Effects and mechanism of morroniside on osteogenic differentiation and proliferation of mouse MC3T3-E1 cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DONG Runbei ¹ , JIA Yutao ² , YANG Houzhi ¹ , LUO Gan ¹ , LI Yuqiao ¹ ,  SUN Tianwei ²

1. Graduate School of Tianjin Medical University, Tianjin, 300070, P. R. China;
2. Department of Spinal Surgery, Tianjin Union Medical Centre, Tianjin, 300121, P. R. China;

Corresponding author：

SUN Tianwei, Email: billsuntw@163.com

Keywords：

Morroniside; osteogenic differentiation; cell proliferation; adenosine A2A receptor; mouse

DOI：

10.7507/1002-1892.202202088

Video：

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Objective To study the effects of morroniside (MOR) on the proliferation and osteogenic differentiation of mouse MC3T3-E1 cells. Methods The 4th generation MC3T3-E1 cells were randomly divided into 6 groups: control group (group A), MOR low dose group (10 μmol/L, group B), MOR medium-low dose group (20 μmol/L, group C), MOR medium dose group (40 μmol/L, group D), MOR medium-high dose group (80 μmol/L, group E), and MOR high dose group (100 μmol/L, group F). The proliferation activity of each group was detected by cell counting kit 8 (CCK-8) assay; the bone differentiation and mineralized nodule formation of each group were detected by alizarin red staining; real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect cyclin-dependent kinase inhibitor 1A (P21), recombinant Cyclin D1 (CCND1), proliferating cell nuclear antigen (PCNA), alkaline phosphatase (ALP), collagen type Ⅰ (COL-1), bone morphogenetic protein 2 (BMP-2), and adenosine A2A receptor (A2AR) mRNA expressions; Western blot was used to detecte the expressions of osteopontin (OPN), Runt-related transcription factor 2 (RUNX2), and adenosine A2AR protein. Results The CCK-8 assay showed that the absorbance (A) values of groups B to F were significantly higher than that of group A at 24 hours of culture, with group C significantly higher than the rest of the groups (P<0.05). The MOR concentration (20 μmol/L) of group C was selected for the subsequent CCK-8 assay; the results showed that the A values of group C were significantly higher than those of group A at 24, 48, and 72 hours of culture (P<0.05). Alizarin red staining showed that orange-red mineralized nodules were visible in all groups and the number of mineralized nodules was significantly higher in groups B and C than in group A (P<0.05). RT-qPCR showed that the relative expressions of P21, CCND1, and PCNA mRNAs were significantly higher in group C than in group A (P<0.05). The expressions of ALP, BMP-2, COL-1, and adenosine A2AR mRNAs in groups B to E were significantly higher than those in group A, with the expressions of ALP, BMP-2, COL-1 mRNAs in group C significantly higher than the rest of the groups (P<0.05). Compared with group A, the expressions of OPN and RUNX2 proteins in groups B and C were significantly increased, while those in group D and E were significantly inhibited (P<0.05). There was no significant difference between groups B and C and between groups D and E (P>0.05). The relative expression of adenosine A2AR protein in groups B to E was significantly higher than that in group A, with group C significantly higher than the rest of the groups (P<0.05). Conclusion MOR can promote the proliferation and osteogenic differentiation of MC3T3-E1 cells; the mechanism of MOR may be achieved by interacting with adenosine A2AR.

Citation： DONG Runbei, JIA Yutao, YANG Houzhi, LUO Gan, LI Yuqiao, SUN Tianwei. Effects and mechanism of morroniside on osteogenic differentiation and proliferation of mouse MC3T3-E1 cells. Chinese Journal of Reparative and Reconstructive Surgery, 2022, 36(7): 889-895. doi: 10.7507/1002-1892.202202088 Copy

