Merlin functions as a critical regulator in Staphylococcus aureus-induced osteomyelitis.

Merlin is known as a tumor suppressor, while its role in osteomyelitis remains unclear. This study aimed to investigate the role of Merlin in Staphylococcus aureus-induced osteomyelitis and its underlying mechanisms. S. aureus-induced osteomyelitis mouse model was established in Merlinfl/fl Lyz2cre/+ and Merlinfl/fl Lyz2+/+ mice. Bone marrow-derived macrophages (BMDMs) were isolated and stimulated by lipopolysaccharide (LPS). Bioassays, including quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot analysis, and enzyme-linked immunosorbent assays, were conducted to determine the levels of target genes or proteins. Immunoprecipitation was applied to determine the interactions between proteins. DCAF1fl/fl mice were further crossed with Lyz2-Cre mice to establish myeloid cell conditional knockout mice (DCAF1fl/fl Lyz2cre/+ ). It was found that the level of Merlin was elevated in patients with osteomyelitis and S. aureus-infected BMDMs. Merlin deficiency in macrophages suppressed the production of inflammatory cytokines and ameliorated the symptoms of osteomyelitis induced by S. aureus. Merlin deficiency in macrophages also suppressed the production of proinflammatory cytokines in BMDMs induced by LPS. The inhibitory effects of Merlin deficiency on the inflammatory response were associated with DDB1-Cul4-associated factor 1 (DCAF1). In summary, Merlin deficiency ameliorates S. aureus-induced osteomyelitis through the regulation of DCAF1.

Involvement of mechanosensitive ion channels in the effects of mechanical stretch induces osteogenic differentiation in mouse bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) can be induced to process osteogenic differentiation with appropriate mechanical and/or chemical stimuli. The present study described the successful culture of murine BMSCs under mechanical strain. BMSCs were subjected to 0%, 3%, 8%, 13%, and 18% cyclic tensile strain at 0.5 Hz for 8 hr/day for 3 days. The expression of osteogenic markers and mechanosensitive ion channels was evaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The expression of alkaline phosphatase (ALP) and matrix mineralization were evaluated with histochemical staining. To investigate the effects of mechanosensitive ion channel expression on cyclic tensile strain-induced osteogenic differentiation, the expression of osteogenic markers was evaluated with real-time RT-PCR in the cells without mechanosensitive ion channel expression. This study revealed a significant augment in osteogenic marker in BMSC strained at 8% compared to other treatments; therefore, an 8% strain was used for further investigations. The ALP expression and matrix mineralization were enhanced in osteogenic induced BMSCs subjected to 8% strain after 7 and 14 days, respectively. Under the same conditions, the osteogenic marker and mechanosensitive ion channel expression were significantly promoted. However, the loss function of mechanosensitive ion channels resulted in the inhibition of osteogenic marker expression. This study demonstrated that strain alone can successfully induce osteogenic differentiation in BMSCs and the expression of mechanosensitive ion channels was involved in the process. The current findings suggest that mechanical stretch could function as efficient stimuli to induce the osteogenic differentiation of BMSCs via the activation of mechanosensitive ion channels.

The promotion of bone regeneration through CS/GP-CTH/antagomir-133a/b sustained release system.

Few studies reported the application of miRNA in bone regeneration. In this study, the expression of miR133a and miR133b in murine BMSCs was inhibited via antagomiR-133a/b and the osteogenic differentiation in murine BMSCs was evaluated. The RT-PCR, flow cytometry, cell counting kit-8, and annexin V-FITC/PI double staining assays were performed. Double knockdown miR133a and miR133b can promote BMSC osteogenic differentiation. At optimum N/P ration (15:1), the loading efficiency can reach over 90%. CTH-antagomiR-133a/b showed no cytotoxicity to BMSCs and diminished miR133a and miR133b expression in BMSCs. Furthermore, chitosan-based sustained delivery system can facilitate continuous dosing of antagomiR-133a/b, which enhanced calcium deposition and osteogenic specific gene expression in vitro. The new bone formation was enhanced after the sustained delivery system containing CTH-antagomiR-133a/b nanoparticles was used in mouse calvarial bone defect model. Our results demonstrate that CTH nanoparticles could facilitate continuous dosing of antagomiR133a/b, which can promote osteogenic differentiation.

Ubiquitin-Specific Peptidase 8 Is Critical for the Onset of Infectious Osteomyelitis by Targeting RIPK2 Ubiquitination.

