IL-6 In Supernatants Research Articles

Innate Immune Reprogramming of Macrophages in the Parous Murine Mammary Gland

Uncomplicated childbirth is associated with an increased risk of developing breast cancer (BC) five-to-ten years postpartum (postpartum breast cancer; PPBC) compared with age-matched nulliparous individuals. PPBC is also associated with a poor prognosis relative to nulliparous individuals with BC. Contrastingly, preeclampsia, an inflammatory pregnancy complication, protects against PPBC development. We hypothesize that differential parity-induced innate immune reprogramming of mammary gland macrophages may be associated with parity-associated increased PPBC risk and preeclampsia-associated protection against PPBC. Female BALB/c mice were mated with male C57Bl/6 mice to establish allogeneic pregnancies. On gestational day (GD) 10.5, dams were injected with saline (controls) or lipopolysaccharide (LPS; 20 μg/kg) to induce inflammatory pregnancy complications. Age-matched nulliparous controls also received LPS or saline. Following birth, parous mice were force-weaned on postnatal day (PD) 7 to induce synchronous involution. Macrophages were isolated from mammary glands upon euthanasia in pregnancy (GD 14.5), lactation (PD 7), involution (PD 10) and regression (PD 28), plated (4x104 cells/well) and exposed to a 23-hour secondary challenge (LPS; 100 ng/ml; Pam3Cys; 10 µg/ml). Cytokine concentrations in cell culture supernatant were assessed using a multiplex assay (Eve Technologies) and data were analyzed by two-way ANOVA with Tukey’s posthoc test. During involution, compared with supernatant from mammary gland macrophages of age-matched nulliparous saline-treated mice, supernatant from macrophages from parous saline-treated mice exhibited significantly increased interleukin (IL)-1β and tumour necrosis factor-α (TNF-α), and significantly decreased IL-6 following secondary challenge. TNF-α, IL-6 and IL-10 in supernatant from macrophages from parous LPS-treated mice was significantly decreased compared with parous saline-treated controls. Analysis of data collected during pregnancy, lactation and regression is ongoing to temporally evaluate reprogramming. These preliminary results demonstrate differential reprogramming in mammary gland macrophages relative to parity and inflammatory pregnancy complication status and rationalize understanding contributions of completion of a prior pregnancy on PPBC risk and progression.

Design, Synthesis, and Antirheumatoid Arthritis Mechanism of TLR4 Inhibitors.

A total of 12 carbonyl compounds were synthesized, their lipopolysaccharide induced inhibition, and activity of RAW264.7 cells was evaluated. The most active compound 3k inhibited RAW264.7 cells with IC50 value of 1.02 ± 0.08 μM. Compound 3k significantly inhibited the release of TNF-α, IL-1β, and IL-6 in supernatant for RAW264.7 cells. In vivo collagen-induced arthritis model tests administered orally, compound 3k showed effects similar to those of methotrexate in the positive control group. The preliminary mechanism study showed that compound 3k had an effect on abnormal expression for TLR4, TNF-α, NF-κB protein, and genes related to inflammation signaling pathway in RAW264.7 cells. Meanwhile, compound 3k showed a good affinity for the TLR4 receptor in molecular docking simulation. Therefore, compound 3k may be a promising lead compound for the treatment of rheumatoid arthritis.

Bacteriophages isolated from mouse feces attenuates pneumonia mice caused by Pseudomonas aeruginosa.

Most of the current bacteriophages (phages) are mostly isolated from environments. However, phages isolated from feces might be more specific to the bacteria that are harmful to the host. Meanwhile, some phages from the environment might affect non-pathogenic bacteria for the host. Here, bacteriophages isolated from mouse feces were intratracheally (IT) or intravenously (IV) administered in pneumonia mice caused by Pseudomonas aeruginosa at 2 hours post-intratracheal bacterial administration. As such, the mice with phage treatment, using either IT or IV administration, demonstrated less severe pneumonia as indicated by mortality, serum cytokines, bacteremia, bacterial abundance in bronchoalveolar lavage fluid (BALF), and neutrophil extracellular traps (NETs) in lung tissue (immunofluorescence of neutrophil elastase and myeloperoxidase). Interestingly, the abundance of phages in BALF from the IT and IV injections was similar, supporting a flexible route of phage administration. With the incubation of bacteria with neutrophils, the presence of bacteriophages significantly improved bactericidal activity, but not NETs formation, with the elevated supernatant IL-6 and TNF-α, but not IL-1β. In conclusion, our findings suggest that bacteriophages against Pseudomonas aeruginosa can be discovered from feces of the host. The phages attenuate pneumonia partly through an enhanced neutrophil bactericidal activity, but not via inducing NETs formation. The isolation of phages from the infected hosts themselves might be practically useful for future treatment. More studies are warranted.

