Guilu Erxian Gum medicated serum mediated osteogenic differentiation of rat bone marrow mesenchymal stem cells

doi:10.12307/2022.327

Abstract

Abstract: BACKGROUND: On the basis of studying the proliferation of bone marrow mesenchymal stem cells, revealing their differentiation mechanism is one of the hot topics in the study of prevention and treatment of osteoporosis.
OBJECTIVE: To explore the relationship between the molecular mechanism of Guilu Erxian Gum medicated serum that induces osteogenic differentiation of bone marrow mesenchymal stem cells to prevent osteoporosis and the activation of extracellular signal-regulated kinase 1/2/E26 transcription factor 1 signaling pathway factors.
METHODS: Passage 3 commercial SD rat bone marrow mesenchymal stem cells were purchased, and divided into fetal bovine serum group, blank serum group, Guilu Erxian Gum medicated serum group, classical induction group, pathway inhibitor group, and Guilu Erxian Gum+pathway inhibitor group. Osteogenic differentiation was conducted according to group conditions, and the fluid was altered every 3 days. At 7 days after intervention, alkaline phosphatase activity detection kit was used to determine the osteogenic differentiation level of bone marrow mesenchymal stem cells in the first four groups. At 14 days after intervention, alizarin red staining was used to verify the level of bone formation and mineralization of bone marrow mesenchymal stem cells in the first four groups. Real-time fluorescent quantitative PCR was used to detect the gene expression levels of alkaline phosphatase, core binding protein 2, peroxisome proliferator activated receptor γ, and E26 transcription factor 1 in bone marrow mesenchymal stem cells of the first four groups. Western blot assay was utilized to determine protein expression of type I collagen, alkaline phosphatase, core binding protein 2, peroxisome proliferator activated receptor γ, E26 transcription factor 1, extracellular signal-regulated kinase 1/2 and phosphorylated extracellular signal-regulated kinase 1/2 in bone marrow mesenchymal stem cells of the six groups.
RESULTS AND CONCLUSION: (1) Compared with the blank serum group, the mRNA expression levels of alkaline phosphatase, core binding protein 2, and E26 transcription factor 1 were significantly higher in the Guilu Erxian Gum medicated serum group, while the mRNA expression levels of the adipogenic differentiation specific index peroxisome proliferator activated receptor γ were inhibited. (2) Compared with the fetal calf serum group and the blank serum group, the expression levels of osteogenic differentiation-related proteins, such as type I collagen, alkaline phosphatase, core binding protein 2, E26 transcription factor 1, and phosphorylated extracellular signal-regulated kinase 1/2 were significantly increased; and the expression level of peroxisome proliferator activated receptor γ protein was also suppressed. The use of MAPK signaling pathway inhibitor PD98059 could down-regulate the expression levels of osteogenic differentiation-related proteins and up-regulate the expression levels of peroxisome proliferator activated receptor γ protein. (3) Compared with the fetal bovine serum group and the blank serum group, the alkaline phosphatase activity (P < 0.05) and calcification capacity (P < 0.05) of bone marrow mesenchymal stem cells were significantly improved in the Guilu Erxian Gum medicated serum group. (4) The results show that Guilu Erxian Gum medicated serum may mediate the osteogenic differentiation of bone marrow mesenchymal stem cells through extracellular signal-regulated kinase 1/2/E26 transcription factor 1 signaling pathway factors, which may be one of its important mechanisms for preventing and treating osteoporosis.

Key words: stem cells, bone marrow mesenchymal stem cells, Guilu Erxian Gum, osteoporosis, osteogenic differentiation, extracellular signal regulated kinase, mechanism

CLC Number:

Chen Jincheng, Zhu Guotao, Qin Xiaofei, Chen Yancheng, Luo Jun, Liu Hongwen, Wu Yijing, Xu Jie. Guilu Erxian Gum medicated serum mediated osteogenic differentiation of rat bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(13): 2020-2026.

