Mechanism of Jiangu Formula in treating osteoporosis based on osteoclast-osteoblast coupling

doi:10.12307/2025.970

Abstract

Abstract: BACKGROUND: Previous studies have found that Jiangu Formula can play a dual role in anti-osteoporosis therapy by inhibiting bone resorption and promoting bone formation, but its mechanism of action has not been elucidated.
OBJECTIVE: To investigate the effect of Jiangu Formula on the coupling mechanism between bone resorption and bone formation at the cellular and molecular levels.
METHODS: Serum containing Jiangu Formula was prepared and its toxicity to bone marrow derived macrophages was determined using MTT assay. The cell proliferation ability of MC3T3-E1 cells that treated Jiangu Formula medicated serum was measured by using cell counting kit-8 method. Tartrate resistant acid phosphatase staining was used to determine the bone resorption inhibition effect of serum containing Jiangu Formula on osteoclast differentiation. The Jiangu Fang conditional culture medium was prepared and the promoting effect of the conditional culture medium on bone formation of MC3T3-E1 was determined through alkaline phosphatase staining. The effect of serum containing Jiangu Formula on the mRNA levels of osteoclast associated sphingosine kinase 2, collagen triple helix repeat containing 1, and slit homolog 3 coupled genes, as well as the effect of conditioned culture medium on the mRNA levels of bone formation related secreted phosphoprotein 1, alkaline phosphatase, Sp7 transcription factor, and secreted protein acidic and rich in cysteine genes, were determined by RT-qPCR method.
RESULTS AND CONCLUSION: (1) The MTT and cell counting kit-8 methods showed that serum containing Jiangu Formula had no significant cytotoxic effects at a concentration of < 5%. (2) Tartrate resistant acid phosphatase staining results indicated that serum containing Jiangu Formula dose-dependently inhibited osteoclast differentiation at 2.5%, 1.25%, and 0.63% concentrations. (3) The alkaline phosphatase staining results showed that the osteoclast conditioned medium of Jiangu Formula promoted osteoblast differentiation in a dose-dependent manner. (4) In addition, RT-qPCR results showed that serum containing 0.63% Jiangu Formula could increase the mRNA levels of osteoclast derived slit homolog 3 and sphingosine kinase 2 coupling factors, and the conditional culture medium could increase the mRNA expression levels of bone formation related secreted phosphoprotein 1, Sp7 transcription factor, and secreted protein acidic and rich in cysteine. To conclude, the serum containing Jiangu Formula can promote bone formation while inhibiting bone resorption, and its mechanism may be related to the mRNA expression of the coupling factors osteoclast derived slit homolog 3 and sphingosine kinase 2 that promote bone resorption and bone formation.

Key words: Jiangu Formula, bone resorption, bone formation, osteoporosis, coupling, osteoclasts, osteoblasts, bone homeostasis, engineered tissue construction

CLC Number:

Lu Xiuli, Xu Huazhen, Chen Yuxing, Yao Nan, Hu Zixuan, Huang Dane. Mechanism of Jiangu Formula in treating osteoporosis based on osteoclast-osteoblast coupling[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(32): 6828-6835.

