Butyrate Reducing Bone Mass Loss by Regulating the Expression of m6A Methyltransferase METTL3 in Implant-Associated Staphylococcus aureus Osteomyelitis

Abstract

Staphylococcus aureus (S. aureus) has been identified as a hindrance to osteoblast differentiation, thereby contributing significantly to the development of osteomyelitis. Consequently, exploring pharmaceutical interventions targeting osteoblast differentiation mediated by S. aureus may present a novel approach for treating osteomyelitis. It has been reported that N6-methyladenosine (m6A) methylation is highly associated with infection. Moreover, studies continue to validate that short-chain fatty acids play an important role in transcriptional modification and have been considered as a potential treatment for S. aureus infection. Our research aimed to examine the impact and underlying mechanism of butyrate, a short-chain fatty acid, in reducing the inhibitory influence exerted by S. aureus on osteoblast differentiation. The concentration of butyrate beneficial to MC3T3-E1 cell viability was screened by Cell Counting Kit-8 (CCK8) assay. Reverse transcription-quantitative PCR (RT-qPCR), Western blotting, and alkaline phosphatase (ALP)staining were used to verify the osteogenic indexes and the expression levels of m6A methylation-related proteins in MC3T3-E1 cells infected with S. aureus at different time points. Besides, the same methods were used to verify the effects of butyrate stimulation and METTL3 knockdown on the osteogenic ability of MC3T3-E1 cells. H&E staining, Goldner staining, and immunohistochemical staining were used to verify the effect of butyrate on mice with endo-plant associated S. aureus osteomyelitis. The potential mechanisms of METTL3 and autophagy in MC3T3-E1 cells were studied by Western blotting. In vitro experiments, we found that butyrate significantly enhanced the expression of osteogenic-related genes down-regulated by infection in MC3T3-E1 cells induced by S. aureus, including RUNX2, OCN, and ALP. In addition, METTL3, an important m6A methyltransferase, was significantly up-regulated in S. aureus-infected MC3T3-E1 cells, but its expression could be down-regulated by butyrate. Inhibiting the expression of METTL3 by siRNA could effectively rescue the osteogenic markers down-regulated by S. aureus infection in MC3T3-E1 cells, which was similar to the results of butyrate treatment. In vivo experiments had shown that butyrate could alleviate inflammation and osteogenic activity in implant-associated osteomyelitis. The construction of bone marrow METTL3 low-expression mice using siRNA also showed similar effects on osteogenic activity. Additionally, Western blotting confirmed that knocking down METTL3 rescued the autophagy imbalance caused by S. aureus infection in MC3T3-E1 cells. In general, our research demonstrated that butyrate effectively alleviated the hindrance of osteoblast activity induced by S. aureus infection by suppressing the expression of METTL3, suggesting that butyrate may be a novel treatment for S. aureus osteomyelitis.