Cytomegalovirus antigen-specific multi-cytokine immune responses in patients with rheumatic diseases under different cytomegalovirus infection status: A case-control study

ObjectiveTo explore Cytomegalovirus (CMV) antigen-specific multi-cytokine immune responses in patients with rheumatic disease (RD) under different CMV infection status. MethodsA total of 60 RD patients in our center from March 2023 to August 2023 were enrolled. The patients were divided into latent CMV infection and active CMV infection, the latter was classified as subclinical CMV infection or CMV disease based on presence or absence of symptoms related to CMV. Whole blood was collected and stimulated with QuantiFERON-CMV antigen. The levels of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-17 and CXCL-2 in supernatant were measured by Luminex Assays. The receiver operating characteristic curve was used to evaluate the diagnostic accuracy of cytokine for distinguishing different CMV infection status. ResultsThe proportion of patients with severe lymphopenia was lowest in the latent CMV infection group, while there were no significant differences in medication usage in different CMV infection status. After stimulation with QF-CMV antigens, the levels of IFN-γ, TNF-α and IL-2 in the CMV disease group were significantly lower than those in the latent CMV infection group. CMV antigen-specific IFN-γ, TNF-α levels and severe lymphopenia together provided the best discriminatory performance for distinguishing between latent and either active CMV infection patients (AUC = 0.854) or CMV disease patients (AUC = 0.935). ConclusionNoninvasive peripheral blood biomarkers (the combination of CMV antigen-specific IFN-γ, TNF-α levels and severe lymphopenia) may have the potential to diferentiate different status of CMV infection in RD population.

Radioprotective effect of polyvinylpyrrolidone modified selenium nanoparticles and its antioxidation mechanism in vitro and in vivo.

Polyvinylpyrrolidone (PVP) is a commonly used biomedical polymer material with good water solubility, biocompatibility, low immunogenicity, and low toxicity. The aim of this study is to investigate the antioxidant mechanism and clinical potential of PVP modified selenium nanoparticles (PVP-Se NPs) as a new radioprotective agent. A laser particle size analyzer and transmission electron microscope were used to characterize PVP-Se nanoparticles prepared by chemical reduction. Human umbilical vein endothelial cells (HUVECs) were used to evaluate the radiation protective effects of PVP-Se NPs. SD rats were employed as an in vivo model to identify the most effective concentration of PVP-Se NPs and assess their potential radioprotective properties. Western blot (WB) was used to detect the expression of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling proteins in human umbilical vein endothelial cells (HUVECs) and rat liver and kidney tissues. PVP-Se NPs could reduce the oxidative stress injury and inflammatory response caused by X-ray irradiation in HUVECs and rats, and inhibit cell apoptosis by modulating NF-κB and MAPK signaling pathways. PVP-Se NPs could increase HUVECs viability, reduce apoptosis, inhibit inflammatory factors IL-1β, IL-6 and TNF-α, improve the survival rate of rats, promote antioxidant enzyme activities in cells and rats, reduce malondialdehyde concentration in serum, and reduce the expression of inflammatory factors such as IL-1β, IL-6 and TNF-α in cell supernatant and liver and kidney tissues. PVP-Se NPs could significantly reduce the phosphorylation levels of NF-κB and MAPK pathway-associated proteins in HUVECs and rat liver and kidney tissues (p < 0.05). PVP-Se NPs can protect against radiation-induced oxidative damage by modulating NF-kB and MAPK pathways, providing a theoretical basis and experimental data for their use as an effective radioprotective agent.

Search in the Periphery for Potential Inflammatory Biomarkers of Dementia with Lewy Bodies and Alzheimer's Disease.

