Structural Analysis of Breast-Milk αS1-Casein: An α-Helical Conformation Is Required for TLR4-Stimulation.

Breast-milk αS1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated αS1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of αS1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V77-E92 of αS1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that αS1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of αS1-casein, as described before, could be regulated by conformational changes induced by phosphorylation.

Polysaccharides from sea buckthorn (Hippophae rhamnoides L.) berries ameliorate cognitive dysfunction in AD mice induced by a combination of d-gal and AlCl3 by suppressing oxidative stress and inflammation reaction.

BACKGROUND: The therapeutic properties of Hippophae rhamnoides L. were known in Ancient Greece and in Tibetan and Mongolian medicine, which commonly used it for the treatment of heart ailments, rheumatism, and brain disorders. Modern studies have indicated that Hippophae rhamnoides L. polysaccharide (HRP) can improve cognitive impairment in mice with Alzheimer's disease (AD) but the specific mechanisms of the protective effect of HRP have not been elucidated fully. RESULTS: Our results showed that Hippophae rhamnoides L. polysaccharide I (HRPI) improved pathological behaviors related to memory and cognition, and reduced 1 Beta-amyloid (Aß) peptide deposition and neuronal cell necrosis. Pretreatment with Hippophae rhamnoides L. polysaccharide I (HRPI) also decreased the level of Toll-like receptor 4 (TLR4) and Myeloid differentiation factor 88 (MyD88), and reduced the release of inflammatory factors Tumor necrosis factor alpha (TNFα) and interleukin 6 (IL-6) in the brains of mice with AD. Treatment with HRPI also suppressed the expression level of Recombinant Kelch Like ECH Associated Protein 1 (KEAP1), and increased the levels of Nuclear factor erythroid 2-Related Factor 2 (Nrf2), antioxidant enzymes Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px) in the brains of AD mice. CONCLUSIONS: On the whole, these findings revealed that HRPI could improve the learning and memory ability and attenuate pathologic impairment in AD mice, and the underlying mechanisms may involve mediating oxidative stress and inflammation, possibly through the regulation of the Keap1/Nrf2 and TLR4/MyD88 signaling pathways. © 2023 Society of Chemical Industry.

Higher Levels of Triglyceride, Fatty Acid Translocase, and Toll-Like Receptor 4 and Lower Level of HDL-C in Pregnant Women with GDM and Their Close Correlation with Neonatal Weight.

OBJECTIVES: In this study, we aimed to compare the levels of maternal blood lipids, placental and venous blood lipid transporters, and inflammatory factor receptors in pregnant women with and without gestational diabetes mellitus (GDM). We also aimed to figure out the relationship between these values and neonatal weight. METHODS: Fifty pregnant women with GDM under blood glucose control belong to the case group, and 50 pregnant women with normal glucose tolerance in concurrent delivery belong to the control group. Fasting venous blood of these pregnant women was taken 2 weeks before delivery, and umbilical cord blood was collected after delivery. The levels of triglyceride (TG), serum total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol (HDL-C) in maternal blood and umbilical cord blood were tested in the laboratory department of our hospital. The level of toll-like receptor 4 (TLR4) in serum of umbilical veins was detected by the double-antibody sandwich ELISA. Western blot and RT-PCR were used to detect the protein and mRNA expressions of TLR4, LPL, and FAT/CD36 in the placenta. RESULTS: The level of TG in maternal blood in the case group was remarkably higher than that in the control group, which was opposite to the level of HDL-C. In the umbilical cord blood of women with GDM, the expression of TLR4 increased and was closely correlated with neonatal weight. In the placenta of women with GDM, the expressions of FAT/CD36 and TLR4 increased, and both of them were closely correlated with neonatal weight. Besides, TLR4 in umbilical cord blood increased and was closely correlated with neonatal weight. Although the expression of LPL in the placenta decreased, it had no obvious correlation with neonatal weight. CONCLUSIONS: TG in maternal blood, TLR4 in the placenta and umbilical cord blood, and FAT/CD36 in the placenta were positively correlated with neonatal weight. However, HDL-C in maternal blood was negatively correlated with neonatal weight. Although the expression of LPL in the placenta reduced due to GDM, it had no correlation with neonatal weight.

Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response.

Ischemic stroke is a leading cause of death among human in the world, and a critical cause for long-term disability. Accumulating studies have indicated that inflammatory response regulated by microglia contributes a lot to neuronal death, but the molecular mechanism still remains unclear. V-set and immunoglobulin domain-containing 4 (Vsig4), a complement receptor of the immunoglobulin superfamily (CRIg) that specifically expresses in resting tissue-resident macrophages, plays a critical role in regulating various inflammatory diseases via multiple signaling pathways. However, the effects of Vsig4 on ischemic stroke have not been investigated. In this study, we identified that Vsig4 expression was decreased after cerebral ischemic injury induced by middle cerebral artery occlusion (MCAO). Immunofluorescence staining showed that Vsig4 was co-localized with Iba1 in microglial cells from the infarct region of MCAO-operated mice. After over-expressing Vsig4 in mice, MCAO-induced infarction area and neurological deficits score were markedly attenuated. In addition, neurological dysfunction due to MCAO surgery was improved by Vsig4 over-expression. Microglial M1 polarization was detected in mice with MCAO surgery, which was markedly inhibited by Vsig4 over-expression, as evidenced by the markedly reduced expression of CD16, CD11b, inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6); however, the expression of M2-like phenotype hallmarks such as arginase 1 (Arg1), CD206, IL-10 and Ym-1 was significantly up-regulated. Mechanistically, the anti-inflammatory role of Vsig4 was mainly through the blockage of toll-like receptor 4/nuclear factor kappa B (TLR4/NF-κB) signaling via the in vivo and in vitro experiments. Also, we found that microglial TLR4 expression in the cerebral infarct area of MCAO mice was highly suppressed by Vsig4 over-expression. In vitro, the neuron-glial mixed culture by fluorescent staining showed that oxygen glucose deprivation (OGD) treatment led to significant cell death, while being attenuated by Vsig4 over-expression in primary microglial cells. Finally, we showed that Vsig4 could interact with TLR4 and repress its expression, subsequently alleviating ischemic stroke. Collectively, our findings demonstrated that microglial Vsig4 protected against post-stroke neuro-inflammation mainly through interacting with TLR4.

Expression profile of Toll-like receptor 4 in rat testis and epididymis throughout postnatal development.

Toll-like receptors (TLRs) belonging to pattern recognition receptors are involved in maintaining testicular and epididymal immune homeostasis. The purpose of the current study was to investigate TLR4 expression in rat testis and epididymis throughout postnatal development. Weak staining was detected in peritubular myoid cells and immature Sertoli cells while no staining was observed in gonocytes during prepubertal period. However, TLR4 expression began to appear in spermatocytes in pubertal period and gradually increased in spermatids. An intense staining was observed in steps 5-19 spermatids in post pubertal and mature periods. Similarly, TLR4 expression in the testes steadily increased from pubertal period to mature period. Puberty also caused a significant increase in TLR4 expression in epididymis. TLR4 expression in cauda epididymis was lower as compared to those of other epididymal segments. The majority of epididymal epithelial cells exhibited apical TLR4 expression, whereas basal cells showed intense intracytoplasmic immunoreaction. We detected an intense staining in epididymal smooth muscle cells. The expression levels of TLR4 showed dynamic changes in both spermatogenic cells, and entire testicular and epididymal tissues during postnatal development. These results suggest that TLR4 expression contributes not only to inflammation but also to the development of spermatogenic cells.

Pharmacological Characters of Oliceridine, a µ-Opioid Receptor G-Protein-Biased Ligand in Mice.

