异泽兰黄素通过调控MAPK和NF-κB信号通路抑制RANKL诱导的破骨细胞分化

doi:10.13701/j.cnki.kqyxyj.2024.09.012

摘要/Abstract

摘要： 目的:探究异泽兰黄素(Eupatilin)对破骨细胞前体细胞向破骨细胞分化的作用和分子机制。方法:CCK-8法检测不同浓度的Eupatilin对破骨细胞前体细胞RAW264.7以及小鼠骨髓巨噬细胞(bone marrow-derived macrophages,BMDMs)细胞活性的影响。用核因子κB 受体活化因子配体(RANKL)诱导RAW264.7和BMDMs分化形成破骨细胞,同时使用不同浓度的Eupatilin(5、10、20 μmol/L)干预,通过TRAP染色和F-actin染色评价Eupatilin对RANKL诱导的破骨细胞形成作用,qRT-PCR和Western blot检测破骨细胞相关标志基因的表达,Western blot检测丝裂原活化蛋白激酶(MAPK)和核因子-κB(NF-κB)信号通路分子的磷酸化水平。结果:20 μmol/L Eupatilin对破骨细胞的分化具有显著的抑制作用,同时对相关标志基因抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphase, TRAP)、基质金属蛋白酶-9(matrix metallopeptidase-9, MMP-9)、组织蛋白酶K(cathepsin K, CTSK)等的表达也表现出有效的下调作用。Western blot结果显示Eupatilin显著抑制了RANKL诱导的MAPK和NF-κB信号通路分子的磷酸化水平。结论:Eupatilin通过调控MAPK和NF-κB信号通路,对RANKL诱导的破骨细胞分化具有显著的抑制作用。

关键词: 异泽兰黄素, 破骨细胞, MAPK信号通路, NF-κB信号通路

Abstract: Objective: To investigate the effects and molecular mechanisms of eupatilin on the differentiation of osteoclast precursor cells to osteoblasts. Methods: CCK-8 assay was used to detect the effects of different concentrations of Eupatilin on the cell activity of osteoclast precursor cells RAW264.7 and mouse bone marrow-derived macrophages (BMDMs). RAW264.7 and BMDMs were induced to differentiate into osteoclasts by receptor activator of nuclear factor-κB ligand (RANKL), and different concentrations of Eupatilin (5, 10, and 20 μmol/L) were used for intervention. The effects of Eupatilin on RANKL-induced osteoclast formation were evaluated through TRAP staining and F-actin staining. The expression of osteoclast-related marker genes was detected by qRT-PCR and Western blot, and the phosphorylation levels of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathway molecules were detected by Western blot. Results: Eupatilin at 20 μmol/L exhibited a significant inhibitory effect on the differentiation of osteoclasts, and also shown an effective downregulation of the expression of related marker genes such as tartrate-resistant acid phosphas (TRAP), matrix metalloproteinase-9 (MMP-9), and cathepsin K (CTSK). Western blot results indicated that Eupatilin significantly inhibited the phosphorylation levels of MAPK and NF-κB signaling pathway molecules induced by RANKL. Conclusion: Eupatilin exerts a significant inhibitory effect on RANKL-induced osteoclast differentiation by regulating the MAPK and NF-κB signaling pathways.

Key words: Eupatilin, osteoclast, MAPK signaling pathway, NF-κB signaling pathway

赵博轩, 李婷, 姚汉涛, 王紫君, 郭海盈, 季耀庭, 杜民权. 异泽兰黄素通过调控MAPK和NF-κB信号通路抑制RANKL诱导的破骨细胞分化[J]. 口腔医学研究, 2024, 40(9): 820-826.

ZHAO Boxuan, LI Ting, YAO Hantao, WANG Zijun, GUO Haiying, JI Yaoting, DU Minquan. Eupatilin Inhibits RANKL-induced Osteoclast Differentiation by Regulating MAPK and NF-κB Signaling Pathways[J]. Journal of Oral Science Research, 2024, 40(9): 820-826.

