Effect of Erxian Decoction on proliferation and bone differentiation of bone marrow mesenchymal stem cells in ovariectomized rats

doi:10.3969/j.issn.2095-4344.0940

Abstract

Abstract:

BACKGROUND: Some studies have shown that bone marrow mesenchymal stem cells (BMSCs) are involved in bone formation. Erxian Decoction can treat osteoporosis, but its relationship with BMSCs in ovariectomized rats is not yet clear.
OBJECTIVE: To reveal the relationship between Erxian Decoction and BMSCs by exploring the effect of Erxian Decoction on proliferation and differentiation of BMSCs in rats with osteoporosis.
METHODS: Sprague-Dawley rats were randomly divided into six groups: sham group, model group, estrogen group, high-dose Erxian Decoction group, middle-dose Erxian Decoction group and low-dose Erxian Decoction groups. Ovariectomy was performed to establish rat osteoporosis models in all the groups except the sham group. The rats in the sham group and model group were given normal saline via gavage, and those in the estrogen group were fed with estradiol valerate (0.08 g/L). Rats in the high-, middle- and low-dose groups were given high (1.2 g/mL), middle (0.8 g/mL) and low (0.4 g/mL) concentrations of Erxian Decoction extracts at a dose of 10 mL/kg, for 90 continuous days. BMSCs were isolated by whole bone marrow adherent method, MTT method was used to draw the growth curve of BMSCs, alkaline phosphates kit was used to detect alkaline phosphatase activity, and alizarin red staining was used to detect calcium nodules in BMSCs.
RESULTS AND CONCLUSION: Compared with the model group, high-, middle -, and low-dose Erxian Decoction significantly increased the proliferative capacity of BMSCs in ovariectomized rats (P < 0.01), and significantly improved alkaline phosphatase activity in BMSCs of ovariectomized rats (P < 0.05, P < 0.0.1). The highest activity of alkaline phosphatase in BMSCs was found after treatment with middle-dose Erxian Decoction (P < 0.01). High- and middle-dose Erxian Decoction also promoted the formation of calcium nodules in BMSCs (P < 0.01). These results indicate that to improve BMSCs proliferation and osteogenic differentiation in ovariectomized rats is one of the intrinsic mechanisms of Erxian Decoction in the treatment of osteoporosis.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Mesenchymal Stem Cells, Alkaline Phosphatase, Tissue Engineering

CLC Number:

R394.2

Wu Qi, Liu Bo, Chen Zhi, Xu Peng. Effect of Erxian Decoction on proliferation and bone differentiation of bone marrow mesenchymal stem cells in ovariectomized rats[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 3963-3968.

Figures/Tables 1

2.1 实验动物数量分析纳入大鼠24只均进入结果分析，无死亡与感染，无脱失值。 2.2 二仙汤能促进SD大鼠BMSCs生长速率从图2中可以看出，与假手术组相比，模型组生长曲线全程较平缓，进入对数生长期耗时较长(第4天起进入对数生长期)，表明骨质疏松可降低大鼠BMSCs增殖能力，呈现衰老现象。从第2天起，各组BMSCs增殖速度加快，生长速率开始出现差异，模型组增殖速率最低，假手术组增殖速率最高，其次是二仙汤低剂量组、二仙汤中剂量组、雌激素组。从第3天起，与模型组相比，各组BMSCs生长速率差异增加(P < 0.01)，细胞进入对数生长期，趋势大致相同。第7天各组BMSCs增殖进入平台期，生长速率趋于平稳，细胞生长速率不再增加，与模型组相比，假手术组和各给药组细胞的生长速率呈差异非常显著(P < 0.01)。 2.3 二仙汤提高骨质疏松大鼠BMSCs碱性磷酸酶活力图3显示，与假手术组相比，模型组碱性磷酸酶活力明显降低，说明骨质疏松可使BMSCs的碱性磷酸酶活力降低。与模型组相比，雌激素组BMSCs碱性磷酸酶活力增加趋势，但无显著性差异。而二仙汤高、中、低剂量组BMSCs碱性磷酸酶活力显著提高(P < 0.05)，尤其以二仙汤中剂量组为甚(P < 0.01)，说明二仙汤能促进BMSCs的骨向分化，其改善骨质疏松症可能与此有关。 2.4 二仙汤提高骨质疏松大鼠BMSCs矿化钙结节形成骨质疏松多表现骨量降低，骨结构破坏，细胞层面则出现骨形成相对减少。模型组BMSCs矿化钙结节形成较低，而与模型组相比，雌激素组、二仙汤高及中剂量组BMSCs矿化钙结节形成均明显增强，其中以雌激素组最为明显,但二仙汤低剂量组BMSCs矿化钙结节形成并无明显提高，见图4，5。这说明一定剂量的二仙汤能促进BMSCs矿化钙结节形成，促进骨形成，改善骨质疏松症。"

References

[1] 吴海洋,索欢,王平.绝经后骨质疏松症的临床中药治疗进展[J].中国骨质疏松杂志,2015,21(2):241-244.

