Gingipains Suppress The Induction Of Macrophages By LPS To M1Phenotypic Polarization By C5a Pathway

Background:Periodontitis is a chronic infectious disease, which involves a series of immuneinflammation. Macrophages play an important role in the immune inflammatoryresponse, but also have a crucial role to play in tissue damage and bone resorption, intissue repair and reconstruction, which is closely related to macrophage polarizationstate. In response to different microenvironment, macrophages undergo M1or M2polarization, M1and M2macrophages play different roles in pro-inflammatory andanti-inflammatory effects respectively.The high expression of IL-12, IL-23, NO andthe low expression of IL-10are M1macrophages markers mainly; the lowexpression of IL-12, IL-23, NO and the high expression of IL-10, Arg-1are M2macrophages markers mainly.Porphyromonas gingivalis (P.g) was significantly associated with the occurrenceand development of periodontitis. The bacteria has a series of virulence factors toevade host defense mechanisms and damage the host organization, includinggingipains, fimbriae, capsule polysaccharide and lipopolysaccharide, they influencethe expression of many inflammatory mediators of tissue cells. There are a lot ofresearch on LPS induced macrophage polarization, but the studies about gingipainsfor macrophage polarization were not found. In order to find a new treatment ofperiodontitis in the macrophage polarization, we explored the polarization ofmacrophage and the induced polarization of macrophage by co-culture with LPS orgingipains in vitro.Methods:1. The preparation of gingipains:The extracellular culture fluid of P.g ATCC33277was acetone precipitated, then theprecipitate was centrifuged. After resuspension and dialysis, the rate of BAPNA hydrolysis was read to determine gingipain activity and10%SDS-PAGE was todetermine the purity and the molecular weight of the sample.2.The experiment was divided into five groups:The first group (blank control group) was set as macrophage separate training；The second group was set as the cocultivation of LPS (1μg/ml) and macrophages；The third group was set as the cocultivation of gingipains (4U/L) andmacrophages；The fourth group was set as the cocultivation of gingipains (4U/L), LPS (1μg/ml)and macrophages；The fifth group was set as the cocultivation of C5aRA(C5a receptor antagonist，1mmol/L), gingipains (4U/L), LPS（1μg/ml）and macrophages.3.The detection of macrophage polarization phenotype markers:The expression of macrophage polarization phenotype markers IL-10, IL-12, IL-23,NO were detected by Realtime-PCR and ELISA method in the gene and protein levelafter24h.Results:1.A single50kDa protein bands was isolated by10%SDS-PAGE,and the activevalue of the sample was16.70U/L.2.Realtime-PCR and ELISA result showed that: the expression IL-12, IL-23, NO inthe LPS group was significantly higher than blank control group (P＜0.05), butLPS+gingipains group was significantly lower than LPS group (P＜0.05), andLPS+gingipains+C5aRA group was significantly higher than LPS+gingipains group(P＜0.05). Compared with the blank control group,there is no significant difference ofIL-10expression in the experimental groups (P＞0.05).Conclusions:1.50KD Rgp can be extracted by acetone precipitation.2.Macrophage phenotypes can be induced to M1polarization by LPS in vitro.3.Gingipains can suppress the LPS induction of macrophages to the M1phenotypicpolarization. 4.Gingipains can suppress the LPS induction of macrophages to the M1phenotypicpolarization by C5a pathway.