Bushen Huoxue decoction (补肾活血汤) attenuates osteoporosis in ovariectomized rats through suppressing osteoclast differentiation by targeting sirtuin 1 to deactivate nuclear factor-κB

doi:10.19852/j.cnki.jtcm.2026.05.007

Journal of Traditional Chinese Medicine ›› 2026, Vol. 46 ›› Issue (5): 1127-1138.DOI: 10.19852/j.cnki.jtcm.2026.05.007

• Original Articles • Previous Articles Next Articles

Bushen Huoxue decoction (补肾活血汤) attenuates osteoporosis in ovariectomized rats through suppressing osteoclast differentiation by targeting sirtuin 1 to deactivate nuclear factor-κB

CHEN Shuai¹, JIN Jie¹, HU Yuwen², TIAN Ningsheng¹, HAN Huawei¹, XU Ziqing¹, WU Lan³(), LI Zhiwei¹()

¹ Department of Orthopaedics, The Second Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210017, China
² School of Chinese Medicine, School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
³ Department of Obstetrics and Gynecology, Women’s Hospital of Nanjing Medical University, Nanjing Women and Children’s Healthcare Hospital, Nanjing 210004, China

Received:2025-05-16 Accepted:2026-01-12 Online:2026-10-15 Published:2026-10-09
Contact: LI Zhiwei, Department of Orthopaedics, the Second Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210017, China. fsyy00797@njucm.edu.cn; Telephone: +86-13512540810; WU Lan, Department of Obstetrics and Gynecology, Women’s Hospital of Nanjing Medical University, Nanjing Women and Children’s Healthcare Hospital, Nanjing 210004, China. 920919625@qq.com,Telephone: +86-15952589935
About author:
CHEN Shuai and JIN Jie are co-first authors and contributed equally to this work
Supported by:
Foundation of Jiangsu Provincial Science and Technology Plan Special Fund Key R&D Plan Social Development Project: Multicenter Study on Traditional Chinese Medicine Preventive Intervention for High-risk Populations of Osteoporosis(BE2023787);Foundation of the Second Affiliated Hospital of Nanjing University of Chinese Medicine: the Role and Mechanism of Bushen Huoxue Decoction in Treating Osteoporosis by Regulating the Ras Homolog Family Member A/Rho-associated Coiled-coil Containing Protein Kinase Pathway to Promote Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells(SEZ202107)

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Abstract

Abstract:

OBJECTIVE: To investigate the anti-osteoporotic effects of Bushen Huoxue decoction (补肾活血汤, BSHXD) and its underlying molecular mechanisms in a postmenopausal osteoporosis (PMOP) model.

METHODS: The model of PMOP was established by bilateral ovariectomy in female rats, and each group were given different doses of BSHXD, or Alendronate by oral gavage. Then, the therapeutic effect of BSHXD on PMOP was estimated using micro-CT analysis, histological staining, and immunohistochemical analysis. In vitro experiment, immunofluorescence analysis, luciferase assay, and co-immunoprecipitation (Co-IP) were used to examine the potential impact and mechanism of BSHXD on osteoclast differentiation. Additionally, Sirtinol was employed to silence Sirt1 and further investigate its involvement in the protective effects of BSHXD.

RESULTS: Our study demonstrated that BSHXD ameliorates bone loss and improves trabecular microstructure in osteoporosis model rats. Simultaneously, histological analysis revealed that BSHXD treatment led to a downregulation of tartrate-resistant acid phosphatase (Trap), matrix metalloproteinase 9, and nuclear factor of activated T cells cytoplasmic 1 expression. In addition, the in vitro experiment provided evidence that BSHXD effectively inhibits the formation of osteoclasts and reduces Trap positive osteoclasts. Luciferase assay and CO-IP analysis revealed that BSHXD decreases the acetylation level of nuclear factor NF-kappa-B p65 subunit (p65) and suppresses the activity of nuclear factor-κB (NF-κB), and significantly promotes the binding of Sirtuin 1 (Sirt1) to p65. Notably, after administration with the Sirt1 inhibitor, the antiosteoporotic effect of BSHXD was reduced and NF‐κB activity was increased.

