Preliminary Study On The Macrophage Exosomes And Its MiRNAs-Mediated Mechanism Underlying The Proinflammatory Activity Of Palmitate

BackgroundChronic non-infectious diseases(NCDs)seriously endanger people’s health,and metabolic inflammation is an important pathological basis for the development of NCDs.Recent studies have shown that exosomes and exosomal miRNAs secreted by macrophages are a new mechanism that regulate metabolic inflammation.Palmitic acid(PA)is a common saturated fatty acid that can induce metabolic inflammation.It is unclear whether macrophage exosomes and their miRNAs can mediate PA-induced metabolic inflammation.ObjectivesIn this study,we conducted a model of macrophage inflammation induced by palmitic acid(PA)to explore the role of inflammatory macrophage exosomes on the expression of molecules in the NF-KB signaling pathway and the role of exosomal miRNAs in this process.The purpose of this study was to investigate the role of exosomes and their miRNAs in the NF-KB signaling pathway,in order to shed new lights on understanding the underlying mechanisms on metabolic inflammation.Methods1.The macrophage exosomes were extracted by differential centrifugation and identified by NTA,TEM and WB;2.Exosomes were labled with PKH26 and co-cultured with macrophages.Then the uptake of exosomes by macrophages can be observed by an inverted fluorescence microscope;3.Macrophages were treated with exosomes secreted by PA-stimulated inflammatory macrophages,and WB was used to detect the expression of NF-κB p65 and p-p65,IκBα,and COX-2 in theae macrophages;4.High-throughput sequencing was used to detect differentially expressed miRNAs in exosomes secreted by PA-stimulated inflammatory macrophages and normal macrophages.Bioinformatics analysis predicted target miRNAs which were then identified with qPCR.Finally,a combination of WB,miRNA mimics and inhibit Agents was used to study and confirm the regulatory effect of target miRNAs on the expression of NF-κB p65 and p-p65,IκBa and COX-2.5.SPSS 20.0 was used for statistical analysis,one-way ANOVA was used to analyze the differences between multiple groups,and T test was used to analyze the differences between the two groups.Results were expressed as Means±SD(means±SD),p<0.05 was statistically significant.Results1.From the culture supernatant of PA treated macrophages by differential centrifugation,a "satellite-like" structural exosomes of approximately 2.9 ×1013 particles/mL could be prepared,which stably express CD9 and Tsg101 with a particle size between 40 nm～160 nm(mean size of 150.7 nm,mode size of 133.1 nm),and the particle size-concentration distribution showed a clear unimodal state.2.After co-culture of PKH26-labeled exosomes with macrophages,a clear red fluorescence could be seen under the microscope,and combined with white light and fluorescent fields,it can be seen that most of the red fluorescence was located in the cells.3.Exosomes(2 μg/105cells)secreted by PA-stimulated inflammatory macrophages treated normal macrophages,and the ratio of NF-κB p-p65/NF-κB p65were significantly increased at 4 and 8 h(p<0.05)after the treatment;The expression of COX-2 was significantly time-dependent(4 h,8 h,16 h,and 24 h)(p<0.05).Exosomes secreted by inflammatory macrophages were treated with normal macrophages for 4 h,and the levels of the ratio of NF-κB p-p65/NF-κB p65 were significantly increased in a concentration-dependent manner(1,2,and 3 μg/105 cells)(p<0.05);In the treatment Exosomes secreted by inflammatory macrophages for 24 h,the expression of COX-2 protein increased significantly at intervention concentrations of 0.5,2 and 3 μg/105 cells(p<0.05).The increased effect of exosomes secreted by inflammatory macrophages on the ratio of NF-κB p-p65/NF-KB p65 in normal macrophages can be significantly reduced by the exosome secretion inhibitor GW4869(p<0.05).4.There were 137 differentially expressed miRNAs in exosomes secreted by PAstimulated inflammatory macrophages compared to normal macrophages exosomal miRNAs,of which 81 were up-regulated and 57 were down-regulated.In all upregulated miRNAs,miR-3064-5p targets the signaling molecule IκBα in the NF-κB pathway.Quantitative qPCR analysis revealed that miR-3064-5p expression in exosomes secreted by PA-stimulated inflammatory macrophages was significantly higher than exosomes secreted by normal macrophages(p<0.05).5.The expression of IκBα protein in miR-3064-5p mimic-transfected macrophages was significantly reduced(p<0.05),and the level of NF-κB p65 phosphorylation was significantly increased(p<0.05).The miR-3064-5p inhibitor was transfected into PA-stimulated inflammatory macrophages.The secreted exosomes were reprocessed to normal macrophages.IκBα protein expression was significantly increased(p<0.05),and the ratio of NF-κB p-p65/NF-KB p65 was significantly Decrease(p<0.05)Conclusions1.Exosomes secreted by PA-stimulated inflammatory macrophages can activate the NF-κB signaling pathway in nonnal macrophages and have pro-inflammatory activity.2.PA-stimulated exosomes secreted by inflammatory macrophages can activate the NF-κB signaling pathway by miR-3064-5p-rich exosomes’ targeting to IκBα in normal macrophages.