Abstract: Objective: To explore the role of gingipain on bacteriostatic cytokines induced by M1 macrophages. Methods: The gingipain was directly extracted from the supernatant of Pg ATCC 33277 and identified by Mass Spectrography. The activity of the gingipain was determined by chromogenic substrate BAPNA. The Chromogenic Limulus Amebocyte Lysate endpoint assay with diazo-coupling reagents test was undertaken to detect the presence of gram-negative bacterial LPS contamination of the purified gingipain. The effects of gingipain on the expression of bacteriostatic cytokines was evaluated in M1 macrophages induced by Escherichia coli lipopolysaccharide (Ec-LPS), including three groups: the negative control group, Ec-LPS group and Ec-LPS+gingipain group. Expressions of IL-12, iNOS and IL-10q were detected by RT-PCR and ELISA in M1 macrophages. Results: The gingipain was characterized as RgpA, the enzymatic activity of gingipain was 20U/L, and the residue of LPS from gingipain was <0.01EU/ml. qRT-PCR and ELISA assays demonstrated that Ec-LPS induced high level of IL-12 and iNOS in RAW264.7, but not IL-10, compared with the control group (P<0.01), which indicated that the M1 macrophages were induced successfully. The gingipain significantly inhibited the level of IL-12 and iNOS in the Ec-LPS treated RAW264.7, but not IL-10, compared with the Ec-LPS group (P<0.01). Conclusion: Gingipain could suppress the bacteriostatic cytokines of IL-12 and iNOS from M1 macrophages induced by Ec-LPS.

Key words: Porphyromonas gingivlis, Gingipain, M1 macrophages, Cytokines