Liuwei Dihuang Pill regulate osteoclast membrane fusion to improve postmenopausal osteoporosis in mice: the CLCF1/NFATc1 signaling axis as a therapeutic target

doi:10.12122/j.issn.1673-4254.2026.08.03

Abstract

Abstract:

Objective To explore the mechanism of Liuwei Dihuang Pill (LWDHW) for improving postmenopausal osteoporosis (PMOP). Methods Thirty female C57BL/6 mice were randomized equally into sham-operated group, PMOP model group, and LWDHW treatment group. The mice in the latter two groups underwent ovariectomy to simulate PMOP, followed by gavage of normal saline and LWDHW (twice daily) for 60 days, respectively. Bone mineral density (BMD) of the mice was detected, and TRAP staining was used to observe osteoclast and osteoclast maturation in the tibia tissue. In RANKL-induced RAW264.7 cells with or without lentivirus-mediated CLCF1 knockdown, the effects of incubation with normal rat serum or LWDHW-medicated rat serum on cell membrane fusion rate and F-actin loop formation were observed using immunofluorescence staining with phalloidin peptide; the mRNA and protein expressions of CLCF1, NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2 were detected using RT-qPCR and Western blotting. Results In PMOP mice, LWDHW treatment significantly increased BMD, reduced osteoclast maturation, osteoclast membrane fusion rate, and F-actin ring formation, upregulated the mRNA and protein expressions of CLCF1 in tibial tissues, and lowered the expressions of NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. In RANKL-induced RAW264.7 cells with CLCF1 knockdown, treatment with LWDHW-medicated rat serum significantly decreased osteoclast membrane fusion rate, F-actin ring formation, and the expressions of NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. Conclusion LWDHW improves PMOP in mice by regulating osteoclast membrane fusion through upregulating CLCF1 expression and downregulating NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2 expressions.

Key words: Liuwei Dihuang Pill, postmenopausal osteoporosis, osteoclasts, membrane fusion

Ruoxi ZHENG, Lihua XIE, Xiaobin HUANG, Jirong GE. Liuwei Dihuang Pill regulate osteoclast membrane fusion to improve postmenopausal osteoporosis in mice: the CLCF1/NFATc1 signaling axis as a therapeutic target[J]. Journal of Southern Medical University, 2026, 46(8): 1743-1753.

Figures/Tables 16

Tab.1 Primer sequences for RT-qPCR

Gene name	Sequence (5'→3')
GAPDH	Forward: TGGAAAGCTGTGGCGTGATG
GAPDH	Reverse: TACTTGGCAGGTTTCTCCAGG
CLCF1	Forward: AACGAGCCTGACTTCAATCC
CLCF1	Reverse: GACTGTACGCCTCATAGTTCTG
NFATc1	Forward: CCGTCACATTCTGGTCCATAC
NFATc1	Reverse: TTCATTCTCCAAGTAACCGTGTAG
DC-STAMP	Forward: GTAGGAACGCTTTGATTGCGG
DC-STAMP	Reverse: TAGATTCAGCGGAGTGGCAAG
OC-STAMP	Forward: CATCCGCTCCCTATTTGTGC
OC-STAMP	Reverse: CACGCACATTGCCTAAGACG
ATP6V0D2	Forward: AGTCTTACCTTGAGGCATTCTACA
ATP6V0D2	Reverse: TCTCCCTGTCTTCTTTGCTTAGT

Fig.1 Tibial TRAP staining in different groups. LWDHW： Liuwei Dihuang Pill group.

Tab.2 Comparison of bone mineral density among the 3 groups(Mean±SD, n=10, g/cm2)

Group	BMD
Sham	0.065±0.02
Model	0.043±0.02*
LWDHW	0.054±0.02^#

Fig.2 Comparison of mRNA expression levels of CLCF1 (A), NFATc1 (B), DC-STAMP (C), OC-STAMP (D), and ATP6V0D2 (E) related to tibial medial membrane fusion in each group of mice (n=3). *P<0.05 vs Sham group; #P<0.05 vs Model group.

Fig.3 Expression levels of membrane fusion-related proteins in the 3 groups of mice. A: Western blotting for detecting expressions of CLCF1, NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 in mouse tibia. B-F: Expression levels of CLCF1, NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 (n=3). *P<0.05 vs Sham group; #P<0.05 vs Model group.

Fig.4 Osteoclast differentiation and maturation in each group (Original magnification: ×200).

Fig.5 Cell membrane fusion rates in each group. *P<0.05 vs Control group; #P<0.05 vs Model group.

Fig.6 Transcriptional levels of the membrane fusion-related genes in the 3 groups. A: CLCF1. B: NFATc1. C: DC-STAMP. D: OC-STAMP. E: ATP6V0D2. *P<0.05 vs Control group; #P<0.05 vs Model group.