参考文献

[1] Kinane DF, Stathopoulou PG, Papapanou PN. Periodontal diseases [J]. Nat Rev Dis Primers, 2017, 3: 17038.
[2] Habashneh RA, Mashal MA, Khader Y, et al. Clinical and biological effects of adjunctive photodynamic therapy in refractory periodontitis [J]. J Lasers Med Sci, 2019, 10(2): 139-145.
[3] Graziani F, Karapetsa D, Alonso B, et al. Nonsurgical and surgical treatment of periodontitis: how many options for one disease? [J]. Periodontol 2000, 2017, 75(1): 152-188.
[4] McGowan NW, Walker EJ, Macpherson H, et al. Cytokine-activated endothelium recruits osteoclast precursors [J]. Endocrinology, 2001, 142(4): 1678-1681.
[5] Teitelbaum SL. Bone resorption by osteoclasts [J]. Science, 2000, 289(5484): 1504-1508.
[6] Xiong J, O'Brien CA. Osteocyte RANKL: new insights into the control of bone remodeling [J]. J Bone Miner Res, 2012, 27(3): 499-505.
[7] Chen K, Qiu P, Yuan Y, et al. Pseurotin a inhibits osteoclastogenesis and prevents ovariectomized-induced bone loss by suppressing reactive oxygen species [J]. Theranostics, 2019, 9(6): 1634-1650.
[8] Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function [J]. Nat Rev Genet, 2003, 4(8): 638-649.
[9] Zhou S, Ji Y, Yao H, et al. Application of ginsenoside Rd in periodontitis with inhibitory effects on pathogenicity, inflammation, and bone resorption [J]. Front Cell Infect Microbiol, 2022, 12: 813953.
[10] Yao H, Du Y, Jiang B, et al. Sulforaphene suppresses RANKL-induced osteoclastogenesis and LPS-induc ed bone erosion by activating Nrf2 signaling pathway [J]. Free Radic Biol Med, 2023, 207: 48-62.
[11] Liao Y, Yan Q, Cheng T, et al. Sulforaphene inhibits periodontitis through regulating macrophage polarization via upregulating dendritic cell immunoreceptor[J]. J Agric Food Chem, 2023, 71(42): 15538-15552.
[12] Nageen B, Sarfraz I, Rasul A, et al. Eupatilin: a natural pharmacologically active flavone compound with it s wide range applications [J]. J Asian Nat Prod Res, 2020, 22(1): 1-16.
[13] Jeong JH, Moon SJ, Jhun JY, et al. Eupatilin exerts antinociceptive and chondroprotective properties in a rat model of osteoarthritis by downregulating oxidative damage and catabolic activity in chondrocytes [J]. PLoS One, 2015, 10(6): e0130882.
[14] Choi EJ, Oh HM, Na BR, et al. Eupatilin protects gastric epithelial cells from oxidative damage and down-regulates genes responsible for the cellular oxidative stress [J]. Pharm Res, 2008, 25(6): 1355-1364.
[15] Zhong W, Wu Z, Chen N, et al. Eupatilin inhibits renal cancer growth by downregulating microRNA-21 through the activation of YAP1 [J]. Biomed Res Int, 2019, 2019: 5016483.
[16] Wu Z, Zou B, Zhang X, et al. Eupatilin regulates proliferation and cell cycle of cervical cancer by regulating hedgehog signalling pathway [J]. Cell Biochem Funct, 2020, 38(4): 428-435.
[17] Kim KJ, Kang NE, Oh YS, et al. Eupatilin alleviates hyperlipidemia in mice by inhibiting HMG-CoA reductase [J]. Biochem Res Int, 2023, 2023: 8488648.
[18] Gong H, Lyu X, Liu Y, et al. Eupatilin inhibits pulmonary fibrosis by activating Sestrin2/PI3K/Akt/mTOR dependent autophagy pathway [J]. Life Sci, 2023, 334: 122218.
