Erxian herbal pair enhances bone formation in infected bone nonunion models and attenuates lipopolysaccharide-induced osteoblastinhibition by regulating miRNA-34a-5p

ABSTRACT

Bacterium-induced inflammatory responses cause bone nonunion. Although antibiotics suppress infection, bone loss after antibacterial treatment remains a critical challenge. Erxian herbal pair (EHP) has been proven effective in promoting bone formation. Our study aimed to investigate the effect of EHP on bone repair after anti-infection treatment, explore its effect on a lipopolysaccharide (LPS)-induced osteoblast. We evaluated effects of EHP on bone repair with Micro-CT, and morphology detecting. Chemical constituents of EHP and EHP-containing serum (EHP-CS) were identified by UHPLC-Q/TOF-MS. In addition, osteoblast induced by LPS was established and administrated with EHP-CS. Cell proliferationwas assessed by MTT. Target prediction identified SMAD2 as a potential target of miRNA-34a-5p. MiRNA mimic, inhibitor and siRNA were transiently transfected into osteoblasts. The mRNA levels and protein expressions of miRNA-34a-5p, BMP2, Runx2, SMAD2 were assessed. The results showed that the main biocactivity ingredients in EHP-CS were Baohuoside Ι and Orcinol Glucoside. EHP could promote bone remolding after anti-infection therapy and restore the activity of LPS-induced osteoblasts. Moreover, miRNA-34a-5p was dramatically downregulated and SMAD2 was upregulated after LPS stimulation, while EHP resisted the inhibition of LPS by promoting miRNA-34a-5p, ALP, and BMP2 expressions. Whereas downregulation of miRNA-34a-5p reversed these effects. Silencing endogenous SMAD2 expression markedly promoted BMP2 and ALP activity and enhanced osteogenesis. Taken together, EHP restored LPS-induced bone loss by regulating miRNA-34a-5p levels and repressing its target gene SMAD2. EHP might be a potential adjuvant herbal remedy for the treatment of bone nonunion, and miRNA-34a-5p is a novel target for controlling bone and metabolic diseases.

Graphical abstract

KEYWORDS:

• Erxian herbal pair
• bone repair
• LPS-induced bone loss
• MiRNA-34a-5p
• osteoblasts

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. For original data, please contact [email protected].

Author contributions

Dan Shou and Yang Zhang conceived and designed the experiments; Li Zhang, Yang Zhang, Maomao Miao, Shaoqi Hu, Xuping Wang, Lisha Zhao, and Xiaowen Huang performed the experiments, analysed the data; Li Zhang and Yang Zhang wrote the manuscript; Dan Shou administered and supervised the experimental work. All authors read and approved the final manuscript.

Abbreviations

BV/TV: Bone volume/tissue volume ratio

BMD: bone mineral density; EHP: Erxian herbal pair

EHP-CS:EHP-containing serum

LPS: lipopolysaccharide; S. aureus: Staphylococcus aureus

TIC: Total ion chromatogram

UPLC-Q/TOF-MS: Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometer

Van-CS: vancomycin-calcium sulfate.

Ethics statement

The animal study was reviewed and approved by The Animal Ethics Committee of Zhejiang Academy of Traditional Chinese Medicine.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21655979.2022.2085388

Additional information

Funding

This work was supported by the National Natural Science Foundation of China (grant nos. 81803808 and 81873062), the Science and technology project of Zhejiang Province (grant no. LGF21H28004), the Zhejiang Provincial Medical and Health Science and Technology Fund (grant no. 2021KY112), and the Zhejiang Provincial Traditional Chinese Medicine Science and Technology Fund (grant no. 2021ZA033, 2020ZQ008