Inflammatory Microenvironment-Responsive Hydrogels Enclosed with Quorum Sensing Inhibitor for Treating Post-Traumatic Osteomyelitis.

Non-antibiotic strategies are desperately needed to treat post-traumatic osteomyelitis (PTO) due to the emergence of superbugs, complex inflammatory microenvironments, and greatly enriched biofilms. Previously, growing evidence indicated that quorum sensing (QS), a chemical communication signal among bacterial cells, can accelerate resistance under evolutionary pressure. This study aims to develop a medical dressing to treat PTO by inhibiting QS and regulating the inflammatory microenvironment, which includes severe oxidative stress and acid abscesses, through a reactive oxygen species (ROS)-responsive bond between N1- (4-borobenzoyl)-N3-(4-borobenzoyl)-the N1, the N1, N3, N3-tetramethylpropane-1,3-diamine (TSPBA) and polyvinyl alcohol (PVA), and the amino side chain of hyperbranched polylysine (HBPL). Physically enclosed QS inhibitors subsequently exerted the antibacterial effects. This hydrogel can scavenge hydrogen peroxide (H2 O2 ), superoxide anion free radical (·O2 - ), hydroxyl radicals (·OH) and 2,2-di(4-tert-octylphenyl)-1-picryl-hydrazyl (DPPH) to reduce oxidative stress and inhibit "bacteria-to-bacteria communication", thus clearing planktonic bacteria and biofilms, accelerating bacterial plasmolysis, reducing bacterial virulence and interfering with membrane transport. After in vivo treatment with hydrogel, nearly all bacteria are eliminated, inflammation is effectively inhibited, and osteogenesis and bone repair are promoted to facilitate recovery from PTO. The work demonstrates the clinical translational potential of the hydrogel in the treatment of drug-resistant bacteria induced PTO.

Proteome analysis develops novel plasma proteins classifier in predicting the mortality of COVID-19.

COVID-19 has been a global concern for 3 years, however, consecutive plasma protein changes in the disease course are currently unclear. Setting the mortality within 28 days of admission as the main clinical outcome, plasma samples were collected from patients in discovery and independent validation groups at different time points during the disease course. The whole patients were divided into death and survival groups according to their clinical outcomes. Proteomics and pathway/network analyses were used to find the differentially expressed proteins and pathways. Then, we used machine learning to develop a protein classifier which can predict the clinical outcomes of the patients with COVID-19 and help identify the high-risk patients. Finally, a classifier including C-reactive protein, extracellular matrix protein 1, insulin-like growth factor-binding protein complex acid labile subunit, E3 ubiquitin-protein ligase HECW1 and phosphatidylcholine-sterol acyltransferase was determined. The prediction value of the model was verified with an independent patient cohort. This novel model can realize early prediction of 28-day mortality of patients with COVID-19, with the area under curve 0.88 in discovery group and 0.80 in validation group, superior to 4C mortality and E-CURB65 scores. In total, this work revealed a potential protein classifier which can assist in predicting the outcomes of COVID-19 patients and providing new diagnostic directions.

Hybrid Biomimetic Membrane Coated Particles-Mediated Bacterial Ferroptosis for Acute MRSA Pneumonia.

Acute methicillin resistant Staphylococcus aureus (MRSA) pneumonia is one of the most frequently seen lung infection diseases with high morbidity and mortality. It is urgent to explore an efficient antibacterial strategy owing to the increase of drug resistance, virulence, and pathogenicity of MRSA. It was found that Fe3O4 can induce ferroptosis in MRSA, but its effect was inhibited by glutathione (GSH) to a certain extent, while cinnamaldehyde (CA) can enhance ferroptosis by consuming GSH. As a bacterial quorum sensing (QS) inhibitor, CA can suppress the QS system and further exert its antibacterial and antibiofilm effects. Here, we developed an Fe3O4-based ferroptosis inducer to promote ferroptosis in MRSA, interrupt the QS, destroy biofilm, and thus effectively treat acute MRSA pneumonia. We used sodium alginate (SA) to wrap Fe3O4 and CA to form particles, and then coated the surface with a hybrid biomimetic membrane composed of an erythrocyte membrane and platelet membrane to obtain lung targeted antibacterial particles (mFe-CA). Under ultrasonic (US) stimulation, mFe-CA can efficiently release Fe3O4 and CA, thereby synergically inducing MRSA death with the characteristics of ferroptosis, including mass ROS production, lipid peroxidation, GSH depletion, and respiratory chain suppression. Furthermore, mFe-CA + US can inhibit the QS system, remove biofilms, and reduce strain virulence. In the mouse model of MRSA pneumonia, mFe-CA + US treatment markedly advanced the survival rate of the mice, reduced the bacterial load in the lungs, and alleviated the inflammatory damage, but there was no obvious toxicity. This study proposes an antibacterial substitute to induce ferroptosis of MRSA, which may provide a foreground for overcoming microbial drug resistance and fighting biofilm-associated infections and also provides a target and theoretical basis for clinical treatment of acute MRSA pneumonia.

