Low-intensity pulsed ultrasound promotes the proliferation of bone marrow mesenchymal stem cells by inducing cyclin D1 up-regulation

doi:10.12307/2022.325

Abstract

Abstract: BACKGROUND: Previous studies found that low-intensity pulsed ultrasound can promote the proliferation of human bone marrow mesenchymal stem cells.
OBJECTIVE: To investigate the mechanism of low-intensity pulsed ultrasound on the proliferation of human bone marrow mesenchymal stem cells via the FAK/PI3K/Akt signaling pathway.
METHODS: The purchased commercial human bone marrow mesenchymal stem cells were resuscitated and cultured to the fourth generation, and they were divided into 0 mW/cm2 ultrasound group (control group), 50 mW/cm2 ultrasound group, and 50 mW/cm2 ultrasound + PF-562271 group. The 50 mW/cm2 ultrasound + PF-562271 group was pretreated with 1 μL FAK/PI3K/Akt signaling pathway inhibitor PF-562271 before ultrasound treatment, once every 24 hours, 5 minutes each time. After three stimuli, cell proliferation was detected by Cell Counting Kit-8 and the cell cycle was detected by flow cytometry. Western blot assay was used to detect the expression of FAK and its downstream target gene and cyclin D1.
RESULTS AND CONCLUSION: (1) Compared with the control group, the cell proliferation ability was significantly enhanced in the 50 mW/cm2 ultrasound group (P < 0.05). After inhibiting the FAK/PI3K/Akt signaling pathway using PF-562271, the cell proliferation ability was significantly reduced (P < 0.001). (2) Compared with the control group, the expression levels of FAK, p-FAK, PI3K, p-PI3K, Akt, p-Akt and cyclin D1 were all up-regulated in the 50 mW/cm2 ultrasound group (P < 0.05). Compared with the 50 mW/cm2 ultrasound group, the above indicators were significantly reduced in the 50 mW/cm2 ultrasound + PF-562271 group (P < 0.05). (3) Compared with the control group, the proportion of cells in the G1 phase decreased in the 50 mW/cm2 ultrasound group (P=0.026), and the proportion of cells in the S phase increased (P=0.002). Compared with the 50 mW/cm2 ultrasound group, the proportion of cells in G1 phase increased (P=0.023), and the proportion of cells in S phase decreased in the 50 mW/cm2 ultrasound + PF-562271 group (P=0.035). (4) These results indicate that low-intensity pulsed ultrasound promotes the proliferation of human bone marrow mesenchymal stem cells via FAK/PI3K/Akt signaling pathway-induced cyclin D1 up-regulation.

Key words: stem cells, human bone marrow mesenchymal stem cells, low-intensity pulsed ultrasound, proliferation, pathway, cell cycle

CLC Number:

Li Zhi, Hua Yongyong, Zhang Jianquan, Fu Zhen. Low-intensity pulsed ultrasound promotes the proliferation of bone marrow mesenchymal stem cells by inducing cyclin D1 up-regulation[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(13): 2006-2011.