Receptor-interacting serine/threonine kinase (RIPK) is associated with cellular inflammation and immune regulation. The current study explored the role of RIPK2 in osteomyelitis and the potential upstream targets of RIPK2. A Staphylococcus aureus-induced osteomyelitis mouse model was established using wild-type (WT) and ubiquitin-specific peptidase 8 (USP8)-deficient (USP-/-) mice, and the osteomyelitis-related symptoms were evaluated. Bone marrow-derived macrophages (BMDMs) were isolated from the WT and USP-/- mice. Enzyme-linked immunosorbent assays, quantitative polymerase chain reaction, and immunoblot analysis were used to determine the levels of target biomarkers, which were induced by lipopolysaccharide (LPS), CpG, or PAM3CSK4. USP8 promoted RIPK2-mediated NF-κB activation. USP8 is indispensable for RIPK2-mediated LPS-induced NF-κB activation in BMDMs. USP8 is required for the production of inflammatory cytokines induced by LPS, CpG, or PAM3CSK4 in BMDMs. In addition, USP-/- mice exhibited ameliorated symptoms, including less body weight and cortical bone loss, and reduced bacterial load and reactive bone formation in the S. aureus-induced osteomyelitis mouse model. USP8 is critical in the S. aureus-induced osteomyelitis mouse model by targeting RIPK2 ubiquitination.

Effects of astragaloside IV on glucocorticoid-induced avascular necrosis of the femoral head via regulating Akt-related pathways.

We investigated the role of astragaloside IV (AS-IV) in preventing glucocorticoid-induced avascular necrosis of the femoral head (ANFH) and the underlying molecular mechanisms. Network pharmacology was used to predict the molecular targets of AS-IV. Molecular dynamic simulations were performed to explore the binding mechanism and interaction mode between AS-IV and Akt. Rat models of glucocorticoid-induced ANFH with AS-IV intervention were established, and osteogenesis, angiogenesis, apoptosis and oxidative stress were evaluated before and after blocking the PI3K/Akt pathway with LY294002. The effects of glucocorticoid and AS-IV on bone marrow mesenchymal stem cells and human umbilical vein endothelial cells incubated with and without LY294002 were determined. Downregulated p-Akt expression could be detected in the femoral heads of glucocorticoid-induced ANFH patients and rats. AS-IV increased trabecular bone integrity and vessel density of the femoral head in the model rats. AS-IV increased Akt phosphorylation and upregulated osteogenesis-, angiogenesis-, apoptosis- and oxidative stress-related proteins and mRNA and downregulated Bax, cleaved caspase-3 and cytochrome c levels. AS-IV promoted human umbilical vein endothelial cell migration, proliferation and tube formation ability; bone marrow mesenchymal stem cell proliferation; and osteogenic differentiation under glucocorticoid influence. AS-IV inhibited apoptosis. LY294002 inhibited these effects. AS-IV prevented glucocorticoid-induced ANFH by promoting osteogenesis and angiogenesis via the Akt/Runx2 and Akt/HIF-1α/VEGF pathways, respectively, and suppressing apoptosis and oxidative stress via the Akt/Bad/Bcl-2 and Akt/Nrf2/HO-1 pathways, respectively.

IL-34 Aggravates Steroid-Induced Osteonecrosis of the Femoral Head via Promoting Osteoclast Differentiation.

IL-34 can promote osteoclast differentiation and activation, which may contribute to steroid-induced osteonecrosis of the femoral head (ONFH). Animal model was constructed in both BALB/c and IL-34 deficient mice to detect the relative expression of inflammation cytokines. Micro-CT was utilized to reveal the internal structure. In vitro differentiated osteoclast was induced by culturing bone marrow-derived macrophages with IL-34 conditioned medium or M-CSF. The relative expression of pro-inflammation cytokines, osteoclast marker genes, and relevant pathways molecules was detected with quantitative real-time RT-PCR, ELISA, and Western blot. Up-regulated IL-34 expression could be detected in the serum of ONFH patients and femoral heads of ONFH mice. IL-34 deficient mice showed the resistance to ONFH induction with the up-regulated trabecular number, trabecular thickness, bone value fraction, and down-regulated trabecular separation. On the other hand, inflammatory cytokines, such as TNF-α, IFN-γ, IL-6, IL-12, IL-2, and IL-17A, showed diminished expression in IL-34 deficient ONFH induced mice. IL-34 alone or works in coordination with M-CSF to promote osteoclastogenesis and activate ERK, STAT3, and non-canonical NF-κB pathways. These data demonstrate that IL-34 can promote the differentiation of osteoclast through ERK, STAT3, and non-canonical NF-κB pathways to aggravate steroid-induced ONFH, and IL-34 can be considered as a treatment target.

