目的 探索牙龈卟啉单胞菌（Pg）来源的外膜囊泡（OMV）对巨噬细胞破骨分化的影响及其作用机制。 方法 使用透射电镜和纳米颗粒追踪分析仪对Pg OMV进行鉴定和表征；使用1、3和10 mg/L的Pg OMV处理破骨前体细胞，分别为1、3和10 mg/L OMV处理组，磷酸盐缓冲液处理破骨前体细胞为对照组，通过抗酒石酸酸性磷酸酶（TRAP）染色、F-肌动蛋白（F-actin）染色和实时荧光定量PCR技术检测破骨细胞形成和破骨相关基因FBJ骨肉瘤癌基因（Fos）和基质金属蛋白酶9（MMP9）的表达；利用多黏菌素B（PMB）封闭脂多糖（LPS）后的Pg OMV处理破骨前体细胞（PMB-OMV处理组），OMV单独处理为对照，通过TRAP和F-actin染色观察破骨细胞及肌动蛋白环的形成情况；使用蛋白质印迹法检测Pg OMV对破骨前体细胞Toll样受体（TLR）2和TLR4表达水平的影响；利用10、50、100和200 μmol/L TLR2抑制剂C29预处理破骨前体细胞后，使用Pg OMV 处理破骨前体细胞，分别为OMV+10 μmol/L、OMV+50 μmol/L、OMV+100 μmol/L和OMV+200 μmol/L C29处理组，未使用C29预处理的OMV处理组为对照，通过TRAP和F-actin染色观察破骨细胞和肌动蛋白环形成情况；使用OMV和PMB孵育的OMV处理破骨前体细胞，分别为OMV处理组和PMB-OMV处理组，通过蛋白质印迹法检测破骨前体细胞TLR2蛋白表达水平。 结果 Pg OMV的平均粒径为179.2 nm，具有典型的囊泡结构；3和10 mg/L OMV 处理组中可见大量肌动蛋白环形成，TRAP阳性破骨细胞面积占比［分别为（22.6±2.1）%、（32.0±2.3）%］均显著高于对照组［（4.9±0.5）%］（P<0.001），3和10 mg/L OMV处理组Fos mRNA表达量（1.491±0.114、1.726±0.254）均显著高于对照组（1.000±0.029）（P=0.013，P=0.001），10 mg/L OMV处理组MMP9 mRNA表达量（2.232±0.097）显著高于对照组（1.007±0.148）（P<0.001）；PMB-OMV处理组中肌动蛋白环形成少于OMV处理组，PMB-OMV处理组TRAP阳性破骨细胞面积占比［（14.8±3.8）%］显著低于OMV处理组［（31.5±6.7）%］（P=0.004）；OMV处理组TLR2表达水平（1.359±0.134）显著高于对照组（1.000±0.000）（t=4.62，P=0.044）；50、100和200 μmol/L C29预处理破骨前体细胞后，TRAP阳性破骨细胞面积占比［分别为（24.1±1.7）%、（18.5±2.1）%、（9.1±1.3）%］均显著小于对照组［（29.4±1.7）%］（P=0.026，P<0.001，P<0.001）；PMB处理后，PMB-OMV处理组TLR2蛋白表达水平（0.780±0.046）显著低于OMV处理组（1.000±0.000）（t=8.32，P=0.001）。 结论 Pg OMV可通过携带LPS显著促进破骨细胞分化，破骨前体细胞表面的TLR2在Pg OMV介导的破骨细胞分化中发挥重要作用。 Objective To investigate the effects of Porphyromonas gingivalis derived outer membrane vesicles (Pg OMV) on osteoclast differentiation of macrophages and its underlying mechanisms. Methods The morphology and the size distribution of Pg OMV were analyzed by transmission electron microscopy and nanoparticle tracing analysis, respectively. The osteoclast precursors were treated with 1, 3 and 10 mg/L Pg OMV (1, 3 and 10 mg/L OMV treatment group) or phosphate buffer solution (PBS)(control group). The formation of osteoclasts was analyzed by tartrate-resistant acid phosphase (TRAP) staining and F-actin staining and real-time quantitative PCR (RT-qPCR) were used to detect the expression of Fos and matrix metallopeptidase 9 (MMP9). Polymyxin B (PMB) was used to block lipopolysaccharide (LPS) and then Pg OMV was used to treat osteoclast precursor (PMB-OMV treatment group), and OMV treatment group was used as control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The effect of Pg OMV on the expression of Toll-like receptor (TLR) 2 and TLR4 in preosteoclasts was detected by Western blotting. The osteoclast precursors were pretreated with 10, 50, 100 and 200 μmol/L C29, an inhibitor of TLR2, and then treated with Pg OMV(OMV+10, 50, 100 and 200 μmol/L C29 treatment group) and OMV treatment group without C29 pretreatment was control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The osteoclast precursor cells were treated with OMV (OMV treatment group) and OMV incubated with PMB (PMB-OMV treatment group) and the expression of TLR2 in osteoclast precursor was detected by Western blotting. Results Pg OMV showed classical vesicular structures, and the average particle size of Pg OMV were 179.2 nm. A large number of actin rings were observed in the 3 and 10 mg/L OMV treatment groups. The percentages of TRAP-positive osteoclast area in 3 mg/L OMV treatment group [(22.6±2.1)%] and 10 mg/L OMV treatment group [(32.0±2.3)%] were significantly increased compared with control group [(4.9±0.5)%] (P<0.001). Compared with the control group (1.000±0.029), the mRNA relative expression of Fos in 3 mg/L OMV treatment group (1.491±0.114) and 10 mg/L OMV treatment group (1.726±0.254) was significantly increased (P=0.013, P=0.001). Compared with the control group (1.007±0.148), the mRNA relative expression of MMP9 in the group of 10 mg/L OMV (2.232±0.097) was significantly increased (P<0.001). Actin ring formation was less in PMB-OMV treatment groups than in OMV treatment groups. The proportion of TRAP-positive osteoclasts area [(14.8±3.8)%] in PMB-OMV treatment group was significantly lower than OMV treatment group [(31.5±6.7) %] (P=0.004). The relative expression of TLR2 in OMV treatment group (1.359±0.134) was significantly higher than that in the control group (1.000±0.000) (t=4.62, P=0.044). Compared with the OMV treatment group [(29.4±1.7)%], 50, 100 and 200 μmol/L C29 significantly decreased the formation of osteoclasts [(24.0±1.7)%, (18.5±2.1)%, (9.1±1.3) %] (P=0.026, P<0.001,P<0.001). TLR2 protein expression in PMB-OMV group (0.780±0.046) was significantly lower than that in OMV group (1.000±0.000)(t=8.32, P=0.001). Conclusions Pg OMV can promote osteoclast differentiation by carrying LPS, TLR2 plays an important role in Pg OMV mediated osteoclast differentiation.