Figures/Tables 5

References

[1] 谢树才,张剑权,蒋锡丽,等.骨髓间充质干细胞诱导分化为肝细胞的方法及机制研究与进展[J].中国组织工程研究,2016,20(50): 7586-7593.
[2] Fleming MM, Liu F, Zhang Y, et al. Model for End-Stage Liver Disease Underestimates Morbidity and Mortality in Patients with Ascites Undergoing Colectomy. World J Surg. 2018;42(10):3390-3397.
[3] Hatzistergos KE, Quevedo H, Oskouei BN, et al. Bone marrow mesenchymal stem cells stimulate cardiac stem cell proliferation and differentiation. Circ Res. 2010;107(7):913-922.
[4] Wu Y, Gao Q, Zhu S, et al. Low-intensity pulsed ultrasound regulates proliferation and differentiation of neural stem cells through notch signaling pathway. Biochem Biophys Res Commun. 2020;526(3):793-798.
[5] Doan N, Reher P, Meghji S, et al. In vitro effects of therapeutic ultrasound on cell proliferation, protein synthesis, and cytokine production by human fibroblasts, osteoblasts, and monocytes. J Oral Maxillofac Surg. 1999;57(4):409-419.
[6] Miyasaka M, Nakata H, Hao J, et al. Low-Intensity Pulsed Ultrasound Stimulation Enhances Heat-Shock Protein 90 and Mineralized Nodule Formation in Mouse Calvaria-Derived Osteoblasts. Tissue Eng Part A. 2015;21(23-24):2829-2839.
[7] Huang D, Gao Y, Wang S, et al. Impact of low-intensity pulsed ultrasound on transcription and metabolite compositions in proliferation and functionalization of human adipose-derived mesenchymal stromal cells. Sci Rep. 2020;10(1):13690.
[8] Gao Q, Walmsley AD, Cooper PR, et al. Ultrasound Stimulation of Different Dental Stem Cell Populations: Role of Mitogen-activated Protein Kinase Signaling. J Endod. 2016;42(3):425-431.
[9] Korstjens CM, van der Rijt RH, Albers GH, et al. Low-intensity pulsed ultrasound affects human articular chondrocytes in vitro. Med Biol Eng Comput. 2008;46(12):1263-1270.
[10] Ishihara Y, Ueki K, Sotobori M, et al. Bone regeneration by statin and low-intensity pulsed ultrasound (LIPUS) in rabbit nasal bone. J Craniomaxillofac Surg. 2014;42(3):185-193.
[11] Sato M, Motoyoshi M, Shinoda M, et al. Low-intensity pulsed ultrasound accelerates nerve regeneration following inferior alveolar nerve transection in rats. Eur J Oral Sci. 2016;124(3):246-250.
[12] 李志,谢树才,张剑权,等.低强度超声波对人骨髓间充质干细胞增殖影响的体外研究[J].中华实验外科杂志,2018,35(11):2006-2009.
[13] Ren C, Chen X, Du N, et al. Low-intensity pulsed ultrasound promotes Schwann cell viability and proliferation via the GSK-3β/β-catenin signaling pathway. Int J Biol Sci. 2018;14(5):497-507.
[14] Xu T, Gu J, Li C, et al. Low-intensity pulsed ultrasound suppresses proliferation and promotes apoptosis via p38 MAPK signaling in rat visceral preadipocytes. Am J Transl Res. 2018;10(3):948-956.
[15] Wu S, Xu X, Sun J, et al. Low-Intensity Pulsed Ultrasound Accelerates Traumatic Vertebral Fracture Healing by Coupling Proliferation of Type H Microvessels. J Ultrasound Med. 2018;37(7):1733-1742.
[16] Coskun ME, Coskun KA, Tutar Y. Determination of Optimum Operation Parameters for Low-Intensity Pulsed Ultrasound and Low-Level Laser Based Treatment to Induce Proliferation of Osteoblast and Fibroblast Cells. Photomed Laser Surg. 2018;36(5):246-252.
[17] Akca H, Demiray A, Tokgun O, et al. Invasiveness and anchorage independent growth ability augmented by PTEN inactivation through the PI3K/AKT/NFkB pathway in lung cancer cells. Lung Cancer. 2011; 73(3):302-309.
[18] Xu R, Chen J, Cong X, et al. Lovastatin protects mesenchymal stem cells against hypoxia- and serum deprivation-induced apoptosis by activation of PI3K/Akt and ERK1/2. J Cell Biochem. 2008;103(1): 256-269.
[19] Aliabouzar M, Lee SJ, Zhou X, et al. Effects of scaffold microstructure and low intensity pulsed ultrasound on chondrogenic differentiation of human mesenchymal stem cells. Biotechnol Bioeng. 2018;115(2):495-506.
[20] Bayat M, Virdi A, Rezaei F, et al. Comparison of the in vitro effects of low-level laser therapy and low-intensity pulsed ultrasound therapy on bony cells and stem cells. Prog Biophys Mol Biol. 2018;133:36-48.