[2] 刘爽,张玉莲,毕聪聪,等.补肾中药对去卵巢大鼠骨质疏松骨代谢指标的影响[J].中国老年学杂志,2017,37(3):529-530.

[3] 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[J].河北中医,2016,38(5):796-800.

[4] 周兴茂,吴俊哲,王伟群,等.二仙汤治疗脊髓损伤后骨质疏松的实验研究[J].中国骨质疏松杂志,2015,(2):223-227.

[5] 张建花,何玉琼,韩婷,等.二仙汤抗骨质疏松有效组分对维甲酸致骨丢失大鼠的影响[J].第二军医大学学报,2018,39(2):165-169.

[6] Fallahnezhad S, Piryaie A, Darbandi H, et al. Effect of low-level laser therapy and oxytocin on osteoporotic bone marrow-derived mesenchymal stem cells. J Cell Biochem. 2018;119(1):983-997.

[7] 宋慧婷,侯建明.骨髓间充质干细胞对骨质疏松的治疗作用概述[J].创伤与急诊电子杂志,2017,5(1):40-43.

[8] 李鹏,黄成硕,高翔,等.大鼠骨髓间充质干细胞体外分离及对原发性骨质疏松性骨折的验研究[J].国际检验医学杂志,2015,36(14): 1984-1987.

[9] Tera Tde M, Nascimento RD, do Prado RF, et al. Immunolocalization of markers for bone formation during guided bone regeneration in osteopenic. Appl Oral Sci. 2014; 22(6):541-553.

[10] Kim J, Ko J. A novel PPARγ2 modulator sLZIP controls the balance between Adipogenesis and osteogenesis during mesenchymal stem cell differentiation. Cell Death Differentiation. 2014;21(10):1642.

[11] Duan Y, Ma W, Li D, et al. Enhanced osseointegration of titanium implants in a rat model of osteoporosis using multilayer bone mesenchymal stem cell sheets. Exp Ther Med. 2017;14(6):5717.

[12] Jafari A, Qanie D, Andersen TL, et al. Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis. Stem Cell Reports. 2017;8(2):373-386.

[13] Jing H, Su X, Gao B, et al. Epigenetic inhibition of Wnt pathway suppresses osteogenic differentiation of BMSCs during osteoporosis. Cell Death Dis. 2018;9(2):176.

[14] 周兴茂,吴俊哲,王伟群,等.二仙汤治疗脊髓损伤后骨质疏松的实验研究[J].中国骨质疏松杂志,2015,21(2):223-227.

[15] 吴俊哲,王伟群,周兴茂,等.二仙汤对脊髓损伤后骨质疏松大鼠神经肽P物质的影响[J].中药材,2015,38(6):1254-1257.

[16] Cao H, Zhang Y, Qian W, et al. Effect of icariin on fracture healing in anovariectomized rat model of osteoporosis. Exp Ther Med. 2017;13(5):2399.

[17] Ramalho-Ferreira G, Faverani LP, Momesso GA, et al. Effect of antiresorptive drugs in the alveolar bone healing. A histometric and immunohistochemical study in ovariectomized rats. Clin Oral Invest. 2016;21(5):1-10.

[18] Macdonald TL, Ritchie KL, Davies S, et al. Exercise training is an effective alternative to estrogen supplementation for improving glucose homeostasis in ovariectomized rats. Physiol Rep. 2015;3(11):e12617.

[19] 刘慧.用二仙汤治疗原发性骨质疏松症的临床效果观察[J].当代医药论丛,2014,12(7):44-45.

[20] Cai Y, Liu T, Fang F, et al. Comparisons of Mouse Mesenchymal Stem Cells in Primary Adherent Culture of Compact Bone Fragments and Whole Bone Marrow. Stem Cells Int. 2015;2015(3):708906.

[21] Zhang W, Zhang F, Shi H, et al. Comparisons of Rabbit Bone Marrow Mesenchymal Stem Cell Isolation and Culture Methods In Vitro. PLoS One. 2014;9(2):e88794.

[22] 宋囡,何文智,王智民,等.左、右归丸及其拆方对骨髓间充质干细胞成骨分化的影响[J].中国病理生理杂志, 2013,29(7): 1268-1274.