Neuroinflammation, with altered peripheral proinflammatory cytokine production, plays a major role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD), while the role of inflammation in dementia with Lewy bodies (DLB) is less known and the results of different studies are often in disagreement. The present study aimed to investigate the levels of TNFα and IL-6 in serum and supernatants, and the related DNA methylation in patients affected by DLB and AD compared to healthy controls (HCs), to clarify the role of epigenetic mechanisms of DNA promoter methylation on of pro-inflammatory cytokines overproduction. Twenty-one patients with DLB and fourteen with AD were frequency-matched for age and sex with eleven HCs. Clinical evaluation, TNFα and IL-6 gene methylation status, cytokine gene expression levels and production in serum and peripheral blood mononuclear cell (PBMC) supernatants were performed. In AD and DLB patients, higher serum levels of IL-6 and TNFα were detected than in HCs. Differences in LPS-stimulated versus spontaneous PBMCs were observed between DLB, AD, and HC in the levels of TNFα (p = 0.027) and IL-6 (p < 0.001). Higher levels were also revealed for sIL-6R in DLB (p < 0.001) and AD (p < 0.001) in comparison with HC.DNA hypomethylation in IL-6 and TNFα CpG promoter sites was detected for DLB and AD patients compared to the corresponding site in HCs. Our preliminary study documented increased levels of IL-6 and TNFα in DLB and AD patients to HCs. This overproduction can be due to epigenetic mechanisms regarding the hypomethylation of DNA promoters.

Human plasma can modulate micronucleus frequency in TK6 and OE33 cells in vitro

In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett’s oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 h (for the micronucleus (Mn) assay and the invasion assay) and 24 h (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment.The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett’s oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24 h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells.The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma’s genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.

Palmatine Attenuated Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting M1 Phenotype Macrophage Polarization via NAMPT/TLR2/CCR1 Signaling.

The present work was conducted to research the potential mechanism of palmatine (PAL) on lipopolysaccharide (LPS)-caused acute lung injury (ALI). Network pharmacology and bioinformatic analyses were carried out. Mice were intragastrically treated with PAL and intratracheally stimulated with LPS. LPS-induced RAW264.7 cells were employed for the in vitro model. The MPO activity, W/D ratio, neutrophils, total cell number in BALF, and histopathological alteration were examined. The levels of TNF-α, IL-1β, IL-6, IL-18, IL-4, and IL-10 in serum, BALF, and supernatant were examined by ELISA. The mRNA expressions of iNOS, CD68, Arg1, Ym1, and CD206 and protein expressions of NAMPT, TLR2, CCR1, and NLRP3 inflammasome were detected by PCR, WB, and immunofluorescence. The NAMPT inhibitor FK866, TLR2 inhibitor C29, CCR1 inhibitor BX471, NAMPT-overexpression (OE) plasmid, and TLR2-OE plasmid were used for mechanism research. As a result, PAL relieved the symptoms of ALI. PAL inhibited M1 phenotype indices and promoted M2 phenotype indices in both LPS-induced mice and RAW264.7 cells. PAL also inhibited the expressions of NAMPT, TLR2, CCR1, and NLRP3 inflammasome. The treatments with FK866, NAMPT-OE plasmid, C29, TLR2-OE plasmid, and BX471 proved that PAL exerted its effect via NAMPT/TLR2/CCR1. Molecular docking suggested that PAL might combine with NAMPT. In conclusion, PAL ameliorated LPS-induced ALI by inhibiting M1 phenotype macrophage polarization via NAMPT/TLR2/CCR1 signaling.

Synergistic effect of Toll-like receptor 2 ligands and alendronate on proinflammatory cytokine production in mouse macrophage-like RAW-ASC cells is accompanied by upregulation of MyD88 expression

