Top

BMC Complementary Medicine and Therapies

Published in:

Open Access 01-12-2016 | Research article

The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells

Authors: In-Ryoung Kim, Seong-Eon Kim, Hyun-Su Baek, Bok-Joo Kim, Chul-Hoon Kim, In-Kyo Chung, Bong-Soo Park, Sang-Hun Shin

Published in: BMC Complementary Medicine and Therapies | Issue 1/2016

Abstract

Background

Kaempferol, a kind of flavonol, has been reported to possess various osteogenic biological activities, such as inhibiting bone resorption of osteoclasts and promoting the differentiation and mineralization of preosteoblasts. However, the precise cellular mechanism of action of kaempferol in osteogenesis is elusive.

Autophagy is a major intracellular degradation system, which plays an important role in cell growth, survival, differentiation and homeostasis in mammals. Recent studies showed that autophagy appeared to be involved in the degradation of osteoclasts, osteoblasts and osteocytes, potentially pointing to a new pathogenic mechanism of bone homeostasis and bone marrow disease. The potential correlation between autophagy, osteogenesis and flavonoids is unclear.

Methods

The present study verified that kaempferol promoted osteogenic differentiation and mineralization and that it elevated osteogenic gene expression based on alkaline phosphatase (ALP) activity, alizarin red staining and quantitative PCR. And then we found that kaempferol induced autophagy by acridine orange (AO) and monodansylcadaverine (MDC) staining and autophagy-related protein expression. The correlation between kaempferol-induced autophagy and the osteogenic process was confirmed by the autophagy inhibitor 3-methyladenine (3-MA).

Results

Kaempferol promoted the proliferation, differentiation and mineralization of osteoblasts at a concentration of 10 μM. Kaempferol showed cytotoxic properties at concentrations above 50 μM. Concentrations above 10 μM decreased ALP activity, whereas those up to 10 μM increased ALP activity. Kaempferol at concentrations up to 10 μM also increased the expression of the osteoblast- activated factors RUNX-2, osterix, BMP-2 and collagen I according to RT-PCR analyses. 10 μM or less, the higher of the concentration and over time, kaempferol promoted the activity of osteoblasts. Kaempferol induced autophagy. It also increased the expression of the autophagy-related factors beclin-1, SQSTM1/p62 and the conversion of LC3-II from LC3-I. The application of 3-MA decreased the activity of ALP and the autophagy induced by kaempferol. In the RT-PCR analysis, the expression of RUNX-2, osterix, BMP-2 and collagen I was decreased.

Conclusion

The present study showed that kaempferol stimulated the osteogenic differentiation of cultured osteoblasts by inducing autophagy.

Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature. 2003;423:337–42.CrossRefPubMed

Rachner TD, Khosla S, Hofbauer LC. Osteoporosis: now and the future. Lancet. 2011;377:1276–87.CrossRefPubMedPubMedCentral

Ikeda K, Takeshita S. Factors and mechanisms involved in the coupling from bone resorption to formation: how osteoclasts talk to osteoblasts. J Bone Metab. 2014;21:163–7.CrossRefPubMedPubMedCentral

Parfitt AM. Bone remodeling and bone loss: understanding the pathophysiology of osteoporosis. Clin Obstet Gynecol. 1987;30:789–811.CrossRefPubMed

Somerset SM, Johannot L. Dietary flavonoid sources in Australian adults. Nutr Cancer. 2008;60:442–9. doi:10.1080/01635580802143836794507408.CrossRefPubMed

Córdoba A, Satué M, Gómez‐Florit M, Hierro‐Oliva M, Petzold C, Lyngstadaas SP, González‐Martín ML, Monjo M, Ramis JM. Flavonoid‐modified surfaces: multifunctional bioactive biomaterials with osteopromotive, anti‐inflammatory, and anti‐fibrotic potential. Adv Healthc Mater. 2015;4:540–9.CrossRefPubMed

Zhang N, Ying M-D, Wu Y-P, Zhou Z-H, Ye Z-M, Li H, Lin D-S. Hyperoside, a flavonoid compound, inhibits proliferation and stimulates osteogenic differentiation of human osteosarcoma cells. PLoS One. 2014;9:e98973.CrossRefPubMedPubMedCentral

Lago J, Toledo-Arruda A, Mernak M, Barrosa K, Martins M, Tibério I, Prado C. Structure-activity association of flavonoids in lung diseases. Molecules. 2014;19:3570–95.CrossRefPubMed

Croft KD. The chemistry and biological effects of flavonoids and phenolic acids. Ann N Y Acad Sci. 1998;854:435–42.CrossRefPubMed

10.

