唑来膦酸调控NLRP3信号通路抑制脂多糖诱导的破骨细胞分化

Zoledronic acid inhibits lipopolysaccharide-induced osteoclast differentiation by regulating NLRP3 signaling pathway

背景:唑来膦酸可抑制骨吸收,但其是否能抑制炎症性骨疾病及其相关机制尚不完全清楚。目的:分析唑来膦酸对脂多糖诱导破骨细胞增殖、分化的影响。方法:利用脂多糖将RAW264.7细胞诱导为破骨细胞,采用抗酒石酸酸性磷酸酶染色和F-actin染色鉴定诱导结果。将RAW264.7细胞分5组培养:空白对照组不进行任何干预,对照组加入脂多糖处理,其余3组在加入脂多糖的基础上分别加入0.1,1,5μmol/L的唑来膦酸处理,培养6 h后,采用ELISA法检测上清液中肿瘤坏死因子α水平,Western Blot检测NLRP3、caspase-1、白细胞介素1β、cleaved-caspase-1及肿瘤坏死因子α的蛋白表达。培养第5天,抗酒石酸酸性磷酸酶染色观察破骨细胞形成情况,F-actin染色观察破骨细胞肌动蛋白环。结果与结论:①抗酒石酸酸性磷酸酶染色和F-actin染色显示,脂多糖可诱导RAW264.7细胞分化为破骨细胞;②对照组肿瘤坏死因子α水平高于空白对照组(P<0.05),0.1,5μmol/L唑来膦酸组肿瘤坏死因子α水平低于对照组(P<0.05);抗酒石酸酸性磷酸酶染色与F-actin染色显示,1,5μmol/L唑来膦酸可抑制脂多糖诱导RAW264.7细胞分化为破骨细胞;③与对照组比较,0.1,1,5μmol/L的唑来膦酸均可抑制NLRP3、caspase-1、白细胞介素1β及肿瘤坏死因子α的蛋白表达(P<0.05),1,5μmol/L的唑来膦酸均可抑制cleaved-caspase-1的蛋白表达(P<0.05);④结果表明,唑来膦酸可抑制脂多糖诱导的破骨细胞分化,该作用可能是通过调控NLRP3信号通路实现的。

BACKGROUND:Zoledronic acid can inhibit bone resorption,but whether it can inhibit inflammatory bone disease and its related mechanisms are completely unclear.OBJECTIVE:To investigate the effect of zoledronic acid on the proliferation and differentiation of osteoclasts induced by lipopolysaccharide.METHODS:RAW264.7 cells were treated with lipopolysaccharide for osteoclastic induction and dyed with tartrate-resistant acid phosphatase and F-actin.RAW264.7 cells were cultured in vitro and divided into blank control group(no intervention),control group(treated with lipopolysaccharide),and 0.1,1,5μmol/L zoledronic acid groups(lipopolysaccharide+0.1,1,5μmol/L zoledronic acid).After 6 hours of culture,the level of tumor necrosis factor α in the supernatant was detected by ELISA,and the protein expression levels of NLRP3,caspase-1,interleukin-1β,cleaved-caspase-1 and tumor necrosis factor α were detected by western blot assay.On the 5^(th) day of culture,the formation of osteoclasts was observed by tartrate-resistant acid phosphatase staining,and the expression of actin rings in osteoclasts was observed by F-actin staining.RESULTS AND CONCLUSION:(1)Lipopolysaccharide could induce RAW264.7 cells to differentiate into osteoclasts.(2)The level of tumor necrosis factor α was higher in the control group than the blank control group(P<0.05)and lower in the 0.1 and 5μmol/L zoledronic acid groups than the control group(P<0.05).Results from tartrate-resistant acid phosphatase and F-actin staining indicated that 1 and 5μmol/L zoledronic acid could inhibit lipopolysaccharide-induced differentiation of RAW264.7 cells into osteoclasts.(3)Compared with the control group,0.1,1,and 5μmol/L zoledronic acid could inhibit the protein expression of NLRP3,caspase-1,interleukin-1β,and tumor necrosis factorα(P<0.05),while 1 and 5μmol/L zoledronic acid could inhibit the protein expression of cleaved-caspase-1(P<0.05).(4)To conclude,zoledronic acid can inhibit osteoclast formation induced by lipopolysaccharide,which acts possibly through regulating the NLRP3 signaling pathway.