on exosomes derived from macrophages provides new ideas for unlocking the regulation of the inflammatory network,and plays an important role in revealing the anti-inflam-matory mechanism of drugs.Due to the complexity of the inflammatory network,finding suitable anti-inflammatory drugs has always been a research hotspot.A large number of studies have shown that Chinese medicine and its compound can adjust the disorder and imbalance of a variety of cytokines,and have a good anti-inflammatory effect.In the preliminary work of our research group,relying on the research platform of anti-inflammatory Chinese medicine components and their compound compatibility built by this research group,based on high-throughput liquid protein chip technology,combined with multi-pharmaceutical data analysis,through the analysis of multiple traditional Chinese medicines Anti-inflammatory activity screening of the components found that Glycyrrhizic acid(GA)and its component compound have a strong anti-inflammatory effect,which can significantly reduce more than ten kinds of inflammations such as macrophage interleukins,chemokines and tumor necrosis factor.It provides a solid foundation for the research of this thesis.Macrophage-derived exosomes play an important role in the regulation of inflammation,and are expected to become important biomarkers for anti-inflammatory therapy and new targets for drug research,and provide new ideas and methods for further uncovering the mechanism of inflammation network regulation.This project takes the intervention of glycyrrhizic acid in macrophage-derived exosomes as the research object,and conducts a preliminary exploration on the effect of the secretion of cytokines and the activation state of the LPS-induced macrophage inflammation model,in order to further study the new anti-inflammatory activity of glycyrrhizic acid.The mechanism of action is to find new ways and new targets for anti-inflammatory treatment of traditional Chinese medicine to provide experimental research basis.Part 1:Study on the effect of traditional Chinese medicine glycyrrhizin against LPS-induced inflammatory injury in RAW264.7 cellsPurpose:This experiment determines the use of exosome-free serum to culture and replicate the inflammation model conditions of RAW264.7 cells,and replicate the anti-inflammatory effect model of glycyrrhizic acid.Methods:In this experiment,ultracentrifugation was used to prepare exosome-free serum,RAW264.7 cells were stimulated with lug/ml LPS concentration,and the RAW264.7 inflammatory cell model was replicated by detecting changes in the level of IL-6 secreted by the cells.Glycyrrhizic acid was used at 400ug/ml.The concentration of ml acts for 24 hours to interfere with the inflammatory cell model to determine the anti-inflammatory effect of glycyrrhizic acid.Results:Under the condition of serum culture without exosomes,RAW264.7 cells grew well and could be induced to become an inflammation model under the stimulation of LPS.After the action of glycyrrhizic acid,the level of IL-6 decreased.Conclusion:The RAW264.7 inflammatory cell model was replicated with exosomes-free serum,the LPS induction dose was lug/ml,and the concentration of glycyrrhizic acid was 400ug/ml.Part 2:Glycyrrhizic acid stimulates the preparation and identification of exosomes derived from macrophagesObjective:To prepare exosomes derived from glycyrrhizic acid to stimulate macrophages and to identify exosomesMethods:In this experiment,the gradient ultracentrifugation method was used to separate and purify the exosomes secreted by glycyrrhizic acid after stimulating macrophages,and the particle size,shape,size,and size were identified by nanoparticle size tracking(NTA),transmission electron microscopy(TEM),and Western Blot detected the marker protein.Results:Nanoparticle size tracking(NTA)showed that the obtained particle size was concentrated around 130nm,and electron microscopy showed that the particle size was a typical cup-shaped structure with double membranes and structures.WB results showed that the exosomes markers CD9,CD63,Does not contain the endoplasmic reticulum protein Calnexin,indicating that the use of gradient ultracentrifugation method can obtain glycyrrhizic acid intervention macrophage-derived exosomes.Conclusion:The obtained particles are in accordance with the characteristics of exosomes.Glycyrrhizic acid can secrete exosomes after stimulating macrophages.Gradient ultracentrifugation can obtain exosomal particles with higher purity.Part Ⅲ:Glycyrrhizic acid interferes with the effect of macrophage-derived exosomes on the function of macrophagesObjective:To study the effect of glycyrrhizic acid intervention on macrophage-derived exosomes on the morphology and function of macrophagesMethods:On the basis of previous experiments,prepare glycyrrhizic acid to interfere with macrophage-derived exosomes(GA-exo),and glycyrrhizic acid to interfere with macrophage-derived exosomes(GA-exo)in different doses of high,medium and low Intervene MO macrophages,4h later use LPS to induce the macrophage inflammation model to polarize MO macrophages into M1 macrophages.After 20 hours,the cells and supernatant were collected,Elisa was used to detect the corresponding inflammatory factors,and Western Blot was used to detect the macrophage activation index iNOS.Observe the effect of glycyrrhizic acid intervention on macrophage-derived exosomes(GA-exo)on the morphology and function of macrophages.Results:Elisa results showed that compared with the control group,after glycyrrhizic acid intervention macrophage-derived exosomes(GA-exo)interfered with the macrophage inflammation model,the IL-6 expression in the cell supernatant of each group in the intervention group decreased.There are statistical differences,and the relative expression of macrophage activation index iNOS decreased,indicating that glycyrrhizic acid intervention in macrophage exosomes(GA-exo)has an inhibitory effect on the macrophage inflammation model.Conclusion:The intervention of oxalic acid in macrophage exosomes(GA-exo)has an inhibitory effect on the macrophage inflammation model.The anti-inflammatory effect of glycyrrhizic acid may be through exosomes.