ObjectivesToll-like receptors (TLRs) recognize whole cells or components of microorganisms. Alendronate (ALN) is an anti-bone-resorptive drug that has inflammatory side effects. The aim in this study was to examine whether ALN augments TLR2 ligand-induced proinflammatory cytokine production using mouse macrophage-like RAW264.7 cells transfected with murine apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) gene (hereafter, referred to as “RAW-ASC cells”). MethodsRAW-ASC cells were pretreated with or without ALN and then incubated with or without TLR2 ligands. The levels of secreted mouse IL-1α, IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in culture supernatants and the activation of activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) were measured using enzyme-linked immunosorbent assay (ELISA). The expressions of myeloid differentiation factor 88 (MyD88), caspase-11, nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), ASC, NF-κB p65, and actin were analyzed via Western blotting. TLR2 expression was analyzed using flow cytometry. ResultsALN substantially upregulated the Pam3CSK4-induced release of IL-1α, IL-1β, IL-6, and TNF-α and MyD88 expression in RAW-ASC cells. ST-2825, a MyD88 inhibitor, inhibited the ALN-augmented release of these cytokines. Pretreatment with ALN augmented Pam3CSK4-induced NF-κB activation in RAW-ASC cells and upregulated AP-1 activation. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein and ALN synergically upregulated the release of IL-1α, IL-1β, IL-6 and TNF-α in RAW-ASC cells. ConclusionsOur findings suggest that ALN augments TLR2 ligand-induced proinflammatory cytokine production via the upregulation of MyD88 expression, and this augmentation is accompanied by the activation of NF-κB and AP-1 in RAW-ASC cells.

A186 INFLAMMATION MODIFIES DOSE-DEPENDENT RESPONSES OF INTESTINAL ANTI-TUMOUR MICRORNAS TO CRANBERRY PROANTHOCYANIDIN AND ITS MICROBIAL METABOLITE 3-(4-HYDROXYPHENYL)-PROPIONIC ACID

Abstract Background Cranberries are a rich source of proanthocyanidins (PAC), a type of polyphenol with anti-cancer properties. We previously found that PAC, along with its microbially-derived metabolite 3-(4-hydroxyphenyl)-propionic acid (HPPA), trigger unique regulatory responses of microRNAs (miRNAs) in intestinal epithelial cells including the upregulation of miR-146a-5p. Both miR-146a-5p and miR363-3p are anti-inflammatory and downregulate anti-tumorigenic signalling pathways such as the interleukin (IL)-17 and wingless-related integration site (Wnt) respectively. miR-146a-5p also attenuates IL-17-promoting cytokines levels (e.g., IL-6). Aims To determine if miR-146a-5p and miR-363-3p respond to different physiologically relevant concentrations of PAC and HPPA in inflammatory and non-inflammatory conditions. Methods Fully differentiated Caco-2BBe1 colonic epithelial cells were treated with two doses of PAC-enriched cranberry extract (50μg/ml, 100μg/ml), HPPA (5μg/ml, 10μg/ml), or with Dulbecco’s Modified Eagle Medium (DMEM; control) for 24 hours, followed by IL-1β (1ng/ml) or mock stimulation for three hours. Human IL-6 homogeneous time-resolved fluorescence (HTRF) kits were used to quantify IL-6 in cell supernatant. RNA was extracted and used for miRNA profiling using Nanostring technology. Statistical analysis was performed in R version 4.2.1 with the R-packages NanostringDiff, NanostringNorm, and Pheatmap. Results At homeostasis, 42 and 2 (miR-146a and miR363-3p) miRNAs, respectively, uniquely responded to increasing PAC or HPPA concentrations. In the inflammatory state, no miRNAs responded to increasing concentrations of PAC and HPPA. However, the expression of miR-363-3p increased (qampersand:003C0.001) in response to 50μg/ml of PAC + IL-1β but decreased in response to 5μg/ml of HPPA + IL-1β , and the expression of miR-146a-5p increased (qampersand:003C0.001) in response to 5μg/ml of HPPA + IL-1β, and 50μg/ml PAC + IL-1β. Predicted miRNA gene-pathway analysis revealed that miR-146a-5p, miR-363-3p, and the other 42 miRNAs commonly target pathways involved in the tumorigenesis of colorectal cancer such as mitogen-activated protein kinases (MAPK), hedgehog, and Wnt pathways. Though, in inflamed cells, only HPPA (5 or 10 μg/ml) attenuated IL-6 secretion (pampersand:003C0.05), which may be driven by increased expression of miR-146a-5p. Conclusions These findings suggest that cranberries proanthocyanidins and their metabolites affect miRNAs involved in cancer related pathways in different manners and concentrations, which may depend on the inflammatory status. The gut microbiota may be partially responsible for unlocking these effects. Funding Agencies Ocean Spray Cranberries, Inc. and the Natural Sciences of Engineering Research Council of Canada (NSERC)