CONCLUSION: BSHXD promoted p65 deacetylation by activating Sirt1, thereby inhibiting the NF-κB pathway and subsequently alleviating osteoclast differentiation and bone resorption.

Key words: osteoporosis, postmenopausal, osteoclasts, Sirtuin 1, acetylation, Bushen Huoxue decoction

CHEN Shuai, JIN Jie, HU Yuwen, TIAN Ningsheng, HAN Huawei, XU Ziqing, WU Lan, LI Zhiwei. Bushen Huoxue decoction (补肾活血汤) attenuates osteoporosis in ovariectomized rats through suppressing osteoclast differentiation by targeting sirtuin 1 to deactivate nuclear factor-κB[J]. Journal of Traditional Chinese Medicine, 2026, 46(5): 1127-1138.

Figures/Tables 4

Figure 1 Effect of BSHXD on bone loss in osteoporosis model rats A: micro-CT with 3D reconstruction was performed, and representative images from each group are displayed; B: quantitative analysis of BMD of femurs; C: quantitative analysis of Tb.N of femurs; D: quantitative analysis of Tb.Th of femurs; E: quantitative analysis of BV/TV of femurs; F: D: quantitative analysis of Tb.Sp of femurs; G: representative HE staining images of femurs from rats (scale bars: 250 μm); A1, A7, G1: sham group; A2, A8, G2: OVX group; A3, A9, G3: BSHXD-L; A4, A10, G4: BSHXD-M; A5, A11, G5: BSHXD-H; A6, A12, G6: Alendronate group. Sham group: the rats received distilled water once every day for 12 weeks; Model group: the rats received distilled water once every day for 12 weeks; BSHXD-L group: the rats received BSHXD at a dose of 5 g/kg once every day for 12 weeks; BSHXD-M group: the rats received BSHXD at a dose of 10 g/kg once every day for 12 weeks; BSHXD-H group: the rats received BSHXD at a dose of 20 g/kg once every day for 12 weeks; Alendronate group: the rats received Alendronate at a dose of 2.5 mg/kg once every day for 12 weeks. BSHXD: Bushen Huoxue decoction; HE: hematoxylin and eosin; OVX: ovariectomized; BMD: bone mineral density; Tb.N: trabecular number, Tb.Th: trabecular thickness; BV/TV: bone volume fraction index; Tb.Sp: trabecular separation. The results are presented as the mean ± standard deviation (n = 6). Statistical analyses were measured using one-way analysis of variance with Tukey’s multiple comparison tests. aP < 0.05, compared to the sham group; bP < 0.05, compared to the OVX group.

Figure 2 BSHXD inhibited osteoclastogenesis in OVX-induced osteoporosis model rats A: representative TRAP staining images of femurs from rats (scale bars: 100 μm); B: quantitative analysis of TRAP staining images of femurs from rats; C: representative immunohistochemistry staining images of MMP9 and NFATc1 in the femur (scale bars: 100 μm); D: quantitative analysis of MMP9 in the femur; E: quantitative analysis of NFATc1 in the femur; A1, C1, C7: sham group; A2, C2, C8: OVX group; A3, C3, C9: BSHXD-L; A4, C4, C10: BSHXD-M; A5, C5, C11: BSHXD-H; A6, C6, C12: Alendronate group. Sham group: the rats received distilled water once every day for 12 weeks; Model group: the rats received distilled water once every day for 12 weeks; BSHXD-L group: the rats received BSHXD at a dose of 5 g/kg once every day for 12 weeks; BSHXD-M group: the rats received BSHXD at a dose of 10 g/kg once every day for 12 weeks; BSHXD-H group: the rats received BSHXD at a dose of 20 g/kg once every day for 12 weeks; Alendronate group: the rats received Alendronate at a dose of 2.5 mg/kg once every day for 12 weeks. BSHXD: Bushen Huoxue decoction; OVX: ovariectomized; Trap: tartrate-resistant acid phosphatase; MMP9: matrix metalloproteinase 9; NFATc1: nuclear factor of activated T cells cytoplasmic 1. The results are presented as the mean ± standard deviation (n = 3). Statistical analyses were measured using one-way analysis of variance with Tukey’s multiple comparison tests. aP < 0.05, compared to the sham group; bP < 0.05, compared to the OVX group.