Figures/Tables 5

2.1 健骨方含药血清对骨髓源巨噬细胞和MC3T3-E1细胞增殖的影响如图1显示，健骨方干预细胞24 h后，与空白组相比，10%，5%的健骨方含药血清组显著抑制了骨髓源巨噬细胞存活率(P < 0.01)，2.5%，1.25%，0.63%和0.33%的健骨方含药血清组对骨髓源巨噬细胞存活率没有显著性影响，提示2.5%健骨方含药血清及以下浓度对骨髓源巨噬细胞生长没有显著影响。如图2所示，健骨方干预细胞24 h后，与空白组相比，10%的健骨方含药血清显著抑制了MC3T3-E1细胞存活率(P < 0.01)，其余各浓度的健骨方含药血清对MC3T3-E1细胞存活率没有影响，提示在5%及以下浓度健骨方含药血清对MC3T3-E1细胞生长没有影响。因此，后续实验选用2.5%、1.25%以及0.63%的健骨方含药血清浓度作为高、中、低剂量组。 2.2 健骨方含药血清对破骨细胞分化的影响 RANKL诱导骨髓源巨噬细胞破骨细胞分化模型的抗酒石酸酸性磷酸酶染色结果如图3所示，与空白组相比，模型组的骨髓源巨噬细胞分化成不规则形状的细胞核数目≥3个的破骨细胞。与模型组相比，阿仑膦酸钠组和健骨方高(2.5%含药血清)、中(1.25%含药血清)、低(0.63%含药血清)剂量组的破骨细胞分化面积明显得到抑制(P < 0.01)，提示阿仑膦酸钠和健骨方能显著抑制破骨细胞生成，并且健骨方高、中、低剂量组呈现剂量依赖性地抑制破骨细胞生成。 2.3 健骨方条件培养基对成骨细胞分化的影响条件培养基是指健骨方含药血清干预破骨细胞成熟后收集的上清液，超滤去无机盐和健骨方化学成分，仅保留有破骨细胞分泌的活性因子的浓缩液。如图4破骨细胞染色所示，给予1 μmol/L阿仑膦酸钠和健骨方2.5%、1.25%和0.63%含药血清干预后，RANKL诱导的骨髓源巨噬细胞分化形成破骨细胞的数量明显减少。使用各组条件培养基干预MC3T3-E1细胞后，其ALP染色结果显示，与空白组相比，模型组成骨细胞分化增加(蓝色染色面积更多)，说明破骨细胞分化后的条件培养基里含有的活性因子有促进成骨细胞分化的作用；与模型组对比，健骨方各剂量组的条件培养基也具有明显促进成骨细胞分化的作用；而阿仑膦酸组的条件培养基则明显抑制成骨细胞分化。这些结果表明，健骨方不仅能够抑制破骨细胞分化，还能够保留破骨细胞偶联成骨细胞分化的能力。 2.4 健骨方含药血清对骨吸收-骨形成相关mRNA的影响为了进一步考察健骨方对骨吸收-骨形成偶联影响的机制，此次研究采用RT-qPCR法测定健骨方含药血清干预破骨细胞分化后破骨细胞源性偶联因子CTHRC1、SLIT3和SPHK2 mRNA表达水平，同时考察健骨方干预破骨细胞分化的条件培养基对成骨细胞分化关键基因ALP、SPP1、SP7和SPARC的mRNA表达情况。结合图3健骨方含药血清对破骨细胞分化抑制结果对偶联因子基因表达结果进行分析。如图5所示，与空白组相比，模型组破骨细胞分化形成，CTHRC1和SPHK2的mRNA表达水平显著上升(P < 0.01，P < 0.05)，且SLIT3表达水平也有上升的趋势，说明经过RANKL诱导的破骨细胞可以表达促进骨形成的偶联基因；与模型组相比，健骨方各剂量组在破骨细胞分化抑制情况下，显著抑制CTHCR1 mRNA表达水平(P < 0.01)，但其高、中剂量组在破骨细胞分化显著抑制的情况下对SLIT3 mRNA水平仍然没有显著影响，且低剂量组对SLIT3 mRNA表达水平有显著提高作用(P < 0.01)；同时中、低剂量组对SPHK2 mRNA表达水平也表现出显著上调的作用(P < 0.01)；而阿仑膦酸钠干预破骨细胞分化后，CTHRC1和SLIT3 mRNA水平显著降低(P < 0.01，P < 0.05)，且SPHK2 mRNA水平有下降的趋势。破骨细胞分化条件培养基对成骨细胞分化关键基因表达的影响如图6所示，与空白组相比，模型组可显著提高成骨细胞分化关键基因ALP、SPP1和SPARC mRNA表达水平(P < 0.05-0.01)。与模型组比较，阿仑膦酸钠组成骨细胞中的ALP和SPARC的mRNA表达水平显著下调(P < 0.01)，而健骨方高剂量组显著提高SPP1 mRNA表达水平(P < 0.05)，且对ALP、SP7和SPARC mRNA表达水平没有显著影响；中剂量组能够显著提高ALP、SPP1、SP7和SPARC mRNA表达水平(P < 0.01)；低剂量组显著提升SPP1、SP7和SPARC mRNA表达水平(P < 0.01)，且对ALP没有显著影响。这些结果表明，健骨方在抑制破骨细胞分化的情况下，可能通过上调SLIT3、SPHK2的mRNA表达发挥偶联促进成骨细胞分化的作用，而阿仑膦酸钠对骨吸收-骨形成偶联的抑制效应与其抑制相关偶联因子的表达水平有关。"

