Puerarin promotes the viability and differentiation of MC3T3‑E1 cells by enhancing LC3B‑mediated autophagy through downregulation of miR‑204

Feng,Qian; Cheng,Song‑Yi; Yang,Rui; Zeng,Xiang‑Wei; Zhao,Feng‑Ming; Zhan,Xiu‑Qin

doi:10.3892/etm.2019.8291

Puerarin promotes the viability and differentiation of MC3T3‑E1 cells by enhancing LC3B‑mediated autophagy through downregulation of miR‑204

Authors:
- Qian Feng
- Song‑Yi Cheng
- Rui Yang
- Xiang‑Wei Zeng
- Feng‑Ming Zhao
- Xiu‑Qin Zhan
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Affiliations: School of Medicine and Life Sciences, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, P.R. China, Department of Cardiology, Nanjing Hospital of Chinese Medicine, Nanjing, Jiangsu 210001, P.R. China, School of Life Sciences, Nanjing University, Nanjing, Jiangsu 210023, P.R. China
Published online on: December 5, 2019 https://doi.org/10.3892/etm.2019.8291
Pages: 883-890
Copyright: © Feng et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Puerarin is a bioactive substance extracted from Pueraria lobata. It is known to promote the viability, differentiation and mineralization of osteoblasts. However, the molecular mechanisms involved in these activities are not well understood. The present study was conducted with the aim of elucidating the effect of puerarin on osteoblasts and to explore the underlying mechanism. CCK‑8 analysis showed that puerarin (0.1, 1 and 10 µM) promoted the viability of osteoblastic MC3T3‑E1 cells, with 1 µM of puerarin exhibiting the strongest effect. Moreover, 1 µM puerarin significantly increased the activity of alkaline phosphatase (ALP) and the formation of mineralized nodules in the MC3T3‑E1 cells. Treatment with 1 µM puerarin for 72 h led to a significant upregulation in the expression level of microtubule‑associated light chain 3 (LC3)B and Beclin1 proteins. This treatment was more effective in promoting LC3B expression than what was observed following treatment with rapamycin (overexpression for autophagy). The bilayer membrane structure of autophagosomes was observed by electron microscopy. Conversely, 3‑methyladenine (3‑MA, inhibitor of autophagy) reduced the cell viability as well as the activity of alkaline phosphatase (ALP) in MC3T3‑E1 cells, although, there was no significant influence on mineralization. Prediction results of the biological information showed that LC3B could be a direct target of microRNA‑204 (miR‑204). In the present study, the expression level of miR‑204 was decreased by puerarin. miR‑204 mimics significantly decreased LC3B expression and inhibited auotophagosome formation, while the miR‑204 inhibitor had the opposite effects. To conclude, the results of the present study suggest that puerarin promotes the viability and differentiation of MC3T3‑E1 cells through autophagy, which is possibly associated with miR‑204‑regulated LC3B upregulation.