Figure 3 BSHXD inhibited the activation of NF-κB by targeting SIRT1 A: quantification of gene expression of NFATc1; B: the expression of p65-Acetyl310 was detected via Western blot; C: relative protein expression levels of p65-Acetyl310; D: representative immunohistochemistry staining images of SIRT1 in the femur (scale bars: 100 μm); E: quantitative analysis of SIRT1 in the femur; F: the expression of SIRT1 was detected via Western blot; G: relative protein expression levels of SIRT1; D1: sham group; D2: OVX group; D3: BSHXD-L; D4: BSHXD-M; D5: BSHXD-H. Sham group: the rats received distilled water once every day for 12 weeks; Model group: the rats received distilled water once every day for 12 weeks; BSHXD-L group: the rats received BSHXD at a dose of 5 g/kg once every day for 12 weeks; BSHXD-M group: the rats received BSHXD at a dose of 10 g/kg once every day for 12 weeks; BSHXD-H group: the rats received BSHXD at a dose of 20 g/kg once every day for 12 weeks. BSHXD: Bushen Huoxue decoction; OVX: ovariectomized; SIRT1: Sirtuin 1; p65: nuclear factor NF-kappa-B p65 subunit. The results are presented as the mean ± standard deviation (n = 3). Statistical analyses were measured using one-way analysis of variance with Tukey’s multiple comparison tests. aP < 0.05, compared to the sham group; bP < 0.05, compared to the OVX group.

Figure 4 BSHXD inhibits osteoclast differentiation by activating SIRT1 A: representative immunofluorescence staining images of SIRT1 (scale bars: 50 μm; green: SIRT1; blue: DAPI); B: quantitative analysis of immunofluorescence staining images of SIRT1; C: the protein expression level of SIRT1 was detected via Western blot; D: the protein expression level of p65 and SIRT1 were detected via Western blot; E: relative protein expression levels of p65/SIRT1; A1, A6, A11: Control group; A2, A7, A12: RANKL group; A3, A8, A13: BSHXD-L group; A4, A9, A14: BSHXD-M group; A5, A10, A15: BSHXD-H group. Control group: RAW 264.7 cells cultured with 30 ng/mL M-CSF; RANKL group: RAW 264.7 cells cultured with 50 ng/mL RANKL and 30 ng/mL M-CSF; BSHXD-L group: RAW 264.7 cells cultured with 50 ng/mL RANKL, 30 ng/mL M-CSF, and 5% BSHXD-containing serum; BSHXD-M group: RAW 264.7 cells cultured with 50 ng/mL RANKL, 30 ng/mL M-CSF, and 10% BSHXD-containing serum; BSHXD-H group: RAW 264.7 cells cultured with 50 ng/mL RANKL, 30 ng/mL M-CSF, and 15% BSHXD-containing serum. BSHXD: Bushen Huoxue decoction; SIRT1: Sirtuin 1; p65: nuclear factor NF-kappa-B p65 subunit. The results are presented as the mean ± standard deviation (n = 3). Statistical analyses were measured using one-way analysis of variance with Tukey’s multiple comparison tests. aP < 0.05, compared to the control group; bP < 0.05, compared to the RANKL group.

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Bushen Huoxue decoction (补肾活血汤) attenuates osteoporosis in ovariectomized rats through suppressing osteoclast differentiation by targeting sirtuin 1 to deactivate nuclear factor-κB

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