Fig.7 Expression levels of membrane fusion-related proteins in each group. A: Western blotting for detecting expressions of CLCF1, NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 in the osteoclasts. B-F: Expression levels of CLCF1, NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. *P<0.05 vs Control group; #P<0.05 vs Model group.

Fig.8 EGFP fluorescence intensity in RAW264.7 cells under different conditions of CLCF1 lentivirus infection (×100). A-C: MOI=100. D-F: MOI=50. G-I: MOI=10. A, D, G: Complete medium. B, E, H: HitransG A. C, F, I. HitransG P.

Fig.9 Effects of different lentiviruses on expression of CLCF1 protein. A: Western blotting for detecting expressions of CLCF1 in the osteoclasts. B: CLCF1. *P<0.05 vs si-NC group; #P<0.05 vs si-CLCF1-2 group.

Fig.10 TRAP staining of RAW264.7 cells in each groups after CLCF1 knockdown (×200).

Fig.11 Osteoclast membrane fusion in each group after CLCF1 knockdown (×200).

Fig.12 Cell membrane fusion in each group after CLCF1 knockdown. *P<0.05 vs si-NC; #P<0.05 vs si-NC+LWDHW; ▲P<0.05 vs si-CLCF1.

Fig.13 Transcriptional levels of genes related to cell membrane fusion after CLCF1 knockdown. A: NFATc1. B: DC-STAMP. C: OC-STAMP. D: ATP6V0D2. *P<0.05 vs si-NC, #P<0.05 vs si-NC+LWDHW, ▲P<0.05 vs si-CLCF1.

Fig.14 Effect of CLCF1 knockdown on expression levels of cell membrane fusion-related proteins. A: Western blotting for detecting expressions of NFATc1, DC-STAMP, OC-STAMP, and ATP6V0D2 in osteoclast. B: NFATc1. C: DC-STAMP. D: OC-STAMP. E: ATP6V0D2. *P<0.05 vs si-NC; #P<0.05 vs si-NC+LWDHW; ▲P<0.05 vs si-CLCF1.

