REGUL ATORY EFFECT OF SIMVASTATIN ON MIDDLE/L ATE STAGES OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS VIA p38MAPK PATHWAY_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGKezhong , LIUGuangyuan ,  TIANFaming , ZHANGLiu

North China University of Science and Technology, Tangshan Hebei, 063000, P. R. China;

Corresponding author：

TIANFaming, Email: tfm9911316@163.com

Keywords：

Simvastatin; Bone marrow mesenchymal stem cells; Osteogenic differentiation; Osteocalcin; Phospho-p38

DOI：

10.7507/1002-1892.20160208

Video：

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Objective To investigate the regulatory effect of simvastatin on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) at middle/late stages by p38MAPK pathway under condition of osteoinductive environment. Methods The bone marrow of bilateral femur and tibia were harvested from 20 4-week-old female Sprague Dawley rats. BMSCs were isolated and cultured with whole bone marrow culture method; the second generation of cells were randomly divided into 5 groups: control group (complete medium, CM), simvastatin group (simvastatin medium, SIM), osteogenic induction group (osteogenic induction medium, OM), simvastatin and osteogenic induction group (simvastatin+osteogenic induction medium, OM+SIM), and blocker group (SB203580+simvastatin+osteogenic induction medium, OM+SIM+SB). MTT assay was used to detect the cell activity in CM group and SIM group at 2, 3, 4, 5, and 6 days, ELISA method to measure the content of alkaline phosphatase (ALP) in OM group and OM+SIM group at 7 and 14 days. The mRNA and protein expressions of osteocalcin (OCN) were detected by real-time quatitative PCR and Western blot after 1, 12, and 24 hours of osteogenic induction at 21 and 28 days. The protein expressions of phospho-p38 (p-p38) and p38 in OM group, OM+SIM group, and OM+SIM+SB group were detected by Western blot at the best induction time of simvastatin. Results MTT assay showed that no significant difference was found in absorbance (A) value between CM group and SIM group at each time point (P > 0.05), indicating no effect of 1×10^-7 mol/L simvastatin on cell viability. ELISA results showed that ALP content significantly increased in OM+SIM group when compared with OM group at 7 and 14 days; the ALP content was significantly higher at 7 days than 14 days in OM group and OM+SIM group (P < 0.05). OCN mRNA and protein expressions at 12 hours were significantly higher than those at other time points in each group (P < 0.05), and the expressions of OM+SIM group was significantly higher than those of OM group (P < 0.05). The best induction time of simvastatin was 12 hours. At 12 hours after blocking intervention, the p-p38/p38 in OM+SIM+SB group was significantly lower than that in OM group and OM+SIM group (P < 0.05), and the p-p38/p38 in OM+SIM group was significantly higher than that in OM group (P < 0.05). Conclusion Simvastatin can increase the mRNA and protein expression levels of OCN and the protein of p-p38 in osteogenic differentiation of BMSCs at middle/ late stages, and its best induction time is 12 hours.

Citation： ZHANGKezhong, LIUGuangyuan, TIANFaming, ZHANGLiu. REGUL ATORY EFFECT OF SIMVASTATIN ON MIDDLE/L ATE STAGES OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS VIA p38MAPK PATHWAY. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(8): 1038-1043. doi: 10.7507/1002-1892.20160208 Copy

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2.	Lee KS, Hong SH, Bae SC. Both the Smad and p38MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-band bone morphogenetic protein. Oncogene, 2002, 21(47):7156-7163.
3.	Yi C, Liu D, Fong CC, et al. Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through p38MAPK pathway. ACS Nano, 2010, 4(11):6439-6448.
4.	Li XD, Liu ZY, Chang B, et al. Panax notoginseng saponins promote osteogenic differentiation of bone marraw stromal cells through the ERK and P38 MAPK signaling pathway. Cell Physiol Biochem, 2011, 28(2):367-376.
5.	Mundy G, Garrett R, Harris S, et al. Stimulation of bone formation in vitro and in rodents by statins. Science, 1999, 286(5446):1946-1949.
6.	Geesink RG, Hoefnagels NH, Bulstra SK. Osteogenic activity of OP-1 bone morphogenetic protein (BMP-7) in a human fibulas defect. J Bone Joint Surg (Br), 1999, 81(4):710-718.
7.	Montazerolghaem M, Ning Y, Engqvist H, et al. Simvastatin and zinc synergistically enhance osteoblasts activity and decrease the acute response of inflammatory cells. J Mater Sci Mater Med, 2016, 27(2):23.
8.	Park JB. Combined effects of simvastatin and fibroblast growth factor-2 on the proliferation and differentiation of preosteoblasts. Biomed Rep, 2013, 1(5):812-814.
9.	Jitumori R, Fernandes D, Jitumori C, et al. Simvastatin protects osteoblasts from the deleterious effects of the liquid milieu of multiple myeloma. West Indian Med J, 2015, 64(3):263-265.
10.	Pullisaar H, Reseland JE, Haugen HJ, et al. Simvastatin coating of TiO? scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells. Biochem Biophys Res Commun, 2014, 447(1):139-144.
11.	王海彬, 何伟, 袁浩.成骨细胞和Ⅰ型胶原.中国中医骨伤科杂志, 2003, 11(3):56-59.
12.	Xue P, Wu X, Zhou L, et al. IGF1 promotes osteogenic differentiation of mesenchymal stem cells derived from rat bone marrow by increasing TAZ expression. Biophys Res Commun, 2013, 433(2):226-231.
13.	白宇, 张柳, 吕志伟, 等.辛伐他汀在人骨髓基质干细胞向成骨细胞分化过程中的作用.中国骨质疏松杂志, 2007, 13(6):381-384.
14.	Walter MS, Frank MJ, Rubert M, et al. Simvastatin-activated implant surface promotes osteoblast differentiation in vitro. J Biomater Appl, 2014, 28(6):897-908.
15.	Liu S, Bertl K, Sun H, et al. Effect of simvastatin on the osteogenetic behavior of alveolar osteoblasts and periodontal ligament cells. Hum Cell, 2012, 25(2):29-35.
16.	Chang HL, Chen CY, Hsu YF, et al. Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade. Biochim Biophys Acta, 2013, 1830(8):4053-4064.
17.	靳文英, 任景怡, 陈红.辛伐他汀抑制人外周血单核巨噬细胞脂蛋白相关磷脂酶A2表达.中华心血管病杂志, 2010, 38(10):923-928.
18.	Greenblatt MB, Shim JH, Zou W, et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J Clin Invest, 2010, 120(7):2457-2473.
19.	Hu N, Feng C, Jiang Y, et al. Regulative effect of mir-205 on osteogenic differentiation of bone mesenchymal stem cells (BMSCs): possible role of SATB2/Runx2 and ERK/MAPK pathway. Int J Mol Sci, 2015, 16(5):10491-1506.
20.	Liao QC, Xiao ZS, Qin YF, et al. Genistein stimulates osteoblastic differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture. Acta Pharmacol Sin, 2007, 28(10):1597-1602.

