Liraglutide Mediates The Anti-inflammatory Effect Of Primary Microglia Via TLR4

Obesity is a chronic metabolic disease caused by the interaction of multiple factors.A long-term high-fat diet induces hypothalamic inflammation in rats and leads to microglia activation.Obesity,like many neurodegenerative diseases,is related to chronic inflammation of the central nervous system and has become one of the underlying mechanisms of disease development.Microglia are immune cells of the brain and the first and most important line of defense in the central nervous system.It plays an important role in hypothalamic inflammation.On the one hand,activated microglia can engulf some foreign toxins,viruses,bacteria,dead cells and tissue debris when they are injured.The large amount of cytotoxins secreted by it include reactive oxygen species(ROS),proteases and cytokines,which can destroy infected tissues and cells.In addition,the good side is that microglia can also secrete anti-inflammatory cytokines and neurotrophic factors to promote the regeneration of nerve tissue.In the process of microglia activation,the participation of Toll-like receptor4(Toll-like recepors4,TLR4)plays an important role in the induction of inflammation.The nuclear transcription factor kappa B(NF-κB)signaling pathway,as an important inflammatory pathway,also regulates the release of microglia inflammatory mediators.Glucagon-like peptide-1(Glucagon-like peptide-1,GLP-1)analogs have anti-inflammatory and neuroprotective effects on microglia in addition to the hypoglycemic effect.GLP-1 is a polypeptide hormone secreted by intestinal L cells.It is not only a commonly used hypoglycemic agent in clinic,but also has a nutritional and protective effect on nerve cells.There are GLP-1 receptors(GLP-1R)on microglia,and GLP-1 analogs can function by binding to GLP-1R on the surface of cell membranes.However,the specific mechanism of the neuroprotective effect of the liraglutide GLP-1 analogue is currently unclear.Therefore,elucidating its mechanism is the premise and research focus of liraglutide as a neuroprotective therapeutic drug.In this project,the primary microglial cells extracted from SD suckling mice within one day of newborn were used to explore the relationship between lipopolysaccharide(LPS)intervention time and expression of inducible nitric oxide synthase(iNOS).Inflammation model.Through the intervention of liraglutide and TLR4 inhibitors in activated hypothalamic microglia,the anti-inflammatory effect of liraglutide through TLR4 on microglia was investigated.The study is introduced in three parts:Part 1 : Isolation,culture and identification of primary microgliaObjective: To cultivate primary microglia to provide a basis for subsequent experiments.Method: Remove SD suckling rats within 1 day of birth,remove brain tissue,digest with trypsin,put into incubator for cultivation,and change the fluid the next day.After overgrowth,the microglial cells are separated from the mixed cells,and the culture is continued.Flow cytometry or immunofluorescence was used to identify cell purity.Results: The purity of microglial cells by flow cytometry was 90.15%,and the purity of microglial cells by confocal microscopy was 95.18%.Conclusion: Primary microglial cells were successfully cultured,which provided a basis for subsequent experiments.Part 2: Study the establishment of primary microglia inflammation model from the expression of iNOSObjective: To explore the relationship between the time of lipopolysaccharide(LPS)interfering with primary microglia and the expression of inducible nitric oxide synthase(iNOS),so as to provide a basis for the establishment of inflammation model of microglia.Methods: Primary microglia cells were randomly divided into experimental group and control group after purification.The experimental group consisted of 4 groups,which were intervened with LPS for 6 h,12 h,24 h and 48 h,respectively,and the control group was LPS without intervention for 0 h.Immunofluorescence and immunoblotting were used to detect the expression of iNOS in primary microglial cells intervened by LPS at different times.Results: After 12 h,24 h and 48 h of LPS interfering with microglia,the expression of iNOS was significantly different from that of the control group(p <0.01),and gradually increased before 24 h,reaching a peak at 24 h,24 After h,it gradually decreased.Conclusion: The time of LPS intervention in primary microglia is related to the expression of iNOS.The expression of iNOS is the highest when LPS intervenes at 24 h.Therefore,it is reasonable to believe that the ideal inflammation model for microglia is LPS intervention at 24 h.Part 3: Liraglutide mediates the anti-inflammatory effect of primary microglia via TLR4Objective: To investigate the anti-inflammatory effects of GLP-1 analogue liraglutide on primary microglia through TLR4 by measuring TLR4 and downstream inflammatory factors iNOS,IL-1β and NF-k B gene expression.Methods: After purification,the cells were divided into 5 groups(control group,LPS group,liraglutide group,LPS + liraglutide group,LPS + TAK-242 group),real-time quantitative PCR detection of TLR4,My D88,TRAF6,COX2 and IL-1β gene expression.Immunofluorescence method was used to locate NF-κB / p65 into the nucleus.Results:1.Immunofluorescence method was used to detect NF-κB / p65 entry into the nucleus.NF-κB / p65 was stained green and the nucleus was stained blue.In the LPS group,P65 is mainly located in the nucleus,while in the control group,liraglutide group,and LPS + liraglutide group,P65 is mainly located in the cytoplasm.2.In the real-time quantitative PCR results,relative to the control group,the relative expression of TLR4,My D88,COX2 and IL-1β in the LPS group increased,and the relative expression of the gene in the liraglutide group did not change significantly,The relative expression of genes decreased in the LPS + liraglutide group and LPS + TAK-242 group.Conclusion:1.In this study,in vitro cell experiments found that after LPS interfered with primary microglia,the expression of TLR4 signaling pathway and downstream inflammatory factors increased.2.Liraglutide can inhibit the nuclear entry of NF-κB / p65 in activated microglia.3.When the GLP-1 analogue liraglutide and the TLR4 inhibitor TAK-242 intervene in the activated microglial cells,the expression results of inflammatory factors are consistent and both are reduced.This shows that liraglutide can reduce the inflammation of microglia by mediating the TLR4 signaling pathway and the release of downstream inflammatory factors.