Translational Potential of Baicalein in Mitigating RSL3-Induced Ferroptosis in Fibroblasts: Implications for Therapeutic Interventions.

Background: Ferroptosis is an iron-driven cell-death mechanism that plays a central role in various diseases. Recent studies have suggested that baicalein inhibits ferroptosis, making it a promising therapeutic candidate. Materials and Methods: Fibroblast cultures were treated with different agents to determine the effects of baicalein on ferroptosis. Ferroptosis-related gene expression, lipid peroxidation, and post-treatment cellular structural changes were measured using real-time quantitative polymerase chain reaction, C11-BODIPY dye, and transmission electron microscopy, respectively. Results: Baicalein significantly inhibited rat sarcoma virus selective lethal 3-induced ferroptosis in fibroblasts. Moreover, in baicalein-treated groups, reduced ferroptosis-related gene expression, decreased lipid peroxidation, and maintained cell structure was observed when compared with those of the controls. Discussion: The ability of baicalein to counteract RSL3-induced ferroptosis underscores its potential protective effects, especially in diseases characterized by oxidative stress and iron overload in fibroblasts. Conclusion: Baicalein may serve as a potent therapeutic agent against conditions in which ferroptosis is harmful. The compound's efficacy in halting RSL3-triggered ferroptosis in fibroblasts paves the way for further in vivo experiments and clinical trials.

Circadian Rhythm Disruption in Hepatocellular Carcinoma Investigated by Integrated Analysis of Bulk and Single-Cell RNA Sequencing Data.

Circadian rhythms are essential regulators of a multitude of physiological and behavioral processes, such as the metabolism and function of the liver. Circadian rhythms are crucial to liver homeostasis, as the liver is a key metabolic organ accountable for the systemic equilibrium of the body. Circadian rhythm disruption alone is sufficient to cause liver cancer through the maintenance of hepatic metabolic disorder. Although there is evidence linking CRD to hepatocarcinogenesis, the precise cellular and molecular mechanisms that underlie the circadian crosstalk that leads to hepatocellular carcinoma remain unknown. The expression of CRD-related genes in HCC was investigated in this study via bulk RNA transcriptomic analysis and single-cell sequencing. Dysregulated CRD-related genes are predominantly found in hepatocytes and fibroblasts, according to the findings. By using a combination of single-cell RNA sequencing and bulk RNA sequencing analyses, the dysregulated CRD-related genes ADAMTS13, BIRC5, IGFBP3, MARCO, MT2A, NNMT, and PGLYRP2 were identified. The survival analysis using the Kaplan-Meier method revealed a significant correlation between the expression levels of BIRC5 and IGFBP3 and the survival of patients diagnosed with HCC.

Effect of extended field-of-view approaches on the accuracy of stopping power ratio estimation for single-energy computed tomography simulators.

BACKGROUND: Extended field-of-view (eFOV) methods have been proposed to generate larger demonstration FOVs for computed tomography (CT) simulators with a limited scanning FOV (sFOV) size in order to ensure accurate dose calculation and patient collision avoidance. Although the efficacy of these strategies has been evaluated for photon applications, the effect of stopping power ratio (SPR) estimation on proton therapy has not been studied. This study investigated the effect of an eFOV approach on the accuracy of SPR to water estimation in homogeneous and heterogeneous phantoms. MATERIALS AND METHODS: To simulate patient geometries, tissue-equivalent material (TEM) and customized extension phantoms were used. The TEM phantom supported various rod arrangements through predefined holes. Images were reconstructed to three FOV sizes using a commercial eFOV technique. A single-energy CT stoichiometric method was used to generate Hounsfield unit (HU) to SPR (HU-to-SPR) conversion curves for each FOV. To investigate the effect of rod location in the sFOV and eFOV regions, eight TEM rods were placed at off-center distances in the homogeneous phantom and scanned individually. Similarly, 16 TEM rods were placed in the heterogeneous TEM phantom and scanned simultaneously. RESULTS: The conversion curves derived from the sFOV and eFOV data were identical. The average SPR differences of soft-tissue, bone, and lung materials for rods placed at various off-center locations were 3.3%, 4.8%, and 39.6%, respectively. In the heterogeneous phantom, the difference was within 1.0% in the absence of extension. However, in the presence of extension, the difference increased to 2.8% for all rods, except for lung materials, whose difference was 4.8%. CONCLUSIONS: When an eFOV method is used, the SPR variation in phantoms considerably increases for all TEM rods, especially for lung TEM rods. This phenomenon may substantially increase the uncertainty of HU-to-SPR conversion. Therefore, image reconstruction with a standard FOV size is recommended.

