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M2-polarized macrophages mediate wound healing by regulating connective tissue growth factor via AKT, ERK1/2, and STAT3 signaling pathways

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Abstract

Background

Timely and sufficient M1 recruitment and M2 polarization are necessary for fibrosis during wound healing. The mechanism of how M2 mediates wound healing is worth exploring. Abnormally up-regulated connective tissue growth factor (CTGF) influences multiple organ fibrosis, including cardiac, pulmonary, hepatic, renal, and cutaneous fibrosis. Previous studies reported that M2 contributed to hepatic and renal fibrosis by secreting CTGF. It is worth discussing if M2 regulates fibrosis through secreting CTGF in wound healing.

Methods and results

We established the murine wound model and inhibited macrophages during proliferation phase with clodronate liposomes in vivo. Macrophages depletion led to down-regulation of wound healing rates, collagen deposition, as well as expression of collagen 1/3 and Ki67. M2 was induced by interleukin-4 (IL-4) and measured by flow cytometry in vitro. Secreted pro-fibrotic and anti-fibrotic factors were tested by enzyme-linked immunosorbent assay (ELISA). M2 was polarized, which producing more CTGF, transforming growth factor-beta1 (TGF-β1), and IL-6, as well as less tumor necrosis factor-α (TNF-α) and IL-10. M2 CTGF gene was blocked using siCTGF. Effects of M2 on fibroblasts activities were detected by cell counting kit 8 (CCK8) and cellular wound healing assay. Expressions of related signaling pathway were assessed by western blotting. Blockade of CTGF in M2 deactivated fibroblasts proliferation and migration by regulating AKT, ERK1/2, and STAT3 pathway. Recombinant CTGF restored these effects.

Conclusions

Our research, for the first time, indicated that M2 promoted wound healing by secreting CTGF, which further mediating proliferation and migration of fibroblasts via AKT, ERK1/2, and STAT3 pathway.

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Data availability

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Acknowledgements

We are indebted to all individuals who participated in or helped with this research project.

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Authors and Affiliations

  1. Department of Plastic and Reconstructive Surgery, Zhongshan Hospital Fudan University, 180 Fenglin Road, Xuhui District, Shanghai, 200032, People’s Republic of China

    Si-Min Zhang, Chuan-Yuan Wei, Qiang Wang, Lu Wang, Lu Lu & Fa-Zhi Qi

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  1. Si-Min Zhang
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  2. Chuan-Yuan Wei
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  3. Qiang Wang
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  4. Lu Wang
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  5. Lu Lu
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  6. Fa-Zhi Qi
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Contributions

All authors participated in the design, interpretation of the studies and analysis of the data and review of the manuscript; SMZ devoted to study design, data collection, and manuscript preparation. CYW and QW devoted to study design and data interpretation. Lu Wang participated in statistical analysis and paper revision. LL participated in statistical analysis and data interpretation. FZQ provided substantial advice in study design, division of labor, manuscript preparation, and funds collection.

Corresponding author

Correspondence to Fa-Zhi Qi.

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All Authors declare that they have no competing interest.

Ethical approval

All manipulations involving C57BL/6 mice were carried out in accordance with the Ethics Committee of the Zhongshan Hospital Fudan University and national ethical guidelines for experimental protocols on animals (Permission No. 2019-067).

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Zhang, SM., Wei, CY., Wang, Q. et al. M2-polarized macrophages mediate wound healing by regulating connective tissue growth factor via AKT, ERK1/2, and STAT3 signaling pathways. Mol Biol Rep 48, 6443–6456 (2021). https://doi.org/10.1007/s11033-021-06646-w

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  • DOI: https://doi.org/10.1007/s11033-021-06646-w

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