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Fufang Zhenshu Tiaozhi capsule enhances bone formation and safeguards against glucocorticoid-induced osteoporosis through innovative Mekk2-mediated β-catenin deubiquitination

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Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

Bone homeostasis depends on the regulation of β-catenin in osteoblasts. Glucocorticoids (GCs) are known to diminish β-catenin activity via Wnt pathway signaling, leading to osteoporosis. Conversely, activating β-catenin in osteoblasts through mitogen-activated protein kinase kinase kinase 2 (Mekk2) offers an innovative approach to combat GC-induced osteoporosis (GIOP). Fufang Zhenshu Tiaozhi (FTZ) capsules have shown effectiveness in treating GIOP, but the mechanisms behind this are still unclear.

Materials and methods

In this study, Mekk2 knockout mice (Mekk2−/−) was generated by CRISPR/Cas9. These mice were then subjected to Alcian Blue-Alizarin Red staining and immunofluorescence to assess their bone and cartilage development. To establish models of GIOP, both Mekk2−/− and wild-type (WT) mice were treated with dexamethasone (DXMS) and subsequently given FTZ capsules. We analyzed the resulting phenotypic changes in these mice using Micro-CT scans and histomorphological studies. Primary osteoblasts, isolated from both Mekk2−/− and WT mice, underwent qRT-PCR to measure key osteogenesis markers, including Runx2, Sp7, Bgalp, Col1a1 and Alp. Cells were then exposed to treatments with either FTZ or Wnt3a and the phosphorylation levels of β-catenin and Mekk2, along with the protein expression of Runx2, were evaluated using Western blotting and immunoprecipitation. Additionally, C3H10T1/2 cells transfected with TOPflash-luciferase and Renilla luciferase reporters were treated with FTZ and Wnt3a to measure β-catenin activity.

Results

In our study, administering FTZ in vivo effectively prevented bone loss typically induced by GCs. However, it's important to note that this protective effect was substantially reduced in mice lacking Mekk2. Additionally, FTZ showed a significant ability to enhance osteogenic differentiation in primary osteoblasts, doing so by altering the expression of Mekk2. Intriguingly, the impact of FTZ on Mekk2 appears to function through a pathway separate from the traditional Wnt signaling route. Furthermore, our findings indicate that FTZ also promotes the deubiquitination of β-catenin, contributing further to its positive effects on bone health.

Conclusions

This study suggests that FTZ plays a significant role in protecting bone mass in cases of GIOP. The mechanism through which FTZ confers this benefit involves the activation of Mekk2/β-catenin signaling pathways, which represents a promising alternative strategy to counteract the deleterious effects of GIOP by augmenting osteoblastogenesis.

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Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

GC:

Glucocorticoid

FTZ:

Fufang Zhenshu Tiaozhi

GIOP:

Glucocorticoid-induced osteoporosis

Mekk2:

Mitogen-activated protein kinase kinase kinase 2

DXMS:

Dexamethasone

WT:

Wild type

ALP:

Alkaline phosphatase

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Acknowledgements

Not applicable.

Funding

This study was supported in part by the National Natural Science Foundation of China (No. 81704098, 82305265), Science and Technology Planning Project of Yunfu (No. 2020A090402), Guangdong Science and Technology Innovation Strategy Special Funding "Great Special Project + Task List" (No. 2020A090402), and China Postdoctoral Science Foundation (grant number: 2023M730809). This sponsor hasn’t participated in the design of the study or collection, analysis, or interpretation of data or in writing manuscript.

Author information

Authors and Affiliations

  1. Department of Orthopaedic Surgery, The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, 510000, People’s Republic of China

    Guoju Hong

  2. The Third Medical Collage, Guangzhou University of Chinese Medicine, Guangzhou, 510000, People’s Republic of China

    Guoju Hong

  3. Department of Endocrinology, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, 510378, People’s Republic of China

    Lin Tang, Tianyu Zhou, Youhong Xie, Jiangyan Wang & Ping Sun

  4. Department of Orthopedic, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, 510378, People’s Republic of China

    Dongdong Ge & Qunwei Dong

  5. Department of Orthopedic, Yunfu Hospital of Traditional Chinese Medicine, Yunfu, 527300, People’s Republic of China

    Qunwei Dong

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  1. Guoju Hong
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  3. Tianyu Zhou
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Contributions

Ping Sun and Qunwei Dong conceived and supervised the study; Guoju Hong designed experiments; Guoju Hong, Jiangyan Wang and Dongdong Ge performed experiments; Lin Tang provided new tools and reagents; Tianyu Zhou and Youhong Xie analysed data; Guoju Hong wrote the manuscript; Ping Sun made manuscript revisions.

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Correspondence to Qunwei Dong or Ping Sun.

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The animal protocol was approved by the animal ethics broad of Guangzhou University of Chinese Medicine where the animal study was performed.

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Hong, G., Tang, L., Zhou, T. et al. Fufang Zhenshu Tiaozhi capsule enhances bone formation and safeguards against glucocorticoid-induced osteoporosis through innovative Mekk2-mediated β-catenin deubiquitination. J Bone Miner Metab (2024). https://doi.org/10.1007/s00774-024-01516-4

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