Cathelicidin Antimicrobial Peptide Acts as a Tumor Suppressor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is associated with high rates of metastasis and recurrence, and is one of the most common causes of cancer-associated death worldwide. This study examined the protein changes within circulating exosomes in patients with HCC against those in healthy people using isobaric tags for a relative or absolute quantitation (iTRAQ)-based quantitative proteomics analysis. The protein levels of von Willebrand factor (VWF), cathelicidin antimicrobial peptide (CAMP), and proteasome subunit beta type-2 (PSMB2) were altered in HCC. The increased levels of VWF and PSMB2 but decreased CAMP levels in the serum of patients with HCC were validated by enzyme-linked immunosorbent assays. The level of CAMP (the only cathelicidin found in humans) also decreased in the circulating exosomes and buffy coat of the HCC patients. The serum with reduced levels of CAMP protein in the HCC patients increased the cell proliferation of Huh-7 cells; this effect was reduced following the addition of CAMP protein. The depletion of CAMP proteins in the serum of healthy people enhances the cell proliferation of Huh-7 cells. In addition, supplementation with synthetic CAMP reduces cell proliferation in a dose-dependent manner and significantly delays G1-S transition in Huh-7 cells. This implies that CAMP may act as a tumor suppressor in HCC.

Efficient method for whole-breast irradiation therapy using Halcyon linear accelerators.

BACKGROUND: The Halcyon is a linear accelerator-based treatment machine designed for a high-throughput simplified workflow. The machine features a compact jawless design, dual-layer multileaf collimators, and a single 6-MV flattening filter-free (FFF) beam. However, the machine's 6-MV FFF beam may restrict its applicability to conventional techniques, such as field-in-field (FiF) radiotherapy, for breast cancer treatment. This study developed a practical and efficient hybrid method for imaging, planning, and irradiation procedures for whole-breast irradiation using Halcyon linear accelerators. MATERIALS AND METHODS: The proposed method involves five major steps: (1) field arrangement, (2) planning target volume (PTV) generation and evaluation, (3) basal plan generation, (4) inverse planning intensity-modulated radiation therapy plan generation, and (5) plan evaluation and irradiation. The PTV is generated using isodose curves plotted on the basis of tangential fields, which are applied to create a basal plan. Subsequently, a basal-dose-compensation approach is applied to further optimize the treatment plan. This efficient workflow necessitates executing only one onboard cone-beam computed tomography procedure. This study included 10 patients with early-stage breast cancer who were treated at our center. The performance of the proposed method was evaluated by comparing its corresponding irradiation time and dose statistics with those derived for a dynamically flattened beam-based FiF (DFB-FiF) method. RESULTS: All plans were normalized to ensure that 98% of the prescribed dose covered 95% of the PTV. On average, the global maximum doses in the proposed and DFB-FiF methods were lower than 106%. The homogeneity index for right-sided (left-sided) breast cancer was 0.053 (0.056) in the proposed method and 0.073 (0.076) in the DFB-FiF method. The dose statistics of normal tissues, including the contralateral breast, heart, and lungs, were comparable between the methods. However, the irradiation time per monitor unit in the proposed method was approximately five times faster than that in the DFB-FiF method, but the planning time and complexity were similar between the methods. CONCLUSIONS: This study developed and evaluated an efficient and practical hybrid method for whole-breast irradiation using the Halcyon. This method can significantly reduce the irradiation time, while providing comparable dose statistics to the DFB-FiF method.

Effects of Mindfulness-Based Therapy for Cancer Patients: A Systematic Review and Meta-analysis.