1.	Compston JE, Mcclung MR, Leslie WD. Osteoporosis. Lancet, 2019, 393(10169): 364-376.
2.	Shoback D, Rosen CJ, Black DM, et al. Pharmacological management of osteoporosis in postmenopausal women: An endocrine society guideline update. J Clin Endocrinol Metab, 2020, 105(3): dgaa048. doi: 10.1210/clinem/dgaa048.
3.	Gupta G, Aronow WS. Treatment of postmenopausal osteoporosis. Compr Ther, Fall 2007, 33(3): 114-119.
4.	Jilka RL, O’Brien CA, Roberson PK, et al. Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age. J Bone Miner Res, 2014, 29(1): 103-117.
5.	Lee WC, Guntur AR, Long F, et al. Energy metabolism of the osteoblast: Implications for osteoporosis. Endocr Rev, 2017, 38(3): 255-266.
6.	Zhang F, Xie J, Wang G, et al. Anti-osteoporosis activity of Sanguinarine in preosteoblast MC3T3-E1 cells and an ovariectomized rat model. J Cell Physiol, 2018, 233(6): 4626-4633.
7.	Liu L, Wang D, Qin Y, et al. Astragalin promotes osteoblastic differentiation in MC3T3-E1 cells and bone formation in vivo. Front Endocrinol (Lausanne), 2019, 10: 228. doi: 10.3389/fendo.2019.00228.
8.	Yamabe N, Kang KS, Goto E, et al. Beneficial effect of Corni Fructus, a constituent of Hachimi-jio-gan, on advanced glycation end-product-mediated renal injury in Streptozotocin-treated diabetic rats. Biol Pharm Bull, 2007, 30(3): 520-526.
9.	Lee NH, Seo CS, Lee HY, et al. Hepatoprotective and antioxidative activities of cornus officinalis against acetaminophen-induced hepatotoxicity in mice. Evid Based Complement Alternat Med, 2012, 2012: 804924. doi: 10.1155/2012/804924.
10.	Xu HQ, Hao HP. Effects of iridoid total glycoside from Cornus officinalis on prevention of glomerular overexpression of transforming growth factor beta 1 and matrixes in an experimental diabetes model. Biol Pharm Bull, 2004, 27(7): 1014-1018.
11.	Pi WX, Feng XP, Ye LH, et al. Combination of morroniside and diosgenin prevents high glucose-induced cardiomyocytes apoptosis. Molecules, 2017, 22(1): 163. doi: 10.3390/molecules22010163.
12.	Sun H, Li L, Zhang A, et al. Protective effects of sweroside on human MG-63 cells and rat osteoblasts. Fitoterapia, 2013, 84: 174-179.
13.	Xia C, Zou Z, Fang L, et al. Bushenhuoxue formula promotes osteogenic differentiation of growth plate chondrocytes through β-catenin-dependent manner during osteoporosis. Biomed Pharmacother, 2020, 127: 110170. doi: 10.1016/j.biopha.2020.110170.
14.	Zhang P, Xu H, Wang P, et al. Yougui pills exert osteoprotective effects on rabbit steroid-related osteonecrosis of the femoral head by activating β-catenin. Biomed Pharmacother, 2019, 120: 109520. doi: 10.1016/j.biopha.2019.109520.
15.	Ye J, Zhang X, Dai W, et al. Chemical fingerprinting of Liuwei Dihuang Pill and simultaneous determination of its major bioactive constituents by HPLC coupled with multiple detections of DAD, ELSD and ESI-MS. J Pharm Biomed Anal, 2009, 49(3): 638-645.
16.	Wang Y, Zhao H, Li X, et al. Formononetin alleviates hepatic steatosis by facilitating TFEB-mediated lysosome biogenesis and lipophagy. J Nutr Biochem, 2019, 73: 108214. doi: 10.1016/j.jnutbio.2019.07.005.
17.	Liu T, Xiang B, Guo D, et al. Morroniside promotes angiogenesis and further improves microvascular circulation after focal cerebral ischemia/reperfusion. Brain Res Bull, 2016, 127: 111-118.
18.	Liu T, Sun F, Cui J, et al. Morroniside enhances angiogenesis and improves cardiac function following acute myocardial infarction in rats. Eur J Pharmacol, 2020, 872: 172954. doi: 10.1016/j.ejphar.2020.172954.
19.	Zeng G, Ding W, Li Y, et al. Morroniside protects against cerebral ischemia/reperfusion injury by inhibiting neuron apoptosis and MMP2/9 expression. Exp Ther Med, 2018, 16(3): 2229-2234.