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1	Suvarna V, Sarkar M, Chaubey P, Khan T, Sherje A, Patel K and Dravyakar B: Bone health and natural products- an insight. Front Pharmacol. 9:9812018. View Article : Google Scholar : PubMed/NCBI
2	Saint-Pastou Terrier C and Gasque P: Bone responses in health and infectious diseases: A focus on osteoblasts. J Infect. 75:281–292. 2017. View Article : Google Scholar : PubMed/NCBI
3	An J, Yang H, Zhang Q, Liu C, Zhao J, Zhang L and Chen B: Natural products for treatment of osteoporosis: The effects and mechanisms on promoting osteoblast-mediated bone formation. Life Sci. 147:46–58. 2016. View Article : Google Scholar : PubMed/NCBI
4	Wei SY, Chen Y and Xu XY: Progress on the pharmacological research of puerarin: A review. Chin J Nat Med. 12:407–414. 2014.PubMed/NCBI
5	Shan Z, Cheng N, Huang R, Zhao B and Zhou Y: Puerarin promotes the proliferation and differentiation of MC3T3-E1 cells via microRNA-106b by targeting receptor activator of nuclear factor-κB ligand. Exp Ther Med. 15:55–60. 2018.PubMed/NCBI
6	Zeng X, Feng Q, Zhao F, Sun C, Zhou T, Yang J and Zhan X: Puerarin inhibits TRPM3/miR-204 to promote MC3T3-E1 cells proliferation, differentiation and mineralization. Phytother Res. 32:996–1003. 2018. View Article : Google Scholar : PubMed/NCBI
7	Suthon S, Jaroenporn S, Charoenphandhu N, Suntornsaratoon P and Malaivijitnond S: Anti-osteoporotic effects of Pueraria candollei var. mirifica on bone mineral density and histomorphometry in estrogen-deficient rats. J Nat Med. 70:225–233. 2016. View Article : Google Scholar : PubMed/NCBI
8	Jiang GM, Tan Y, Wang H, Peng L, Chen HT, Meng XJ, Li LL, Liu Y, Li WF and Shan H: The relationship between autophagy and the immune system and its applications for tumor immunotherapy. Mol Cancer. 18:172019. View Article : Google Scholar : PubMed/NCBI
9	Shen G, Ren H, Shang Q, Qiu T, Yu X, Zhang Z, Huang J, Zhao W, Zhang Y, Lian g and Jiang X: Autophagy as a target for glucocorticoid-induced osteoporosis therapy. Cell Mol Life Sci. 75:2683–2693. 2018. View Article : Google Scholar : PubMed/NCBI
10	Hurley JH and Young LN: Mechanisms of autophagy initiation. Annu Rev Biochem. 86:225–244. 2017. View Article : Google Scholar : PubMed/NCBI
11	Lee YK and Lee JA: Role of the mammalian ATG8/LC3 family in autophagy: Differential and compensatory roles in the spatiotemporal regulation of autophagy. BMB Rep. 49:424–430. 2016. View Article : Google Scholar : PubMed/NCBI
12	Schaaf MB, Keulers TG, Vooijs MA and Rouschop KM: LC3/GABARAP family proteins: Autophagy-(un)related functions. FASEB J. 30:3961–3978. 2016. View Article : Google Scholar : PubMed/NCBI
13	Moulis M and Vindis C: Autophagy in metabolic age-related human diseases. Cells. 7:E1492018. View Article : Google Scholar : PubMed/NCBI
14	Zhan XQ, Zeng XW, Zhang YY, Feng Q, Zhao FM, Jiang ZQ and Sun C: Puerarin promotes the viability and differentiation of MC3T3-E1 cells by miR-204-regulated Runx2 upregulation. Mol Med Rep. 16:6262–6268. 2017. View Article : Google Scholar : PubMed/NCBI
15	Mikhaylova O, Stratton Y, Hall D, Kellner E, Ehmer B, Drew AF, Gallo CA, Plas DR, Biesiada J, Meller J and Czyzyk-Krzeska MF: VHL-regulated MiR-204 suppresses tumor growth through inhibition of LC3B-mediated autophagy in renal clear cell carcinoma. Cancer Cell. 21:532–546. 2012. View Article : Google Scholar : PubMed/NCBI
16	Chen J, Lan Y, He Y, He C, Xu F, Zhang Y, Zhao Y and Liu Y: 99Tc-MDP-induced human osteoblast proliferation, differentiation and expression of osteoprotegerin. Mol Med Rep. 16:1801–1809. 2017. View Article : Google Scholar : PubMed/NCBI
17	Sharma U, Pal D and Prasad R: Alkaline phosphatase: An overview. Indian J Clin Biochem. 29:269–278. 2014. View Article : Google Scholar : PubMed/NCBI
18	Reggiori F and Ungermann C: Autophagosome maturation and fusion. J Mol Biol. 429:486–496. 2017. View Article : Google Scholar : PubMed/NCBI
19	Barbosa MC, Grosso RA and Fader CM: Hallmarks of aging: An autophagic perspective. Front Endocrinol (Lausanne). 9:7902019. View Article : Google Scholar : PubMed/NCBI
20	Kang C, Wei L, Song B, Chen L, Liu J, Deng B, Pan X and Shao L: Involvement of autophagy in tantalum nanoparticle-induced osteoblast proliferation. Int J Nanomedicine. 12:4323–4333. 2017. View Article : Google Scholar : PubMed/NCBI
21	Darcy A, Meltzer M, Miller J, Lee S, Chappell S, Ver Donck K and Montano M: A novel library screen identifies immunosuppressors that promote osteoblast differentiation. Bone. 50:1294–1303. 2012. View Article : Google Scholar : PubMed/NCBI
22	Cheng Y, Zhang W, Fan H and Xu P: Water-soluble Nano-pearl powder promotes MC3T3-E1 cell differentiation by enhancing autophagy via the MEK/ERK signaling pathway. Mol Med Rep. 18:993–1000. 2018.PubMed/NCBI
23	Kim IR, Kim SE, Baek HS, Kim BJ, Kim CH, Chung IK, Park BS and Shin SH: The role of kaempferol-induced autophagy on differentiation and mineralization of osteoblastic MC3T3-E1 cells. BMC Complement Altern Med. 16:3332016. View Article : Google Scholar : PubMed/NCBI
24	Sun M, Zhou X, Chen L, Huang S, Leung V, Wu N, Pan H, Zhen W, Lu W and Peng S: The regulatory roles of microRNAs in bone remodeling and perspectives as biomarkers in osteoporosis. BioMed Res Int. 2016:16524172016. View Article : Google Scholar : PubMed/NCBI
25	Wang FE, Zhang C, Maminishkis A, Dong L, Zhi C, Li R, Zhao J, Majerciak V, Gaur AB, Chen S and Miller SS: MicroRNA-204/211 alters epithelial physiology. FASEB J. 24:1552–1571. 2010. View Article : Google Scholar : PubMed/NCBI
26	Wang P, Liang J, Li Y and Li J, Yang X, Zhang X, Han S, Li S and Li J: Down-regulation of miRNA-30a alleviates cerebral ischemic injury through enhancing beclin 1-mediated autophagy. Neurochem Res. 39:1279–1291. 2014. View Article : Google Scholar : PubMed/NCBI
27	Zheng B, Zhu H, Gu D, Pan X, Qian L, Xue B, Yang D, Zhou J and Shan Y: MiRNA-30a-mediated autophagy inhibition sensitizes renal cell carcinoma cells to sorafenib. Biochem Biophys Res Commun. 459:234–239. 2015. View Article : Google Scholar : PubMed/NCBI