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

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. <i>Target prediction identified SMAD2 as a</i> <i>potential target of miRNA-34a-5p.</i> 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.