BACKGROUND: A major advancement in the field of analgesic pharmacology has been the development of G-protein-biased opioid agonists that display less respiratory depression than conventional drugs. It is uncertain, however, whether these new drugs cause less tolerance, hyperalgesia, and other maladaptations when administered repeatedly. METHODS: The archetypical µ-opioid receptor agonist morphine and, separately, the G-protein-biased µ-opioid receptor agonist oliceridine were administered to mice. These drugs were used in models of acute analgesia, analgesic tolerance, opioid-induced hyperalgesia, reward, and physical dependence. In addition, morphine and oliceridine were administered for 7 days after tibia fracture and pinning; mechanical allodynia and gait were followed for 3 weeks. Finally, the expression of toll-like receptor-4 and nacht domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NALP3) and interleukin-1ß mRNA were quantified in spinal tissue to measure surgical and drug effects on glia-related gene expression. RESULTS: We observed using the tail flick assay that oliceridine was a 4-fold more potent analgesic than morphine, but that oliceridine treatment caused less tolerance and opioid-induced hyperalgesia than morphine after 4 days of ascending-dose administration. Using similar analgesic doses, morphine caused reward behavior in the conditioned place preference assay while oliceridine did not. Physical dependence was, however, similar for the 2 drugs. Likewise, morphine appeared to more significantly impair the recovery of nociceptive sensitization and gait after tibial fracture and pinning than oliceridine. Furthermore, spinal cord toll-like receptor-4 levels 3 weeks after fracture were higher in fracture mice given morphine than those given oliceridine. CONCLUSIONS: Aside from reduced respiratory depression, G-protein-biased agonists such as oliceridine may reduce opioid maladaptations and enhance the quality of surgical recovery.

Knockdown XIST alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via targeting miR-370-3p/TLR4 in acute pneumonia.

Pneumonia is an inflammatory disease that occurs in the lungs associated with pathogens or other factors. It has been well established that long noncoding RNA X inactivate-specific transcript (XIST) is involved in several cancers. The present study focused on the effect and detailed mechanism of XIST in lipopolysaccharide (LPS)-induced injury in pneumonia. Here, XIST was silenced by transfection with XIST-targeted siRNA, and then, mRNA expression, cell viability, apoptosis, and protein expression were, respectively, assessed by qRT-PCR, CCK-8, flow cytometry, and Western blotting. Luciferase reporter, RIP, and RNA pull-down assays were used to detect the combination of miR-370-3p and XIST. Besides, the tested proinflammatory factors were analysed by qRT-PCR and Western blot, and their productions were quantified by ELISA. The results showed that XIST expression was robustly increased in serum of patients with acute-stage pneumonia and LPS-induced WI-38 human lung fibroblasts cells. Functional analyses demonstrated that knockdown of XIST remarkably alleviated LPS-induced cell injury through increasing cell viability and inhibiting apoptosis and inflammatory cytokine levels. Mechanistically, XIST functioned as a competitive endogenous RNA (ceRNA) by effectively binding to miR-370-3p and then restoring TLR4 expression. More importantly, miR-370-3p inhibitor abolished the function of XIST knockdown on cell injury and JAK/STAT and NF-κB pathways. Taken together, XIST may be involved in progression of cell inflammatory response, and XIST/miR-370-3p/TLR4 axis thus may shed light on the development of novel therapeutics to the treatment of acute stage of pneumonia. SIGNIFICANCE OF THE STUDY: Our study demonstrated that XIST was highly expressed in patients with acute stage of pneumonia. Knockdown of XIST remarkably alleviated LPS-induced cell injury through increasing cell viability and inhibiting apoptosis and inflammatory cytokine levels through regulating JAK/STAT and NF-κB pathways.

Intrarenal Toll-like receptor 4 and Toll-like receptor 2 expression correlates with injury in antineutrophil cytoplasmic antibody-associated vasculitis.

In antineutrophil cytoplasmic antibody-associated vasculitis (AAV), Toll-like receptors (TLRs) may be engaged by infection-associated patterns and by endogenous danger signals, linking infection and innate inflammation with this autoimmune disease. This study examined intrarenal TLR2, TLR4, and TLR9 expression and renal injury in AAV, testing the hypothesis that increased TLR expression correlates with renal injury. Patients with AAV exhibited both glomerular and tubulointerstitial expression of TLR2, TLR4, and TLR9, with TLR4 being the most prominent in both compartments. Glomerular TLR4 expression correlated with glomerular segmental necrosis and cellular crescents, with TLR2 expression correlating with glomerular segmental necrosis. The extent and intensity of glomerular and tubulointerstitial TLR4 expression and the intensity of glomerular TLR2 expression inversely correlated with the presenting estimated glomerular filtration rate. Although myeloid cells within the kidney expressed TLR2, TLR4, and TLR9, TLR2 and TLR4 colocalized with endothelial cells and podocytes, whereas TLR9 was expressed predominantly by podocytes. The functional relevance of intrarenal TLR expression was further supported by the colocalization of TLRs with their endogenous ligands high-mobility group box 1 and fibrinogen. Therefore, in AAV, the extent of intrarenal TLR4 and TLR2 expression and their correlation with renal injury indicates that TLR4, and to a lesser degree TLR2, may be potential therapeutic targets in this disease.

Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.

BACKGROUND: Trauma is the leading cause of death and disability in patients aged 1-46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood. METHODS AND FINDINGS: We developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean [95% CI] 106.4 µl [88.5-124.3] for fresh RBCs and 192.5 µl [140.9-244.0] for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean [95% CI] 1.2 × 10(7) [-1.0 × 10(7) to 2.5 × 10(7)] for fresh RBCs and 3.6 × 10(7) [2.5 × 10(7) to 4.7 × 10(7)] for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa-induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean [95% CI] 15.4 ng/ml [6.7-24.0] for fresh RBCs and 50.3 ng/ml [12.3-88.2] for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar-millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients. CONCLUSIONS: We provide evidence that large volume resuscitation with stored blood, compared to fresh blood, in mice increases mortality from subsequent pneumonia, which occurs via mechanisms sensitive to hemopexin and TLR4 and HMGB1 inhibition.

Ectodomain Shedding of RAGE and TLR4 as a Negative Feedback Regulation in High-Mobility Group Box 1-Activated Aortic Endothelial Cells.

BACKGROUND/AIMS: High-mobility group box 1 (HMGB1) elicits inflammatory responses through interactions with the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4). We investigated how RAGE and TLR4 expressions are regulated after HMGB1 stimulation in cultured human aortic endothelial cells (HAECs). METHODS: RAGE and TLR4 expressions were analyzed by Western blot analysis and immunofluorescence staining. A disintegrin and metalloprotease 17 (ADAM17) activity was measured using a fluorogenic substrate. RESULTS: Upon treatment with HMGB1, both RAGE and TLR4 began to decrease in cell lysate and remained decreased up to 24 h. The decrease in cellular RAGE and TLR4 was accompanied by an increase of N-terminal fragment of RAGE and TLR4 in culture supernatant, indicating ectodomain shedding of the receptors. HMGB1 activated p38 mitogen-activated protein kinase (p38 MAPK) and ADAM17, while HMGB1-induced ADAM17 activation was inhibited by SB203580, a p38 MAPK inhibitor. HMGB1-induced ectodomain shedding of RAGE and TLR4 was prevented by siRNA depletion of ADAM17 as well as TAPI-2, an inhibitor of ADAM family, and SB203580. HMGB1 pretreatment abolished p38 MAPK activation in response to 2nd HMGB1 stimulation. In the cells depleted of ADAM17, HMGB1-induced p38 MAPK activation was prolonged. siRNA depletion of RAGE, but not TLR4, suppressed HMGB1-induced p38 MAPK activation. CONCLUSION: In response to HMGB1 stimulation, HAECs rapidly undergo ectodomain shedding of RAGE and TLR4, and thereby become insensitive to further HMGB1 stimulation. ADAM17, activated through RAGE-p38 MAPK pathway, is implicated in the ectodomain cleavage of the receptors.

Glycyrrhizin Treatment Facilitates Extinction of Conditioned Fear Responses After a Single Prolonged Stress Exposure in Rats.