Dai J, Li Y, Zhou H, Chen J, Chen M, Xiao Z. Genistein promotion of osteogenic differentiation through BMP2/SMAD5/RUNX2 signaling. Int J Biol Sci. 2013;9:1089–98.CrossRefPubMedPubMedCentral

11.

Ma XN, Zhou J, Ge BF, Zhen P, Ma HP, Shi WG, Cheng K, Xian CJ, Chen KM. Icariin induces osteoblast differentiation and mineralization without dexamethasone in vitro. Planta Med. 2013;79:1501–8.CrossRefPubMed

12.

Satue M, Arriero Mdel M, Monjo M, Ramis JM. Quercitrin and taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells. Biochem Pharmacol. 2013;86:1476–86.CrossRefPubMed

13.

Sharan K, Siddiqui JA, Swarnkar G, Maurya R, Chattopadhyay N. Role of phytochemicals in the prevention of menopausal bone loss: evidence from in vitro and in vivo, human interventional and pharma-cokinetic studies. Curr Med Chem. 2009;16:1138–57.CrossRefPubMed

14.

Huang L, Yagura T, Chen S. Sedative activity of hexane extract of Keampferia galanga L. and its active compounds. J Ethnopharmacol. 2008;120:123–5.CrossRefPubMed

15.

Wattel A, Kamel S, Mentaverri R, Lorget F, Prouillet C, Petit JP, Fardelonne P, Brazier M. Potent inhibitory effect of naturally occurring flavonoids quercetin and kaempferol on in vitro osteoclastic bone resorption. Biochem Pharmacol. 2003;65:35–42.CrossRefPubMed

16.

Miyake M, Arai N, Ushio S, Iwaki K, Ikeda M, Kurimoto M. Promoting effect of kaempferol on the differentiation and mineralization of murine pre-osteoblastic cell line MC3T3-E1. Biosci Biotechnol Biochem. 2003;67:1199–205.CrossRefPubMed

17.

Prouillet C, Maziere JC, Maziere C, Wattel A, Brazier M, Kamel S. Stimulatory effect of naturally occurring flavonols quercetin and kaempferol on alkaline phosphatase activity in MG-63 human osteoblasts through ERK and estrogen receptor pathway. Biochem Pharmacol. 2004;67:1307–13.CrossRefPubMed

18.

Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010;12:814–22.CrossRefPubMedPubMedCentral

19.

Ha SW, Weitzmann MN, Beck Jr GR. Bioactive silica nanoparticles promote osteoblast differentiation through stimulation of autophagy and direct association with LC3 and p62. ACS Nano. 2014;8:5898–910.CrossRefPubMedPubMedCentral

20.

Hocking LJ, Whitehouse C, Helfrich MH. Autophagy: a new player in skeletal maintenance? J Bone Miner Res. 2012;27:1439–47. doi:10.1002/jbmr.1668.CrossRefPubMed

21.

Maeda T, Matsunuma A, Kawane T, Horiuchi N. Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys Res Commun. 2001;280:874–7. http://dx.doi.org/10.1006/bbrc.2000.4232.CrossRefPubMed

22.

Biederbick A, Kern H, Elsässer H. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol. 1995;66:3–14.PubMed

23.

Paglin S, Hollister T, Delohery T, Hackett N, McMahill M, Sphicas E, Domingo D, Yahalom J. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61:439–44.PubMed

24.

Amic D, Davidovic-Amic D, Beslo D, Rastija V, Lucic B, Trinajstic N. SAR and QSAR of the antioxidant activity of flavonoids. Curr Med Chem. 2007;14:827–45.CrossRefPubMed

25.

Srivastava S, Bankar R, Roy P. Assessment of the role of flavonoids for inducing osteoblast differentiation in isolated mouse bone marrow derived mesenchymal stem cells. Phytomedicine. 2013;20:683–90. http://dx.doi.org/10.1016/j.phymed.2013.03.001.CrossRefPubMed

26.

Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147:728–41. doi:10.1016/j.cell.2011.10.026.CrossRefPubMed

27.

Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42. doi:10.1016/j.cell.2007.12.018.CrossRefPubMedPubMedCentral

28.

Liu F, Fang F, Yuan H, Yang D, Chen Y, Williams L, Goldstein SA, Krebsbach PH, Guan JL. Suppression of autophagy by FIP200 deletion leads to osteopenia in mice through the inhibition of osteoblast terminal differentiation. J Bone Miner Res. 2013;28:2414–30. doi:10.1002/jbmr.1971.CrossRefPubMedPubMedCentral

29.

