Puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway

doi:10.12307/2024.595

Abstract

Abstract: BACKGROUND: Previous studies have shown that puerarin can inhibit the differentiation of osteoclasts, and the expression of Notch signaling pathway-related proteins such as Notch1, HES1, and Jagged1 is decreased. However, the specific mechanism of the Notch1 signaling pathway for the inhibition of osteoclast differentiation by puerarin is not clear.
OBJECTIVE: To explore the effect of Notch signaling pathway on puerarin inhibiting the differentiation of mouse macrophage Raw264.7 into osteoclasts.
METHODS: Raw264.7 cells were divided into seven groups for intervention culture. Blank control group was cultured in high-sugar DMEM medium; the osteoclast induction group was cultured in osteoclast induction medium; the puerarin intervention group was cultured with 50 μmol/L puerarin at the same time of osteoclast induction; Notch1 siRNA control group, Notch1 siRNA group, Notch1 overexpression control group and Notch1 overexpression group were transfected with Notch1 siRNA control sequence, Notch1 siRNA, Notch1 overexpression control plasmid and Notch1 overexpression plasmid, respectively, and then cultured with osteoclast induction medium and puerarin. The number and size of osteoclasts were observed by tartrate-resistant acid phosphatase staining, the skeleton formation of osteoclasts was observed by F-actin staining, and the gene expression level of osteoclast formation markers was detected by RT-PCR.
RESULTS AND CONCLUSION: Tartrate-resistant acid phosphatase staining results showed that puerarin intervention could inhibit the generation of osteoclasts, Notch1 silencing could further reduce the number of osteoclasts, while the number of osteoclasts in the osteoclast-induced group increased significantly after Notch1 overexpression. The results of F-actin showed that Raw264.7 cells could form a well-defined F-actin ring after osteoclast induction. Puerarin intervention would inhibit the formation of cytoskeleton, and Notch1 silencing could aggravate the inhibitory effect of cytoskeleton formation, while Notch1 overexpression could alleviate this inhibitory effect of puerarin. RT-PCR results showed that puerarin could inhibit the mRNA expression levels of tartrate-resistant acid phosphatase, Cathepsin K and c-Fos, the expression of the above-mentioned three factors decreased significantly after Notch1 gene silencing, and Notch1 overexpression could upregulate the expression of these factors. These finding indicate that puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway.

Key words: puerarin, Raw264.7 cells, Notch1, overexpression, silence, osteoclast induction, osteoclast, periodontitis

CLC Number:

Liu Chunli, Yan Yujuan, Mo Liwen, Wu Zhijie, Zhang Li. Puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(35): 5636-5641.

Figures/Tables 6

References

[1] JEONG J, KIM HS, LEE D, et al. Association between Four Dietary Patterns and the Risk of Periodontal Diseases: A Systematic Review and Meta-Analysis. Nutrients. 2022;14(20):4362.
[2] MA Q, LIANG M, WU Y, et al. Osteoclast-derived apoptotic bodies couple bone resorption and formation in bone remodeling. Bone Res. 2021;9(1):5.
[3] 刘艳,格根塔娜.细胞因子在慢性牙周炎骨吸收中的作用[J].医学信息,2021,34(21):36-38.
[4] 王东红,王春爱,薛建军.葛根素的研究进展[J].西部中医药,2017, 30(1):139-142.
[5] 闫波.葛根素抑制PMMA颗粒诱导破骨细胞形成及骨溶解的机制研究[D].郑州:郑州大学,2020.
[6] 杨一秋,李兰,解继胜,等.葛根素介导PTEN-PI3K-AKT信号通路抑制绝经后骨质疏松症的机制研究[J].中国骨质疏松杂志,2022, 28(3):347-351.
[7] 詹乐,马瑞堉,万妮,等.葛根素对牙周炎大鼠Th17/Treg细胞免疫平衡及相关转录因子表达的影响[J].昆明医科大学学报,2022, 43(11):36-43.
[8] ZHAO B, GRIMES SN, LI S, et al. TNF-induced osteoclastogenesis and inflammatory bone resorption are inhibited by transcription factor RBP-J. J Exp Med. 2012;209(2):319-334.
[9] FUJIWARA T, ZHOU J, YE S, et al. RNA-binding protein Musashi2 induced by RANKL is critical for osteoclast survival. Cell Death Dis. 2016;7(7): e2300.
[10] CANALIS E, PARKER K, FENG JQ, et al. Osteoblast lineage-specific effects of notch activation in the skeleton. Endocrinology. 2013;154(2):623-634.
[11] 乔松,许琦,叶子青,等.骨保护素对高龄骨质疏松症患者骨代谢的影响[J].解放军医学杂志,2015,40(6):472-474.
[12] 刘春丽,闫雨娟,莫礼文,等.葛根素对Raw264.7细胞破骨分化的影响[J].中国组织工程研究,2023,27(32):5114-5119.
[13] LI X, LI B, SHI Y, et al. Targeing reactive oxygen species in stem cells for bone therapy. Drug Discov Today. 2021;26(5):1226-1244.
[14] LUNDBERG M, SUN S, EBETINO FH, et al. Osteoclasts recycle via osteomorphs during RANKL-stimulated bone resorption. Cell. 2021; 184(5):1330-1347.
[15] ABDELMAGID SM, SONDAG GR, MOUSSA FM, et al. Mutation in Osteoactivin Promotes Receptor Activator of NFκB Ligand (RANKL)-mediated Osteoclast Differentiation and Survival but Inhibits Osteoclast Function. J Biol Chem. 2015;290(33):20128-20146.
[16] KIM H, LEE K, KIM JM, et al. Selenoprotein W ensures physiological bone remodeling by preventing hyperactivity of osteoclasts. Nat Commun. 2021;12(1):2258.
[17] VAN DEN MOLEN E. Penicilline en streptomycine bij periodontitis en paradentitis [Penicillin and streptomycin in the treatment of periodontitis and paradentitis]. Tijdschr Tandheelkd. 1950;57(5):376-379.
[18] 路瑞芳,徐莉,冯向辉,等.侵袭性牙周炎基础治疗中不同时机口服抗生素的短期疗效观察[J].中华口腔医学杂志,2012,47(11): 666-670.
[19] 梁向阳,李春年.雷尼酸锶对实验性牙周炎大鼠牙槽骨改建的影响[J].口腔医学研究,2021,37(4):310-313.
[20] KULCZYŃSKI B, GRAMZA-MICHAŁOWSKA A, SULIBURSKA J, et al. Puerarin-an isoflavone with beneficial effects on bone health. Front Biosci (Landmark Ed). 2021;26(12):1653-1667.
[21] ZHOU YX, ZHANG H, PENG C. Puerarin: a review of pharmacological effects. Phytother Res. 2014;28(7):961-975.
[22] 朱庆磊,吕欣然,何爱霞.葛根素对氧自由基的清除和抗氧化性损伤作用[J].解放军药学学报,2001,17(1):1-3,13.
[23] MIZUKAMI J, TAKAESU G, AKATSUKA H, et al. Receptor activator of NF-kappaB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Mol Cell Biol. 2002;22(4):992-1000.