This meta-analysis was a systematic review of evidence on the effects of mindfulness-based stress reduction (MBSR) and mindfulness-based cognitive therapy (MBCT) on quality of life (QOL), pain, fatigue, anxiety, and depression in cancer patients. Until July 2020, PubMed, Cochrane Library, and Embase were searched for randomized controlled trials (RCTs). The study included 18 RCTs. The MBSR/MBCT intervention resulted in a significant effect on QOL (SMD 0.80, CI 0.28, 1.32, I2 = 94%). In subgroup analysis, MBSR/MBCT interventions had a significant effect in the early cancer stage on anxiety (SMD - 3.48, CI - 4.07, - 2.88), and QOL (SMD 4.30, CI 3.62, 4.99); in alleviating decreasing pain (SMD - 0.42, CI - 0.70, - 0.14) within 4 weeks after the end of intervention, and alleviating fatigue in younger participants (SMD - 0.64, CI - 1.09, - 0.19). MBSR/MBCT has short-term effects on cancer patients, especially in younger patients and early cancer stages.

Identification and characterization of hADSC-derived exosome proteins from different isolation methods.

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.

Subpopulations of exosomes purified via different exosomal markers carry different microRNA contents.

The heterogeneity of exosome populations presents a great challenge to their study. The current study was designed to investigate the potential heterogeneity miRNA contents in circulating exosomes purified via different exosomal markers. In this study, exosomes from the serum of C57BL/6 mice after cecum ligation and perforation (CLP) or sham operation were isolated by precipitation using ExoQuick-TC and affinity purified with anti-Rab5b, anti-CD9, anti-CD31, and anti-CD44 antibodies using the Exo-Flow Exosome Capture kit to collect exosome subpopulations. RNA extracted from the exosomes isolated by ExoQuick-TC were profiled by next-generation sequencing (NGS). Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also employed to determine the expression profiles of four representative exosomal miRNAs (mmu-miR-486-5p, mmu-miR-10a-5p, mmu-miR-143-3p, and mmu-miR-25-3p) selected from the NGS analysis. The results revealed that the expression patterns of these miRNAs in exosomes isolated by ExoQuick-TC as determined by RT-qPCR and NGS were similar, showing upregulation of mmu-miR-10a-5p and mmu-miR-143-3p but downregulation of mmu-miR-25-3p and mmu-miR-486-5p following CLP when compared to the levels in exosomes from sham control mice. However, their expression levels in the antibody-captured exosome subpopulations varied. The miRNAs in the exosomes captured by anti-Rab5b or anti-CD9 antibodies were more similar to those isolated by ExoQuick-TC than to those captured by anti-CD44 antibodies. However, there were no significant differences in these four miRNAs in CD31-captured exosomes. This study demonstrated that purification with different exosomal markers allows the collection of different exosome subpopulations with various miRNA contents. The results of this study demonstrate the heterogeneity of circulating exosomes and suggest the importance of stratifying exosome subpopulations when using circulating exosomes as biomarkers or investigating exosome function. In addition, this study also emphasized the necessity of using a consistent exosome marker across different samples as detecting biomarkers.

Long-term in vitro degradation and in vivo evaluation of resorbable bioceramics.

An essential criterion for the selection of resorbable bioceramics is their ability to degrade inside human body within a reasonable time frame. Furthermore, if the bioceramic can release beneficial ions, such as strontium, as it degrades, recovery time might be shortened. The present study demonstrates that strontium-containing calcium sulfate (Sr,Ca)SO4 can fulfill these criteria. A long-term in vitro degradation analysis for 12 weeks using sintered (Sr,Ca)SO4 discs in phosphate buffered solution (PBS) was conducted. The sintered (Sr,Ca)SO4 disc was then implanted into defects in the distal femur of rats. The degradation rate of (Sr,Ca)SO4 discs showed a strong dependence on the Sr content. Similar results were observed between the long-term in vitro degradation analysis and the in vivo evaluation. The sintered (3.8%Sr,Ca)SO4 disc lost more than 80% of its initial weight after soaking in PBS with shaking at 37 °C for 12 weeks. After 12 weeks in vivo, the remaining volume of the (3.8%Sr,Ca)SO4 disc within the bone defect was ~25%. Over the same time period, new bone was formed at a relative volume of 40%. This study demonstrates the potential of (Sr,Ca)SO4 bioceramic, and the benefits of using a long-term degradation test during the evaluation of resorbable bioceramics.

Exosomes Secreted by Adipose-Derived Stem Cells Following FK506 Stimulation Reduce Autophagy of Macrophages in Spine after Nerve Crush Injury.

