Effects Of DHA On The Secretion Of Macrophage Inflammatory Cytokines Induced By Porphyromonas Gingivalis Lipopolysaccharide

Background and objective:Implant dentures are increasingly becoming an indispensable replacement for missing teeth,with a 10-year survival rate of 97%。However,compared with natural teeth,the epithelial sealing around implant is less effective,which makes it easier for bacteria to break through the epithelial barrier and causes immune inflammation in deep connective tissue and even bone tissue,finally leading to peri-implant mucositis and peri-implantitis.The tissue damage around the implant is not directly caused by the pathogen itself,but by the immune response around it.Macrophages can specifically recognize the lipopolysaccharide(LPS)on the surface of Poryromonas gingivlis(P.gingivlis)through Toll-like receptors,and activate the transformation of macrophages to the M1pro-inflammatory phenotype,which significantly upregulate the release of intracellular lysosomes,reactive oxygen species(ROS),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)and other cytokines.On the one hand,it can phagocyte,kill and remove pathogenic bacterias.On the other hand,excessive and uncontrolled inflammatory response will destroy the homeostasis of the tissues around the implant and cause dysfunction of the immune response of the body,triggering the inflammatory damage around the implant.Inhibition of inflammatory response by reducing the polarization of macrophages to pro-inflammatory phenotypes is expected to become an important target for the treatment of peri-implantitis.As an important component of cell membrane phospholipids,docosahexaenoic acid(DHA)binds to cell membrane and then plays a variety of physiological functions such as anti-inflammatory,antimicrobial and anti-cancer by regulating cell signaling and gene expression.DHA has been applied in the treatment of diseases related to inflammation and bacterial dysregulation.Therefore,we took macrophage RAW264.7 as the research object to explore the regulation effect of DHA on the secretion of macrophage inflammatory cytokines induced by P.gingivlis-LPS,the main pathogenic pathogen of peri-implantitis,so as to provide a preliminary research basis and theoretical basis for the treatment of peri-implantitis with DHA.Methods:1.Mouse mononuclear/macrophage line RAW264.7 cells were cultured,and setting experiment groups.P.gingivlis-LPS stimulation group was stimulated with 1 mg/L P.gingivlis-LPS for 24 hours,and then the RAW264.7 cells were cultured in DMEM medium for 24 hours.RAW264.7 cells were cultured with 25,50,75 and 100 μmol/L DHA for24 hours after stimulated with 1 mg/L P.gingivlis-LPS for 24 hours in DHA treatment groups,while the negative control group was cultured with the same amount of DMEM medium for 48 hours.CCK-8 was used to detect the proliferation of RAW264.7 cells in each group,so as to screen the safe concentration of DHA for subsequent experiments.2.The experimental groups were determined according to the cytotoxicity results,and the total RNA of each experimental group was extracted.The m RNA expressions of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in RAW264.7 cells of each experimental group were detected by real-time fluorescence quantitative PCR(qRT-PCR).3.The supernatants of cells in each experimental group were collected,and the protein secretions of TNF-α,IL-1β and IL-6 in the supernatants were detected by enzyme-linked immunosorbent assay(ELISA).4.ROS of RAW264.7 cells in the above experimental groups were labeled with DCFH-DA fluorescent probe,and the fluorescence intensity of each group was observed under a fluorescence microscope,and the fluorescence intensity was analyzed with Image J software.Results:1.Compared with negative control group,1 mg/L of P.gingivlis-LPS had no toxicity on macrophages induced by P.gingivlis-LPS(P>0.05),while 100 μmol/L DHA had significant toxicity on RAW264.7 cells stimulated by P.gingivlis-LPS(P<0.05).2.The m RNA expressions of TNF-α,IL-1β and IL-6 in P.gingivlis-LPS stimulation group were significantly higher than those in negative control group(P<0.05);Compared with P.gingivlis-LPS stimulation group,the m RNA expressions of TNF-α,IL-1β and IL-6 were decreased by 25,50 and 75 μmol/L DHA(P<0.05).3.The levels of TNF-α,IL-1β and IL-6 in P.gingivlis-LPS stimulation group were significantly higher than those in negative control group(P<0.05);Compared with P.gingivlis-LPS stimulation group,25,50 and 75μmol/L DHA decreased the syntheses of TNF-α and IL-6(P<0.05);The secretion of IL-1β was not detected in the supernatant of cells in each experimental group.4.The ROS production of P.gingivlis-LPS stimulation group was significantly higher than that of negative control group.Compared with P.gingivlis-LPS stimulation group,25,50 and 75 μmol/L DHA could reduce the production of ROS to a certain extent(P<0.05),and the inhibition degree was concentration dependent.Conclusion:1.1 mg/L P.gingivlis-LPS can induce the inflammatory response of macrophages.2.DHA(≥100 μmol/L)had a toxic effect on macrophages induced by P.gingivlis-LPS.3.DHA can effectively inhibit the inflammatory response of macrophages induced by P.gingivlis-LPS.