Host-Defense-Peptide-Mimicking ß-Peptide Polymer Acting as a Dual-Modal Antibacterial Agent by Interfering Quorum Sensing and Killing Individual Bacteria Simultaneously.

Host defense peptides (HDPs) are one of the potentially promising agents for infection diseases due to their broad spectrum and low resistance rate, but their clinical applications are limited by proteolytic instability, high-cost, and complicated synthesis process. Here, we report a host-defense-peptide-mimicking ß-peptide polymer that resists proteolysis to have enhanced the activity under physiological conditions, excellent antimicrobial efficiency even at high density of bacteria, and low cost for preparation. The ß-peptide polymer demonstrated quorum sensing (QS) interference and bactericidal effect against both bacterial communities and individual bacterium to simultaneously block bacterial communication and disrupt bacterial membranes. The hierarchical QS network was suppressed, and main QS signaling systems showed considerably down-regulated gene expression, resulting in excellent biofilm eradication and virulence reduction effects. The dual-modal antibacterial ability possessed excellent therapeutic effects in Pseudomonas aeruginosa pneumonia, which could inhibit biofilm formation and exhibit better antibacterial and anti-inflammatory efficiency than clinically used antibiotics, levofloxacin. Furthermore, the ß-peptide polymer also showed excellent therapeutic effect Escherichia coli pyogenic liver abscess. Together, we believed that the ß-peptide polymer had a feasible clinical potential to treat bacterial infection diseases.

FBXO6 regulates the antiviral immune responses via mediating alveolar macrophages survival.

Inducing early apoptosis in alveolar macrophages is one of the strategies influenza A virus (IAV) evolved to subvert host immunity. Correspondingly, the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 is reported to interact with virus polymerase basic protein 1-frame 2 (PB1-F2) accessory protein to counteract virus-induced apoptosis. Herein, we report that one of the F-box proteins, FBXO6, promotes proteasomal degradation of NLRX1, and thus facilitates IAV-induced alveolar macrophages apoptosis and modulates both macrophage survival and type I interferon (IFN) signaling. We observed that FBXO6-deficient mice infected with IAV exhibited decreased pulmonary viral replication, alleviated inflammatory-associated pulmonary dysfunction, and less mortality. Analysis of the lungs of IAV-infected mice revealed markedly reduced leukocyte recruitment but enhanced production of type I IFN in Fbxo6-/- mice. Furthermore, increased type I IFN production and decreased viral replication were recapitulated in FBXO6 knockdown macrophages and associated with reduced apoptosis. Through gain- and loss-of-function studies, we found lung resident macrophages but not bone marrow-derived macrophages play a key role in the differences FBXO6 signaling pathway brings in the antiviral immune response. In further investigation, we identified that FBXO6 interacted with and promoted the proteasomal degradation of NLRX1. Together, our results demonstrate that FBXO6 negatively regulates immunity against IAV infection by enhancing the degradation of NLRX1 and thus impairs the survival of alveolar macrophages and antiviral immunity of the host.

Emodin Combined with Multiple-Low-Frequency, Low-Intensity Ultrasound To Relieve Osteomyelitis through Sonoantimicrobial Chemotherapy.

