| Background and purpose of the researchMetabolic inflammation(metainflammation)is one of the key factors to promote the occurrence and development of chronic noncommunicable diseases(NCDs)which threats seriously human health.Palmitic acid(PA)is one of the most important saturated fatty acids that can induce metainflammation.Recent studies have shown that macrophages and its exosomes can mediate metainflammation.Our previous studies showed that exosomes secreted from the PA-stimulated macrophage and its contained miR-3064-5p mediated the production of inflammation in normal macrophages.However,it is not clear whether the exosomes and its miR-3064-5p could mediate the production of inflammation in metabolic organs or tissues(liver,fat,muscle).Therefore,this study further investigated the role of exosomes secreted from the PA-stimulated primary macrophages and its contained miR-3064-5p in the mediation of metainflammation in primary hepatocytes,fat cells and muscle cells.This study aimed to provide a new mechanism for explaining the metainflammation induced by palmitic acid.Results of this study will provide new idea for the prevention and treatment of metainflammation-associated NCDs.Methoh1.Six weeks old C57 mice were intraperitoneally injected with mercaptoacetate culture medium,and then performed the peritoneal lavage to collect the macrophages which’s purity were determined by flow cytometry.2.Exosomes secreted by the primary mice peritoneal macrophages were extracted by using the ExoQuick TC exosome kit,and were identified by transmission electron microscopy,nanoparticle tracking analysis(NTA)and western blot.3.Primary mouse hepatocytes were extracted by using two-step perfusion method from the 10 weeks old C57 mice.Primary mouse muscle satellite cells were extracted by using the methods of collagenase digestion combined with tissue mass crawling from the 6 weeks old C57 mice,and identified by detecting the specific protein MyoD with immunofluorescence,and finally differentiated into muscle tube cells by the stimulation of horse serum.Mouse preprimary adipocytes were extracted by collagenase digestion from the 10 weeks old C57 mice and differentiated into adipocytes which were identified by oil red O staining..4.The exosomes secreted by the PA-stimulated mouse primary macrophages were used to treat the primary mouse macrophages,hepatocytes,fat cells and muscle cells.The exosomes were first labeled with PKH26,and the uptake of exosomes by the four types of primary cells was observed under an inverted fluorescence microscope.In the four types of primary cells,the expression levels of miR-3064-5p were detected by qPCR,and the expression levels of the molecules in the NF-κB signaling pathway were detected by western blot.5.The primary mouse macrophages,hepatocytes,fat cells and muscle cells were transfected with miR-3064-5p mimics and inhibitor.Then,the expression level of miR-3064-5p was detected by qPCR,and the expression level of molecules in the NF-κB signaling pathway was detected by western blot.The exosome secretion inhibitor GW4869 was used as a control.6.SPSS 20.0 statistical software was used for the T-test of two independent samples or one-way analysis of variance between multiple groups.The results were expressed as mean±SD and p<0.05 was statistically significant.Result1.Macrophages with a purity of(99.17±0.65)%and a number of about 5×106 were extracted from the abdominal cavity of each mouse.The exosomes secreted by the primary mouse macrophages showed typical saucer-like vesicle morphology under electron microscope,and had a size of between 100～200 nm,and expressed the exosome-specific markers of CD63,CD9,and TSG101.2.The mouse primary hepatocytes were round shape with a biconuclear or multiconuclear structure.The mouse primary muscle cells could be induced to differentiate into muscle tube cells.The mouse fat cells could be induced to secrete lipid droplets.3.Following the treatment of Pkh26-labeled exosomes,the fluorescence were presented in the cytoplasm of all the four types of mouse primary cells.4.Exosomes from the PA-stimualted primary mouse macrophages significcanlty increased(p<0.05)the levels of miR-3064-5p,COX-2,TNF-α and p-p65,and at the same time significantly decreased the the level of IκBα in all the four types of primary mouse cells.5.The miR-3064-5p mimics significcanlty increased(p<0.05)the levels of miR-3064-5p,COX-2,TNF-α and p-p65,and decreased the the level of IκBα in all the four types of primary mouse cells.However,exosome extracts from the GW4869-treated primary mouse macrophages had no effect on the four types of primary mouse cells.Conclusion1.The ExoQuick TC kit method can be used to extract the exosomes with high purity from the supernatants of primary mouse macrophages.2.The primary mouse macrophages,hepatocytes,fat cells and muscle cells can take up the exosomes secreted from the primary mouse macrophages.3.Exosomes secreted from the PA-stimulated primary mouse macrophages enrich miR-3064-5p,and can target to inhibit IκBα and activate the NF-κB signaling pathway,and thus mediate the production of metainflammation in the primary mouse macrophages,hepatocytes,fat cells and muscle cells. |
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