| Objective:Studies have shown that there is a close relationship between hypothalamic inflammation and obesity,and inhibiting hypothalamic inflammation is helpful to resist the occurrence and development of obesity.Our team earlier studies have found that n-3 polyunsaturated fatty acids(n-3 PUFAs)can inhibit hypothalamus inflammation and fight obesity,but the specific mechanism is unclear.Exosomal miRNAs plays an important role in the occurrence and development of diseases,so we hypothesized that the increase of n-3 PUFAs may affect the expression of exosomal miRNAs derived from hypothalamic neural stem cells in mice,so we chose to analyze the exosomal miRNAs derived from hypothalamic neural stem cells and their target genes.Furthermore,the mechanism of n-3 PUFAs inhibiting hypothalamic inflammation and obesity was explored.Methods:The hypothalamic neural stem cells of neonatal fat-1 transgenic mice that could spontaneously produce n-3 PUFAs and wild-type mice in the same nest were taken for primary culture,and exosomes were collected from cell supernatant by overspeed differential centrifugal method for identification,The samples were identified and tested according to the international common identification method for studying exosomes to determine whether the samples were exosomes.Examining samples for classic exosome structures by Transmission Electron Microscope(TEM),Nanoparticle Tracking Analysis(NTA)technology is used to detect the overall particle size distribution of nanoparticles in samples and Western Blotting assay was used to detect the expression of CD63 in the samples.Total RNA was extracted from hypothalamic neural stem cell-derived exosomes,and a library was constructed for miRNA high-throughput sequencing.After sequencing,bioinformatics technology was used to analyze the high-throughput sequencing results to screen significantly differentially expressed miRNAs and make cluster heat map,scatter map and volcano map of differentially expressed miRNAs.The differentially expressed miRNAs with large degree of difference were selected for target gene prediction in the miRNA target gene databases Target Scan,miRDB and mi Tar Base.According to the prediction results of differentially expressed miRNA target Genes,the function enrichment analysis of target Genes was performed in GO(Gene Ontology)database and KEGG(Kyoto Encyclopedia of Genes and Genomes)database,signaling pathways related to hypothalamic inflammation and fatty acid metabolism were investigated,and representative and strongly correlated miRNAs differentially expressed were selected.According to the prediction results of target genes of related miRNAs,q PCR verification was performed to analyze the influence of the increase of endogenous n-3PUFas on the exosome miRNAs derived from hypothalamus neural stem cells,and the regulatory effect of these differentially expressed miRNAs on downstream target genes.so as to explore the relationship between n-3 PUFAs and hypothalamic inflammation or other functions.Results:The hypothalamic neural stem cells were successfully cultured and identified by morphological and WB markers.Exosomes were extracted and identified successfully.The typical double vesicle exosome structure was detected by Transmission Electron Microscopy(TEM).Nanoparticle Tracking Analysis(NTA)technique showed that the particle size of the samples was concentrated at about120 nm,accounting for more than 97.5%,which was consistent with the characterization of the particle size of exosomes.Western Blotting showed that exosome surface marker protein CD63 was expressed in the samples.Total RNA was successfully extracted from the exosome samples,and the RNA samples met the requirements for sequencing database construction,which could be used for the next step of high-throughput sequencing.The quantitative analysis of miRNA high-fluence sequencing results showed that compared with the hypothalamic neural stem cell exosome miRNA of wild-type C57BL/6 mice,105 miRNAs in the Fat-1 transgenic mice group were differentially expressed(P < 0.05 and FC value ≥1.5),among which65 were up-regulated significantly and 40 were down-regulated significantly.The bioinformatics technology was used to deeply screen miRNAs with significant differential expression,and it was found that 8 of them were in key positions in hypothalamic inflammatory pathway and fatty acid metabolism pathway.After in-depth screening,eight important differentially expressed miRNAs were found to be miR-122-3p,miR-694-5p,miR-17-92 gene cluster,miR-3683b-3p,miR-493-5p,miR-300-5p,miR-217-5p and miR-31-3p,respectively.By analyzing the target genes of the above miRNA,14 important genes were found,including STAT3,IL-1α,IL-6,TLR-4,Elovl2,Elovl5,Srebf1,Scd1,Acot13,Ffar1,Ffar2,Fads3,ACSL3,Hsd17b4.The function of these genes is closely related to the expression of inflammatory factors and fatty acid metabolism.The above genes were up-regulated or down-regulated by q PCR,indicating that these genes were regulated.Conclusions: The increase of endogenous n-3 PUFAs can regulate the expression of miRNAs in the exosomes of hypothalamus neural stem cells.Among them,the miRNAs with significant differences are related to hypothalamus inflammation and fatty acid metabolism,and their target genes are at the key position of relevant pathways.These results suggest that endogenous n-3PUFAs can regulate the expression of miRNAs in the exosomes of hypothalamic neural stem cells,and then regulate the expression of genes related to inflammatory response and lipid metabolism,thus inhibiting hypothalamic inflammation and fighting obesity. |