20.	Lee CG, Kim J, Yun SH, et al. Anti-osteoporotic effect of morroniside on osteoblast and osteoclast differentiation in vitro and ovariectomized mice in vivo. Int J Mol Sci, 2021, 22(19): 10642. doi: 10.3390/ijms221910642.
21.	Liu H, Li X, Lin J, et al. Morroniside promotes the osteogenesis by activating PI3K/Akt/mTOR signaling. Biosci Biotechnol Biochem, 2021, 85(2): 332-339.
22.	Xiao Z, Wang J, Chen S, et al. Autophagy promotion enhances the protective effect of Morroniside on human OA chondrocyte. Biosci Biotechnol Biochem, 2020, 84(5): 989-996.
23.	Sun Y, Zhu Y, Liu X, et al. Morroniside attenuates high glucose-induced BMSC dysfunction by regulating the Glo1/AGE/RAGE axis. Cell Prolif, 2020, 53(8): e12866. doi: 10.1111/cpr.12866.
24.	Duan FX, Shi YJ, Chen J, et al. The neuroprotective role of morroniside against spinal cord injury in female rats. Neurochem Int, 2021, 148: 105105. doi: 10.1016/j.neuint.2021.105105.
25.	Gfeller D, Grosdidier A, Wirth M, et al. SwissTargetPrediction: a web server for target prediction of bioactive small molecules. Nucleic Acids Res, 2014, 42(Web Server issue): W32-W38.
26.	Yu H, Yao S, Zhou C, et al. Morroniside attenuates apoptosis and pyroptosis of chondrocytes and ameliorates osteoarthritic development by inhibiting NF-κB signaling. J Ethnopharmacol, 2021, 266: 113447. doi: 10.1016/j.jep.2020.113447.
27.	杜昕楠, 刘维. 补肾活血中药治疗原发性骨质疏松症临床研究进展. 河北中医, 2016, 38(5): 796-800.
28.	Yu B, Wang W. Cardioprotective effects of morroniside in rats following acute myocardial infarction. Inflammation, 2018, 41(2): 432-436.
29.	Tang X, Wu H, Mao X, et al. The GLP-1 receptor herbal agonist morroniside attenuates neuropathic pain via spinal microglial expression of IL-10 and β-endorphin. Biochem Biophys Res Commun, 2020, 530(3): 494-499.
30.	Borhani S, Corciulo C, Larranaga-Vera A, et al. Adenosine A 2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts. FASEB J, 2019, 33(6): 7555-7562.
31.	He W, Mazumder A, Wilder T, et al. Adenosine regulates bone metabolism via A1, A2A, and A2B receptors in bone marrow cells from normal humans and patients with multiple myeloma. FASEB J, 2013, 27(9): 3446-3454.
32.	Mediero A, Frenkel SR, Wilder T, et al. Adenosine A2A receptor activation prevents wear particle-induced osteolysis. Sci Transl Med, 2012, 4(135): 135ra65. doi: 10.1126/scitranslmed.3003393.
33.	Mediero A, Kara FM, Wilder T, et al. Adenosine A(2A) receptor ligation inhibits osteoclast formation. Am J Pathol, 2012, 180(2): 775-786.
34.	Yasuda H, Shima N, Nakagawa N, et al. A novel molecular mechanism modulating osteoclast differentiation and function. Bone, 1999, 25(1): 109-113.
35.	Mediero A, Perez-Aso M, Cronstein BN. Activation of adenosine A(2A) receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation. Br J Pharmacol, 2013, 169(6): 1372-1388.
36.	Mediero A, Wilder T, Perez-Aso M, et al. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2. FASEB J, 2015, 29(4): 1577-1590.
37.	Mediero A, Wilder T, Shah L, et al. Adenosine A 2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis. FASEB J, 2018, 32(7): 3487-3501.
38.	Mediero A, Cronstein BN. Adenosine and bone metabolism. Trends Endocrinol Metab, 2013, 24(6): 290-300.
39.	Costa MA, Barbosa A, Neto E, et al. On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells. J Cell Physiol, 2011, 226(5): 1353-1366.
40.	Gharibi B, Abraham AA, Ham J, et al. Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes. J Bone Miner Res, 2011, 26(9): 2112-2124.
41.	Katebi M, Soleimani M, Cronstein BN. Adenosine A2A receptors play an active role in mouse bone marrow-derived mesenchymal stem cell development. J Leukoc Biol, 2009, 85(3): 438-444.