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 a 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 antibacterial and antibiofilm effects. The efficiencies of different intensities of ultrasound, including single (S-LFLIU, 15 min) and multiple ultrasound (M-LFLIU, 3 times for 5 min at 4-h intervals), against bacteria and biofilms were compared, contributing to developing the best treatment regimen. Our results demonstrated that EM plus S-LFLIU or M-LFLIU (EM+S-LFLIU or EM+M-LFLIU) had significant combined bactericidal and antibiofilm effects, with EM+M-LFLIU in particular exhibiting superior antibiofilm performance. Furthermore, it was suggested that EM+M-LFLIU could produce a large amount of reactive oxygen species (ROS), destroy the integrity of the bacterial membrane and cell wall, and downregulate the expression of genes involved in oxidative stress, membrane wall synthesis, and bacterial virulence, as well as that of other related genes (agrB, pbp3, sgtB, gmk, zwf, and msrA). In vivo studies, micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and bacterial quantification of bone tissue indicated that EM+M-LFLIU could also relieve osteomyelitis due to MRSA infection. Our work proffers an original approach to bacterial osteomyelitis treatment that weakens drug-resistant bacteria and suppresses and degrades biofilm formation through SACT, which may provide new prospects for clinical treatment. IMPORTANCE Antibiotic therapy is the first choice for clinical treatment of osteomyelitis, but the formation of bacterial biofilms and the emergence of many drug-resistant strains also create an urgent need to find an alternative treatment to effectively eliminate the infection. Recently, LFLIU has come to be considered a safe and promising method of debridement and antibacterial therapy. In this study, we found that ultrasound and EM have a significant combined antibacterial effect in vivo and in vitro, which may play an antibacterial role by stimulating the production of ROS, destroying the bacterial cell wall, and inhibiting the expression of related genes. Our study expands the body of knowledge on the antibacterial effect of drugs-specifically emodin (EM)-through combined physiotherapy. If successfully integrated into clinical practice, these methods may reduce the burden of high concentrations of drugs needed to treat bacterial biofilms and avoid the growing resistance of bacteria to antibiotics.

Wound microenvironment-responsive glucose consumption and hydrogen peroxide generation synergistic with azithromycin for diabetic wounds healing.

Rationale: Chronic wounds are one of the common complications of diabetes. Due to the physiological conditions of diabetic patients, these wounds are more susceptible to bacterial infections and the formation of bacterial biofilms, leading to the inefficiency of conventional antibiotic treatment. Methods: Here, hollow mesoporous silica nanoparticles (HMSN) were used as the nanocarriers for co-delivery of azithromycin (AZM) and glucose oxidase (GOX), achieving a remarkable synergistic effect in chronic diabetic wounds. GOX possesses the catalytic ability to consume glucose and produce H2O2 in the diabetic wound area. The down-regulation of local glucose could effectively improve the chronic diabetic wound microenvironment. Meanwhile, the generated H2O2 effectively inhibits bacterial growth and eradicates bacterial biofilms with the synergism of antibiotics AZM. Results: In the bacteria-infected diabetic cutaneous wound models, the reduction of glucose, generation of H2O2, and release of AZM could effectively reduce the bacterial infection and promote the wounds healing. Moreover, there is no obvious toxicity behavior after the treatment. Conclusions: Therefore, the designed nanosystem could effectively accelerate the diabetic wound healing process by the amelioration of the hyperglycemia microenvironment and the eradication of bacterial biofilms around the wounds, making them promising candidates for clinical transformation.

Yangonin treats inflammatory osteoporosis by inhibiting the secretion of inflammatory factors and RANKL expression.

OBJECTIVES: As the main cause of osteoporosis, abnormal activity of osteoclasts could disrupt the balance between bone resorption and formation. Moreover, up-regulation of nuclear factor-kappa ligand (RANKL) expression by chronic inflammation-mediated inflammatory factors might contribute to the differentiation of osteoclast precursor cells. Therefore, an anti-inflammatory agent named yangonin was presented for inhibiting osteoclast and relieving inflammatory osteoporosis through down-regulating inflammatory factors. METHODS: We established a model of macrophage inflammation and then verified the anti-inflammatory effect of yangonin. The inhibitory effect of yangonin on osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining, Western blotting and quantitative real-time PCR (qRT-PCR). Finally, micro-CT, TRAP and hematoxylin-eosin (HE) staining were used to show the effect of yangonin on inflammatory osteoporosis in vivo. RESULTS: Our results suggested that yangonin was able to reduce the secretion of inflammatory factors, down-regulate osteoclast-related genes such as TRAP, RANKL, cathepsin K (CTSK) and nuclear factor-activated T-cell 1 (NFATc1). Furthermore, it was demonstrated that yangonin could suppress the function of inflammatory cytokines in osteoclast differentiation and reporting, wherein NF-κB, AKT and downstream c-Fos/NFATc1 signaling pathways were involved. In an in vivo study, we implied that yangonin has a relieving effect on inflammatory osteoporosis. CONCLUSION: Our research shows that yangonin down-regulates inflammatory factors and inhibits the bone-breaking effect of inflammation through NF-κB, AKT and downstream c-Fos/NFATc1 signaling pathways to achieve the purpose of treating inflammatory osteoporosis.

Pulsatilla saponin E suppresses viability, migration, invasion and promotes apoptosis of NSCLC cells through negatively regulating Akt/FASN pathway via inhibition of flotillin-2 in lipid raft.