Anti-bacterial and anti-inflammatory properties of ethanol extract of Melicope pteleifolia, an ethnomedicine in Southwest China

BackgroundTraditionally, Melicope pteleifolia has been used for heat clearing and detoxifying, traumatic injuries, bruise, and snake or insect bite. Up to now, its effect on sepsis remains unknown. MethodsMinimum inhibitory concentration (MIC) and dynamic antibacterial curve, and bacteria-induced sepsis mouse models were used to assess the anti-bacterial effect of Melicope pteleifolia ethanol extract (MPE) in vitro and in vivo. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells and cecum ligation and puncture (CLP)-induced sepsis mouse model were used to assess the anti-inflammatory effect of MPE in vitro and in vivo. ResultsMPE treatment significantly inhibited the growth of some pathogenic bacteria and increased survival rates of the sepsis mice challenged with the bacteria. In addition, MPE markedly reduced increased supernatant TNF-α and IL-6 levels. Further, it remarkably downregulated protein expressions of TLR4, MyD88, and NF-κB in the LPS-treated cells. Similarly, MPE significantly increased the survival rate, reduced the serum TNF-α and IL-6 levels of the CLP-induced sepsis mice. Importantly, it attenuated the CLP-induced pathological injuries of small intestines. Moreover, the anti-inflammatory effects of MPE in vitro and in vivo were not significantly abolished in presence of TAK-242 or JSH-23. In addition, MPE changed composition of intestinal flora at phylum and genus levels. Further, MPE affect some metabolism pathways such as carbohydrate, lipid metabolism, other amino acids metabolism, drug resistance, and others. Kaempferol (KAF), quercetin (QC), and N-p-coumaroyl-tyramine (NPC) were characteristic components possessing moderate binding to pathogenic associated molecular patterns (PAMPs) kdo2-Lipid A ammonium (KLA) and ODN 1826. ConclusionIn summary, MPE exerts anti-bacterial and anti-inflammatory effects, and its anti-inflammatory activity is independent of the TLR4/MyD88/NF-κB pathway.

Multiple cytokine analysis based on QuantiFERON-TB gold plus in different tuberculosis infection status: an exploratory study

BackgroundMore efficient and convenient diagnostic method is a desperate need to reduce the burden of tuberculosis (TB). This study explores the multiple cytokines secretion based on QuantiFERON-TB Gold Plus (QFT-Plus), and screens for optimal cytokines with diagnostic potential to differentiate TB infection status.MethodsTwenty active tuberculosis (ATB) patients, fifteen patients with latent TB infection (LTBI), ten patients with previous TB and ten healthy controls (HC) were enrolled. Whole blood samples were collected and stimulated by QFT-Plus TB1 and TB2 antigens. The levels of IFN-γ, TNF-α, IL-2, IL-6, IL-5, IL-10, IP-10, IL-1Ra, CXCL-1 and MCP-1 in supernatant were measured by Luminex bead-based multiplex assays. The receiver operating characteristic curve was used to evaluate the diagnostic accuracy of cytokine for distinguishing different TB infection status.ResultsAfter stimulation with QFT-Plus TB1 and TB2 antigens, the levels of all cytokines, except IL-5 in TB2 tube, in ATB group were significantly higher than that in HC group. The levels of IL-1Ra concurrently showed the equally highest AUC for distinguishing TB infection from HC, followed by the levels of IP-10 in both TB1 tube and TB2 tube. Moreover, IP-10 levels displayed the largest AUC for distinguishing ATB patients from non-ATB patients. Meanwhile, the levels of IP-10 also demonstrated the largest AUC in both TB1 tube and TB2 tube for distinguishing ATB patients from LTBI.ConclusionsIn addition to conventional detection of IFN-γ, measuring IP-10 and IL-1Ra based on QFT-Plus may have the more tremendous potential to discriminate different TB infection status.

Regulatory effects of mangiferin on LPS-induced inflammatory responses and intestinal flora imbalance during sepsis.