Gallium Porphyrin and Gallium Nitrate Reduce the High Vancomycin Tolerance of MRSA Biofilms by Promoting Extracellular DNA-Dependent Biofilm Dispersion.

Biofilms, structured communities of bacterial cells embedded in a self-produced extracellular matrix (ECM) which consists of proteins, polysaccharide intercellular adhesins (PIAs), and extracellular DNA (eDNA), play a key role in clinical infections and are associated with an increased morbidity and mortality by protecting the embedded bacteria against drug and immune response. The high levels of antibiotic tolerance render classical antibiotic therapies impractical for biofilm-related infections. Thus, novel drugs and strategies are required to reduce biofilm tolerance and eliminate biofilm-protected bacteria. Here, we showed that gallium, an iron mimetic metal, can lead to nutritional iron starvation and act as dispersal agent triggering the reconstruction and dispersion of mature methicillin-resistant Staphylococcus aureus (MRSA) biofilms in an eDNA-dependent manner. The extracellular matrix, along with the integral bacteria themselves, establishes the integrated three-dimensional structure of the mature biofilm. The structures and compositions of gallium-treated mature biofilms differed from those of natural or antibiotic-survived mature biofilms but were similar to those of immature biofilms. Similar to immature biofilms, gallium-treated biofilms had lower levels of antibiotic tolerance, and our in vitro tests showed that treatment with gallium agents reduced the antibiotic tolerance of mature MRSA biofilms. Thus, the sequential administration of gallium agents (gallium porphyrin and gallium nitrate) and relatively low concentrations of vancomycin (16 mg/L) effectively eliminated mature MRSA biofilms and eradicated biofilm-enclosed bacteria within 1 week. Our results suggested that gallium agents may represent a potential treatment for refractory biofilm-related infections, such as prosthetic joint infections (PJI) and osteomyelitis, and provide a novel basis for future biofilm treatments based on the disruption of normal biofilm-development processes.

Astragaloside IV ameliorates steroid-induced osteonecrosis of the femoral head by repolarizing the phenotype of pro-inflammatory macrophages.

Osteonecrosis of the femoral head (ON-FH) is a common complication of steroid use. Pro-inflammatory macrophages play a crucial role in the apoptosis of osteocytes. The objective of the study was to evaluate a plant extract astragaloside IV (AS-IV) in treating ON-FN. Bone-marrow-derived macrophages (BMDMs) were treated with lipopolysaccharides (LPS), IFN-γ or IL-4 to induce M1 and M2-like phenotypes. Quantitative real-time PCR and Western blot were used to examine M1 and M2 phenotypic markers. Flow cytometry was used to analyze MHC II, CD206, F4/80, and CD11b levels and cell apoptosis. Glucocorticoid was used to induce ON-FN in mice. TNF-α and IL-1ß levels in femoral head were determined using enzyme-linked immunosorbent assay. AS-IV repolarized macrophages from M1 to M2 phenotypes. Culture medium from AS-IV treated M1 macrophages induced less cell apoptosis osteocytes compared to that from untreated M1 macrophages. In ON-FH mice, the ratio of M1 macrophages was decreased in the femoral head by AS-IV, concomitant with a decrease in TNF-α and IL-1ß levels. AS-IV is effective in alleviating ON-FH through its effects in repolarizing macrophages from M1-like phenotype to M2-like phenotype, promoting survival of osteocytes, reducing arthritic symptoms, and decreasing inflammatory cytokines.

Tumor necrosis factor-α promotes Staphylococcus aureus-induced osteomyelitis through downregulating endothelial nitric oxide synthase.