[21] Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997;64(2):278-294.
[22] Baker N, Sohn J, Tuan RS. Promotion of human mesenchymal stem cell osteogenesis by PI3-kinase/Akt signaling, and the influence of caveolin-1/cholesterol homeostasis. Stem Cell Res Ther. 2015;6:238.
[23] Liu Y, Zhang Y, Lin L, et al. Effects of bone marrow-derived mesenchymal stem cells on the axonal outgrowth through activation of PI3K/AKT signaling in primary cortical neurons followed oxygen-glucose deprivation injury. PLoS One. 2013;8(11):e78514.
[24] Xu P, Gul-Uludag H, Ang WT, et al. Low-intensity pulsed ultrasound-mediated stimulation of hematopoietic stem/progenitor cell viability, proliferation and differentiation in vitro. Biotechnol Lett. 2012;34(10):1965-1973.
[25] Zhang J, Guan J, Qi X, et al. Dimethyloxaloylglycine Promotes the Angiogenic Activity of Mesenchymal Stem Cells Derived from iPSCs via Activation of the PI3K/Akt Pathway for Bone Regeneration. Int J Biol Sci. 2016;12(6):639-652.
[26] Presneau N, Shalaby A, Idowu B, et al. Potential therapeutic targets for chordoma: PI3K/AKT/TSC1/TSC2/mTOR pathway. Br J Cancer. 2009;100(9):1406-1414.
[27] Liu H, Xu J, Zhou L, et al. Hepatitis B virus large surface antigen promotes liver carcinogenesis by activating the Src/PI3K/Akt pathway. Cancer Res. 2011;71(24):7547-7557.
[28] Cheng K, Xia P, Lin Q, et al. Effects of low-intensity pulsed ultrasound on integrin-FAK-PI3K/Akt mechanochemical transduction in rabbit osteoarthritis chondrocytes. Ultrasound Med Biol. 2014;40(7):1609-1618.
[29] Liu XB, Jiang J, Gui C, et al. Angiopoietin-1 protects mesenchymal stem cells against serum deprivation and hypoxia-induced apoptosis through the PI3K/Akt pathway. Acta Pharmacol Sin. 2008;29(7):815-822.
[30] Matsuo T, Sato K, Matsui T, et al. Inhibitory effects of low-intensity pulsed ultrasound sonication on the proliferation of osteosarcoma cells. Oncol Lett. 2017;14(3):3071-3076.
[31] Jung YJ, Kim R, Ham HJ, et al. Focused low-intensity pulsed ultrasound enhances bone regeneration in rat calvarial bone defect through enhancement of cell proliferation. Ultrasound Med Biol. 2015; 41(4):999-1007.
[32] Uddin SM, Richbourgh B, Ding Y, et al. Chondro-protective effects of low intensity pulsed ultrasound. Osteoarthritis Cartilage. 2016;24(11):1989-1998.
[33] Puts R, Rikeit P, Ruschke K, et al. Functional regulation of YAP mechanosensitive transcriptional coactivator by Focused Low-Intensity Pulsed Ultrasound (FLIPUS) enhances proliferation of murine mesenchymal precursors. PLoS One. 2018;13(10):e0206041.
[34] Li L, Yang Z, Zhang H, et al. Low-intensity pulsed ultrasound regulates proliferation and differentiation of osteoblasts through osteocytes. Biochem Biophys Res Commun. 2012;418(2):296-300.
[35] Go MJ, Takenaka C, Ohgushi H. Effect of forced expression of basic fibroblast growth factor in human bone marrow-derived mesenchymal stromal cells. J Biochem. 2007;142(6):741-748.
[36] Jäger M, Feser T, Denck H, et al. Proliferation and osteogenic differentiation of mesenchymal stem cells cultured onto three different polymers in vitro. Ann Biomed Eng. 2005;33(10):1319-1332.
[37] Song G, Ju Y, Soyama H, et al. Regulation of cyclic longitudinal mechanical stretch on proliferation of human bone marrow mesenchymal stem cells. Mol Cell Biomech. 2007;4(4):201-210.
[38] Zhong C, Zhang X, Xu Z, et al. Effects of low-intensity electromagnetic fields on the proliferation and differentiation of cultured mouse bone marrow stromal cells. Phys Ther. 2012;92(9): 1208-1219.
[39] Tsai MT, Li WJ, Tuan RS, et al. Modulation of osteogenesis in human mesenchymal stem cells by specific pulsed electromagnetic field stimulation. J Orthop Res. 2009;27(9):1169-1174.
[40] 蒋柳宏,郑有华,张志光,等.bFGF基因转染对兔骨髓间充质干细胞生物学特性的影响[J].中山大学学报(医学科学版), 2009,30(S2): 41-46.
[41] 朱磊,沈洪,刘丽,等.健脾补肾、清肠化湿方对骨髓间充质干细胞增殖迁移的影响[J].中国中西医结合杂志,2016,36(2):191-195.