Anti-inflammatory Effect Of C5a Receptor Antagonist PMX205 In Mouse Macrophages

Background:Porphyromonas gingivalis(Pg)is considered a keystone pathogen in the pathogenic progression of periodontitis.Gingipain,a major virulence factor of Pg,can trigger C5a-C5 a receptor(C5aR)signaling pathway.The crosstalk impairs periodontal tissue immunity inflammation and leads to periodontal tissue destruction.PMX205,a synthetic cyclic hexapeptide,is a C5 aR antagonist,which can block C5a-C5 aR axis by simulating C5 a active C-terminal amino acid residues and exert good anti-inflammatory effect.Presumably,this hexapeptide can be used to treat periodontitis.Macrophages,an important part of the body's innate immune system,plays an important role in periodontal immune inflammatory response.Macrophages display strong plasticity,they can be effective under external microenvironment changes to induce a variety of reactions and differentiations into M1 or M2 macrophages.M1 macrophages,which induces and expands inflammation,release bacteriostatic substances of reactive oxygen species(ROS)and nitric oxide(NO),increase the production of IL-1?,IL-6 and tumor necrosis factor-?(TNF-?)act as proinflammatory and stimulate osteoclasts,promote bone destruction at the same time.M2 phenotype is characterized with low NO production,an increased production of anti-inflammatory cytokines IL-10,transforming growth factor-?1(TGF-?1)and arginase-1(Arg-1).The macrophages found in the gingival tissue of Pg-infected mice are predominantly M1 macrophages(CD86+).Macrophage depletion using clodronate liposomes significantly reduces the level of Pg infection with significantly less Pg-induced bone resorption.The effect of PMX205 on gingipain extracts-or Pg-induced macrophage polarization has not been reported yet.Therefore,the anti-inflammatory effect of PMX205 was observed in an vitro cell model experiment from the perspective of macrophage polarization.Methods:1.Cytotoxicity assay of PMX205PMX205 was co-cultured with mouse macrophages(RAW264.7)and human osteoblasts(MG63).The effect of PMX205 on cell survival and proliferation was examined by MTT assay,and the cytotoxicity of PMX205 was evaluated.2.Effect of PMX205 on gingipain-induced inflammation modelRAW264.7 and gingipains were co-cultured in vitro to simulate the inflammatory environment,and the anti-inflammatory effect of PMX205 was observed.The experiment was divided into 6 groups: negative control group,G group,G+PMX205(0.1,1,5,10 ?g/ml)group.After 24 h,Real-time PCR,ELISA and Griess method were used to detect the expression of M1 macrophage markers(iNOS(NO),IL-6 and TNF-?),M2 macrophage cytokines(IL-10 and TGF-?1)at gene and protein levels.Flow cytometry was used to detect the M1 macrophage surface molecule CD86 and M2 macrophage marker CD206.Phagocytosis assay was used to detect the effect of PMX205 on phagocytosis capacity of RAW264.7.3.Effect of PMX205 on Porphyromonas gingivalis-induced inflammation modelThe experiment was divided into 3 groups: negative control group,Pg group and Pg+PMX205 group.After co-cultured for 24 h,the expression level of macrophage related cytokines was detected by Real-time PCR,ELISA and Griess method.Results:1.MTT results showed that the light absorption(A)of PMX205(0.001,0.01,0.1,1 and 5 ?g/ml)were increased with the prolonged time,and the cytotoxicity rate for RAW264.7 was 0 or 1.However,at 10 ?g/ml,the observed macrophage toxicity at 24 h was 2,statistically different from the BL group(P<0.05).The cytotoxicity of PMX205 for MG63 was 0 or 1 in each concentration group.2.Effect of PMX205 on gingipain-induced inflammation modelThe Real-time PCR test results showed that after 24 h,the IL-6 and TNF-? expression levels of G group increase than the negative control group,IL-10 expression levels decreased(P<0.01 or P<0.05).The IL-6 and TNF-? expression of G+PMX205 group levels reduce compared to G group(P<0.01),but iNOS,TGF-?1,IL-10 expression levels of G+PMX205 group increase compared with G group(P<0.01).The optimal concentration(1 ?g/ml)was determined and the concentration of PMX205 was used in RAW264.7 cells.The results of ELISA and Griess showed that the expression of IL-6,TNF-?,NO and IL-10 in G group was significantly higher than that in the negative control group(P<0.01);the expression of TNF-? in G+PMX205 group was significantly lower(P<0.01)and the expression of IL-10 and TGF-?1 was higher than that in the G group(P<0.01).Flow cytometry results showed that compared with the negative control group,the expression of M1 macrophage marker single positive rate of CD86 was increased in the G group,while the expression of M2 macrophage marker single positive rate of CD206 was decreased.The expression of single positive rate of CD86 in G+PMX205 group was lower than that in G group,but the expression of single positive rate of CD206 was increased.Macrophage pretreatment with gingipains increased the phagocytosis for Escherichia coli when compared with untreated cells.Macrophage pretreatment with gingipains plus PMX205 phagocytosed Escherichia coli more than that of simple G group.3.Effect of PMX205 on Porphyromonas gingivalis-induced inflammation modelData showed that Pg resulted in an increased gene expression levels of IL-6,TNF-? and IL-10 and high protein production of IL-6,TNF-? and NO.PMX205 enhanced the effect of Pg on the gene expression level of iNOS,IL-10 and increased the NO production.PMX205 further weakened the effects of Pg on TNF-? and IL-6 at gene and protein levels.Conclusions:1.PMX205 have low toxicity and good biosafety;2.PMX205 has anti-inflammatory effects by regulating macrophage polarization on inflammatory response induced by gingipains;3.PMX205 has anti-inflammatory effects on inflammatory reactions caused by Porphyromonas gingivalis.