Gingival Mesenchymal Stem Cells Attenuate Pro-inflammatory Macrophages Stimulated With Oxidized Low-density Lipoprotein And Modulate Lipid Metabolism

OBJECTGingival mesenchymal stem cells(GMSCs)are stem cells with multi-directional differentiation potential,which can regulate immune responses through various mechanisms and exert biological therapeutic effects.Macrophages(M?)have potent immunoregulatory functions and play an important role in anti-inflammation and tissue repair processes.In response to different microenvironmental signals,macrophages could be polarized into two types,namely M1 macrophages(M1 M?)which promote inflammation and M2 macrophages(M2 M?)which promote tissue repair.In this study,we selected the most suitable macrophage polarization method by comparing the biological characteristics of human macrophages from different sources,and preliminarily studied the regulation effect of GMSCs on inflammatory macrophages under oxidized low-density lipoprotein(ox-LDL)stimulation to search a basis and new idea for the clinical application of GMSCs in the treatment of periodontal disease with hyperlipidemia.METHODS1.Peripheral blood mononuclear cells(PBMC),isolated using density gradient centrifugation and magnetic cell sorting methods,were cultured and differentiated into PBMC M? under the induction of Macrophage colony-stimulating factor(M-CSF).The human myeloid leukemia mononuclear cells(THP-1)differentiated into THP-1 M? under the induction of phorbol 12-myristate 13-acetate(PMA).Then the PBMC M? and THP-1 M? were further induced to M1 M? and M2 M? respectively.The expressions of M1 and M2 polarized markers in macrophages were detected by flow cytometry and real-time quantitative PCR(qPCR).The supernatants of cytokines were detected by enzyme-linked immunosorbent assay(ELISA).2.Healthy gingival connective tissues were collected during periodontal surgery or tooth extraction.GMSCs were isolated by enzyme digestion method.The stem cell characteristics of gingiva were identified by colony forming unit-fibroblast(CFU-F),osteogenic / adipogenic differentiation assay and flow cytometry.GMSCs were co-cultured with M1 M? for 24 hours in the presence of oxidized low-density lipoprotein(ox-LDL)in the transwell system.The supernatants were collected for ELISA.M1 and M2 markers of macrophages were analyzed by flow cytometry and PCR,and lipid accumulation was assessed by oil red O staining.RESULTS1.It was showed that after M1 polarization,the mRNA levels of TNF-? and IL-1? were markedly up-regulated(P<0.05),the expression of CD86 was enhanced(P<0.05),and the secretions of IL-1? and TNF-? in THP-1 M? were higher than PBMC M?(P<0.05);after M2 polarization,the mRNA levels of MRC1 and IL-10 were increased(P<0.05),and the expression of CD206 was significantly up-regulated in PBMC M?(P<0.05),however,there was no difference in THP-1 M?(P>0.05).2.The primary GMSCs were successfully isolated with stable morphology and continuously subcultured in vitro.GMSCs highly expressed mesenchymal stem cell markers including CD73,CD90 and CD105 by flow cytometry,but not CD45.CFU-F assay showed that GMSCs possess strong colony forming ability.Under specific induction conditions,GMSCs showed the ability of multidirectional differentiations.After the coculture of GMSCs with M1 macrophages,it was found that the number of macrophages with radial and dendritic morphology decreased significantly and the shape of macrophages became rounded and blunt.When co-cultured with GMSCs,the M1 markers of TNF-?,IL-6 and IL-1? were significantly reduced(P<0.05).By contrast,M2 markers such as IL-10 and CD206 were moderately increased(P<0.05).The level of TNF-? in the supernatant decreased significantly(P<0.05),while the level of IL-6 and IL-1? increased significantly(P<0.05).Flow analysis showed that GMSCs significantly reduced the expression of CD86 and HLA-DR in macrophages(P<0.05).Oil red O staining showed that lipid droplets accumulated less in co-cultured macrophages compared with M1 macrophages(P<0.05).CONCLUSIONSTHP-1 M? have similar polarization expression with PBMC M?,while the THP-1 are appropriate for M1 polarization rather than M2 polarization;GMSCs isolated from gingival tissues by enzymatic digestion have been identified possessing stem cell characteristics;GMSCs inhibit the activation of ox-LDL induced M1 macrophages,and further reduce inflammatory response and regulate lipid metabolism.