白杨素抑制核因子κB受体活化因子配体诱导的小鼠破骨细胞生成
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摘要
背景:白杨素存在于多种天然植物提取物的黄酮醇类化合物,具有广泛的治疗作用并且参与机体内的炎症反应,而炎症反应可增强破骨细胞生成从而导致骨侵蚀。目的:探究白杨素在炎症环境和非炎症环境下对破骨细胞分化的影响以及对骨侵蚀的保护作用。方法:选择RAW264.7细胞为种子细胞,首先,通过核因子κB受体活化因子配体(50μg/L)、巨噬细胞集落刺激因子(25μg/L)将细胞诱导分化为破骨细胞后,以0,20,40,60μg/L白杨素干预。其次,通过脂多糖模拟炎症环境,并将RAW264.7细胞在脂多糖诱导的炎症环境下诱导分化为破骨细胞,观察0,20,40,60μg/L白杨素在炎症环境下对破骨细胞分化的影响。结果与结论:(1)白杨素有效抑制破骨细胞分化,在60μg/L时抑制效果达到最大;(2)白杨素显著抑制了破骨细胞的骨吸收功能,提示白杨素对破骨细胞造成的骨侵蚀具有保护作用;(3)白杨素通过核因子κB信号通路,抑制多种破骨细胞分化关键蛋白和基因表达;(4)白杨素对炎症有明显的抑制作用,并对炎症环境导致的破骨细胞分化有强烈的抑制作用。
BACKGROUND: Chrysin(5,7-dihydroxyflavone) is a flavonol in many natural plant extracts. It has a wide range of therapeutic effects and is involved in inflammatory reactions in the body that can enhance osteoclast formation and lead to bone erosion.OBJECTIVE: To investigate the effects of chrysin on osteoclast differentiation and its protective effect on bone erosion in inflammatory and non-inflammatory environments.METHODS: RAW264.7 cells were selected as seed cells. First, the RAW264.7 cells were induced with receptor activator of nuclear factor kappa B ligand(50 μg/L) and macrophage colony-stimulating factor(25 μg/L) to generate osteoclasts. The cells were randomly divided into four groups according to chrysin concentration(0, 20, 40, and 60 μg/L). Second, lipopolysaccharide was used to simulate the inflammatory environment. RAW264.7 cells were induced by lipopolysaccharide to differentiate into osteoclasts, and the effect of different concentrations of chrysin(0, 20, 40, 60 μg/L) on osteoclast differentiation was observed in the same way.RESULTS AND CONCLUSION: Chrysin effectively inhibited osteoclast differentiation, with the maximum effect at 60 μg/L. Chrysin significantly inhibited the bone absorption function of osteoclasts, suggesting that chrysin has a protective effect on bone erosion caused by osteoclasts. Chrysin suppressed the protein and gene expression related to osteoclast differentiation by nuclear factor kappa B signaling pathway. Therefore, chrysin has an anti-inflammatory effect and it is also powerful to inhibit osteoclast differentiation in an inflammatory environment.
BACKGROUND: Chrysin(5,7-dihydroxyflavone) is a flavonol in many natural plant extracts. It has a wide range of therapeutic effects and is involved in inflammatory reactions in the body that can enhance osteoclast formation and lead to bone erosion.OBJECTIVE: To investigate the effects of chrysin on osteoclast differentiation and its protective effect on bone erosion in inflammatory and non-inflammatory environments.METHODS: RAW264.7 cells were selected as seed cells. First, the RAW264.7 cells were induced with receptor activator of nuclear factor kappa B ligand(50 μg/L) and macrophage colony-stimulating factor(25 μg/L) to generate osteoclasts. The cells were randomly divided into four groups according to chrysin concentration(0, 20, 40, and 60 μg/L). Second, lipopolysaccharide was used to simulate the inflammatory environment. RAW264.7 cells were induced by lipopolysaccharide to differentiate into osteoclasts, and the effect of different concentrations of chrysin(0, 20, 40, 60 μg/L) on osteoclast differentiation was observed in the same way.RESULTS AND CONCLUSION: Chrysin effectively inhibited osteoclast differentiation, with the maximum effect at 60 μg/L. Chrysin significantly inhibited the bone absorption function of osteoclasts, suggesting that chrysin has a protective effect on bone erosion caused by osteoclasts. Chrysin suppressed the protein and gene expression related to osteoclast differentiation by nuclear factor kappa B signaling pathway. Therefore, chrysin has an anti-inflammatory effect and it is also powerful to inhibit osteoclast differentiation in an inflammatory environment.