Studies suggest that mangiferin (MAF) has good therapeutic effects on chronic bronchitis and hepatitis. Also, it is one of the antiviral ingredients in Anemarrhena asphodeloides Bunge. However, its effect on the LPS-induced inflammation and intestinal flora during sepsis remains unclear yet. In the present study, LPS-stimulated inflammation RAW264.7 cells and LPS-induced sepsis mice were used to evaluate the efficacy of MAF invitro and invivo. 16S rDNA sequencing was performed to analyze the characteristics of intestinal flora of the sepsis mice. It has been demonstrated that MAF (12.5 and 25 μg/mL) significantly inhibited protein expressions of TLR4, MyD88, NF-κB, and TNF-α in the LPS-treated cells and reduced the supernatant TNF-α and IL-6 levels. Invivo, MAF (20 mg/kg) markedly protected the sepsis mice and reduced the serum TNF-α and IL-6 levels. Also, MAF significantly downregulated the protein expressions of TLR4, NF-κB, and MyD88 in the livers. Importantly, MAF significantly attenuated the pathological injuries of the livers and small intestines. Further, MAF significantly increased proportion of Bacteroidota and decreased the proportions of Firmicutes, Desulfobacterota, Actinobacteriota, and Proteobacteria at phylum level, and it markedly reduced the proportions of Escherichia-Shigella, Pseudoalteromonas, Staphylococcus at genus level. Moreover, MAF affects some metabolism-related pathways such as citrate cycle (TCA cycle), lipoic acid metabolism, oxidative phosphorylation, bacterial chemotaxis, fatty acid biosynthesis, and peptidoglycan biosynthesis of the intestinal flora. Thus, it can be concluded that MAF as a treatment reduces the inflammatory responses invitro and invivo by inhibiting the TLR4/ MyD88/NF-κB pathway, and corrects intestinal flora imbalance during sepsis to some degree.

Long Non-coding RNA SNHG1 Suppresses the Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Binding with HMGB1.

Osteoporosis (OP) has a significant detrimental impact on the health of the elder. Long-term clinical effectiveness of current drugs used for OP treatment is limited. Therefore, it is very important to explore novel treatment targets for OP. The expression of SNHG1, HMGB1, OCN and OPN in gene level was measured using RT-qPCR, and the protein expression was determined by Western blotting assay. The concentration of IL-1β and IL-18 in supernatant of the bone marrow mesenchymal stem cells (BMSCs) was measured by ELISA. The interaction between SNHG1 and HMGB1 was confirmed by RNA pull down. Besides, alizarin red staining was performed to evaluate the differentiation of BMSCs into osteoblast. SNHG1 and HMGB1 were found to be upregulated in the serum of OP patients. During the osteogenic differentiation of BMSCs, the expression of osteoblastogenesis markers (OCN and OPN) and the activity of ALP were upregulated, while the expression levels of SNHG1 and HMGB1 were decreased in a time-dependent manner. In addition, the interaction between SNHG1 and HMGB1, expression of pyroptosis-associated factors (caspase-1 p20 and GSDMD-N), and secretion of IL-1β and IL-18 were also decreased during osteogenic differentiation. Interestingly, increasing SNHG1 promoted HMGB1 expression, activated pyroptosis, but inhibited osteogenic differentiation. Silencing HMGB1 or inhibiting caspase-1 partially rescued the inhibitory effect of SNHG1 on osteogenic differentiation. Our findings indicate that SNHG1 suppresses the osteogenic differentiation of BMSCs by activating pyroptosis through interaction with HMGB1 and promotion of HMGB1 expression. Our work provides further evidence supporting SNHG1 acts as a potential target for OP treatment, and reveals for the first time that SNHG1 regulates osteogenic differentiation by affecting pyroptosis.

Salidroside Ameliorates Ischemia/Reperfusion-Induced Human Cardiomyocyte Injury by Inhibiting the Circ_0097682/miR-671-5p/USP46 Pathway.

