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. 2023 Jul 18;18(1):508.
doi: 10.1186/s13018-023-03842-1.

Exploring the role and mechanism of Fuzi decoction in the treatment of osteoporosis by integrating network pharmacology and experimental verification

Fudong Li #  1 , Chuan Guo #  2 , Shikai Zhang  3 , Bing Zheng  4 , Kaiqiang Sun  5 , Jiangang Shi  6
Affiliations

Exploring the role and mechanism of Fuzi decoction in the treatment of osteoporosis by integrating network pharmacology and experimental verification

Fudong Li et al. J Orthop Surg Res. .

Abstract

Background: Fuzi decoction (FZD), a traditional Chinese medicine formula, was used to treat musculoskeletal diseases by warming channels, strengthening yang and dispelling pathogenic cold and dampness. In clinical practice, FZD has been used to treat rheumatoid arthritis and osteoarthritis. It alleviated osteoarticular disorders through ameliorating the degradation of cartilage and improving meniscal damage in osteoarthritis, while its roles and mechanisms in the treatment of bone loss diseases remain unclear. This study aims to investigate the underlying mechanisms of FZD in treating osteoporosis using an integrative method of network pharmacology and experimental study.

Methods: In this study, network pharmacology was used to predict the core targets and potential pathways of the bioactive ingredients of FZD to attenuate osteoporosis. Molecular docking was performed to evaluate the interactions between core compounds and key targets. In addition, both cell and animal experiments were carried out to validate the role and potential mechanism in treating osteoporosis.

Results: In the present study, data revealed that kaempferol, beta-sitosterol, stigmasterol, fumarine, and (+)-catechin may be the primary bioactive ingredients of FZD in the treatment of osteoporosis, which were closely associated with the osteoporosis-related targets. And the KEGG results indicated that the NF-κB pathway was closely associated with the function of FZD in treating osteoporosis. In addition, in vivo demonstrated that FZD ameliorated osteoporosis. In vitro experiments showed that the pro-apoptotic factors indicators including CASP3 and BAX were decreased by FZD and the anti-apoptotic factor BCL2 was increased by FZD. In addition, FZD significantly suppressed the osteoclast differentiation in culture and the expression levels of osteoclast-related genes including TRAF6, CTSK, and MMP9. And the NF-κB pathway was confirmed, via in vitro experiment, to be involved in osteoclast differentiation.

Conclusions: This study demonstrated that FZD played a pivotal role in suppressing the osteoclast differentiation via regulating the NF-κB pathway, indicating that FZD could be a promising antiosteoporosis drug and deserve further investigation.

Keywords: Fuzi decoction; NF-κB pathway; Network pharmacology; Osteoclast; Osteoporosis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1
Fig. 1
Selection of the targets of osteoporosis and the common targets between FZD and osteoporosis. A The number of genes related to osteoporosis were retrieved from five databases. B The intersection between targets of FZD and genes of osteoporosis
Fig. 2
Fig. 2
The core targets and key ingredients of FZD in treating osteoporosis. A The network between the common targets and the ingredients. B The core targets and key ingredients selected by degree. C The core targets and key ingredients selected by MCC. The darker the color, the more important the term
Fig. 3
Fig. 3
The PPI network and the core proteins selected from the PPI network. A The PPI network obtained using the STRING database. B The potential core targets of FZD in treating osteoporosis were selected using CytoNCA. C, D The core targets selected by degree or MCC. The darker the color, the more important the term
Fig. 4
Fig. 4
GO enrichment and KEGG pathway analysis. A The results of GO enrichment. B The results of KEGG analysis
Fig. 5
Fig. 5
Molecular docking results. A Heat map of the binding energies. B–G The visualized molecular docking results
Fig. 6
Fig. 6
The biological effect of FZD on the OVX-induced osteoporosis in rats. A The images of micro-CT. B–I The quantitative analysis of Micro-CT results. J, K The H&E staining of the femurs and the quantitative analysis results. L, M The results of TRAP staining and the quantitative analysis results. NS indicates no significance. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 7
Fig. 7
The CCK-8 result and the anti-apoptosis effect of FCS on BMMs. A FCS not more than 200 nM could not negatively affect the cell viability of BMMs. B The western blot results of the apoptosis-related indicators including CASP3, BAX, and BCL2. C–E Semi-quantitative analysis of western blot results on the expression of CASP3, BAX, and BCL2. F–H Semi-quantitative analysis of the mRNA levels of CASP3, BAX, and BCL2. NS indicates no significance. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 8
Fig. 8
The results of RANKL-induced osteoclastogenesis and the formation of F-actin ring. A The images of TRAP staining. B The quantitative analysis results of the number of TRAP+ OCs in per well. C The semi-quantitative analysis results of the area of OCs. D The images of the F-actin ring of the RANKL-induced OCs. E The quantitative analysis results of the number of F-actin rings in per well. F The quantitative analysis results of the number of nuclei in OCs. G The semi-quantitative analysis results of the area of F-actin rings. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 9
Fig. 9
The effect of FCS on the expression levels of osteoclastogenesis-associated indicators. A The western blot results of TARF6, CTSK and MMP9. B–D The semi-quantitative analysis results of the protein levels of TARF6, CTSK and MMP9. E–G The semi-quantitative analysis results of the mRNA levels of TARF6, CTSK and MMP9. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 10
Fig. 10
The RANKL-induced activation of NF-κB pathway and the downstream factors were suppressed by FZD. A The western blot results of p-p65, p65, NFATc1 and c-FOS. B–E The semi-quantitative analysis results of the protein levels of p-p65, p65, NFATc1 and c-FOS. F–G The semi-quantitative analysis results of the mRNA levels of NFATc1 and c-FOS. Values are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001
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