Porphyromonas gingivalis outer membrane vesicles activate Toll-like receptor 2 to promote osteoclast differentiation by carrying lipopolysaccharide
Objective To investigate the effects of Porphyromonas gingivalis derived outer membrane vesicles (Pg OMV) on osteoclast differentiation of macrophages and its underlying mechanisms. Methods The morphology and the size distribution of Pg OMV were analyzed by transmission electron microscopy and nanoparticle tracing analysis, respectively. The osteoclast precursors were treated with 1, 3 and 10 mg/L Pg OMV (1, 3 and 10 mg/L OMV treatment group) or phosphate buffer solution (PBS)(control group). The formation of osteoclasts was analyzed by tartrate-resistant acid phosphase (TRAP) staining and F-actin staining and real-time quantitative PCR (RT-qPCR) were used to detect the expression of Fos and matrix metallopeptidase 9 (MMP9). Polymyxin B (PMB) was used to block lipopolysaccharide (LPS) and then Pg OMV was used to treat osteoclast precursor (PMB-OMV treatment group), and OMV treatment group was used as control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The effect of Pg OMV on the expression of Toll-like receptor (TLR) 2 and TLR4 in preosteoclasts was detected by Western blotting. The osteoclast precursors were pretreated with 10, 50, 100 and 200 μmol/L C29, an inhibitor of TLR2, and then treated with Pg OMV(OMV+10, 50, 100 and 200 μmol/L C29 treatment group) and OMV treatment group without C29 pretreatment was control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The osteoclast precursor cells were treated with OMV (OMV treatment group) and OMV incubated with PMB (PMB-OMV treatment group) and the expression of TLR2 in osteoclast precursor was detected by Western blotting. Results Pg OMV showed classical vesicular structures, and the average particle size of Pg OMV were 179.2 nm. A large number of actin rings were observed in the 3 and 10 mg/L OMV treatment groups. The percentages of TRAP-positive osteoclast area in 3 mg/L OMV treatment group [(22.6±2.1)%] and 10 mg/L OMV treatment group [(32.0±2.3)%] were significantly increased compared with control group [(4.9±0.5)%] (P<0.001). Compared with the control group (1.000±0.029), the mRNA relative expression of Fos in 3 mg/L OMV treatment group (1.491±0.114) and 10 mg/L OMV treatment group (1.726±0.254) was significantly increased (P=0.013, P=0.001). Compared with the control group (1.007±0.148), the mRNA relative expression of MMP9 in the group of 10 mg/L OMV (2.232±0.097) was significantly increased (P<0.001). Actin ring formation was less in PMB-OMV treatment groups than in OMV treatment groups. The proportion of TRAP-positive osteoclasts area [(14.8±3.8)%] in PMB-OMV treatment group was significantly lower than OMV treatment group [(31.5±6.7) %] (P=0.004). The relative expression of TLR2 in OMV treatment group (1.359±0.134) was significantly higher than that in the control group (1.000±0.000) (t=4.62, P=0.044). Compared with the OMV treatment group [(29.4±1.7)%], 50, 100 and 200 μmol/L C29 significantly decreased the formation of osteoclasts [(24.0±1.7)%, (18.5±2.1)%, (9.1±1.3) %] (P=0.026, P<0.001,P<0.001). TLR2 protein expression in PMB-OMV group (0.780±0.046) was significantly lower than that in OMV group (1.000±0.000)(t=8.32, P=0.001). Conclusions Pg OMV can promote osteoclast differentiation by carrying LPS, TLR2 plays an important role in Pg OMV mediated osteoclast differentiation.
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