References

[1] DU J, YANG J, HE Z, et al. Osteoblast and Osteoclast Activity Affect Bone Remodeling Upon Regulation by Mechanical Loading-Induced Leukemia Inhibitory Factor Expression in Osteocytes. Front Mol Biosci. 2020;7:585056.
[2] WANG L, YOU X, ZHANG L, et al. Mechanical regulation of bone remodeling. Bone Res. 2022;10(1):16.
[3] JOHNELL O, KANIS JA. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures. Osteoporos Int. 2006;17(12):1726-1733.
[4] 白璧辉, 谢兴文, 李鼎鹏, 等. 我国近5年来骨质疏松症流行病学研究现状[J]. 中国骨质疏松杂志,2018,24(2):253-258.
[5] 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.
[6] RUIZ-ESTEVES KN, TEYSIR J, SCHATOFF D, et al. Disparities in osteoporosis care among postmenopausal women in the United States. Maturitas. 2022;156:25-29.
[7] VEIS DJ, O’BRIEN CA. Osteoclasts, Master Sculptors of Bone. Annu Rev Pathol. 2023;18:257-281.
[8] COMPSTON JE, MCCLUNG MR, LESLIE WD. Osteoporosis. Lancet. 2019; 393(10169):364-376.
[9] FOESSL I, DIMAI HP, OBERMAYER-PIETSCH B. Long-term and sequential treatment for osteoporosis. Nat Rev Endocrinol. 2023;19(9):520-533.
[10] 于龙, 王亮. 老年骨质疏松症现状及进展[J]. 中国临床保健杂志, 2022,25(1): 6-11.
[11] REID IR, BILLINGTON EO. Drug therapy for osteoporosis in older adults. Lancet. 2022;399(10329):1080-1092.
[12] TAKESHITA S, FUMOTO T, MATSUOKA K, et al. Osteoclast-secreted CTHRC1 in the coupling of bone resorption to formation. J Clin Invest. 2013;123(9):3914-3924.
[13] KIM BJ, LEE YS, LEE SY, et al. Osteoclast-secreted SLIT3 coordinates bone resorption and formation. J Clin Invest. 2018;128(4):1429-1441.
[14] GREWE JM, KNAPSTEIN PR, DONAT A, et al. The role of sphingosine-1-phosphate in bone remodeling and osteoporosis. Bone Res. 2022; 10(1):34.
[15] 任明诗, 丁羽, 李子涵, 等. 成骨细胞与破骨细胞相互调节作用的研究进展[J]. 中国药理学通报,2022,38(6):822-827.
[16] BORCIANI G, MONTALBANO G, BALDINI N, et al. Co-culture systems of osteoblasts and osteoclasts: Simulating in vitro bone remodeling in regenerative approaches. Acta Biomater. 2020;108:22-45.
[17] SIMS N, MARTIN TJ. Osteoclasts Provide Coupling Signals to Osteoblast Lineage Cells Through Multiple Mechanisms. Annu Rev Physiol. 2020; 82:507-529.
[18] PARK-MIN KH, LORENZO J. Osteoclasts: Other functions. Bone. 2022; 165:116576.
[19] SHI S, DUAN H, OU X. Targeted delivery of anti-osteoporosis therapy: Bisphosphonate-modified nanosystems and composites. Biomed Pharmacother. 2024;175:116699.
[20] BROWN SA, GUISE TA. Drug insight: the use of bisphosphonates for the prevention and treatment of osteoporosis in men. Nat Clin Pract Urol. 2007;4(6):310-320.