BACKGROUND: Infections of Staphylococcus aureus (S. aureus) often result in osteomyelitis, which is the acute or chronic infections of the bone marrow or bones. TNF-α is long recognized as a key factor contributing to the pathogenesis of osteomyelitis, but little is known about the underlying molecular mechanism. METHODS: Expression levels of TNF-α, and several candidate genes, including endothelial nitric oxide synthase (eNOS), known to be downregulated by TNF-α were analysed in MC3T3-E1 cells with S. aureus infection and osteomyelitis patient blood. MicroRNA(miR)-129-5p was predicted and experimentally verified to target eNOS. Alizarin red sulfate (ARS) and alkaline phosphatase (ALP) staining assays were conducted on MC3T3-E1 cells with S. aureus infection to assess the role of TNF-α/miR-129-5p/eNOS on mineralization defect. RESULTS: TNF-α and miR-129-5p were upregulated while eNOS was downregulated in MC3T3-E1 cells with S. aureus infection and osteomyelitis patients, showing inversely correlated expression profiles. MiR-129-5p directly binds to the 3'-UTR of eNOS mRNA to suppress eNOS expression in MC3T3-E1 cells. TNF-α blocker inhibited miR-129-5p and elevated eNOS expression, likely contributing to rescued mineralization defect in S. aureus-infected MC3T3-E1 cells. During S. aureus infection, upregulated TNF-α increases endogenous miR-129-5p expression, which in turn inhibits eNOS, contributing to osteomyelitis. CONCLUSION: Our study thereby proposes a novel signalling cascade involving TNF-α/miR-129-5p/eNOS in the pathogenesis of osteomyelitis, which may also serve as therapeutic targets.

Bone morphogenetic protein 2 controls steroid-induced osteonecrosis of the femoral head via directly inhibiting interleukin-34 expression.

Increased inflammatory response is one of the major characteristics of osteonecrosis of the femoral head (ONFH). We aimed to investigate the function of bone morphogenetic protein 2 (BMP-2)/interleukin (IL)-34 axis in the inflammatory responses of ONFH. The systemic and local expression of BMPs in ONFH patients was detected by qRT-PCR and ELISA. In vitro osteoclast differentiation and ONFH mouse models, induced by 20 mg/kg methylprednisolone through i.m. injection, were established using WT and BMP-2-/- mice to explore the regulatory role of BMP-2 in pro-inflammatory responses and bone defects of ONFH. IL-34 expression and function were examined in vitro and in vivo through qRT-PCR, tartrate-resistant acid phosphatase (TRAP) staining, and gene knockout. The systemic and local expression of BMPs was elevated in ONFH patients. BMP-2 reduced the production of pro-inflammatory cytokines and inhibited the differentiation of osteoclasts. Mechanistically, BMP-2 inhibited osteoclasts formation through suppressing IL-34 expression and then promoted bone repair and alleviated ONFH. In conclusion, our study reveals that BMP-2 inhibits inflammatory responses and osteoclast formation through downregulating IL-34.

Ddb1-Cullin4-Associated-Factor 1 in Macrophages Restricts the Staphylococcus aureus-Induced Osteomyelitis.

INTRODUCTION: Ddb1-cullin4-associated-factor 1 (DCAF1) is known to regulate protein ubiquitination, while the roles of DCAF1 in osteomyelitis remain unknown. This study aims to investigate the effects of DCAF1 deficiency in macrophages on osteomyelitis and elucidate the molecular mechanism. METHODS: Staphylococcus aureus-induced mouse model of osteomyelitis was established on the DCAF1fl/flLyz2cre/+ and DCAF1fl/flLyz2+/+ (control) mice. Flow cytometry was conducted to analyze the populations of adaptive and innate immune cells. Lipopolysaccharides (LPS)-induced bone marrow-derived macrophages (BMDMs) were established. qRT-PCR and immunoblot analysis were used to determine the levels of inflammation-related biomarkers. ELISA was used to determine the release of inflammatory cytokines including IL-1ß, IL-6, and TNF. RESULTS: The populations of immune cells in the bone marrow and spleen were not affected due to DCAF1 deficiency in macrophages. DCAF1 suppressed inflammatory cytokines in LPS-induced BMDMs. Additionally, DCAF1 deficiency in macrophages induced severe symptoms including less bacterial load in the femur, cortical bone loss, and reactive bone formation. Mechanistic study revealed that DCAF1 deficiency induced p38 hyperactivation. DISCUSSION: DCAF1 in macrophages suppressed the Staphylococcus aureus-induced mouse model of osteomyelitis.

Machine learning-guided evolution of BMP-2 knuckle Epitope-Derived osteogenic peptides to target BMP receptor II.

