Prophylactic Effects of NFκB Essential Modulator-Binding Domain Peptides on Bone Infection: An Experimental Study in a Rabbit Model.

Introduction: Osteomyelitis is characterized by intensive inflammatory bone disease and remains a clinical challenge in orthopedic surgery, despite the advances made in medical and surgical therapies. Staphylococcus aureusis a major causative agent of osteomyelitis, causing the progressive inflammatory destruction of bone. Prophylaxis of osteomyelitis during orthopedic surgery is necessary. NFκB essential modulator-binding domain (NBD) peptides are cell-permeable peptide inhibitors of the IκB-kinase complex. The prophylactic effect of NBD peptides in relieving inflammation and inhibiting bone defects in osteomyelitis is still under investigation. Our purpose was to determine the preventive effect of NBD peptides in S. aureus infection-induced bone defects in osteomyelitis. Methods: An S. aureus osteomyelitis rabbit model was used in this study. The rabbits were divided into four groups: NBD, cefazolin, control, and PBS. Clinical and laboratory indicators of erythrocyte-sedimentation rate, CRP, and TNFα levels were assessed to monitor systemic reactions. The efficacy of NBD peptides in S. aureus-induced osteomyelitis was evaluated by radiological, histological, and microbiological examinations, immunohistochemistry, immunofluorescence, and micro-CT scans. Results: In general, NBD peptides effectively reduced clinical signs in rabbits when compared with the control group. Radiography indicated that there was more severe osteomyelitis in the bacterium-infection control group. There was no significance between cefazolin- and NBD-group average scores. The histological results of the lesion slices further confirmed different severity among the groups. Additionally, significant pathological differences were found between the cefazolin and NBD groups, and the PBS group showed no obvious pathological changes. Conclusion: Prophylactic administration of NBD peptides to bone-defect areas inhibited bacterial spread and promoted bone regeneration, making NBD peptides a possible treatment option for prophylaxis in bone infections.

Novel Elongator Protein 2 Inhibitors Mitigating Tumor Necrosis Factor-α Induced Osteogenic Differentiation Inhibition.

Tumor necrosis factor-α is a common cytokine that increases in inflammatory processes, slows the differentiation of bone formation, and induces osteodystrophy in the long-term inflammatory microenvironment. Our previous study confirmed that the Elongation protein 2 (ELP2) plays a significant role in osteogenesis and osteogenic differentiation, which is considered a drug discovery target in diseases related to bone formation and differentiation. In this study, we applied an in silico virtual screening method to select molecules that bind to the ELP2 protein from a chemical drug molecule library and obtained 95 candidates. Then, we included 11 candidates by observing the docking patterns and the noncovalent bonds. The binding affinity of the ELP2 protein with the candidate compounds was examined by SPR analysis, and 5 out of 11 compounds performed good binding affinity to the mouse ELP2 protein. After in vitro cell differentiation assay, candidates 2# and 5# were shown to reduce differentiation inhibition after tumor necrosis factor-α stimulation, allowing further optimization and development for potential clinical treatment of inflammation-mediated orthopedic diseases.

Efficacy of NEMO-binding domain peptide used to treat experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus: an in-vivo study.

