Preliminary Study Of Resolvin D1 Regulates Monocyte Macrophage Polarization Influence On Differentiation Capacity Of Hμman Periodontal Cells

Background:Enhancement of pro-inflammatory M1 macrophages and/or impairment of anti-inflammatory M2 macrophages in the monocyte-macrophage system as the main pathogenesis of periodontitis,affecting periodontal tissue regeneration by inhibiting the differentiation capacity of periodontal ligament cells.Resolvin D1（Rv D1）,a metabolite derived from omega-3 long-chain polyunsaturated fatty acids,has shown potent anti-inflammatory effects in many animal models of inflammation.However,the mechanism by which Rv D1 regulates the phenotypic polarization of monocyte-macrophages remains unclear in the field of periodontitis treatment.Objective:The purpose of this study is to verify whether Rv D1 can improve the inflammatory microenvironment of periodontal tissue by regulating the phenotypic polarization of monocytes/macrophages,and to preliminarily explore the effect of Rv D1 on the effect of macrophages of different phenotypes on hμman periodontal ligament.The effect of osteogenic and cementoblast differentiation ability of Hμman Periodontal Ligament Cells（h PDLCs）.Methods:This experimental design is divided into three parts:in the first part,hμman monocytes U937 were induced by Phorbol 12-myristate 13-acetate（PMA）,and the morphological changes of the cells were observed under a light microscope,the nμmber of adherent cells was quantitatively analyzed,and the quantitative analysis of real-time reverse transcription（Quantitative real Time polymerase chain reaction,q RT-PCR）to detect the expression of CD68 m RNA,a characteristic marker of macrophages,to identify whether the resting/unpolarized macrophages（MФ）were successfully obtained.The second part treated MФwith Escherichia coli lipopolysaccharides（LPS）and/or Rv D1 and divided them into the following four groups:Rv D1^-LPS^-,Rv D1⁺LPS^-,LPS⁺Rv D1^-and LPS⁺Rv D1⁺groups.The changes of cell morphology were observed under microscope,the concentration difference of pro-inflammatory factors and anti-inflammatory factors was detected by enzyme-linked immunosorbent assay（ELISA）,and the M1 type and M2 type were detected by q RT-PCR and flow cytometry.To further explore the effect of Rv D1 on the phenotypic polarization of monocytes/macrophages stimulated by LPS.Part III:The primary h PDLCs were isolated from the premolars of orthodontic patients using the tissue block method combined with enzymatic digestion for subculture,and the cell source was identified by hμman vimentin staining and hμman keratin staining.The supernatants of macrophages with different phenotypes obtained in the second part of the experiment were removed,and the supernatants of each group were collected after re-adding fresh culture mediμm to prepare different conditioned media（Conditioned Media,CM）,Cell counting kit-8（CCK-8）was used to analyze the effect of different CMs on the proliferation of h PDLCs,and q RT-PCR was used to analyze the m RNA expressions of osteogenesis and cementogenesis-related markers of h PDLCs by different CMs.To further analyze the effect of Rv D1 on the cytokines secreted by macrophages stimulated by LPS on the osteogenic and cementogenic differentiation of h PDLCs.Results:Part 1:The morphology of U937 cells induced by PMA changed from round to long-spindle,and the cytoskeleton became irregular.Quantitative analysis showed that compared with the control group,the nμmber of adherent cells in the PMA group increased significantly（P<0.001）,at the same time,the q RT-PCR results showed that the expression of CD68 m RNA,a characteristic marker of macrophages in the PMA group was significantly higher than that in the control group（P<0.01）.Part II:Under light microscope,it was found that the cells in LPS⁺Rv D1^-group changed from spindle shape to elongated shape,and the pseudopodia were obviously elongated.ELISA results showed that compared with LPS⁺Rv D1^-,LPS⁺Rv D1⁺group significantly decreased the concentration of pro-inflammatory factor TNF-a（P<0.05）,and increased the concentration of anti-inflammatory factor IL-10（P<0.01）.q RT-PCR results showed that the LPS⁺Rv D1⁺group significantly decreased the m RNA expression of M1 macrophage markers（TNF-a and IL-6）（P<0.05）,and increased the M2 macrophage marker（Arg-1 and IL-10）m RNA expression（P<0.001）compared with LPS⁺Rv D1^-.Flow cytometry results showed that LPS⁺Rv D1^-group significantly up-regulated the expression level of M1 macrophage marker CD86,and LPS⁺Rv D1⁺group significantly up-regulated the expression level of M2 macrophage marker CD163.Part 3:The h PDLCs were observed to grow radially under the light microscope.Immunofluorescence detection showed that hμman vimentin staining was positive,and hμman keratin staining was negative,proving that the cells were derived from mesenchyme.The results of CCK-8 showed that different CMs could promote the growth of h PDLCs and there was no significant difference among the groups.q RT-PCR results showed that compared with the LPS⁺Rv D1^-group,the LPS⁺Rv D1⁺group significantly increased the m RNA of osteogenic markers（ALP and Runx-2）and cementogenetic markers（CAP and CEMP-1）of h PDLCs expression level.Conclusion:In this study,hμman monocyte-derived MФmacrophages were successfully obtained,indicating that Rv D1 exerts an anti-inflammatory effect by promoting the polarization of monocyte/macrophage phenotype from M1 to M2 type,improving periodontal inflammation microenvironment,and enhancing h PDLCs These results provide theoretical guidance for Rv D1 to regulate periodontal inflammation and promote periodontal regeneration.