Liberal J, Francisco V, Costa G, Figueirinha A, Amaral MT, Marques C, Girão H, Lopes MC, Cruz MT, Batista MT. Bioactivity of Fragaria vesca leaves through inflammation, proteasome and autophagy modulation. J Ethnopharmacol. 2014;158:113–22.CrossRefPubMed

30.

Aiyer HS, Warri AM, Woode DR, Hilakivi-Clarke L, Clarke R. Influence of berry polyphenols on receptor signaling and cell-death pathways: implications for breast cancer prevention. J Agric Food Chem. 2012;60:5693–708.CrossRefPubMedPubMedCentral

31.

Filomeni G, Graziani I, De Zio D, Dini L, Centonze D, Rotilio G, Ciriolo MR. Neuroprotection of kaempferol by autophagy in models of rotenone-mediated acute toxicity: possible implications for Parkinson’s disease. Neurobiol Aging. 2012;33:767–85.CrossRefPubMed

32.

Quarles LD, Yohay DA, Lever LW, Caton R, Wenstrup RJ. Distinct proliferative and differentiated stages of murine MC3T3-E1 cells in culture: an in vitro model of osteoblast development. J Bone Miner Res. 1992;7:683–92.CrossRefPubMed

33.

Podder B, Song KS, Song H-Y, Kim Y-S. Cytoprotective effect of kaempferol on paraquat-exposed BEAS-2B cells via modulating expression of MUC5AC. Biol Pharm Bull. 2014;37:1486–94.CrossRefPubMed

34.

Guo AJ, Choi RC, Zheng KY, Chen VP, Dong TT, Wang Z-T, Vollmer G, Lau DT, Tsim KW-k. Kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling. Chin Med. 2012;7:1–7.CrossRef

35.

Trivedi R, Kumar S, Kumar A, Siddiqui JA, Swarnkar G, Gupta V, Kendurker A, Dwivedi AK, Romero JR, Chattopadhyay N. Kaempferol has osteogenic effect in ovariectomized adult Sprague–Dawley rats. Mol Cell Endocrinol. 2008;289:85–93.CrossRefPubMed

36.

Chin A, Yang Y, Chai L, Wong RW, Rabie AB. Effects of medicinal herb salvia miltiorrhiza on osteoblastic cells in vitro. J Orthop Res. 2011;29:1059–63.CrossRefPubMed

37.

DeSelm CJ, Miller BC, Zou W, Beatty WL, van Meel E, Takahata Y, Klumperman J, Tooze SA, Teitelbaum SL, Virgin HW. Autophagy proteins regulate the secretory component of osteoclastic bone resorption. Dev Cell. 2011;21:966–74. doi:10.1016/j.devcel.2011.08.016.CrossRefPubMedPubMedCentral

38.

Zheng YT, Shahnazari S, Brech A, Lamark T, Johansen T, Brumell JH. The adaptor protein p62/SQSTM1 targets invading bacteria to the autophagy pathway. J Immunol. 2009;183:5909–16.CrossRefPubMed

39.

Kim H, Moon JY, Ahn KS, Cho SK. Quercetin induces mitochondrial mediated apoptosis and protective autophagy in human glioblastoma U373MG cells. Oxid Med Cell Longev. 2013;2013:596496. doi:10.1155/2013/596496.PubMedPubMedCentral

Title: The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells
Authors: In-Ryoung Kim
Seong-Eon Kim
Hyun-Su Baek
Bok-Joo Kim
Chul-Hoon Kim
In-Kyo Chung
Bong-Soo Park
Sang-Hun Shin
Publication date: 01-12-2016
Publisher: BioMed Central
Published in: BMC Complementary Medicine and Therapies / Issue 1/2016
Electronic ISSN: 2662-7671
DOI: https://doi.org/10.1186/s12906-016-1320-9

Keynote webinar | Spotlight on medication adherence

Springer Medicine

The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells

Abstract

Background

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 1/2016

Anti-inflammatory effect and action mechanisms of traditional herbal formula Gamisoyo-san in RAW 264.7 macrophages

Antibacterial and antibiotic-resistance modifying activity of the extracts and compounds from Nauclea pobeguinii against Gram-negative multi-drug resistant phenotypes

Anthelmintic activity of silver-extract nanoparticles synthesized from the combination of silver nanoparticles and M. charantia fruit extract

Beneficial effects of cocoa, coffee, green tea, and garcinia complex supplement on diet induced obesity in rats

Antidiabetic activity of the ethyl acetate fraction of Ficus lutea (Moraceae) leaf extract: comparison of an in vitro assay with an in vivo obese mouse model

Long term follow-up of cervical intervertebral disc herniation inpatients treated with integrated complementary and alternative medicine: a prospective case series observational study