Effects Of Glycine On The Secretion Of Pro-inflammatory Cytokines In Macrophages Stimulated By Porphyromonas Gingivalis LPS

Background and Objective:Bacterium adhesion and accumulation of biofilms are one of the initiating factors of peri-implantitis.Toxins produced by bacterium directly stimulate healthy tissues around the implant,and then trigger inflammatory reactions,eventually lead to a series of pathological changes,such as mucosal redness and swelling,loss of connective tissue adhesion,and bone absorption.During these processes,macrophages can produce a series of related inflammatory factors such as TNF-?,L-6,IL-1?,etc.For resisting external stimulation,M1 macrophage polarization is mainly involved in killing pathogenic microorganisms,while causing connective tissue and bone tissue destruction at the same time.It has been found that glycine is an anti-inflammatory and immunoregulatory agent.Therefore,the aim of this experiment is to explore whether glycine can regulate the production and secretion of macrophage M1-type polarization-related inflammatory cytokines,so as to provide theoretical basis for therapy of peri-implantitis.Methods:1.Mice macrophages RAW264.7 were cultured.The experiment group was added with glycine at different concentrations,and in the control group,the equal amount of H-DMEM medium was added.At 24 h,48h and 72 h,the effects of different concentrations of glycine on the proliferative activity of macrophages were detected by CCK-8 method,in order to screen out the safe concentration of glycine on macrophages for subsequent experiments.2.Experimental grouping: The negative control group was that H-DMEM medium cultured macrophages for 48 h,the stimulation group and the test group were H-DMEM medium and safe concentrations of glycine relatively pretreated RAW264.7 for 24 h,and then after 24 h stimulation with P.gingivalis LPS,the growth status and morphological changes of cells in each group were observed under microscope.3.The experimental groups were the same as above.After 48 h of culture in each group,cells were collected,and the gene expression levels of TNF-?,IL-6 and IL-1? were detected by quantitative real-time PCR technology.4.The experimental groups were the same as above.After 48 h of culture in each group,the cell supernatants were collected,and the protein expression levels of TNF-?,IL-6,IL-1? were detected by ELISA technology.Results:1.The CCK-8 assay showed that at 72 h,the effects of 4,8,16mmol/L glycine on cell proliferation was comparable to the control group with no significant difference,as the concentrations of glycine in subsequent experiments.2.Under microscope we observed that the macrophages changed their morphology after being stimulated by P.gingivalis LPS,and the macrophages pretreated with a safe concentration of glycine have changed smaller than those in the stimulation group.3.The q RT-PCR results showed that the gene expression levels of TNF-?,IL-6 and IL-1? in the stimulation group were higher than those in the negative control group(P<0.05);in the test group(4,8,16 mmol/L glycine),the genes expression levels of TNF-?,IL-6 and IL-1? were reduced compared with the stimulated group(P<0.05),16 mmol/L glycine decreased gene expression most significantly(P<0.05).4.ELISA results showed that the protein expression levels of TNF-? and IL-6 in the stimulation group were higher than those in the negative control group(P<0.05);the protein expression levels of TNF-? and IL-6 in the test group(4,8,16 mmol/L / L glycine)were reduced compared with the stimulation group(P<0.05),16 mmol/L glycine decreased the protein expression most significantly(P < 0.05),and we did not detect IL-1?.Conclusion:1.?16mmol/L glycine has no significantly effect on mice macrophage RAW 264.7 cell proliferation,but ? 32 mmol/L glycine inhibits cell proliferation significantly.2.Glycine(?16mmol/L)inhibits the secretion of pro-inflammatory cytokines in macrophage RAW264.7 stimulated by Porphyromonas gingivalis LPS.