1. Compston JE, Mcclung MR, Leslie WD. Osteoporosis. Lancet, 2019, 393(10169): 364-376.
2. Shoback D, Rosen CJ, Black DM, et al. Pharmacological management of osteoporosis in postmenopausal women: An endocrine society guideline update. J Clin Endocrinol Metab, 2020, 105(3): dgaa048. doi: 10.1210/clinem/dgaa048.
3. Gupta G, Aronow WS. Treatment of postmenopausal osteoporosis. Compr Ther, Fall 2007, 33(3): 114-119.
4. Jilka RL, O’Brien CA, Roberson PK, et al. Dysapoptosis of osteoblasts and osteocytes increases cancellous bone formation but exaggerates cortical porosity with age. J Bone Miner Res, 2014, 29(1): 103-117.
5. Lee WC, Guntur AR, Long F, et al. Energy metabolism of the osteoblast: Implications for osteoporosis. Endocr Rev, 2017, 38(3): 255-266.
6. Zhang F, Xie J, Wang G, et al. Anti-osteoporosis activity of Sanguinarine in preosteoblast MC3T3-E1 cells and an ovariectomized rat model. J Cell Physiol, 2018, 233(6): 4626-4633.
7. Liu L, Wang D, Qin Y, et al. Astragalin promotes osteoblastic differentiation in MC3T3-E1 cells and bone formation in vivo. Front Endocrinol (Lausanne), 2019, 10: 228. doi: 10.3389/fendo.2019.00228.
8. Yamabe N, Kang KS, Goto E, et al. Beneficial effect of Corni Fructus, a constituent of Hachimi-jio-gan, on advanced glycation end-product-mediated renal injury in Streptozotocin-treated diabetic rats. Biol Pharm Bull, 2007, 30(3): 520-526.
9. Lee NH, Seo CS, Lee HY, et al. Hepatoprotective and antioxidative activities of cornus officinalis against acetaminophen-induced hepatotoxicity in mice. Evid Based Complement Alternat Med, 2012, 2012: 804924. doi: 10.1155/2012/804924.
10. Xu HQ, Hao HP. Effects of iridoid total glycoside from Cornus officinalis on prevention of glomerular overexpression of transforming growth factor beta 1 and matrixes in an experimental diabetes model. Biol Pharm Bull, 2004, 27(7): 1014-1018.
11. Pi WX, Feng XP, Ye LH, et al. Combination of morroniside and diosgenin prevents high glucose-induced cardiomyocytes apoptosis. Molecules, 2017, 22(1): 163. doi: 10.3390/molecules22010163.
12. Sun H, Li L, Zhang A, et al. Protective effects of sweroside on human MG-63 cells and rat osteoblasts. Fitoterapia, 2013, 84: 174-179.
13. Xia C, Zou Z, Fang L, et al. Bushenhuoxue formula promotes osteogenic differentiation of growth plate chondrocytes through β-catenin-dependent manner during osteoporosis. Biomed Pharmacother, 2020, 127: 110170. doi: 10.1016/j.biopha.2020.110170.
14. Zhang P, Xu H, Wang P, et al. Yougui pills exert osteoprotective effects on rabbit steroid-related osteonecrosis of the femoral head by activating β-catenin. Biomed Pharmacother, 2019, 120: 109520. doi: 10.1016/j.biopha.2019.109520.
15. Ye J, Zhang X, Dai W, et al. Chemical fingerprinting of Liuwei Dihuang Pill and simultaneous determination of its major bioactive constituents by HPLC coupled with multiple detections of DAD, ELSD and ESI-MS. J Pharm Biomed Anal, 2009, 49(3): 638-645.
16. Wang Y, Zhao H, Li X, et al. Formononetin alleviates hepatic steatosis by facilitating TFEB-mediated lysosome biogenesis and lipophagy. J Nutr Biochem, 2019, 73: 108214. doi: 10.1016/j.jnutbio.2019.07.005.
17. Liu T, Xiang B, Guo D, et al. Morroniside promotes angiogenesis and further improves microvascular circulation after focal cerebral ischemia/reperfusion. Brain Res Bull, 2016, 127: 111-118.
18. Liu T, Sun F, Cui J, et al. Morroniside enhances angiogenesis and improves cardiac function following acute myocardial infarction in rats. Eur J Pharmacol, 2020, 872: 172954. doi: 10.1016/j.ejphar.2020.172954.
19. Zeng G, Ding W, Li Y, et al. Morroniside protects against cerebral ischemia/reperfusion injury by inhibiting neuron apoptosis and MMP2/9 expression. Exp Ther Med, 2018, 16(3): 2229-2234.
20. Lee CG, Kim J, Yun SH, et al. Anti-osteoporotic effect of morroniside on osteoblast and osteoclast differentiation in vitro and ovariectomized mice in vivo. Int J Mol Sci, 2021, 22(19): 10642. doi: 10.3390/ijms221910642.