Purpose: Treatment of chronic osteomyelitis (bone infection) remains a clinical challenge. Our previous study had demonstrated that NEMO-binding domain (NBD) peptide effectively ameliorates the inhibition of osteoblast differentiation by TNF-α in vitro. In this work, NBD peptide was evaluated in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model. Methods: Tibial osteomyelitis was induced in 50 New Zealand white rabbits by tibial canal inoculation of MRSA strain. After 3 weeks, 45 rabbits with osteomyelitis were randomly divided into four groups that correspondingly received the following interventions: 1) Control group (9 rabbits, no treatment); 2) Van group (12 rabbits, debridement and parenteral treatment with vancomycin); 3) NBD + Van group (12 rabbits, debridement and local NBD peptide injection, plus parenteral treatment with vancomycin); 4) NBD group (12 rabbits, debridement and local NBD peptide injection). Blood samples were collected weekly for the measurement of leucocyte count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. The rabbits in all four groups were sacrificed 6 weeks after debridement; the anti-infective efficacy was evaluated by radiological, histological, and microbiological examination, and promotion of bone remodeling was quantified by micro-CT using the newly formed bone. Results: Except two rabbits in the Control group and one in the NBD group that died from severe infection before the end point, the remaining 42 animals (7, 12, 12, 11 in the Control, Van, NBD + Van, and NBD group respectively) were sacrificed 6 weeks after debridement. In general, there was no significant difference in the leucocyte count, and ESR and CRP levels, although there were fluctuations throughout the follow-up period after debridement. MRSA was still detectable in bone tissue samples of all animals. Interestingly, treatment with NBD peptide plus vancomycin significantly reduced radiological and histological severity scores compared to that in other groups. The best therapeutic efficacy in bone defect repair was observed in the NBD peptide + Van group. Conclusions: In a model of osteomyelitis induced by MRSA, despite the failure in demonstrating antibacterial effectiveness of NBD peptide in vivo, the results suggest antibiotics in conjunction with NBD peptide to possibly have promising therapeutic potential in osteomyelitis.

ELP2 negatively regulates osteoblastic differentiation impaired by tumor necrosis factor α in MC3T3-E1 cells through STAT3 activation.

Tumor necrosis factor-α (TNF-α) is a pluripotent signaling molecule. The biological effect of TNF-α includes slowing down osteogenic differentiation, which can lead to bone dysplasia in long-term inflammatory microenvironments. Signal transducer and activator of transcription 3 (STAT3)-interacting protein 1 (StIP1, also known as elongator complex protein 2, ELP2) play a role in inhibiting TNF-α-induced osteoblast differentiation. In the present study, we investigated whether and how ELP2 activation mediates the effects of TNF-α on osteoblastic differentiation. Using in vitro cell cultures of preosteoblastic MC3T3-E1 cells, we found that TNF-α inhibited osteoblastic differentiation accompanied by an increase in ELP2 expression and STAT3 activation. Forced ELP2 expression inhibited osteogenic differentiation of MC3T3-E1 cells, with a decrease in the expression of osteoblast marker genes, alkaline phosphatase activity, and matrix mineralization capacity. In contrast, ELP2 silencing ameliorated osteogenic differentiation in MC3T3-E1 cells, even after TNF-α stimulation. The TNF-α-induced inhibitory effect on osteoblastic differentiation was therefore mediated by ELP2, which was associated with Janus kinase 2 (JAK2)/STAT3 activation. These results suggest that ELP2 is upregulated at the differentiation of MC3T3-E1 cells into osteoblasts and inhibits osteogenic differentiation in response to TNF-α through STAT3 activation.

Discovery of novel elongator protein 2 inhibitors by compound library screening using surface plasmon resonance.

Tumour necrosis factor-α (TNF-α) is a pleiotropic cytokine that becomes elevated in chronic inflammatory states, including slowing down osteogenic differentiation, which leads to bone dysplasia in long-term inflammatory microenvironments. The elongator complex plays a role in gene regulation and association with various cellular activities, including the downstream signal transduction of TNF-α in osteogenic cells. To find an inhibitor of Elongator Protein 2 (Elp2), we performed a compound library screen and verified the pharmaceutical effects of candidate compounds on the mouse myoblast cell (C2C12) and mouse osteoblastic cells (MC3T3-E1). The commercial FDA-approved drug (FD) library and the bioactive compound (BC) library were used as candidate libraries. After a label-free, high-throughput affinity measurement with surface plasmon resonance (SPRi), seven kinds of compounds showed binding affinity with mouse Elp2 protein. The seven candidates were then used to perform an inhibition test with TNF-α-induced C2C12 and MC3T3-E1 cell lines. One candidate compound reduced the differentiation suppression caused by TNF-α with resuscitated alkaline phosphatase (ALP) activity, mineralization intensity and expression of osteogenic differentiation marker genes. The results of our study provide a competitive candidate to mitigate the TNF-α-induced osteogenic differentia.