[23] 肖仕辉,韦庆军,赵劲民,等.全骨髓贴壁法培养兔骨髓间充质干细胞体外定向成骨诱导分化及鉴定[J].中国组织工程研究, 2013, 17(6):1069-1074.

[24] 王云,鲍小明,侯永新,等.去卵巢骨质疏松大鼠骨髓间充质干细胞的体外成骨诱导[J].中国组织工程研究, 2013,17(36): 6423-6429.

[25] Cheng W, Meng H, Xin W, et al. Differentiation of Bone Marrow Mesenchymal Stem Cells in Osteoblasts and Adipocytes and its Role in Treatment of Osteoporosis. Med Sci Monit. 2016;22:226-233.

[26] 范金柱,杨柳,罗卓荆,等.雌激素对绝经后骨质疏松患者骨髓间充质干细胞Notch信号通路的影响[J].中华骨质疏松和骨矿盐疾病杂志,2013,6(3):232-239.

[27] 赖满香,廖利平,谭玮璐,等.“肾精-骨质疏松-骨髓间充质干细胞”理论探讨[J].中医杂志,2018,59(2):100-103.

[28] 龚理,索有军.骨质疏松大鼠骨髓间充质干细胞的生物学特性[J]. 中国组织工程研究与临床康复,2010,14(19):3458-3460.

[29] 明溪,虞坚尔,李刚,等.基于中医“肾精”学说与间充质干细胞的内在相关性探讨补肾药在哮喘治疗中的作用机理[J].中医杂志, 2016,57(16):1358-1362.

[30] 陈辉,王新杰,袁盛平,等.二仙汤经皮给药治疗老年人骨质疏松症的临床研究[J].中国医药科学,2016,6(5):65-67.

[31] 叶丙霖,李盛华,李淑玲,等.二仙汤联合依降钙素治疗绝经期妇女骨质疏松症临床研究[J].中国中医药信息杂志, 2016,23(6): 23-26.

[32] 陈苑妮.加味二仙汤联合阿仑膦酸钠维D3片治疗绝经后骨质疏松症的临床研究[J].中医临床研究,2017,9(17):11-13.

[33] 蔡榕琪,李建军,孟依临,等.冬季服用补肾养精中药对绝经后骨质疏松症患者骨质的影响[J].长春中医药大学学报, 2016,32(6): 1222-1224.

[34] 高翔,陈广谋,黄成硕,等.骨髓间充质干细胞对继发性骨质疏松大鼠骨缺损愈合的影响[J].海南医学,2016,27(20):3269-3272.

[35] Cui GS, Zeng JY, Zhang J, et al. Effect of nerve growth factor on osteogenic potential of type 2 diabetic mice bone marrow stromal cell in vitro. Zhonghua Kou Qiang Yi Xue Za Zhi. 2018; 53(2):97-102.

[36] Hou Q, Huang Y, Luo Y, et al. MiR-351 negatively regulates osteoblast differentiation of MSCs induced by (+)-cholesten-3-one through targeting VDR. Am J Transl Res. 2017;9(11):4963-4973.

[37] Wang Q, Li Y, Zhang Y, et al. LncRNA MEG3 inhibited osteogenic differentiation of bone marrow mesenchymal stem cells from postmenopausal osteoporosis by targeting miR-133a-3p. Biomed Pharmacother. 2017;89:1178-1186.

[38] Xu JC, Wu GH, Zhou LL, et al. Leptin improves osteoblast differentiation of human bone marrow stroma stem cells. Eur Rev Med Pharmacol Sci. 2016;20(16):3507-3513.

[39] Fei W, Huiyu Z, Yuxin D, et al. Calcitonin gene-related peptide-induced osteogenic differentiation of mouse bone marrow stromal cells through Hippo pathway in vitro. Hua Xi Kou Qiang Yi Xue Za Zhi. 2016;34(3):286-290.

[40] Yang B, Lin X, Tan J, et al. Root bark of Sambucus Williamsii Hance promotes rat femoral fracture healing by the BMP-2/Runx2 signaling pathway. J Ethnopharmacol. 2016; 191:107-114.

[41] Kong X, Li X, Zhang C, et al. Aqueous Fraction of Huogu Formula Promotes Osteogenic Differentiation of Bone Marrow Stromal Cells Through the BMP and Wnt Signaling Pathways. Rejuvenation Res. 2016;19(6):509-520.

[42] Zhang Q, Ge K, Ren H, et al. Effects of Cerium Oxide Nanoparticles on the Proliferation, Osteogenic Differentiation and Adipogenic Differentiation of Primary Mouse Bone Marrow Stromal Cells In Vitro. J Nanosci Nanotechnol. 2015; 15(9):6444-6451.