引文
[1]Song D,Meng T,Xu W,et al.5-Fluoruracil blocked giant cell tumor progression by suppressing osteoclastogenesis through NF-kappaB signals and blocking angiogenesis.Bone.2015;78:46-54.
[2]Wei CM,Liu Q,Song FM,et al.Artesunate inhibits RANKL-induced osteoclastogenesis and bone resorption in vitro and prevents LPS-induced bone loss in vivo.J Cell Physiol.2018;233(1):476-485.
[3]Zhang T,Zhao K,Han W,et al.Deguelin inhibits RANKL-induced osteoclastogenesis in vitro and prevents inflammation-mediated bone loss in vivo.J Cell Physiol.2019;234(3):2719-2729.
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[5]Thummuri D,Naidu VGM,Chaudhari P.Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling.J Mol Med(Berl).2017;95(10):1065-1076.
[6]Wang W,Yang L,Zhang D,et al.MicroRNA-218 Negatively Regulates Osteoclastogenic Differentiation by Repressing the Nuclear Factor-κB Signaling Pathway and Targeting Tumor Necrosis Factor Receptor 1.Cell Physiol Biochem.2018;48(1):339-347.
[7]Song F,Wei C,Zhou L,et al.Luteoloside prevents lipopolysaccharide-induced osteolysis and suppresses RANKL-induced osteoclastogenesis through attenuating RANKL signaling cascades.J Cell Physiol.2018;233(2):1723-1735.
[8]Kim HJ,Park C,Kim GY,et al.Sargassum serratifolium attenuates RANKL-induced osteoclast differentiation and oxidative stress through inhibition of NF-κB and activation of the Nrf2/HO-1 signaling pathway.Biosci Trends.2018;12(3):257-265.
[9]Sapkota M,Li L,Kim SW,et al.Thymol inhibits RANKL-induced osteoclastogenesis in RAW264.7 and BMMcells and LPS-induced bone loss in mice.Food Chem Toxicol.2018;120:418-429.
[10]Choi JK,Jang YH,Lee S,et al.Chrysin attenuates atopic dermatitis by suppressing inflammation of keratinocytes.Food Chem Toxicol.2017;110:142-150.
[11]Zeinali M,Rezaee SA,Hosseinzadeh H.An overview on immunoregulatory and anti-inflammatory properties of chrysin and flavonoids substances.Biomed Pharmacother.2017;92:998-1009.
[12]Ramírez-Espinosa JJ,Salda?a-Ríos J,García-Jiménez S,et al.Chrysin Induces Antidiabetic,Antidyslipidemic and Anti-Inflammatory Effects in Athymic Nude Diabetic Mice.Molecules.2017;23(1):E67.
[13]George MY,Esmat A,Tadros MG,et al.In vivo cellular and molecular gastroprotective mechanisms of chrysin;Emphasis on oxidative stress,inflammation and angiogenesis.Eur JPharmacol.2018;818:486-498.
[14]Mani R,Natesan V,Arumugam R.Neuroprotective effect of chrysin on hyperammonemia mediated neuroinflammatory responses and altered expression of astrocytic protein in the hippocampus.Biomed Pharmacother.2017;88:762-769.
[15]Zheng W,Tao Z,Cai L,et al.Chrysin Attenuates IL-1β-Induced Expression of Inflammatory Mediators by Suppressing NF-κB in Human Osteoarthritis Chondrocytes.Inflammation.2017;40(4):1143-1154.
[16]Jiang Y,Gong FL,Zhao GB,et al.Chrysin suppressed inflammatory responses and the inducible nitric oxide synthase pathway after spinal cord injury in rats.Int J Mol Sci.2014;15(7):12270-12279.
[17]Eldutar E,Kandemir FM,Kucukler S,et al.Restorative effects of Chrysin pretreatment on oxidant-antioxidant status,inflammatory cytokine production,and apoptotic and autophagic markers in acute paracetamol-induced hepatotoxicity in rats:An experimental and biochemical study.J Biochem Mol Toxicol.2017;31(11):e21960.
[18]Chen Y,Dou C,Yi J,et al.Inhibitory effect of vanillin on RANKL-induced osteoclast formation and function through activating mitochondrial-dependent apoptosis signaling pathway.Life Sci.2018;208:305-314.
[19]Tang R,Yi J,Yang J,et al.Interleukin-37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction.J Cell Physiol.2019;234(5):7645-7658.
