West China School of Public Health and West China Fourth Hospital, Sichuan University, Chengdu, 610041, PR China; Food Safety Monitoring and Risk Assessment Key Laboratory of Sichuan Province, Department of Public Health Laboratory Sciences, West China School of Public Health, Sichuan University, Chengdu, 610041, PR China.
College of Medical Technology, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, PR China.
J Ethnopharmacol. 2022 May 10;289:115094. doi: 10.1016/j.jep.2022.115094. Epub 2022 Feb 8.
Zanthoxylum bungeanum Maxim (ZBM), a traditional Chinese medicine, is traditionally used for osteoporosis treatment recorded in ancient Chinese medicine work Benjingshuzheng and reported to have the anti-bone loss activity in recent studies. However, the anti-osteoporotic activities of the seed of ZBM have not been elucidated yet. Our previous study found that Zanthoxylum bungeanum Maxim seed oil (ZBSO) was rich in polyunsaturated fatty acids (PUFAs), which were reported to prevent bone loss. Thus, we propose a hypothesis that ZBSO could be a potential natural resource for anti-bone loss.
To investigate whether ZBSO could prevent bone loss by targeting osteoclastogenesis and investigate the potential mechanisms in receptor-activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells.
RAW264.7 cells were treated with RANKL in the presence or absence of ZBSO. The effect of ZBSO on osteoclast differentiation and bone resorption activity of RAW264.7 cells were evaluated by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, and bone resorption assay. Differentially expression genes (DEGs) and relevant pathways of different cell groups were obtained from RNA sequencing and protein-protein interaction (PPI) network analysis followed by KEGG pathway enrichment analysis. The effect of ZBSO on the RANKL-induced cell cycle change was analyzed by flow cytometry assay, and the expression of genes and proteins related to the selected pathways was further verified by RT-qPCR and western blot analysis.
The inhibitory effects of ZBSO on osteoclast differentiation and bone resorption activity in a dose-dependent manner were demonstrated by TRAP staining, F-actin ring staining, and bone resorption assay in RANKL-induced RAW264.7 cells. Osteoclast differentiation and cell cycle pathways were the most enriched pathways based on DEGs enrichment analysis among different cell groups. The reversion effect of ZBSO on the RANKL-induced RAW264.7 cell cycle arrest at the G1 phase was observed by flow cytometry assay. Western blot results showed that ZBSO markedly decreased RANKL-induced activation of ERK, as well as the phosphorylation of c-JUN and NFATc1 expression, and subsequently suppressed osteoclast-specific genes, such as Ctsk, Trap, and Dc-stamp.
ZBSO exhibited an inhibitory effect on osteoclastogenesis via suppressing the ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest induced by RANKL, suggesting that ZBSO may serve as a promising agent for anti-bone loss.
花椒（ZBM）是一种传统的中药，在中国古代医学著作《本草纲目》中被记载用于治疗骨质疏松症，最近的研究表明其具有抗骨丢失活性。然而，花椒种子的抗骨质疏松活性尚未得到阐明。我们之前的研究发现，花椒籽油（ZBSO）富含多不饱和脂肪酸（PUFAs），据报道可预防骨丢失。因此，我们提出假设，花椒籽油可能是一种具有抗骨丢失潜力的天然资源。
研究花椒籽油是否可以通过靶向破骨细胞分化来预防骨丢失，并探讨其在核因子κB 受体激活剂配体（RANKL）诱导的 RAW264.7 细胞中的潜在机制。
用 RANKL 处理 RAW264.7 细胞，同时存在或不存在 ZBSO。通过抗酒石酸酸性磷酸酶（TRAP）染色、F-肌动蛋白环染色和骨吸收试验评估 ZBSO 对 RAW264.7 细胞破骨细胞分化和骨吸收活性的影响。通过 RNA 测序和蛋白质-蛋白质相互作用（PPI）网络分析获得不同细胞组的差异表达基因（DEGs）和相关途径，然后进行 KEGG 途径富集分析。通过流式细胞术分析 ZBSO 对 RANKL 诱导的细胞周期变化的影响，并通过 RT-qPCR 和 Western blot 分析进一步验证与所选途径相关的基因和蛋白质的表达。
TRAP 染色、F-肌动蛋白环染色和骨吸收试验结果表明，ZBSO 以剂量依赖的方式抑制 RANKL 诱导的 RAW264.7 细胞中的破骨细胞分化和骨吸收活性。在不同细胞组的 DEGs 富集分析中，破骨细胞分化和细胞周期途径是最富集的途径。流式细胞术结果表明，ZBSO 可逆转 RANKL 诱导的 RAW264.7 细胞在 G1 期的细胞周期阻滞。Western blot 结果表明，ZBSO 显著降低了 RANKL 诱导的 ERK 激活以及 c-JUN 和 NFATc1 表达的磷酸化，从而抑制了破骨细胞特异性基因，如 Ctsk、Trap 和 Dc-stamp。
ZBSO 通过抑制 ERK/c-JUN/NFATc1 通路和调节 RANKL 诱导的细胞周期阻滞，对破骨细胞发生具有抑制作用，提示 ZBSO 可能是一种有前途的抗骨丢失药物。
