BushenHuoxue decoction suppresses M1 macrophage polarization and prevents LPS induced inflammatory bone loss by activating AMPK pathway

Shuangshuang Chen; Lihong Tao; Feng Zhu; Zhifang Wang; Qi Zhuang; Yajun Li; Yunshang Yang; Chengcheng Feng; Haiwei Shi; Jiandong Shi; Like Zhu; Long Xiao; Dechun Geng; Zhirong Wang

doi:10.1016/j.heliyon.2023.e15583

BushenHuoxue decoction suppresses M1 macrophage polarization and prevents LPS induced inflammatory bone loss by activating AMPK pathway

Heliyon. 2023 Apr 19;9(5):e15583. doi: 10.1016/j.heliyon.2023.e15583. eCollection 2023 May.

Authors

Shuangshuang Chen^{1

2}, Lihong Tao², Feng Zhu³, Zhifang Wang⁴, Qi Zhuang¹, Yajun Li^{1

4}, Yunshang Yang^{1

4}, Chengcheng Feng¹, Haiwei Shi⁴, Jiandong Shi⁴, Like Zhu¹, Long Xiao^{1

3

4}, Dechun Geng³, Zhirong Wang^{1

4}

Affiliations

¹ Translational Medical Innovation Center, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, 215600, China.
² Department of Rheumatology, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, 215600, China.
³ Department of Orthopedics, The First Affiliated Hospital of Soochow University, Suzhou, 215006, China.
⁴ Department of Orthopedics, Zhangjiagang TCM Hospital Affiliated to Nanjing University of Chinese Medicine, Zhangjiagang, 215600, China.

Abstract

Abnormal bone metabolism and subsequence osteoporotic fractures are common complications of chronic inflammatory diseases. No effective treatment for these bone-related complications is available at present. The chronic inflammatory state in these diseases has been considered as a key factor of bone loss. Therefore, the combination of inflammation inhibition and bone loss suppression may be an important strategy for reducing bone damage associated with inflammatory diseases. Bushen Huoxue Decoction (BSHXD) is a traditional Chinese herbal compound that has demonstrated the ability to improve bone quality and increase bone density. However, the efficacy of BSHXD on inflammatory bone loss and its underlying mechanisms remain unclear. This study aimed to investigate whether BSHXD inhibits inflammatory bone loss in mice and its potential molecular mechanisms. In the present study, the effect of BSHXD on lipopolysaccharide (LPS)-induced M1 polarization of RAW264.7 macrophage and on local inflammatory bone loss model of mouse skull was determined. The results showed that after treating RAW264.7 cells with LPS for 24 h, the expression levels of IL-1β (39.42 ± 3.076 ng/L, p < 0.05), IL-6 (49.24 ± 1.766 mg/L, p < 0.05) and TNF-α (286.3 ± 27.12 ng/L, p < 0.05) were significantly increased. The addition of BSHXD decreased the expression levels of IL-1β, IL-6, and TNF-α to 31.55 ± 1.296 ng/L, 37.94 ± 0.8869 mg/L, and 196.4 ± 25.25 ng/L, respectively (p < 0.05). The results of immunofluorescence staining, Western blotting (WB) and flow cytometry indicated that the proportion of M1 macrophages in RAW264.7 cells treated with BSHXD for 24 h was significantly lower than that in the LPS group (13.36% ± 0.9829% VS 24.80% ± 4.619%, p < 0.05). The evidence from in-vitro experiments showed that the immunomodulatory ability of BSHXD may be associated with the activation of AMP-dependent protein kinase (AMPK) pathway in LPS-treated macrophages. In addition, the results of micro-CT, H&E staining, immunohistochemical staining and immunofluorescence staining of mouse skull further demonstrated that BSHXD treatment significantly alleviated LPS-induced local bone loss and inflammatory damage in mouse skull model. All results indicated that BSHXD significantly inhibited inflammatory factors release and M1 polarization of macrophage through AMPK signaling pathway. Therefore, BSHXD may be a promising drug for the treatment of inflammatory bone loss.

Keywords: Bone immune regulation; Bushen huoxue decoction; Inflammation; Inflammatory bone loss; Macrophage polarization.