Emodin Combined with Multiple Low-frequency Low-intensity Ultrasound to Relieve Osteomyelitis Through Sonoantimicrobial Chemotherapy

ABSTRACT Treatment of osteomyelitis is still challenging as conventional antibiotic therapy is limited by the emergence of resistant strains and the formation of biofilms. Sonoantimicrobial chemotherapy (SACT) is a novel therapy of low-frequency and low-intensity ultrasound (LFLIU) combined with sonosensitizer. Therefore, in our study, a sonosensitizer named emodin (EM) was proposed to be combined with LFLIU to relieve acute osteomyelitis caused by Methicillin-resistant Staphylococcus aureus (MRSA) through synergistic antibacterial and anti-biofilm effects. The efficiency of different intensities of ultrasound, single (S-LFLIU, 15 min) and multiple ultrasound (M-LFLIU, 5 min every 4 h, three times) against bacteria and biofilm was compared, contributing to develop the best treatment regimen. Our results demonstrated that EM plus S-LFLIU or M-LFLIU (EM+S-LFLIU or EM+M-LFLIU) have significant synergetic bactericidal and anti-biofilm effects and EM+M-LFLIU exhibits superior performance in anti-biofilm. Furthermore, it was suggested that EM+M-LFLIU could produce a large amount of reactive oxygen species (ROS), destroy the integrity of bacterial membrane and wall, down-regulate the expression of oxidative stress, membrane wall synthesis, bacterial virulence and other related genes ( agrB, PBP3, sgtB, GMK, zwf, msrA ). In vivo study, micro-CT, H&E staining, ELISA assay and bacterial quantification of bone tissue indicated that EM+M-LFLIU could also relieve osteomyelitis of MRSA infection. Our work proffers an original treating bacterial osteomyelitis approach that weakens drug-resistant bacterial and suppresses biofilm formation through SACT, which may provide new prospects for clinical treatment.