BACKGROUND/AIMS: Impaired fear memory extinction is widely considered a key mechanism of post-traumatic stress disorder (PTSD). Recent studies have suggested that neuroinflammation after a single prolonged stress (SPS) exposure may play a critical role in the impaired fear memory extinction. Studies have shown that high mobility group box chromosomal protein 1 (HMGB-1) is critically involved in neuroinflammation. However, the role of HMGB-1 underlying the development of impairment of fear memory extinction is still not known. METHODS: Thus, we examined the levels of HMGB-1 in the basolateral amygdala (BLA) following SPS using Western blot and evaluated the levels of microglia and astrocytes activation in the BLA after SPS using immunohistochemical staining. We then examined the effects of pre-SPS intra-BLA administration of glycyrrhizin, an HMGB1 inhibitor, or LPS-RS, a competitive TLR4 antagonist, on subsequent post-SPS fear extinction. RESULTS: We found that SPS treatment prolonged the extinction of contextual fear memory after the SPS. The impairment of SPS-induced extinction of contextual fear memory was associated with increased HMGB1 and Toll-like receptor 4 (TLR4) levels in the BLA. Additionally, the impairment of SPS-induced extinction of contextual fear memory was associated with increased activation of microglia and astrocyte in the BLA. Intra-BLA administrations of glycyrrhizin (HMGB-1 inhibitor) or LPS-RS (TLR4 antagonist) can prevent the development of SPS-induced fear extinction impairment. CONCLUSION: Taken together, these results suggested that SPS treatment may not only produce short term effects on the HMGB1/TLR4-mediated pro-inflammation, but alter the response of microglia and astrocytes to the exposure to fear associated contextual stimuli.

Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor-Adaptor Molecular Dynamics.

Single-molecule imaging (SMI) has been widely utilized to investigate biomolecular dynamics and protein-protein interactions in living cells. However, multicolor SMI of intracellular proteins is challenging because of high background signals and other limitations of current fluorescence labeling approaches. To achieve reproducible intracellular SMI, a labeling probe ensuring both efficient membrane permeability and minimal non-specific binding to cell components is essential. We developed near-infrared fluorescent probes for protein labeling that specifically bind to a mutant ß-lactamase tag. By structural fine-tuning of cell permeability and minimized non-specific binding, SiRcB4 enabled multicolor SMI in combination with a HaloTag-based red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar concentrations, single-molecule imaging revealed the dynamics of TLR4 and its adaptor protein, TIRAP, which are involved in the innate immune system. Statistical analysis of the quantitative properties and time-lapse changes in dynamics revealed a protein-protein interaction in response to ligand stimulation.

Plasminogen Activator Inhibitor 1 Promotes Immunosuppression in Human Non-Small Cell Lung Cancers by Enhancing TGF-Β1 Expression in Macrophage.

BACKGROUND: Plasminogen activator inhibitor-1 (PAI-1) has been regarded as a risk factor for thrombosis and atherosclerosis. Since it has been shown that PAI-1 can activate macrophages through Toll-like receptor-4, we sought to investigate the role of PAI-1 in the tumor microenvironment. METHODS: The expression and distribution patterns of PAI-1 and transforming growth factor beta (TGF-ß) were measured in 60 non-small cell lung cancer (NSCLC) tumors. A statistical correlation analysis was performed between PAI-1 and TGF-ß expression and distribution in each tumor. The distribution of tumor-associated macrophages (TAMs) was also measured and its correlation to PAI-1 levels was analyzed. Levels of secreted CCL-17, CCL-22, IL-6 and TGF-ß were measured in cell cultures of human macrophage cell lines THP-1 and U937 treated with PAI-1. Levels of secreted PAI-1 were monitored in cell cultures of human NSCLCs cell lines 95D and A549 treated with TGF-ß. Secreted proteins were measured in cell culture supernatants using ELISA. Changes in downstream signaling pathways were investigated using western blot. RESULTS: PAI-1 and TGF-ß were found to be overexpressed in human NSCLCs. PAI-1 expression was tightly correlated to TGF-ß expression as well as the percentage of TAMs. PAI-1 treatment increased the expression of TAM-associated cytokines and chemokines, including CCL-17, CCL-22, and IL-6. PAI-1 treatment was also observed to enhance TGF-ß expression in macrophage cell lines through an IL-6 autocrine/paracrine manner. The effects on TGF-ß expression were blocked by NF-κB and STAT3 inhibition. Interestingly, TGF-ß also increased levels of secreted PAI-1 in NSCLC cells through SMAD3-dependent signaling, therefore resulting in a feed-forward loop. However, this loop could be blocked by NF-κB, STAT3 and SMAD3 signaling inhibition, as well as treatment with a high concentration of TGF-ß. CONCLUSION: PAI-1 and TGF-ß promote NSCLC tumor cells and TAMs and might be valuable targets for cancer immunosuppression.

Knockdown of Angiopoietin-Like Protein 2 Ameliorates Diabetic Nephropathy by Inhibiting TLR4.

