自噬在结核菌素诱导破骨细胞形成中的作用及机制

Role and mechanism of autophagy in osteoclast formation induced by tuberculin

目的研究自噬在结核菌素纯化蛋白洐生物(PPD)诱导破骨细胞形成中的作用及机制。方法培养小鼠单核巨噬细胞(RAW264.7细胞),用不同浓度(1.0、2.5、5.0、10.0 IU/mL)PPD及自噬激动剂雷帕霉素、自噬抑制剂3-甲基腺苷(3-MA)处理。处理24 h后,检测细胞活力;处理7 d后,检测破骨细胞数量、抗酒石酸酸性磷酸酶(TRAP)活力、自噬小体数目及自噬标志基因Beclin-1、LC3-Ⅱ/LC3-Ⅰ的表达水平。结果与对照组相比,10.0 IU/mL PPD组细胞活力降低,差异有统计学意义(P<0.05),1.0、2.5、5.0 IU/mL PPD组细胞活力无明显变化。与对照组相比,2.5、5.0、10.0 IU/mL PPD组破骨细胞数量、TRAP活力、自噬小体数目及Beclin-1、LC3-Ⅱ/LC-Ⅰ的表达水平均明显增加,差异有统计学意义(P<0.05)。与5.0 IU/mL PPD组相比,5.0 IU/mL PPD+激动剂组破骨细胞数量、TRAP活力、自噬小体数目及Beclin-1、LC3-Ⅱ/LC-Ⅰ的表达水平均明显增加,差异有统计学意义(P<0.05);细胞活力无明显变化。与5.0 IU/mL PPD组相比,5.0 IU/mL PPD+抑制剂组破骨细胞数量、TRAP活力、自噬小体数目及Beclin-1、LC3-Ⅱ/LC-Ⅰ的表达水平均明显降低,差异有统计学意义(P<0.05);细胞活力无明显变化。结论PPD能够诱导破骨细胞形成,其机制与自噬激活有关。

Objective To investigate the role and mechanism of autophagy in osteoclast formation induced by tuberculin purified protein derivatives(PPD).Methods Monocyte macrophages(RAW264.7 cells)were cultured and treated with different concentrations of PPD(1.0,2.5,5.0 and 10.0 IU/mL),autophagy agonist rapamycin and autophagy inhibitor 3-methyladenosine(3-MA).After 24 h of treatment,the cells viability was detected;after 7 d of treatment,the number of osteoclasts,the activity of tartrate resistant acid phosphatase(TRAP),the number of autophagosome and the expression of autophagy marker genes Beclin-1 and LC3-Ⅱ/LC3-Ⅰwere detected.Results Compared with the control group,the cells viability in 10.0 IU/mL PPD group was decreased,and the difference was statistically significant;but it had no significant change in 1.0,2.5 and 5.0 IU/mL PPD groups.Compared with the control group,the number of osteoclasts,the activity of TRAP,the number of autophagosome,and the expression levels of Beclin-1 and LC3-Ⅱ/LC-Ⅰin 2.5,5.0 and 10.0 IU/mL PPD groups were significantly increased,and the differences were statistically significant(P<0.05).Compared with 5.0 IU/mL PPD group,the number of osteoclasts,the activity of TRAP,the number of autophagosome,and the expression levels of Beclin-1 and LC3-Ⅱ/LC-Ⅰin 5.0 IU/mL PPD+agonist group were significantly increased,and the differences were statistically significant(P<0.05);the cell viability had no significant change.Compared with 5.0 IU/mL PPD group the number of osteoclasts,the activity of TRAP,the number of autophagosome,and the expression levels of Beclin-1 and LC3-Ⅱ/LC-Ⅰin 5.0 IU/mLPPD+inhibitor group were significantly decreased,and the differences were statistically significant(P<0.05);the cell viability had no significant change(P>0.05).Conclusion PPD can induce osteoclast formation,and its mechanism is related to the activation of autophagy.