| BACKGROUND AND OBJECTPeriodontitis is a chronic destructive disease caused by plaque.The ideal goal of treating periodontitis is to completely eliminate inflammation and obtain tissue regeneration.Recently,the high correlation between hyperlipidemia and periodontitis has been recognized by scholars,the vicious circle formed by the two becomes the difficulty in the treatment of hyperlipidemic periodontitis.Macrophages are one of the main immune cells in early periodontitis,and involved in the process of damage and repair.Due to the heterogeneity and plasticity of macrophages,they can polarize into pro-inflammatory(M1)and anti-inflammatory(M2)phenotypes in a complex local environment.Gingival mesenchymal stem cells(GMSCs)possess ideal immunomodulatory and lipid-lowering abilities,they have attracted much attention in the field of stem cells.As the paracrine products from cells,exosomes possess the properties of their parental cells.Cell-free therapy based on mesenchymal stem cells derived exosomes(MSCs-Exos)exerts the powerful immune regulation ability of stem cells,and avoids the shortcomings of cell therapy.In this study,we investigated the effects of gingival mesenchymal stem cellderived exosomes(GMSCs-Exos)on the polarization of inflammatory macrophages in a high-lipid microenvironment.The aim of the research was to provide a new idea for the treatment of hyperlipidemic periodontitis.METHODSGMSCs were isolated from the connective tissue of human gingival tissues by enzymatic digestion combined with limited dilution.The characteristics of GMSCs were identified.GMSCs-Exos were isolated from GMSCs-conditioned medium by ultracentrifugation.The particle size,morphology and surface markers of GMSCs-Exos were observed and identified by nanoparticle tracking analysis,transmission electron microscopy and Western blot.The blank group was produced by inducing THP-1 into na(?)ve macrophages(M0)for24h.Inflammatory macrophages(M1)were made by activating M0 macrophages with LPS(Lipopolysaccharide)/IFN-γ(Interferon-gamma).These M1 macrophages were treated with oxidized low-density lipoprotein(ox-LDL)to create the control group.The inflammatory macrophages in high-lipid microenvironment were co-incubated with GMSCs-Exos to construct the experimental group.The cytokine concentration in the macrophage supernatant were analyzed by enzyme-linked immunosorbent assay.The expression levels of polarization markers in macrophages were analyzed by flow cytometry;Real-time quantitative PCR was used to analyzed the m RNA levels of M1 and M2 polarization markers.Oil red O staining was used to detect lipid accumulation level in macrophages RESULTSGMSCs were successfully obtained from gingival connective tissue by enzymatic digestion combined with limited dilution.The cells showed obvious monoclonal colony capacity and multiple differentiation potentials into osteoblasts and adipocytes.Flow cytometry analysis showed that the cells highly expressed CD73,CD90 and CD105,and negatively expressed CD45.Products obtained by ultracentrifugation showed exosome typical cup-like morphology under transmission electron microscope and positively expressed the exosome marker CD9、CD63、CD81 and HSP70.The particle size was approximately 100 nm.Compared with the blank group,the m RNA expression of inflammation markers TNF-α,IL-1β and IL-6 in the control group was significantly increased,and the m RNA expression of lipid regulation related gene PPAR-γ was significantly decreased.Flow cytometry analysis showed that M1 polarization marker CD86 was significantly increased(P < 0.05).The number of lipid droplets in macrophages was significantly increased by the inverted microscope.The concentration of inflammatory cytokine TNF-α in supernatant was significantly increased by ELISA(P < 0.05).Compared with the control group,the m RNA expression of inflammation markers TNF-α,IL-1β and IL-6 in the experimental group were significantly decreased(P<0.05),the m RNA expression of lipid regulation related gene PPAR-γ and M2 polarization marker IL-10 were significantly increased(P<0.05).Flow cytometry analysis showed that M1 polarization marker CD86 was significantly decreased(P<0.05),while M2 polarization marker CD163 showed no significant difference between the control group and the experimental group(P>0.05).The number of lipid droplets in macrophages was significantly reduced,and the level of lipid accumulation was significantly decreased(P<0.05).The concentration of TNF-α in the supernatant was significantly decreased,and IL-10 was increased significantly.CONCLUSIONSGMSCs-Exos inhibit the inflammatory response and M1 polarization of macrophages in a high-lipid microenvironment,promote the M2 polarization of macrophages,and regulate the lipid metabolism of macrophages. |