21. Liu H, Li X, Lin J, et al. Morroniside promotes the osteogenesis by activating PI3K/Akt/mTOR signaling. Biosci Biotechnol Biochem, 2021, 85(2): 332-339.
22. Xiao Z, Wang J, Chen S, et al. Autophagy promotion enhances the protective effect of Morroniside on human OA chondrocyte. Biosci Biotechnol Biochem, 2020, 84(5): 989-996.
23. Sun Y, Zhu Y, Liu X, et al. Morroniside attenuates high glucose-induced BMSC dysfunction by regulating the Glo1/AGE/RAGE axis. Cell Prolif, 2020, 53(8): e12866. doi: 10.1111/cpr.12866.
24. Duan FX, Shi YJ, Chen J, et al. The neuroprotective role of morroniside against spinal cord injury in female rats. Neurochem Int, 2021, 148: 105105. doi: 10.1016/j.neuint.2021.105105.
25. Gfeller D, Grosdidier A, Wirth M, et al. SwissTargetPrediction: a web server for target prediction of bioactive small molecules. Nucleic Acids Res, 2014, 42(Web Server issue): W32-W38.
26. Yu H, Yao S, Zhou C, et al. Morroniside attenuates apoptosis and pyroptosis of chondrocytes and ameliorates osteoarthritic development by inhibiting NF-κB signaling. J Ethnopharmacol, 2021, 266: 113447. doi: 10.1016/j.jep.2020.113447.
27. 杜昕楠, 刘维. 补肾活血中药治疗原发性骨质疏松症临床研究进展. 河北中医, 2016, 38(5): 796-800.
28. Yu B, Wang W. Cardioprotective effects of morroniside in rats following acute myocardial infarction. Inflammation, 2018, 41(2): 432-436.
29. Tang X, Wu H, Mao X, et al. The GLP-1 receptor herbal agonist morroniside attenuates neuropathic pain via spinal microglial expression of IL-10 and β-endorphin. Biochem Biophys Res Commun, 2020, 530(3): 494-499.
30. Borhani S, Corciulo C, Larranaga-Vera A, et al. Adenosine A 2A receptor (A2AR) activation triggers Akt signaling and enhances nuclear localization of β-catenin in osteoblasts. FASEB J, 2019, 33(6): 7555-7562.
31. He W, Mazumder A, Wilder T, et al. Adenosine regulates bone metabolism via A1, A2A, and A2B receptors in bone marrow cells from normal humans and patients with multiple myeloma. FASEB J, 2013, 27(9): 3446-3454.
32. Mediero A, Frenkel SR, Wilder T, et al. Adenosine A2A receptor activation prevents wear particle-induced osteolysis. Sci Transl Med, 2012, 4(135): 135ra65. doi: 10.1126/scitranslmed.3003393.
33. Mediero A, Kara FM, Wilder T, et al. Adenosine A(2A) receptor ligation inhibits osteoclast formation. Am J Pathol, 2012, 180(2): 775-786.
34. Yasuda H, Shima N, Nakagawa N, et al. A novel molecular mechanism modulating osteoclast differentiation and function. Bone, 1999, 25(1): 109-113.
35. Mediero A, Perez-Aso M, Cronstein BN. Activation of adenosine A(2A) receptor reduces osteoclast formation via PKA- and ERK1/2-mediated suppression of NFκB nuclear translocation. Br J Pharmacol, 2013, 169(6): 1372-1388.
36. Mediero A, Wilder T, Perez-Aso M, et al. Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2. FASEB J, 2015, 29(4): 1577-1590.
37. Mediero A, Wilder T, Shah L, et al. Adenosine A 2A receptor (A2AR) stimulation modulates expression of semaphorins 4D and 3A, regulators of bone homeostasis. FASEB J, 2018, 32(7): 3487-3501.
38. Mediero A, Cronstein BN. Adenosine and bone metabolism. Trends Endocrinol Metab, 2013, 24(6): 290-300.
39. Costa MA, Barbosa A, Neto E, et al. On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells. J Cell Physiol, 2011, 226(5): 1353-1366.
40. Gharibi B, Abraham AA, Ham J, et al. Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes. J Bone Miner Res, 2011, 26(9): 2112-2124.
41. Katebi M, Soleimani M, Cronstein BN. Adenosine A2A receptors play an active role in mouse bone marrow-derived mesenchymal stem cell development. J Leukoc Biol, 2009, 85(3): 438-444.

Chinese Journal of Reparative and Reconstructive Surgery

Effects and mechanism of morroniside on osteogenic differentiation and proliferation of mouse MC3T3-E1 cells

Abstract Full text Figures/Tables Video References Cited by

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