[20]Dharmapatni AASSK,Algate K,Coleman R,et al.Osteoclast-Associated Receptor(OSCAR)Distribution in the Synovial Tissues of Patients with Active RA and TNF-αand RANKL Regulation of Expression by Osteoclasts In Vitro.Inflammation.2017;40(5):1566-1575.
[21]Crockett JC,Rogers MJ,Coxon FP,et al.Bone remodelling at a glance.J Cell Sci.2011;124(Pt 7):991-998.
[22]Suda T,Nakamura I,Jimi E,et al.Regulation of osteoclast function.J Bone Miner Res.1997;12(6):869-879.
[23]Muruganandan S,Dranse HJ,Rourke JL,et al.Chemerin neutralization blocks hematopoietic stem cell osteoclastogenesis.Stem Cells.2013;31(10):2172-2182.
[24]Wu X,Chim SM,Kuek V,et al.HtrA1 is upregulated during RANKL-induced osteoclastogenesis,and negatively regulates osteoblast differentiation and BMP2-induced Smad1/5/8,ERK and p38 phosphorylation.FEBS Lett.2014;588(1):143-150.
[25]Kim JY,Min JY,Baek JM,et al.CTRP3 acts as a negative regulator of osteoclastogenesis through AMPK-c-Fos-NFATc1signaling in vitro and RANKL-induced calvarial bone destruction in vivo.Bone.2015;79:242-251.
[26]Jeong E,Choi HK,Park JH,et al.STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex.Cell Death Differ.2018;25(8):1364-1374.
[27]Park JH,Lee NK,Lee SY.Current Understanding of RANKSignaling in Osteoclast Differentiation and Maturation.Mol Cells.2017;40(10):706-713.
[28]Obaid R,Wani SE,Azfer A,et al.Optineurin Negatively Regulates Osteoclast Differentiation by Modulating NF-κBand Interferon Signaling:Implications for Paget's Disease.Cell Rep.2015;13(6):1096-1102.
[29]Krauss JL,Zeng R,Hickman-Brecks CL,et al.NLRP12provides a critical checkpoint for osteoclast differentiation.Proc Natl Acad Sci U S A.2015;112(33):10455-10460.
[30]Gu Z,Wang H,Xia J,et al.Decreased ferroportin promotes myeloma cell growth and osteoclast differentiation.Cancer Res.2015;75(11):2211-2221.
[31]Jie Z,Shen S,Zhao X,et al.Activatingβ-catenin/Pax6 axis negatively regulates osteoclastogenesis by selectively inhibiting phosphorylation of p38/MAPK.FASEB J.2018.doi:10.1096/fj.201801977R.
[32]Zhang HQ,Wang YJ,Yang GT,et al.Taxifolin Inhibits Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis of Human Bone Marrow-Derived Macrophages in vitro and Prevents Lipopolysaccharide-Induced Bone Loss in vivo.Pharmacology.2019;103(1-2):101-109.
[33]Wang W,Huang M,Hui Y,et al.Cryptotanshinone inhibits RANKL-induced osteoclastogenesis by regulating ERK and NF-κB signaling pathways.J Cell Biochem.2018.doi:10.1002/jcb.28008.
[34]Rehman MU,Ali N,Rashid S,et al.Alleviation of hepatic injury by chrysin in cisplatin administered rats:probable role of oxidative and inflammatory markers.Pharmacol Rep.2014;66(6):1050-1059.
[35]Kats A,Gerasimcik N,N?reoja T,et al.Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS-stimulated co-cultures of periodontal ligament and RAW 264.7 cells,and RANKL-mediated osteoclastogenesis and bone resorption in PBMCs.J Cell Mol Med.2019;23(2):1152-1163.
[36]Kim HJ,Yoon HJ,Kim SY,et al.A medium-chain fatty acid,capric acid,inhibits RANKL-induced osteoclast differentiation via the suppression of NF-κB signaling and blocks cytoskeletal organization and survival in mature osteoclasts.Mol Cells.2014;37(8):598-604.
[37]Kaneko K,Miyamoto Y,Tsukuura R,et al.8-Nitro-cGMP is a promoter of osteoclast differentiation induced by RANKL.Nitric Oxide.2018;72:46-51.
[38]Chai L,Zhou K,Wang S,et al.Psoralen and Bakuchiol Ameliorate M-CSF Plus RANKL-Induced Osteoclast Differentiation and Bone Resorption Via Inhibition of AKT and AP-1 Pathways in Vitro.Cell Physiol Biochem.2018;48(5):2123-2133.