Macrophages emerge in the milieu around innervated neurons after nerve injuries. Following nerve injury, autophagy is induced in macrophages and affects the regulation of inflammatory responses. It is closely linked to neuroinflammation, while the immunosuppressive drug tacrolimus (FK506) enhances nerve regeneration following nerve crush injury and nerve allotransplantation with additional neuroprotective and neurotrophic functions. The combined use of FK506 and adipose-derived stem cells (ADSCs) was employed in cell therapy for organ transplantation and vascularized composite allotransplantation. This study aimed to investigate the topical application of exosomes secreted by ADSCs following FK506 treatment (ADSC-F-exo) to the injured nerve in a mouse model of sciatic nerve crush injury. Furthermore, isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile the potential exosomal proteins involved in autophagy. Immunohistochemical analysis revealed that nerve crush injuries significantly induced autophagy in the dorsal root ganglia and dorsal horn of the spinal segments. Locally applied ADSC-F-exo significantly reduced autophagy of macrophages in the spinal segments after nerve crush injury. Proteomic analysis showed that of the 22 abundant exosomal proteins detected in ADSC-F-exo, heat shock protein family A member 8 (HSPA8) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) are involved in exosome-mediated autophagy reduction.

Enhanced Nerve Regeneration by Exosomes Secreted by Adipose-Derived Stem Cells with or without FK506 Stimulation.

Exosomes secreted by adipose-derived stem cells (ADSC-exo) reportedly improve nerve regeneration after peripheral nerve injury. Herein, we investigated whether pretreatment of ADSCs with FK506, an immunosuppressive drug that enhances nerve regeneration, could secret exosomes (ADSC-F-exo) that further augment nerve regeneration. Designed exosomes were topically applied to injured nerve in a mouse model of sciatic nerve crush injury to assess the nerve regeneration efficacy. Outcomes were determined by histomorphometric analysis of semi-thin nerve sections stained with toluidine blue, mouse neurogenesis PCR array, and neurotrophin expression in distal nerve segments. Isobaric tags for relative and absolute quantitation (iTRAQ) were used to profile potential exosomal proteins facilitating nerve regeneration. We observed that locally applied ADSC-exo and ADSC-F-exo significantly enhanced nerve regeneration after nerve crush injury. Pretreatment of ADSCs with FK506 failed to produce exosomes possessing more potent molecules for enhanced nerve regeneration. Proteomic analysis revealed that of 192 exosomal proteins detected in both ADSC-exo and ADSC-F-exo, histone deacetylases (HDACs), amyloid-beta A4 protein (APP), and integrin beta-1 (ITGB1) might be involved in enhancing nerve regeneration.

Increased Angiogenesis by Exosomes Secreted by Adipose-Derived Stem Cells upon Lipopolysaccharide Stimulation.

Exosomes secreted by adipose-derived stem cells (ADSCs) enhance angiogenesis and wound healing. However, in clinical settings, wounds may be infected by various bacteria or pathogens. We investigated whether human ADSCs stimulated with lipopolysaccharide (LPS) secrete exosomes (ADSC-LPS-exo) that augment the angiogenesis of human umbilical vein endothelial cells (HUVECs). ExoQuick-TC exosome precipitation solution was used to purify exosomes from human ADSC culture media in the presence or absence of 1 µg/mL LPS treatment for 24 h. The uptake of ADSC-LPS-exo significantly induced the activation of cAMP response element binding protein (CREB), activating protein 1 (AP-1), and nuclear factor-κB (NF-κB) signaling pathways and increased the migration of and tube formation in HUVECs. RNA interference with CREB, AP-1, or NF-κB1 significantly reduced the migration of and tube formation in HUVECs treated with ADSC-LPS-exo. An experiment with an antibody array for 25 angiogenesis-related proteins revealed that only interleukin-8 expression was significantly upregulated in HUVECs treated with ADSC-LPS-exo. In addition, proteomic analysis revealed that eukaryotic translation initiation factor 4E, amyloid beta A4 protein, integrin beta-1, and ras-related C3 botulinum toxin substrate 1 may be potential candidates involved in ADSC-LPS-exo-mediated enhanced angiogenesis.

Septic arthritis significantly increased the long-term mortality in geriatric patients.