[24] XIE BP, SHI LY, LI JP, et al. Oleanolic acid inhibits RANKL-induced osteoclastogenesis via ER alpha/miR-503/RANK signaling pathway in RAW264.7 cells. Biomed Pharmacother. 2019;117:109045.
[25] SANO T, AKEDA K, YAMADA J, et al. Expression of the RANK/RANKL/OPG system in the human intervertebral disc: implication for the pathogenesis of intervertebral disc degeneration. BMC Musculoskelet Disord. 2019;20(1):225.
[26] ARTAVANIS-TSAKONAS S, RAND MD, LAKE RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999; 284(5415):770-776.
[27] HAYMAN AR. Tartrate-resistant acid phosphatase (TRAP) and the osteoclast/immune cell dichotomy. Autoimmunity. 2008;41(3): 218-223.
[28] 刘官娟,宋娜,霍花,等.唑来膦酸调控NLRP3信号通路抑制脂多糖诱导的破骨细胞分化[J].中国组织工程研究,2023,27(29): 4677-4683.
[29] MATSUNO K. Notch signaling. Dev Growth Differ. 2020;62(1):3.
[30] PENTON AL, LEONARD LD, SPINNER NB. Notch signaling in human development and disease. Semin Cell Dev Biol. 2012;23(4):450-457.
[31] EHEBAUER M, HAYWARD P, MARTINEZ-ARIAS A. Notch signaling pathway. Sci STKE. 2006;2006(364):cm7.
[32] FANG ZQ, RUAN B, LIU JJ, et al. Notch-triggered maladaptation of liver sinusoidal endothelium aggravates nonalcoholic steatohepatitis through endothelial nitric oxide synthase. Hepatology. 2022;76(3): 742-758.
[33] YOSHIDA G, KAWABATA T, TAKAMATSU H, et al. Degradation of the NOTCH intracellular domain by elevated autophagy in osteoblasts promotes osteoblast differentiation and alleviates osteoporosis. Autophagy. 2022;18(10):2323-2332.
[34] 胡超,李适廷,张纲,等.NICD过表达对人牙髓干细胞细胞增殖及细胞迁移的影响[J].牙体牙髓牙周病学杂志,2014,24(4):196-201.
[35] 龙晏,邱申彩,李淑慧,等.转化生长因子-β1对人牙周膜干细胞生物学行为的影响[J].中华实用诊断与治疗杂志,2018,32(11): 1044-1046.
[36] FILIPOVIĆ M, FLEGAR D, ŠUĆUR A, et al. Inhibition of Notch Signaling Stimulates Osteoclastogenesis From the Common Trilineage Progenitor Under Inflammatory Conditions. Front Immunol. 2022;13:902947.
[37] BALLHAUSE TM, JIANG S, BARANOWSKY A, et al. Relevance of Notch Signaling for Bone Metabolism and Regeneration. Int J Mol Sci. 2021; 22(3):1325.
[38] 康鑫.葛根素促进成骨细胞增殖及信号调控机制的实验研究[D].兰州:兰州大学,2015.
[39] 冯燕陵. FoxO1介导白藜芦醇、葛根素抑制破骨细胞生成和活性的机制研究[D].兰州:兰州大学,2018.
[40] 曾芳馨,熊中云.抗酒石酸酸性磷酸酶与骨代谢[J].华西医学,2005, 20(3):545.
[41] JULES J, CHEN W, FENG X, et al. C/EBPα transcription factor is regulated by the RANK cytoplasmic 535IVVY538 motif and stimulates osteoclastogenesis more strongly than c-Fos. J Biol Chem. 2018;293(4): 1480-1492.
[42] ARMSTRONG AP, TOMETSKO ME, GLACCUM M, et al. A RANK/TRAF6-dependent signal transduction pathway is essential for osteoclast cytoskeletal organization and resorptive function. J Biol Chem. 2002; 277(46):44347-44356.