[2]Wei CM,Liu Q,Song FM,et al.Artesunate inhibits RANKL-induced osteoclastogenesis and bone resorption in vitro and prevents LPS-induced bone loss in vivo.J Cell Physiol.2018;233(1):476-485.
[3]Zhang T,Zhao K,Han W,et al.Deguelin inhibits RANKL-induced osteoclastogenesis in vitro and prevents inflammation-mediated bone loss in vivo.J Cell Physiol.2019;234(3):2719-2729.
[4]Chen Y,Wang X,Yang M,et al.miR-145-5p Increases Osteoclast Numbers In Vitro and Aggravates Bone Erosion in Collagen-Induced Arthritis by Targeting Osteoprotegerin.Med Sci Monit.2018;24:5292-5300.
[5]Thummuri D,Naidu VGM,Chaudhari P.Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling.J Mol Med(Berl).2017;95(10):1065-1076.
[6]Wang W,Yang L,Zhang D,et al.MicroRNA-218 Negatively Regulates Osteoclastogenic Differentiation by Repressing the Nuclear Factor-κB Signaling Pathway and Targeting Tumor Necrosis Factor Receptor 1.Cell Physiol Biochem.2018;48(1):339-347.
[7]Song F,Wei C,Zhou L,et al.Luteoloside prevents lipopolysaccharide-induced osteolysis and suppresses RANKL-induced osteoclastogenesis through attenuating RANKL signaling cascades.J Cell Physiol.2018;233(2):1723-1735.
[8]Kim HJ,Park C,Kim GY,et al.Sargassum serratifolium attenuates RANKL-induced osteoclast differentiation and oxidative stress through inhibition of NF-κB and activation of the Nrf2/HO-1 signaling pathway.Biosci Trends.2018;12(3):257-265.
[9]Sapkota M,Li L,Kim SW,et al.Thymol inhibits RANKL-induced osteoclastogenesis in RAW264.7 and BMMcells and LPS-induced bone loss in mice.Food Chem Toxicol.2018;120:418-429.
[10]Choi JK,Jang YH,Lee S,et al.Chrysin attenuates atopic dermatitis by suppressing inflammation of keratinocytes.Food Chem Toxicol.2017;110:142-150.
[11]Zeinali M,Rezaee SA,Hosseinzadeh H.An overview on immunoregulatory and anti-inflammatory properties of chrysin and flavonoids substances.Biomed Pharmacother.2017;92:998-1009.
[12]Ramírez-Espinosa JJ,Salda?a-Ríos J,García-Jiménez S,et al.Chrysin Induces Antidiabetic,Antidyslipidemic and Anti-Inflammatory Effects in Athymic Nude Diabetic Mice.Molecules.2017;23(1):E67.
[13]George MY,Esmat A,Tadros MG,et al.In vivo cellular and molecular gastroprotective mechanisms of chrysin;Emphasis on oxidative stress,inflammation and angiogenesis.Eur JPharmacol.2018;818:486-498.
[14]Mani R,Natesan V,Arumugam R.Neuroprotective effect of chrysin on hyperammonemia mediated neuroinflammatory responses and altered expression of astrocytic protein in the hippocampus.Biomed Pharmacother.2017;88:762-769.
[15]Zheng W,Tao Z,Cai L,et al.Chrysin Attenuates IL-1β-Induced Expression of Inflammatory Mediators by Suppressing NF-κB in Human Osteoarthritis Chondrocytes.Inflammation.2017;40(4):1143-1154.
[16]Jiang Y,Gong FL,Zhao GB,et al.Chrysin suppressed inflammatory responses and the inducible nitric oxide synthase pathway after spinal cord injury in rats.Int J Mol Sci.2014;15(7):12270-12279.
[17]Eldutar E,Kandemir FM,Kucukler S,et al.Restorative effects of Chrysin pretreatment on oxidant-antioxidant status,inflammatory cytokine production,and apoptotic and autophagic markers in acute paracetamol-induced hepatotoxicity in rats:An experimental and biochemical study.J Biochem Mol Toxicol.2017;31(11):e21960.
[18]Chen Y,Dou C,Yi J,et al.Inhibitory effect of vanillin on RANKL-induced osteoclast formation and function through activating mitochondrial-dependent apoptosis signaling pathway.Life Sci.2018;208:305-314.
[19]Tang R,Yi J,Yang J,et al.Interleukin-37 inhibits osteoclastogenesis and alleviates inflammatory bone destruction.J Cell Physiol.2019;234(5):7645-7658.