Figures/Tables 8

References

[1] Consensus development conference: diagnosis, prophylaxis, and treatment of osteoporosis. Am J Med. 1993;94(6):646-650.
[2] SIRIS ES, ADLER R, BILEZIKIAN J, et al. The clinical diagnosis of osteoporosis: a position statement from the National Bone Health Alliance Working Group. Osteoporos Int. 2014;25(5):1439-1443.
[3] 马远征,王以朋,程晓光,等.中国老年骨质疏松症诊疗指南(2018)[J].中国实用内科杂志,2019,39(1):38-61.
[4] 牛素生,林建华,李楠,等.龟鹿二仙胶对去卵巢骨质疏松大鼠腰椎骨强度的影响及机制[J].福建中医药大学学报,2014,24(3):5-7．
[5] 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):10491-10506.
[6] ZHENG JM, KONG YY, LI YY, et al. MagT1 regulated the odontogenic differentiation of BMMSCs induced byTGC-CM via ERK signaling pathway. Stem Cell Res Ther. 2019;10(1):48.
[7] 冯倩,曾祥伟,詹秀琴,等.骨髓间充质干细胞成骨分化相关信号通路及其与BMP-2关系的研究进展[J].山东医药,2017,57(48):97-99.
[8] 史冬雪.葛根素激活P38、ERK1/2信号通路促进MC3T3-E1细胞增殖的研究[D].兰州:甘肃农业大学,2020.
[9] SUN T, YAO S, LIU M, et al. Composite Scaffolds of Mineralized Natural Extracellular Matrix on True Bone Ceramic Induce Bone Regeneration Through Smad1/5/8 and ERK1/2 Pathways. Tissue Eng Part A. 2018; 24(5-6):502-515.
[10] 黄继汗,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[11] 牛素生,李楠,张燕,等.龟鹿二仙胶诱导大鼠骨髓基质干细胞成骨分化作用及对Wnt通路的影响[J].中国中西医结合杂志,2017, 37(1):72-78.
[12] SI L, WINZENBERG TM, JIANG Q, et al. Projection of osteoporosis-related fractures and costs in China: 2010-2050. Osteoporos Int. 2015; 26(7):1929-1937.
[13] 李春岭,王德惠,李普宏.骨质疏松症的中医病名辨析[J].云南中医中药杂志,2017,38(7):13-15.
[14] 陈文辉,蒋云霞,白蕊,等.中医药治疗老年性骨质疏松症研究进展[J].辽宁中医杂志,2020,48(12):1-10.
[15] CURTIS EM, MOON RJ, DENNISON EM, et al. Recent advances in the pathogenesis and treatment of osteoporosis. Clin Med (Lond). 2016; 16(4):360-364.
[16] KOHLI P, WHELTON SP, HSU S, et al. Clinician’s guide to the updated ABCs of cardiovascular disease prevention. J Am Heart Assoc. 2014; 3(5):e001098.
[17] 梁文娜,沈建英,闵莉.绝经后骨质疏松症肾虚证与骨髓基质干细胞分化失衡内在联系的探微[J].中华中医药杂志,2020,35(10): 4794-4796.
[18] 陆吴超,钱伟宏,姚志宏,等.补肾、活血中药及补肾活血复方对骨髓间充质干细胞增殖、定向迁移及成骨分化的影响及作用机制的研究进展[J].中医正骨,2018,30(7):36-38.
[19] 李祥雨,姜劲挺,李建国,等.骨质疏松症中药防治研究进展[J].中国骨质疏松杂志,2018,24(2):270-275.
[20] TANG DZ, HOU W, ZHOU Q, et al. Osthole stimulates osteoblast differentiation and bone formation by activation of beta-catenin-BMP signaling. J Bone Miner Res. 2010;25(6):1234-1245.
[21] ZHAO J, OHBA S, SHINKAI M, et al. Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner. Biochem Biophys Res Commun. 2008;369(2):444-448.
[22] IWAMA H, AMAGAYA S, OGIHARA Y. Effect of shosaikoto, a Japanese and Chinese traditional herbal medicinal mixture, on the mitogenic activity of lipopolysaccharide: a new pharmacological testing method. J Ethnopharmacol. 1987;21(1):45-53.