牙龈蛋白酶对M1型巨噬细胞表达抑菌细胞因子的调控作用

目的:探讨牙龈蛋白酶对M1型巨噬细胞表达抑菌细胞因子的调控作用。方法:采用sheets改良法从Pg ATCC 33277培养物上清中制备牙龈蛋白酶,应用质谱法鉴定牙龈蛋白酶,底物发色法测定酶活性,鲎试剂检测牙龈蛋白酶中LPS残留量。体外细胞模型实验评价牙龈蛋白酶对大肠杆菌脂多糖(Ec-LPS)诱导的M1型巨噬细胞表达抑菌细胞因子的影响。实验分为3组:阴性对照组、Ec-LPS组和Ec-LPS+牙龈蛋白酶组,采用qRT-PCR和ELISA法检测M1型巨噬细胞表达的抑菌细胞因子白细胞介素12(IL-12)、一氧化氮合酶(iNOS)和白细胞介素10(IL-10)在基因和蛋白水平的表达情况。结果:制备的牙龈蛋白酶为RgpA,活性20 U/L,LPS残留量<0.01EU/mL。qRT-PCR和ELISA结果显示,与阴性对照组相比,Ec-LPS组IL-12和iNOS基因和蛋白的表达水平明显升高,IL-10的表达降低,即成功诱导M1型巨噬细胞,组间有显著差异(P<0.01);Ec-LPS+牙龈蛋白酶组IL-12、iNOS和IL-10基因和蛋白的表达较Ec-LPS组显著降低,但高于阴性对照组,组间有显著差异(P<0.01)。结论:牙龈蛋白酶具有抑制M1型巨噬细胞表达抑菌细胞因子IL-12、iNOS的作用。

Effects of Gingipains on the Expression of Bacteriostatic Cytokines in M1 Polarized Mouse Macrophages

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.