Bone morphogenetic protein-2 (BMP-2) is a key regulator of bone formation, growth and regeneration, which contains a conformational wrist epitope and a linear knuckle epitope that are functionally responsible for the protein by mediating its interaction with type-I and type-II receptors, respectively. Previously, a long (19-mer) knuckle peptide derived from the knuckle epitope region (residues 73-92) has been found to promote osteogenesis and bone repair. Here, we attempt to rationally redesign the knuckle peptide by using bioinformatics and machine learning-guided evolution to obtain structurally simplified, potent osteogenic peptides that are capable of targeting type-II receptor. Complex analysis reveals that only a fraction of the epitope region can directly interact with type-II receptor, which represents a small (12-mer) knuckle-derived peptide (KDP0 peptide). Glycine scanning further identifies three KDP0 anchor residues Ser88, Leu90 and Tyr91 that are fundamentally important in the peptide-receptor binding. Systematic mutation, amino acid combination and uniform design of other nine KDP0 non-anchor residues generate 32 new knuckle-derived peptides (KDP1-KDP32); their binding affinities to recombinant protein of human type-II receptor are determined using fluorescence spectroscopy assay. The resulting affinity values (Kd) are used to train six regression models developed by combination of two machine learning methods and three amino acids descriptors. The best SVM/VHSE predictor is then utilised to guide the genetic evolution of a knuckle-derived peptide population. Eight peptides (KDP33-KDP40) with high affinity scores are selected from the improved population, and their osteogenic activities on bone marrow stromal cells are measured using alkaline phosphatase assay. Consequently, six out of the 8 tested peptides exhibit increased activity relative to KDP0 peptide. The KDP34 (DFQTWSFLYVEN) is found as the most potent peptide with APL activities of 195% and 279% at 0.01 and 0.1 µg/ml, respectively, which shares a similar binding mode with the native knuckle epitope and can form diverse nonbonded interactions of hydrogen bond, hydrophobic contact, cation-π/π-π stacking and salt bridge with type-II receptor.

miR-26a Attenuated Bone-Specific Insulin Resistance and Bone Quality in Diabetic Mice.

Diabetes mellitus is a prevalent disease result in several complications, including bone problems. Previous studies have shown that microRNA (miR)-26a regulates glucose metabolism and plays a protective role in diabetes. However, whether miR-26a also affects bone quality in diabetes remains unknown. In the present study, we evaluated the potential effects of miR-26a on bone in diabetic mice. We administrated miR-26a in streptozotocin-induced diabetic mice. The metabolic parameters, bone quality, osteoblast and osteoclast markers, and insulin signaling activation were measured. miR-26a ameliorated insulin resistance and glucose tolerance, improved bone microarchitecture and quality, increased osteoblasts and bone formation, decreased osteoclasts, and promoted the insulin signaling pathway in diabetic mice. These effects were abolished in insulin receptor-compromised Col1a1-Insr+/- mice. In conclusion, miR-26a could ameliorate bone-specific insulin resistance and bone quality in diabetic mice, which depended on the insulin receptors on osteoblasts. Our findings highlight the potential of miR-26a as a therapeutic target for diabetes mellitus-related bone metabolism and diseases.

5-ARI induces autophagy of prostate epithelial cells through suppressing IGF-1 expression in prostate fibroblasts.

OBJECTIVES: 5α-reductase inhibitor (5-ARI) is a commonly used medicine in the treatment of lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH). Our study mainly focuses on the mechanism of BPH development after 5ARI treatment. MATERIALS AND METHODS: Prostate specimens from patients were collected. Insulin-like growth factor 1 (IGF-1), Beclin-1, LC3 levels, was analysed by immunohistochemistry. The role IGF-1 on autophagic flux in prostate epithelial cells was studied. Additionally, effect of autophagy on recombinant grafts consisting of prostate stromal and epithelial cells in nude mice was investigated. RESULTS: We demonstrated that IGF-1 expression is down-regulated in prostate fibroblasts after long-term 5-ARI application. A decrease in IGF-1 levels was found to activate autophagic flux through the mTOR pathway in prostate epithelial cells, while the inhibition of IGF-1 receptor function induced autophagy in prostate epithelial cells. In addition, we revealed that blocking autophagic flux initiation can reduce the volume of recombinant grafts in vivo. Finally, our findings suggest that long-term 5-ARI application reduces IGF-1 secretion by prostatic stromal cells, thereby inducing autophagy of prostatic epithelial cells, which is one of the mechanisms underlying BPH pathogenesis and progression. CONCLUSIONS: Focusing on the autophagy induced by low levels of IGF-1 in prostatic epithelial cells, after elucidating AR signalling impairment of prostate stromal cells, might provide a novel strategy for the treatment and prevention of BPH development.