Aloperine inhibits RANKL-induced osteoclast differentiation via suppressing the MAPK signaling pathways

Xiang Wu, Caiyun Fu, Xuefeng He, Shaolei Wang
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Abstract

To figure out the molecular mechanism of aloperine (ALO) on receptor activator of nuclear factor (NF)-kappa-B (κb) ligand (RANKL)-caused osteoclast differentiation. The histopathological analysis and tartrate-resistant acid phosphatase (TRAP) staining assays were applied to check the extent of bone loss of the femur. Then, the protein expressions of nitric oxide synthase 2, glutathione reductase, nuclear erythroid 2-related factor 2, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1, and catalase were checked in RAW264.7, a macrophage cell line, by enzyme-linked-immunosorbent serologic assay and Western blot analysis. Further, the TRAP staining and quantitative polymerase chain reaction assay were applied to characterize the level of RAW264.7 osteoclast differentiation. Besides, Western blot assay was used to check the protein expressions of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and P38 in RAW264.7. Finally, ERK activation blocker U0126 was used to inhibit mitogen-activated protein kinase (MAPK) signaling pathway to determine the effect of MAPK signaling pathway on RAW264.7 osteoclast differentiation. In this study, the results demonstrated that ALO could inhibit lipopolysaccharide-induced bone loss in vivo in a dose-dependent manner, with significant inhibitory effects at high doses. Further research indicated that ALO could inhibit RANKL-induced oxidative stress and osteoclast differentiation of RAW264.7. Then, ALO could inactivate MAPK signaling pathway. Finally, the results showed that inhibition of MAPK signaling pathway could increase ALO inhibition of RANKL-caused osteoclast differentiation. ALO inhibits RANKL-caused osteoclast differentiation by suppressing the MAPK signaling pathways.
Aloperine通过抑制MAPK信号通路抑制rankl诱导的破骨细胞分化
探讨aloperine (ALO)对核因子受体激活剂(NF)- κb (κb)配体(RANKL)诱导破骨细胞分化的分子机制。采用组织病理学分析和抗酒石酸酸性磷酸酶(TRAP)染色检测股骨骨丢失程度。采用酶联免疫吸附血清学和Western blot检测巨噬细胞RAW264.7中一氧化氮合酶2、谷胱甘肽还原酶、核红细胞2相关因子2、血红素加氧酶-1、烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶1和过氧化氢酶的蛋白表达。进一步,采用TRAP染色和定量聚合酶链反应技术表征RAW264.7破骨细胞分化水平。Western blot检测RAW264.7细胞外调节蛋白激酶(ERK)、c-Jun n -末端激酶(JNK)、P38蛋白的表达情况。最后,利用ERK激活阻断剂U0126抑制丝裂原活化蛋白激酶(MAPK)信号通路,确定MAPK信号通路对RAW264.7破骨细胞分化的影响。本研究结果表明,在体内,ALO能够以剂量依赖的方式抑制脂多糖诱导的骨质流失,且在高剂量时具有明显的抑制作用。进一步研究表明,ALO可抑制rankl诱导的RAW264.7氧化应激和破骨细胞分化。因此,ALO可以使MAPK信号通路失活。最后,结果表明,抑制MAPK信号通路可以增加ALO对rankl诱导的破骨细胞分化的抑制。ALO通过抑制MAPK信号通路抑制rankl引起的破骨细胞分化。
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