Effects of Porphyromonas gingivalis outer membrane vesicles on the pro-inflammatory capacity of macrophages
ZHU Yue1, LIN Jie1, HONG Liang2, YU Lin2
1. Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, Hangzhou, Zhejiang 310013, China; 2. The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou,Zhejiang 310009, China
Abstract：Objective To investigate the effect of Porphyromonas gingivalis outer membrane vesicles (OMV) on the pro-inflammatory capacity of macrophages and the underlying mechanisms, so as to provide a theoretical basis for the prevention and treatment of chronic periodontitis. Methods OMV were isolated and purified from Porphyromonas gingivalis culture supernatants using the ExoBacteriaTM OMV Isolation Kit, and characterized by transmission electron microscopy and GroEL detection. THP-1 cells were differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA) stimulation. Protein and RNA fractions extracted from the OMV were separately incubated with THP-1 macrophages, with untreated cells serving as the control group. The levels of cytokines including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were measured by flow cytometric fluorescence luminescence method, and the phosphorylation levels of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase (JNK), and p65 phosphorylation were determined by Western blotting. Results From 100 mL of Porphyromonas gingivalis culture supernatant, (0.87±0.14) mg of OMV were isolated. Transmission electron microscopy revealed a typical cup-shaped or spherical morphology, and the presence of the hallmark chaperone protein GroEL was confirmed. The OMV group exhibited significantly higher levels of TNF-α and IL-6 at (8 309.97±0.15) pg/mL and (5.13±0.93) pg/mL, respectively, compared to the control group at (52.78±4.59) pg/mL and (0.37±0.10) pg/mL (both P<0.05). The OMV protein fraction induced TNF-α and IL-6 secretion by THP-1 macrophages at (2 614.67±536.10) pg/mL and (2.45±0.41) pg/mL, respectively, higher than those in the control group at (296.51±26.02) pg/mL and (0.63±0.04) pg/mL (both P<0.05). No statistically significant differences in TNF-α or IL-6 levels were observed between the OMV RNA fraction-treated group and the control group (all P>0.05). Western blot analysis showed that, compared with the control group, the OMV group had increased p38 phosphorylation level, while the levels of JNK and p65 phosphorylation remained unchanged. Inhibition of p38 activity resulted in decreased TNF-α and IL-6 levels in the OMV-treated group (both P<0.05). ConclusionPorphyromonas gingivalis OMV may enhance macrophage secretion of the pro-inflammatory cytokines TNF-α and IL-6 through activation of the p38 MAPK pathway.
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