[19] Bai D, Sun T, Lu F, et al. Eupatilin Suppresses OVA-induced asthma by inhibiting NF-κB and MAPK and activating Nrf2 signaling pathways in mice [J]. Int J Mol Sci, 2022, 23(3): 1582.
[20] Novack DV. Role of NF-κB in the skeleton [J]. Cell Res, 2011, 21(1): 169-182.
[21] Rosen CJ, Kessenich CR. The pathophysiology and treatment of postmenopausal osteoporosis. An e vidence-based approach to estrogen replacement therapy [J]. Endocrinol Metab Clin North Am, 1997, 26(2): 295-311.
[22] Cicalău GIP, Babes PA, Calniceanu H, et al. Anti-inflammatory and antioxidant properties of carvacrol and magnolol, in periodontal disease and diabetes mellitus [J]. Molecules, 2021, 26(22):6899.
[23] Jegal KH, Ko HL, Park SM, et al. Eupatilin induces Sestrin2-dependent autophagy to prevent oxidative stress [J]. Apoptosis, 2016, 21(5): 642-656.
[24] Park BB, Yoon Js, Kim Es, et al. Inhibitory effects of eupatilin on tumor invasion of human gastric cancer MKN-1 cells [J]. Tumour Biol, 2013, 34(2): 875-885.
[25] Choi EJ, Lee S, Chae JR, et al. Eupatilin inhibits lipopolysaccharide-induced expression of inflammatory mediators in macrophages [J]. Life Sci, 2011, 88(25-26): 1121-1126.
[26] Min SW, Kim NJ, Baek NI, et al. Inhibitory effect of eupatilin and jaceosidin isolated from Artemisia princeps on carrageenan-induced inflammation in mice [J]. J Ethnopharmacol, 2009, 125(3): 497-500.
[27] Jeon JI, Ko SH, Kim YJ, et al. The flavone eupatilin inhibits eotaxin expression in an NF-κB-dependent and STAT6-independent manner [J]. Scand J Immunol, 2015, 81(3): 166-176.
[28] Kim MJ, Kim DH, Na HK, et al. TNF-α induces expression of urokinase-type plasminogen activator and β-catenin activation through generation of ROS in human breast epithelial cells [J]. Biochem Pharmacol, 2010, 80(12): 2092-2100.
[29] Cai M, Phan PT, Hong JG, et al. The neuroprotective effect of eupatilin against ischemia/reperfusion-induced delayed neuronal damage in mice [J]. Eur J Pharmacol, 2012, 689(1-3): 104-110.
[30] Sapkota A, Gaire BP, Cho KS, et al. Eupatilin exerts neuroprotective effects in mice with transient focal cerebral ischemia by reducing microglial activation [J]. PLoS One, 2017, 12(2): e0171479.
[31] Je HD, Kim HD, Jeong JH. The inhibitory effect of eupatilin on the agonist-induced regulation of vascular contractility [J]. Korean J Physiol Pharmacol, 2013, 17(1): 31-36.
[32] Yang H, Lee DY, Jeon M, et al. Determination of five active compounds in Artemisia princeps and A. capillaris based on UPLC-DAD and discrimination of two species with multivariate analysis [J]. Arch Pharm Res, 2014, 37(5): 617-625.
[33] Soysa NS, Alles N. Osteoclast function and bone-resorbing activity: An overview [J]. Biochem Biophys Res Commun, 2016, 476(3): 115-120.
[34] Kong L, Wang B, Yang X, et al. Integrin-associated molecules and signalling cross talking in osteoclast cytoskeleton regulation [J]. J Cell Mol Med, 2020, 24(6): 3271-3281.
[35] Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation [J]. Nature, 2003, 423(6937): 337-342.
[36] Wada T, Nakashima T, Hiroshi N, et al. RANKL-RANK signaling in osteoclastogenesis and bone disease [J]. Trends Mol Med, 2006, 12(1): 17-25.