BACKGROUND: The elderly are predisposed to septic arthritis (SA) because of the aging nature and increasing comorbidities. SA may in turn increase the long-term mortality in the geriatric patients; however, it remains unclear. We conducted this prospective nationwide population-based cohort study to clarify this issue. METHODS: Using Taiwan National Health Insurance Research Database (NHIRD), we identified 1667 geriatric participants (≥ 65 years) with SA and 16,670 geriatric participants without SA matched at a ratio of 1:10 by age, sex, and index date between 1999 and 2010. A comparison of the long-term mortality between the two cohorts through follow-up until 2011 was performed. RESULTS: Geriatric participants with SA had a significantly increased mortality than those without SA [Adjusted hazard ratio (AHR): 1.49, 95% confidence interval (CI): 1.34-1.66], particularly the old elderly (≥ 85 years, AHR: 2.12, 95% CI: 1.58-2.84) and males (AHR: 1.54, 95% CI: 1.33-1.79). These results were stated after adjustment for osteoarthritis, diabetes, gout, renal disease, liver disease, cancer, rheumatoid arthritis, systemic lupus erythematosus, alcoholism, and human immunodeficiency virus infection. The increased mortality risk was highest in the first month (AHR: 3.93, 95% CI: 2.94-5.25) and remained increased even after following up for 2-4 years (AHR: 1.30, 95% CI: 1.03-1.65). After Cox proportional hazard regression analysis, SA (AHR: 1.37, 95% CI: 1.20-1.56), older age (≥ 85 years, AHR: 1.79, 95% CI: 1.59-2.02, 75-84 years, AHR: 1.65, 95% CI: 1.53-1.78), male sex, diabetes, renal disease, liver disease, cancer, and gout were independent mortality predictors. There was no significant difference in the mortality for SA between upper limb affected and lower limb affected. CONCLUSIONS: This study delineated that SA significantly increased the long-term mortality in geriatric participants. For the increasing aging population worldwide, strategies for the prevention and treatment of SA and concomitant control of comorbidities are very important.

InGaN light emitting diodes with a nanopipe layer formed from the GaN epitaxial layer.

A Si-heavy doped GaN:Si epitaxial layer is transformed into a directional nanopipe GaN layer through a laser-scribing process and a selectively electrochemical (EC) etching process. InGaN light-emitting diodes (LEDs) with an EC-treated nanopipe GaN layer have a high light extraction efficiency. The direction of the nanopipe structure was directed perpendicular to the laser scribing line and was guided by an external bias electric field. An InGaN LED structure with an embedded nanopipe GaN layer can enhance external quantum efficiency through a one-step epitaxial growth process and a selective EC etching process. A birefringence optical property and a low effective refractive index were observed in the directional-nanopipe GaN layer.

Weight-reduction through a low-fat diet causes differential expression of circulating microRNAs in obese C57BL/6 mice.

BACKGROUND: To examine the circulating microRNA (miRNA) expression profile in a mouse model of diet-induced obesity (DIO) with subsequent weight reduction achieved via low-fat diet (LFD) feeding. RESULTS: Eighteen C57BL/6NCrl male mice were divided into three subgroups: (1) control, mice were fed a standard AIN-76A (fat: 11.5 kcal %) diet for 12 weeks; (2) DIO, mice were fed a 58 kcal % high-fat diet (HFD) for 12 weeks; and (3) DIO + LFD, mice were fed a HFD for 8 weeks to induce obesity and then switched to a 10.5 kcal % LFD for 4 weeks. A switch to LFD feeding led to decreases in body weight, adiposity, and blood glucose levels in DIO mice. Microarray analysis of miRNA using The Mouse & Rat miRNA OneArray® v4 system revealed significant alterations in the expression of miRNAs in DIO and DIO + LFD mice. Notably, 23 circulating miRNAs (mmu-miR-16, mmu-let-7i, mmu-miR-26a, mmu-miR-17, mmu-miR-107, mmu-miR-195, mmu-miR-20a, mmu-miR-25, mmu-miR-15b, mmu-miR-15a, mmu-let-7b, mmu-let-7a, mmu-let-7c, mmu-miR-103, mmu-let-7f, mmu-miR-106a, mmu-miR-106b, mmu-miR-93, mmu-miR-23b, mmu-miR-21, mmu-miR-30b, mmu-miR-221, and mmu-miR-19b) were significantly downregulated in DIO mice but upregulated in DIO + LFD mice. Target prediction and function annotation of associated genes revealed that these genes were predominantly involved in metabolic, insulin signaling, and adipocytokine signaling pathways that directly link the pathophysiological changes associated with obesity and weight reduction. CONCLUSIONS: These results imply that obesity-related reductions in the expression of circulating miRNAs could be reversed through changes in metabolism associated with weight reduction achieved through LFD feeding.