[20]Dharmapatni AASSK,Algate K,Coleman R,et al.Osteoclast-Associated Receptor(OSCAR)Distribution in the Synovial Tissues of Patients with Active RA and TNF-αand RANKL Regulation of Expression by Osteoclasts In Vitro.Inflammation.2017;40(5):1566-1575.
[21]Crockett JC,Rogers MJ,Coxon FP,et al.Bone remodelling at a glance.J Cell Sci.2011;124(Pt 7):991-998.
[22]Suda T,Nakamura I,Jimi E,et al.Regulation of osteoclast function.J Bone Miner Res.1997;12(6):869-879.
[23]Muruganandan S,Dranse HJ,Rourke JL,et al.Chemerin neutralization blocks hematopoietic stem cell osteoclastogenesis.Stem Cells.2013;31(10):2172-2182.
[24]Wu X,Chim SM,Kuek V,et al.HtrA1 is upregulated during RANKL-induced osteoclastogenesis,and negatively regulates osteoblast differentiation and BMP2-induced Smad1/5/8,ERK and p38 phosphorylation.FEBS Lett.2014;588(1):143-150.
[25]Kim JY,Min JY,Baek JM,et al.CTRP3 acts as a negative regulator of osteoclastogenesis through AMPK-c-Fos-NFATc1signaling in vitro and RANKL-induced calvarial bone destruction in vivo.Bone.2015;79:242-251.
[26]Jeong E,Choi HK,Park JH,et al.STAC2 negatively regulates osteoclast formation by targeting the RANK signaling complex.Cell Death Differ.2018;25(8):1364-1374.
[27]Park JH,Lee NK,Lee SY.Current Understanding of RANKSignaling in Osteoclast Differentiation and Maturation.Mol Cells.2017;40(10):706-713.
[28]Obaid R,Wani SE,Azfer A,et al.Optineurin Negatively Regulates Osteoclast Differentiation by Modulating NF-κBand Interferon Signaling:Implications for Paget's Disease.Cell Rep.2015;13(6):1096-1102.
[29]Krauss JL,Zeng R,Hickman-Brecks CL,et al.NLRP12provides a critical checkpoint for osteoclast differentiation.Proc Natl Acad Sci U S A.2015;112(33):10455-10460.
[30]Gu Z,Wang H,Xia J,et al.Decreased ferroportin promotes myeloma cell growth and osteoclast differentiation.Cancer Res.2015;75(11):2211-2221.
[31]Jie Z,Shen S,Zhao X,et al.Activatingβ-catenin/Pax6 axis negatively regulates osteoclastogenesis by selectively inhibiting phosphorylation of p38/MAPK.FASEB J.2018.doi:10.1096/fj.201801977R.
[32]Zhang HQ,Wang YJ,Yang GT,et al.Taxifolin Inhibits Receptor Activator of NF-κB Ligand-Induced Osteoclastogenesis of Human Bone Marrow-Derived Macrophages in vitro and Prevents Lipopolysaccharide-Induced Bone Loss in vivo.Pharmacology.2019;103(1-2):101-109.
[33]Wang W,Huang M,Hui Y,et al.Cryptotanshinone inhibits RANKL-induced osteoclastogenesis by regulating ERK and NF-κB signaling pathways.J Cell Biochem.2018.doi:10.1002/jcb.28008.
[34]Rehman MU,Ali N,Rashid S,et al.Alleviation of hepatic injury by chrysin in cisplatin administered rats:probable role of oxidative and inflammatory markers.Pharmacol Rep.2014;66(6):1050-1059.
[35]Kats A,Gerasimcik N,N?reoja T,et al.Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS-stimulated co-cultures of periodontal ligament and RAW 264.7 cells,and RANKL-mediated osteoclastogenesis and bone resorption in PBMCs.J Cell Mol Med.2019;23(2):1152-1163.
[36]Kim HJ,Yoon HJ,Kim SY,et al.A medium-chain fatty acid,capric acid,inhibits RANKL-induced osteoclast differentiation via the suppression of NF-κB signaling and blocks cytoskeletal organization and survival in mature osteoclasts.Mol Cells.2014;37(8):598-604.
[37]Kaneko K,Miyamoto Y,Tsukuura R,et al.8-Nitro-cGMP is a promoter of osteoclast differentiation induced by RANKL.Nitric Oxide.2018;72:46-51.
[38]Chai L,Zhou K,Wang S,et al.Psoralen and Bakuchiol Ameliorate M-CSF Plus RANKL-Induced Osteoclast Differentiation and Bone Resorption Via Inhibition of AKT and AP-1 Pathways in Vitro.Cell Physiol Biochem.2018;48(5):2123-2133.