References 28

[1]	谢丽华, 柴昊, 叶云金, 等. 续苓健骨颗粒治疗肾虚血瘀型绝经后骨质疏松症的疗效及转录机制研究[J]. 中国骨质疏松杂志, 2022, 28(4): 558-61, 600.
[2]	陈远程, 吴文, 许翎, 等. 地舒单抗治疗的原发性骨质疏松症患者血钙及骨代谢标志物的动态变化[J]. 南方医科大学学报, 2025, 45(4): 760-4.
[3]	Walker MD, Shane E. Postmenopausal osteoporosis[J]. N Engl J Med, 2023, 389(21): 1979-91. doi：10.1056/nejmcp2307353
[4]	陈梓锋, 李胜发, 张祐鸣, 等. 脂质运载蛋白2自限性抑制间充质干细胞的成骨细胞分化[J]. 南方医科大学学报, 2023, 43(8): 1339-44.
[5]	Fischer V, Haffner-Luntzer M. Interaction between bone and immune cells: Implications for postmenopausal osteoporosis[J]. Semin Cell Dev Biol, 2022, 123: 14-21. doi：10.1016/j.semcdb.2021.05.014
[6]	Wan YM, Nemoto YL, Oikawa T, et al. Mechanical control of osteoclast fusion by membrane-cortex attachment and BAR proteins[J]. J Cell Biol, 2025, 224(7): e202411024. doi：10.1083/jcb.202411024
[7]	Ge JR, Xie LH, Chen J, et al. Liuwei Dihuang pill (六味地黄丸) treats postmenopausal osteoporosis with Shen (kidney) Yin deficiency via Janus kinase/signal transducer and activator of transcription signal pathway by up-regulating cardiotrophin-like cytokine factor 1 expression[J]. Chin J Integr Med, 2018, 24(6): 415-22. doi：10.1007/s11655-016-2744-2
[8]	Gao S, Li NH, Qi LJ, et al. Myokines and osteokines in aging-related degenerative diseases: Regulatory networks in the muscle-bone-brain axis[J]. Cytokine Growth Factor Rev, 2025, 86: 17-28. doi：10.1016/j.cytogfr.2025.09.001
[9]	Lampiasi N, Russo R, Kireev I, et al. Osteoclasts differentiation from murine RAW 264.7 cells stimulated by RANKL: timing and behavior[J]. Biology, 2021, 10(2): 117. doi：10.3390/biology10020117
[10]	黄小彬, 葛继荣, 李生强, 等. 不同滋阴补肾法干预去势大鼠破骨通路的机制[J]. 中国组织工程研究, 2025, 29(6): 1214-9.
[11]	魏伟, 吴希美, 李元建. 药理实验方法学. 4版[M]. 北京: 人民卫生出版社, 2010: 71-72.
[12]	Verma SK, Chernomordik LV, Melikov K. An improved metrics for osteoclast multinucleation[J]. Sci Rep, 2018, 8: 1768. doi：10.1038/s41598-018-20031-x
[13]	叶云金, 黄小彬, 薛立鹏, 等. 肾骨胶囊治疗绝经后骨质疏松症(肝肾不足证)的临床研究[J]. 中国骨质疏松杂志, 2025, 31(6): 812-7, 830. doi：10.3969/j.issn.1006-7108.2025.06.005
[14]	Miyamoto H, Suzuki T, Miyauchi Y, et al. Osteoclast stimulatory transmembrane protein and dendritic cell-specific transmembrane protein cooperatively modulate cell-cell fusion to form osteoclasts and foreign body giant cells[J]. J Bone Miner Res, 2012, 27(6): 1289-97. doi：10.1002/jbmr.1575
[15]	Lavialle C, Cornelis G, Dupressoir A, et al. Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[J]. Philos Trans R Soc Lond B Biol Sci, 2013, 368(1626): 20120507. doi：10.1098/rstb.2012.0507
[16]	Jansen IDC, Vermeer JAF, Bloemen V, et al. Osteoclast fusion and fission[J]. Calcif Tissue Int, 2012, 90(6): 515-22. doi：10.1007/s00223-012-9600-y
[17]	Verma SK, Leikina E, Melikov K, et al. Cell-surface phosphatidylserine regulates osteoclast precursor fusion[J]. J Biol Chem, 2018, 293(1): 254-70. doi：10.1074/jbc.m117.809681
[18]	Witwicka H, Hwang SY, Reyes-Gutierrez P, et al. Studies of OC-STAMP in osteoclast fusion: a new knockout mouse model, rescue of cell fusion, and transmembrane topology[J]. PLoS One, 2015, 10(6): e0128275. doi：10.1371/journal.pone.0128275
[19]	Kim HJ, Seong HS, Choi Y, et al. Letrozole suppresses the fusion of osteoclast precursors through inhibition of p38-mediated DC-STAMP pathway[J]. Int J Mol Sci, 2020, 21(21): 8396. doi：10.3390/ijms21218396
[20]	Stegen S, Moermans K, Stockmans I, et al. The serine synthesis pathway drives osteoclast differentiation through epigenetic regulation of NFATc1 expression[J]. Nat Metab, 2024, 6(1): 141-52. doi：10.1038/s42255-023-00948-y
[21]	Matsuo K, Galson DL, Zhao C, et al. Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-fos[J]. J Biol Chem, 2004, 279(25): 26475-80. doi：10.1074/jbc.m313973200
[22]	Bae S, Kim K, Kang K, et al. RANKL-responsive epigenetic mechanism reprograms macrophages into bone-resorbing osteoclasts[J]. Cell Mol Immunol, 2023, 20(1): 94-109. doi：10.1038/s41423-022-00959-x
[23]	Asagiri M, Sato K, Usami T, et al. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis[J]. J Exp Med, 2005, 202(9): 1261-9. doi：10.1084/jem.20051150
[24]	Kim SS, Jeong SP, Park BS, et al. Melatonin attenuates RANKL-induced osteoclastogenesis via inhibition of Atp6v0d2 and DC-STAMP through MAPK and NFATc1 signaling pathways[J]. Molecules, 2022, 27(2): 501. doi：10.3390/molecules27020501
[25]	Yuan LC, Zhao N, Wang JY, et al. Major vault protein (MVP) negatively regulates osteoclastogenesis via calcineurin-NFATc1 pathway inhibition[J]. Theranostics, 2021, 11(15): 7247-61. doi：10.7150/thno.58468
[26]	Han SY, Kim YK. Coptidis rhizoma water extract attenuates RANKL-induced osteoclast differentiation via MAPK, Akt, and NF-κB pathways and prevents ovariectomy (OVX)-mediated bone loss[J]. Int J Mol Sci, 2025, 26(17): 8707. doi：10.3390/ijms26178707
[27]	Yokota S, Matsumae G, Shimizu T, et al. Cardiotrophin Like Cytokine Factor 1 (CLCF1) alleviates bone loss in osteoporosis mouse models by suppressing osteoclast differentiation through activating interferon signaling and repressing the nuclear factor-κB signaling pathway[J]. Bone, 2021, 153: 116140. doi：10.1016/j.bone.2021.116140
[28]	陈娟, 叶云金, 谢丽华, 等. Raji-MG63细胞共培养体系中心肌营养蛋白样细胞因子1(CLCF1)对RANKL/OPG比值及成骨细胞分化的影响[J]. 中国骨质疏松杂志, 2021, 27(2): 157-61.