Profiling the circulating miRNAs in mice exposed to gram-positive and gram-negative bacteria by Illumina small RNA deep sequencing.

BACKGROUND: We profiled the expression of circulating microRNAs (miRNAs) in mice using Illumina small RNA deep sequencing in order to identify the miRNAs that may potentially be used as biomarkers to distinguish between gram-negative and gram-positive bacterial infections. RESULTS: Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 10(8) bacteria/100 µL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14 + Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection. CONCLUSIONS: This study identified mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p as potential circulating miRNAs for gram-positive bacterial infections.

Altered exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury.

BACKGROUND: The NF-κB signaling pathway plays a role in local and remote tissue damage following ischemia-reperfusion (I/R) injury to skeletal muscles. Evidence suggests that exosomes can act as intercellular communicators by transporting active proteins to remote cells and may play a role in regulating inflammatory processes. This study aimed to profile the exosomal protein expression in the serum of NF-κB knockout mice following skeletal muscle ischemia-reperfusion injury. RESULTS: To investigate the potential changes in protein expression mediated by NF-κB in secreted exosomes in the serum following I/R injury, the levels of circulating exosomal proteomes in C57BL/6 and NF-κB(-/-) mice were compared using two dimensional differential in-gel electrophoresis (2-DE), liquid chromatography tandem mass spectrometry (LC-MS/MS), and proteomic analysis. In C57BL/6 mice, the levels of circulating exosomal proteins, including complement component C3 prepropeptide, PK-120 precursor, alpha-amylase one precursor, beta-enolase isoform 1, and adenylosuccinate synthetase isozyme 1, increased following I/R injury. However, in the NF-κB(-/-) mice, the expression of the following was upregulated in the exosomes: protease, serine 1; glyceraldehyde-3-phosphate dehydrogenase-like isoform 1; glyceraldehyde-3-phosphate dehydrogenase; and pregnancy zone protein. In contrast, the expression of apolipoprotein B, complement component C3 prepropeptide, and immunoglobulin kappa light chain variable region was downregulated in NF-κB(-/-) mice. Bioinformatic annotation using the Protein Analysis Through Evolutionary Relationships (PANTHER) database revealed that the expression of the exosomal proteins that participate in metabolic processes and in biological regulation was lower in NF-κB(-/-) mice than in C57BL/6 mice, whereas the expression of proteins that participate in the response to stimuli, in cellular processes, and in the immune system was higher. CONCLUSIONS: The data presented in this study suggest that NF-κB might regulate exosomal protein expression at a remote site via circulation following I/R injury.

Regulatory and Effector Helper T-Cell Profile after Nerve Xenografting in the Toll-Like Receptor-Deficient Mice.

INTRODUCTION: The balance between regulatory T cells (Tregs) and effector T help cells (Th cells) is critical for the control of adaptive immune response during nerve transplantation. However, whether the homeostasis of immune regulation between Tregs and Th cells requires toll-like receptor (TLR) signaling is unclear. The aim of this study is to profile the distribution of spleen Tregs and Th cells in a mouse model of nerve xenografting in the TLR2 and NF-κB gene knockout mice. METHODS: The sciatic nerve was taken from a SD rat or an allogeneic mouse and transplanted to a right back leg of recipient C57BL/6, TLR2(-/-), or NF-κB(-/-) mice by subcutaneous transplantation. After 7 days, the T lymphocytes were then isolated from spleen, stained with phenotyping kits, and analyzed by flow cytometry. RESULTS: The results showed that Tregs were decreased after nerve xenografting in the recipient C57BL/6 mouse. In addition, nerve xenografting also increased the Th1 and Th17 but not the Th2 cell populations. In contrast, amelioration of the Tregs elimination was found in TLR2(-/-) and NF-κB(-/-) mice after transplantation of the nerve xenograft. Moreover, the mice lacking TLR2 or NF-κB showed attenuation of the increase in Th1 and Th17 cells after nerve xenografting. CONCLUSIONS: TLR signaling is involved in T cell population regulation during tissue transplantation. Knock-out of TLR2 and NF-κB prevented Tregs elimination and inhibited Th1- and Th17-driven immune response after nerve xenografting. This study highlighted the potential of inhibiting TLR signaling to modulate T cell-mediated immune regulation to facilitate tolerance to nerve transplantation.