1. Han J, Lee JD, Bibbs L, et al. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science, 1994, 265(5173): 808-811.
2. Lee KS, Hong SH, Bae SC. Both the Smad and p38MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-band bone morphogenetic protein. Oncogene, 2002, 21(47):7156-7163.
3. Yi C, Liu D, Fong CC, et al. Gold nanoparticles promote osteogenic differentiation of mesenchymal stem cells through p38MAPK pathway. ACS Nano, 2010, 4(11):6439-6448.
4. Li XD, Liu ZY, Chang B, et al. Panax notoginseng saponins promote osteogenic differentiation of bone marraw stromal cells through the ERK and P38 MAPK signaling pathway. Cell Physiol Biochem, 2011, 28(2):367-376.
5. Mundy G, Garrett R, Harris S, et al. Stimulation of bone formation in vitro and in rodents by statins. Science, 1999, 286(5446):1946-1949.
6. Geesink RG, Hoefnagels NH, Bulstra SK. Osteogenic activity of OP-1 bone morphogenetic protein (BMP-7) in a human fibulas defect. J Bone Joint Surg (Br), 1999, 81(4):710-718.
7. Montazerolghaem M, Ning Y, Engqvist H, et al. Simvastatin and zinc synergistically enhance osteoblasts activity and decrease the acute response of inflammatory cells. J Mater Sci Mater Med, 2016, 27(2):23.
8. Park JB. Combined effects of simvastatin and fibroblast growth factor-2 on the proliferation and differentiation of preosteoblasts. Biomed Rep, 2013, 1(5):812-814.
9. Jitumori R, Fernandes D, Jitumori C, et al. Simvastatin protects osteoblasts from the deleterious effects of the liquid milieu of multiple myeloma. West Indian Med J, 2015, 64(3):263-265.
10. Pullisaar H, Reseland JE, Haugen HJ, et al. Simvastatin coating of TiO? scaffold induces osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells. Biochem Biophys Res Commun, 2014, 447(1):139-144.
11. 王海彬, 何伟, 袁浩.成骨细胞和Ⅰ型胶原.中国中医骨伤科杂志, 2003, 11(3):56-59.
12. Xue P, Wu X, Zhou L, et al. IGF1 promotes osteogenic differentiation of mesenchymal stem cells derived from rat bone marrow by increasing TAZ expression. Biophys Res Commun, 2013, 433(2):226-231.
13. 白宇, 张柳, 吕志伟, 等.辛伐他汀在人骨髓基质干细胞向成骨细胞分化过程中的作用.中国骨质疏松杂志, 2007, 13(6):381-384.
14. Walter MS, Frank MJ, Rubert M, et al. Simvastatin-activated implant surface promotes osteoblast differentiation in vitro. J Biomater Appl, 2014, 28(6):897-908.
15. Liu S, Bertl K, Sun H, et al. Effect of simvastatin on the osteogenetic behavior of alveolar osteoblasts and periodontal ligament cells. Hum Cell, 2012, 25(2):29-35.
16. Chang HL, Chen CY, Hsu YF, et al. Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade. Biochim Biophys Acta, 2013, 1830(8):4053-4064.
17. 靳文英, 任景怡, 陈红.辛伐他汀抑制人外周血单核巨噬细胞脂蛋白相关磷脂酶A2表达.中华心血管病杂志, 2010, 38(10):923-928.
18. Greenblatt MB, Shim JH, Zou W, et al. The p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice. J Clin Invest, 2010, 120(7):2457-2473.
19. Hu N, Feng C, Jiang Y, et al. Regulative effect of mir-205 on osteogenic differentiation of bone mesenchymal stem cells (BMSCs): possible role of SATB2/Runx2 and ERK/MAPK pathway. Int J Mol Sci, 2015, 16(5):10491-1506.
20. Liao QC, Xiao ZS, Qin YF, et al. Genistein stimulates osteoblastic differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture. Acta Pharmacol Sin, 2007, 28(10):1597-1602.

Chinese Journal of Reparative and Reconstructive Surgery

REGUL ATORY EFFECT OF SIMVASTATIN ON MIDDLE/L ATE STAGES OSTEOGENIC DIFFERENTIATION OF BONE MARROW MESENCHYMAL STEM CELLS VIA p38MAPK PATHWAY

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