Salidroside shows an inhibitory effect on myocardial ischemia/reperfusion (I/R) injury; however, the underlying mechanism remains to be explored. The present work analyzes the mechanism that drives salidroside to ameliorate I/R-induced human cardiomyocyte injury. Human cardiomyocytes were subjected to I/R treatment to simulate a myocardial infarction cell model. Cell viability, cell proliferation, and cell apoptosis were analyzed by CCK-8 assay, EdU assay, and flow cytometry analysis, respectively. RNA expression levels of circ_0097682, miR-671-5p, and F-box and ubiquitin-specific peptidase 46 (USP46) were detected by qRT-PCR. Protein expression was measured by Western blotting assay. The levels of IL-6, IL-1β, and TNF-α in cell supernatant were detected by enzyme-linked immunosorbent assays. Salidroside treatment relieved I/R-induced inhibitory effect on AC16 cell proliferation and promoting effects on cell apoptosis, inflammation, and oxidative stress. Salidroside inhibited circ_0097682 expression in I/R-treated AC16 cells. Salidroside-mediated inhibition of I/R-induced cell injury involved the downregulation of circ_0097682 expression. In addition, circ_0097682 bound to miR-671-5p in AC16 cells, and miR-671-5p inhibitors rescued salidroside pretreatment-mediated effects in I/R-treated AC16 cells. Moreover, miR-671-5p targeted USP46 in AC16 cells, and USP46 introduction partially relieved circ_0097682 depletion or salidroside pretreatment-induced effects in I/R-treated AC16 cells. Salidroside ameliorated I/R-induced AC16 cell injury by inhibiting the circ_0097682/miR-671-5p/USP46 pathway.

HDAC3-mediated lncRNA ZFAS1 inhibited IL-13-induced secretion of proinflammatory cytokines in nasal epithelial cells by regulating the miR-7-5p/SIRT1 pathway.

Allergic rhinitis (AR) is a disease that is difficult to cure and accompanies the patient's life. Proinflammatory cytokines (GM-CSF and eotaxin) and MUC5AC are key mediators promoting AR progression. Herein, the function of lncRNA ZFAS1 in AR was investigated. Nasal epithelial cells (NECs) were subjected to 50ng/mL IL-13 for 24h to construct an AR cell model. The mRNA and protein expressions were assessed using qRT-PCR and western blot. The levels of GM-CSF, eotaxin, IL-1β, IL-6, TNF-α and MUC5AC in cell supernatant were examined by ELISA. The binding relationships between HDAC3, ZFAS1, miR-7-5p and SIRT1 were analysed using dual luciferase reporter or ChIP assays. Herein, our results displayed that ZFAS1 and SIRT1 were lowly expressed in AR, while miR-7-5p and HDAC3 were highly expressed. Functional experiments displayed that ZFAS1 overexpression suppressed IL-13-induced proinflammatory cytokines and mucin production in NECs. The highly expressed HDAC3 in AR inhibited ZFAS1 expression by binding with ZFAS1 promoter. In addition, our experiments revealed that ZFAS1 targeted miR-7-5p, and miR-7-5p targeted SIRT1. As expected, miR-7-5p overexpression or SIRT1 silencing abrogated ZFAS1 upregulation's repression on IL-13-induced proinflammatory cytokines and MUC5AC secretory levels in NECs. ZFAS1 suppressed proinflammatory cytokines, inflammatory cytokines, and MUC5AC secretory levels in AR by regulating the miR-7-5p/SIRT1 axis. Thus, our work suggested that ZFAS1 might serve as a novel target for AR treatment and prevention.

Role and significance of SIRT1 in regulating the LPS-activated pyroptosis pathway in children with congenital hydronephrosis

ObjectiveTo explore the characteristics and mechanism of sirtuin 1 (SIRT1) in lipopolysaccharide (LPS)-activated pyroptosis in the renal tissue of children with congenital hydronephrosis (CHn).MethodsWe detected the expression characteristics and clinical...

Down-regulation of Toll-like Receptor 2 Via HMGB1 Blocking With Kaempferol on Kupffer Cells From Hepatocellular Carcinoma Patients

Background: Kaempferol is a pharmacological kaempferol that can inhibit many inflammatory factors implicated in cancer. Methods: The present study focused on kaempferol function on the immunity signaling pathway. In this study, Kupffer cells were also isolated with new methods. Results: Our results indicated that the levels of interleukin 17 (IL-17), IL-33, and IL-6 in supernatants from cultured Kupffer cells from patients were increased after stimulation with high mobility group box-1 (HMGB1), but the levels of these cytokines decreased after using kaempferol to prevent inflammation. Conclusion: The findings of this study imply that the reduction of the Toll-like receptor 2 (TLR2) pathway and T helper 17 cell polarization may be due to HMGB1 blocking with Kaempferol in hepatocellular carcinoma.