Effect of Weight-Reduction in Obese Mice Lacking Toll-Like Receptor 5 and C57BL/6 Mice Fed a Low-Fat Diet.

BACKGROUND: This study aims to investigate the effect of feeding low-fat diet (LFD) to diet-induced obesity (DIO) mice lacking TLR5 (TLR5(-/-)), which have a tendency to develop glucose intolerance with increased adiposity, compared to that in C57BL/6 mice. RESULTS: TLR5(-/-) and C57BL/6 male mice were divided into three subgroups: (1) control, mice were fed a standard AIN-76A (fat: 11.5 kcal%) diet for 12 weeks; (2) DIO, mice were fed a 58 kcal% high-fat diet (HFD) for 12 weeks; and (3) diet, mice were fed a HFD for 8 weeks to induce obesity and then switched to a 10.5 kcal% LFD for 4 weeks. The glucose intolerance in DIO TLR5(-/-) mice was more significant than that in DIO C57BL/6 mice and was not attenuated by a switch to the LFD. Weight-reduction with LFD had significantly decreased the epididymal fat mass in C57BL/6 mice but not in TLR5(-/-) mice. In addition, the LFD-fed TLR5(-/-) mice showed significantly higher expression of ghrelin in the serum and resistin in the epididymal fat than that in C57BL/6 mice. CONCLUSIONS: This study demonstrated that TLR5 gene knockout impairs some effects of weight-reduction in DIO.

Inhibition of the phosphoinositide 3-kinase pathway decreases innate resistance to lipopolysaccharide toxicity in TLR4 deficient mice.

BACKGROUND: Upon lipopolysaccharide (LPS) stimulation, activation of both the Toll-like receptor 4 (TLR4) and phosphoinositide 3-kinase (PI3K) pathways serves to balance proinflammatory and anti-inflammatory responses. Although the antagonist to TLR4 represents an emerging promising target for the treatment of sepsis; however, the role of the PI3K pathway under TLR4-null conditions is not well understood. This goal of this study was to investigate the effect of inhibition of PI3K on innate resistance to LPS toxicity in a murine model. RESULTS: The overall survival of the cohorts receiving intraperitoneal injections of 100, 500, or 1000 µg LPS from Escherichia coli serotype 026:B6 after 7 d was 100%, 10%, and 10%, respectively. In contrast, no mortality was noted after 500-µg LPS injection in Tlr4-/- mice. When the PI3K inhibitor LY294002 was injected (1 mg/25 g body weight) 1 h prior to the administration of LPS, the overall survival of the Tlr4-/- mice was 30%. In the Tlr4-/- mice, the LPS injection induced no NF-κB activation but an increased Akt phosphorylation in the lung and liver, when compared to that of the C57BL/6 mice. Injection of 500 µg LPS led to a significant induction in O2⁻ detected by electron paramagnetic resonance (EPR) spin trapping spectroscopy in the lung and liver at 3 and 6 h in C57BL/6 but not Tlr4-/- mice. Addition of LY294002 only significantly increased the O2⁻ level in the lung and liver of the Tlr4-/- mice but not in the C57BL/6 mice following 500-µg LPS injection. In addition, the serum IL-1ß and IL-2 levels were more elevated in C57BL/6 mice than in Tlr4-/- mice. Notably, IL-1ß and IL-2 were significantly increased in Tlr4-/- mice but not in the C57BL/6 mice when the PI3K pathway was inhibited by LY294002 prior to LPS injection. CONCLUSIONS: In this study, we demonstrate that innate resistance to LPS toxicity in Tlr4-/- mice is impaired by inhibition of the PI3K pathway, with a corresponding increase in mortality and production of tissue O2⁻ and inflammatory cytokines.