[21] ROSINI S, ROSINI S, BERTOLDI I, et al. Understanding bisphosphonates and osteonecrosis of the jaw: uses and risks. Eur Rev Med Pharmacol Sci. 2015;19(17):3309-3317.
[22] JENSEN PR, ANDERSEN TL, CHAVASSIEUX P, et al. Bisphosphonates impair the onset of bone formation at remodeling sites. Bone. 2021; 145:115850.
[23] XU H, LU X, LI M, et al. Jiangu formula: A novel osteoclast-osteoblast coupling agent for effective osteoporosis treatment. Phytomedicine. 2024;128:155501.
[24] 许华珍, 黄丹娥, 郑柳怡, 等. 健骨方对破骨细胞形成和成骨细胞增殖分化的影响[J]. 中国骨质疏松杂志,2022,28(12):1728-1734.
[25] 许金松, 邓娜, 张潇. 骨碎补影响破骨细胞分化的程度与含药血清浓度有关[J]. 中国组织工程研究,2020,24(29):4620-4625.
[26] HUANG D, LUO X, YIN Z, et al. Diterpenoids from the aerial parts of Flueggea acicularis and their activity against RANKL-induced osteoclastogenesis. Bioorg Chem. 2020;94:103453.
[27] UDAGAWA N, KOIDE M, NAKAMURA M, et al. Osteoclast differentiation by RANKL and OPG signaling pathways. J Bone Miner Metab. 2021; 39(1):19-26.
[28] TSURUKAI T, UDAGAWA N, MATSUZAKI K, et al. Roles of macrophage-colony stimulating factor and osteoclast differentiation factor in osteoclastogenesis. J Bone Miner Metab. 2000;18(4):177-184.
[29] VIMALRAJ S. Alkaline phosphatase: Structure, expression and its function in bone mineralization. Gene. 2020;754:144855.
[30] JIANG Q, NAGANO K, MORIISHI T, et al. Roles of Sp7 in osteoblasts for the proliferation, differentiation, and osteocyte process formation. J Orthop Translat. 2024;47:161-175.
[31] HOJO H, OHBA S. Gene regulatory landscape in osteoblast differentiation. Bone. 2020;137:115458.
[32] LIU Y, CHEN Y, LI XH, et al. Dissection of Cellular Communication between Human Primary Osteoblasts and Bone Marrow Mesenchymal Stem Cells in Osteoarthritis at Single-Cell Resolution. Int J Stem Cells. 2023;16(3):342-355.
[33] KIM JM, LIN C, STAVRE Z, et al. Osteoblast-Osteoclast Communication and Bone Homeostasis. Cells. 2020;9(9):2073.
[34] KIM BJ, KOH JM. Coupling factors involved in preserving bone balance. Cell Mol Life Sci. 2019;76(7):1243-1253.
[35] PEDERSON L, RUAN M, WESTENDORF JJ, et al. Regulation of bone formation by osteoclasts involves Wnt/BMP signaling and the chemokine sphingosine-1-phosphate. Proc Natl Acad Sci U S A. 2008; 105(52):20764-20769.
[36] MATSUOKA K, PARK KA, ITO M, et al. Osteoclast-derived complement component 3a stimulates osteoblast differentiation. J Bone Miner Res. 2014;29(7):1522-1530.
[37] SALHOTRA A, SHAH HN, LEVI B, et al. Mechanisms of bone development and repair. Nat Rev Mol Cell Biol. 2020;21(11):696-711.
[38] XU X, HAN Y, ZHU T, et al. The role of SphK/S1P/S1PR signaling pathway in bone metabolism. Biomed Pharmacother. 2023;169:115838.