Effect of Duanteng Yimu Decoction on RANKL Signaling Pathway and MMP-9 in Osteoclasts

CAI Xu-dong; LIN Chang-song; PAN Dong-mei; LI Nan; JIANG Yu-bao; QIU Wen-yu; GUO Tian-feng

doi:10.13422/j.cnki.syfjx.20181023

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Effect of Duanteng Yimu Decoction on RANKL Signaling Pathway and MMP-9 in Osteoclasts

更新时间：2024-01-04

- Effect of Duanteng Yimu Decoction on RANKL Signaling Pathway and MMP-9 in Osteoclasts
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 24, Issue 12, Pages: 68-73(2018)
- 作者机构：
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20181023
  CLC： R285
- Published：2018
- 稿件说明：
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CAI Xu-dong, LIN Chang-song, PAN Dong-mei, et al. Effect of Duanteng Yimu Decoction on RANKL Signaling Pathway and MMP-9 in Osteoclasts[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(12): 68-73.
DOI：

CAI Xu-dong, LIN Chang-song, PAN Dong-mei, et al. Effect of Duanteng Yimu Decoction on RANKL Signaling Pathway and MMP-9 in Osteoclasts[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(12): 68-73. DOI： 10.13422/j.cnki.syfjx.20181023.

摘要

目的：探讨断藤益母汤（DTYMD）对破骨细胞（OC）核转录因子-κB （NF-κB）受体活化因子配体（RANKL）信号通路及基质金属蛋白酶-9（MMP-9）的影响及其骨保护的作用机制。方法：通过诱导RAW264.7巨噬细胞建立体外破骨细胞培养体系；设空白组，模型组，DTYMD （400，600，800，1 000 mg·L-1）组和甲氨蝶呤（2 mg·L-1）组，然后通过抗酒石酸酸性磷酸酶（TRAP）染色对破骨细胞进行鉴定；细胞增殖检测（CCK-8）各组细胞生存率；蛋白免疫印迹法（Western blot）检测RANKL信号通路关键蛋白表达；酶联免疫吸附法（ELISA）检测各组细胞MMP-9表达水平的变化。结果： TRAP染色显示，与模型组相比，DTYMD 400，600 mg·L-1组融合细胞数量有所减少，但差异无统计学差异；DTYMD 800，1 000 mg·L-1组融合细胞数目显著减少（P<0.01）。CCK-8结果显示，与模型组比较，DTYMD 600，800，1 000 mg·L-1组和甲氨蝶呤2 mg·L-1组生存率明显下降（P<0.05，P<0.01）。Western blot结果显示，与模型组相比，DTYMD 1 000 mg·L-1组、甲氨蝶呤2 mg·L-1组中RANKL蛋白表达有所下降（P<0.05）。DTYMD各组中的OPG蛋白表达水平无明显变化。ELISA结果显示，与模型组相比，DTYMD各组及甲氨蝶呤组中的MMP-9表达水平均有所下降，且DTYMD质量浓度越高，下降越明显（P<0.05，P<0.01）。结论：断藤益母汤可能是通过下调RANKL和MMP-9的表达以及抑制破骨细胞的分化和增殖而起到骨保护作用。

Abstract

Objective: To investigate the effect of Duanteng Yimu decoction (DTYMD) on the expression of NF-κB receptor activator ligand (RANKL) pathway and matrix metalloproteinase-9 (MMP-9) in osteoclasts and the mechanism of bone protection. Method: In vitro osteoclast culture system was established by inducing RAW264.7 macrophages. Blank group

model group

methotrexate group (2 mg·L-1) and DTYMD groups (400

600

800

1 000 mg·L-1) were set up. The osteoblasts were identified by anti-tartaric acid phosphatase (TRAP) staining. The survival rate of each group was detected by cell counting kit-8 (CCK-8) method. The protein expression of RANKL signal pathway was detected by Western blot. The expression of MMP-9 was detected by enzyme-linked immunosorbent assay (ELISA). Result: Compared with model group

the number of fusion cells in DTYMD 400

600 mg·L-1 concentration group decreased

but the difference was not statistically significant; the number of fusion cells was significantly decreased in DTYMD 800

1 000 mg·L-1 concentration groups (P<0.01). CCK-8 results showed significant decreases in survival rates of DTYMD (600

800

1 000 mg·L-1) and methotrexate (2 mg·L-1) compared with model group (P<0.05

P<0.01). Western blot results showed that the protein expression of RANKL decreased in DTYMD 1 000 mg·L-1 and methotrexate 2 mg·L-1 groups (P<0.05) compared with model group. The protein expression of OPG in DTYMD group had no significant change. The results of ELISA showed that the expression of MMP-9 decreased in DTYMD concentration group and methotrexate group compared with the model group; and the higher the DTYMD concentration was

the more significantly decreased(P<0.05). Conclusion: DTYMD may protect bone cells by down-regulating the expressions of RANKL and MMP-9 and inhibiting the differentiation and proliferation of osteoclasts.

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