Lactobacillus Probiotic Strains Differ in Their Ability to Adhere to Human Lung Epithelial Cells and to Prevent Adhesion of Clinical Isolates of Pseudomonas aeruginosa from Cystic Fibrosis Lung.

The field of probiotic applications is rapidly expanding, including their use for the control of respiratory tract infections. Nevertheless, probiotics ability to colonize the lung environment and to compete with pulmonary pathogens is still a poorly investigated research area. In this study, we aimed to evaluate the adhesion ability of a number of commercial probiotic strains to the human lung epithelial cell line A549. Furthermore, we assessed probiotic ability to prevent host cell adhesion of one of the major lung pathogens in cystic fibrosis, Pseudomonas aeruginosa, and to reduce the pathogen-induced inflammatory response of human peripheral blood mononuclear cells (PBMCs) in terms of cytokine release. Lactobacillus acidophilus displayed the highest adhesion ability to A549 cells evaluated as percent of adhered bacteria compared to the inoculum. In agreement with such an observation, L. acidophilus was the most efficient in preventing adhesion to A549 cells of a P. aeruginosa isolate from CF sputum. Three-color fluorescence labeling of A549 cells, P. aeruginosa, and L. acidophilus, and confocal microcopy image analyses revealed a likely exclusion effect played by both live and UV-killed L. acidophilus towards P. aeruginosa. Such results were confirmed by CFU count. When co-cultured with PBMCs, both live and UV-killed L. acidophilus reduced the amount of IL-1β and IL-6 in culture supernatants in a statistically significant manner. Overall, the results obtained point to L. acidophilus as an interesting candidate for further studies for a potential aerogenous administration to control P. aeruginosa infections.

1496-P: Resatorvid, an Inhibitor of Toll-Like Receptor-4, Protects Allogenic Islets from Inflammatory and Immune Response

Purpose: Loss of islet mass by instant blood mediated inflammatory reaction (IBMIR) and immunological rejection are major hurdles currently facing the field of islet transplantation. Toll-like receptor 4 (TLR4) plays a central role in inflammation and shaping adaptive immune response. We investigated the protective effects of resatorvid (TAK-242) using in vitro models of islet transplantation. Methods: Soluble TAK-242 or TAK-242 conjugated to the surface of islets utilizing copper-free click chemistry with a cleavable chemical linker were used. Islets were mixed with allogenic blood for a period of 3 hours and plasma levels of miRNA-375 and miRNA-200c were analyzed by RT-qPCR. Allogenic peripheral blood mononuclear cells were cultured with islets. T cell and monocyte activation were monitored by flow cytometry. Cytokine and miRNA levels in culture supernatant were measured by Luminex assay and RT-qPCR respectively. Results: The presence of TAK-242 in soluble form as well as released by cleavable linker from islet surface significantly reduced the release of miRNA-375 (p&amp;lt;0.01) and miRNA-200c (p&amp;lt;0.01) indicating less islet damage. In allogenic co-culture of islets and PBMCs, TAK-242 inhibited the expression of CD69 in T cells as well as decreased (p&amp;lt;0.05) the expression of CD86 and CD80 in monocytes. The production of cytokines IL-1β (p&amp;lt;0.001), IL-6 (p=0.01), IL-8 (p&amp;lt;0.05), IP10 (p&amp;lt;0.05), MCP-1 (p&amp;lt;0.001) and TNFα (p=0.005) in culture supernatant was decreased in the presence of soluble TAK-242 as well as TAK-242 released from islet surfaces. Conclusions: Inhibition of TLR4 using small molecule, TAK-242, protected islets from innate damage due to IBMIR as well as adaptive responses of activation of T cells and monocytes. These results validate the future study of inhibition of TLR4 using in vivo transplant model(s) to further investigate the efficacy of TLR4 inhibition to improve islet transplant survival and function. Disclosure J.D.Mattke: None. B.Naziruddin: None. B.Kane: None.