PURPOSE: Pulsatilla saponins from pulsatilla chinensis (Bunge) Regel have potential anti-tumor activities to certain human cancers. However, the roles of pulsatilla saponin E separated from pulsatilla saponins in non-small cell lung cancer (NSCLC) have not been reported. MATERIALS AND METHODS: After treating NSCLC cells by pulsatilla saponin E at different concentrations, cell viability was measured by MTT and CCK-8 assays, and cell migration, invasion and apoptosis were detected by scratch wound-healing, transwell and flow cytometry assays. The contents of free cholesterol (FC) and total cholesterol (TC) were measured by high performance liquid chromatography (HPLC). The expression levels of flotillin-1, flotillin-2, Akt, fatty acid synthase (FASN) were detected by qRT-PCR and Western blot assays. RESULTS: Pulsatilla saponin E suppressed viability, migration, invasion and promoted apoptosis of NSCLC cells followed by regulation of apoptosis-related proteins, reduced contents of FC and TC, and the expression levels of flotillin-1, flotillin-2, Akt, and FASN in a concentration-dependent manner. However, the inhibitory effects of pulsatilla saponin E on viability, migration, invasion of A549 cells and the expression levels of flotillin-1, flotillin-2, Akt, and FASN were reversed by flotillin-2 overexpression. CONCLUSIONS: Our study revealed that pulsatilla saponin E suppressed migration, invasion and promoted apoptosis of NSCLC cells through negatively regulating Akt/FASN signaling pathway via the inhibition of flotillin-2 in lipid raft (LR). The current findings could be explored for developing a novel therapeutic drug for NSCLC treatment.

Lung-Targeting Lysostaphin Microspheres for Methicillin-Resistant Staphylococcus aureus Pneumonia Treatment and Prevention.

Multifunctional antimicrobial strategies are urgently needed to treat methicillin-resistant Staphylococcus aureus (MRSA) caused pneumonia due to its increasing resistance, enhanced virulence, and high pathogenicity. Here, we report that lysostaphin, a bacteriolytic enzyme, encapsulated within poly(lactic-co-glycolic acid) microspheres (LyIR@MS) specially treats planktonic MRSA bacteria, mature biofilms, and related pneumonia. Optimized LyIR@MS with suitable diameters could deliver a sufficient amount of lysostaphin to the lung without a decrease in survival rate after intravenous injection. Furthermore, the degradable properties of the carrier make it safe for targeted release of lysostaphin to eliminate MRSA, repressing the expression of virulence genes and improving the sensitivity of biofilms to host neutrophils. In the MRSA pneumonia mouse model, treatment or prophylaxis with LyIR@MS significantly improved survival rate and relieved inflammatory injury without introducing adverse events. These findings suggest the clinical translational potential of LyIR@MS for the treatment of MRSA-infected lung diseases.

MitoQ protects against hyperpermeability of endothelium barrier in acute lung injury via a Nrf2-dependent mechanism.

Recently, numerous evidence has revealed that excessive reactive oxygen species (ROS) production and mitochondrial disruption during acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS) will aggravate the inflammatory process. To identify whether antioxidation can be one of the treatment strategies during this progress, we chose mitoQ, a mitochondria-targeted antioxidant that was proved to be effective in reducing ROS generated in mitochondria, as a ROS scavenger to investigate the role of antioxidation in ALI. We demonstrated that overoxidation occurred during the process of ALI, which could be reduced by mitoQ. In the meantime, apoptosis of endothelial cells of ALI mice, accompanied by hyperpermeability of pulmonary vascular and impaired pulmonary function, was partially reversed following an intraperitoneal injection of mitoQ. Moreover, in in vitro study, lipopolysaccharides (LPS) induced excessive ROS production, mitochondrial dysfunction and apoptosis in human pulmonary microvascular endothelial cells (HPMECs), which were rectified by mitoQ. To explore underlying mechanisms, we proceeded RNA-sequencing and found significantly upregulated expression of musculoaponeurotic fibrosarcoma F (MafF) in mitoQ treated group. Additionally, mitoQ inhibited the degradation and increased nuclear translocation of NF-E2-related factor 2 (Nrf2) and upregulated its downstream antioxidant response elements (AREs), such as heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase (NQO)-1. This effect was abolished by transfecting HPMECs with Nrf2 or Maff siRNA. In Nrf2 deficient mice, the protective effects of mitoQ on LPS model of ALI were largely vanished. Taken together, these results provide insights into how antioxidation exerts beneficial effects on ALI via maintaining mitochondrial hemostasis, inhibiting endothelial cells apoptosis, attenuating the endothelial disruption and regulating lung inflammation via Nrf2-MafF/ARE pathway.