Objective: To investigate the molecular mechanism of osteoclast differentiation induced by Staphylococcal peptidoglycan (PGN-sa).
Methods: Raw264.7 cells were stimulated with PGN-sa and with PGN-sa+SC75741[a potent inhibitor of nuclear factor κB (NF-κB) activation] in a concentration of 200 ng/mL. The protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) was tested at 0, 1, 2, and 3 days; the proteins related to osteoclast differentiation of extracellular regulated protein kinases (ERK), p38, c-Jun N-terminal kinase (JNK), NF-κB, inhibitor of NF-κB (IκB-α), Akt, and the phosphorylation forms of p38, ERK, JNK, Akt, NF-κB were measured at 0, 5, 10, 20, 40, and 60 minutes by Western blot. In addition, Raw264.7 cells were stimulated with PGN-sa in the concentrations of 100 ng/mL (group A), 200 ng/mL (group B), 400 ng/mL (group C), and with PBS (group D) for 1, 2, and 3 days; the expression levels of tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), and IL-6 were detected by ELISA.
Results: The results of Western blot showed that the expression of NFATc1 increased gradually with time, showing significant difference between different time points (P<0.05). However, after SC75741 was added, the expression of NFATc1 was inhibited at 2 and 3 days, showing significant difference when compared with no addition of SC75741 (P<0.001). After stimulation of PGN-sa, the expression of IkB-α decreased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes. Meanwhile, the expression of p-NF-κB increased significantly at 5 and 10 minutes when compared with those at the other time points (P<0.001), and returned to normal at 20 minutes; and the expression of p-NF-κB at 5 minutes was significantly higher than that at 10 minutes (P<0.001). After the addition of SC75741, there was no change in the expressions of IκB-α and p-NF-κB, showing no significant difference between different time points P>0.05). Moreover, the expressions of ERK, p38, JNK, NF-κB, Akt, p-p38, p-ERK, p-JNK, and p-Akt showed no significant change between different time points P>0.05). ELISA results showed that there were no expressions of TNF-α and IL-1α in groups A-D at different time points. The expression of IL-6 had an increasing trend with time prolonged in each group, showing significant differences between different time points (P<0.05). Moreover, at 1 day after culture, the expression of IL-6 showed no significant difference among groups P>0.05). At 2 and 3 days after culture, the expression of IL-6 in groups A-C showed an increasing trend and was significantly higher than that in group D, showing significant difference among groups (P<0.05).
Conclusions: PGN-sa can promote osteoclast differentiation through NF-κB signaling pathway, and IL-6 may play a role in this process.
目的: 探讨金黄色葡萄球菌肽聚糖（Staphylococcal peptidoglycan，PGN-sa）促进破骨细胞分化的分子机制。.
方法: Western blot检测：取Raw264.7细胞，以PGN-sa或SC75741（NF-κB激活的强效抑制剂）+PGN-sa分别作用0、1、2、3 d，检测活化T细胞核因子1（nuclear factor of activated T-cells cytoplasmic 1，NFATc1）蛋白表达；分别作用0、5、10、20、40、60 min，检测破骨细胞分化信号通路相关蛋白[细胞外调节蛋白激酶（extracellular regulated protein kinases，ERK）、p38、c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）、NF-κB、NF-κB抑制蛋白（inhibitor of NF-κB，IκB-α）、Akt及磷酸化蛋白p-p38、p-ERK、p-JNK、p-Akt、p-NF-κB]表达。ELISA检测：取Raw264.7细胞分为4组，分别以100（A组）、200（B组）、400 ng/mL PGN-sa（C组）及PBS（D组）作用1、2、3 d，检测TNF-α、IL-1α及IL-6的蛋白表达量。.
结果: Western blot检测：随培养时间延长，NFATc1蛋白表达量呈逐渐增加趋势，各时间点间比较差异均有统计学意义（P＜0.05）；但加入SC75741后，NFATc1蛋白表达在2、3 d明显受到抑制，与未加入SC75741前比较差异有统计学意义（P＜0.001）。PGN-sa刺激后5、10 min时IκB-α蛋白表达量明显降低，p-NF-κB蛋白表达量明显增高，均于20 min后恢复正常，5、10 min时蛋白表达量与其余时间点比较差异均有统计学意义（P＜0.001），且5 min时p-NF-κB蛋白表达量高于10 min时（P＜0.001）。加入SC75741后，IκB-α及p-NF-κB的蛋白表达量无变化，各时间点间比较差异均无统计学意义（P＞0.05）。其他蛋白（ERK、p38、JNK、NF-κB、Akt及p-p38、p-ERK、p-JNK、p-Akt）表达量在各时间点均无明显变化（P＞0.05）。ELISA检测：各时间点A～D组TNF-α及IL-1α均未见表达。各组IL-6的表达随时间延长呈递增趋势，各时间点间比较差异均有统计学意义（P＜0.05）。培养1 d时，各组间比较IL-6蛋白表达差异无统计学意义（P＞0.05）；2、3 d时，A、B、C组IL-6蛋白表达显著高于D组，A、B、C组间呈递增趋势，各组间比较差异均有统计学意义（P＜0.05）。.
结论: PGN-sa通过NF-κB信号通路促进破骨细胞的分化形成，IL-6在这一过程中可能起一定作用。.
Keywords: Molecular mechanism; Nuclear factor κB; Osteoclast differentiation; Osteomyelitis; Staphylococcal peptidoglycan.