BACKGROUND/AIMS: Angiopoietin-like protein 2 (ANGPTL2) was reported to be implicated in the pathogenesis of inflammatory disease. Its role in diabetic nephropathy (DN) remained illdefined. METHODS: qRT-PCR and western blot analysis were performed to detect the expressions of ANGPTL2 or TLR4 in streptozotocin (STZ)-induced DN rats and HG-stimulated podocytes. The renal injury index including 24-h proteinuria, blood glucose level, serum creatinine and blood urea nitrogen were measured in DN rats using corresponding commercial kits. The effect of ANGPTL2 knockdown on the secretion or expression of inflammatory cytokines was detected by ELISA or qRT-PCR analysis. The effect of ANGPTL2 knockdown on extracellular matrix (ECM) accumulation was determined by testing TGF-ß1, Collagen-IV, fibronectin (FN) and PTEN expression via western blot. RESULTS: ANGPTL2 and TLR4 were both highly expressed in DN rats compared with control group. ANGPTL2 knockdown alleviated renal injury in STZ-induced DN rat model. ANGPTL2 knockdown also suppressed inflammatory cytokines (IL-6, TNF-α, MCP-1, IL-1ß) expression and ECM accumulation (TGF-ß1, Collagen-IV, FN, PTEN) in HG-induced podocytes. Moreover, ANGPTL2 knockdown led to a significant decrease of TLR4 expression in both DN rat and cell model. Furthermore, TAK-242 treatment exacerbated the inhibitory effect of ANGPTL2 knockdown on inflammatory cytokines expression and ECM accumulation in HG-induced podocytes. CONCLUSION: ANGPTL2 knockdown ameliorates DN by inhibiting TLR4 expression, an observation contributing to a better understanding of DN pathogenesis.

Effect of Shikonin on Spinal Cord Injury in Rats Via Regulation of HMGB1/TLR4/NF-kB Signaling Pathway.

BACKGROUND/AIMS: Shikonin, a compound extracted from Zicao, has been demonstrated to hold anti-bacterial, anti-inflammatory, and anti-tumor activities in various diseases and it has been shown to protect human organs from injuries. However, the effect of shikonin on the recovery of spinal cord injury (SCI) remains unknown. This study was designed to estimate the potential therapeutic effect and underlying mechanism of shikonin on SCI in vivo. METHODS: In the study, we used HE staining, ELISA assay, transfection assay, TUNEL assay, real time PCR and Western blot to detect the effects of shikonin on spinal cord injury in rats. RESULTS: we showed that shikonin could promote the recovery of motor function and tissue repair after SCI treatment in rats SCI model. Moreover, we demonstrated that shikonin inhibited the spinal cord edema in SCI model of rats. According to further investigation, shikonin induced the reduction of inflammatory response through decreasing the expression levels of HMGB1, TLR4 and NF-κB after SCI injury. In addition, we also found that shikonin could suppress the apoptosis and expression of caspase-3 protein in SCI model of rats. CONCLUSION: Our results demonstrated that shikonin induced the recovery of tissue repair and motor function via inactivation of HMGB1/TLR4/NF-κB signaling pathway in SCI model of rats. Meanwhile, shikonin regulated the inflammation response in SCI by suppressing the HMGB1/TLR4/NF-κB signaling pathway. The described mechanism sheds novel light on molecular signaling pathway in spinal cord injury and secondary injury including inflammatory response.

Farnesoid X receptor agonist GW4064 ameliorates lipopolysaccharide-induced ileocolitis through TLR4/MyD88 pathway related mitochondrial dysfunction in mice.

Inflammatory bowel disease (IBD) is a complex and relapsing inflammatory condition of the gastro intestinal tract characterized by diarrhoea and abdominal pain. Farnesoid X receptor (FXR) plays an important role in enteroprotection and mucosal injury by regulating inflammatory responses and barrier function in the intestinal tract. Here we show the mechanisms of FXR agonist, GW4064, inhibits mucosal injury in ileum caused by lipopolysaccharides (LPS). Ileum injury was induced by intraperitoneal injection of LPS in Wild-type (WT) and FXR knockout (KO) mice. GW4064 alleviates LPS-mediated tight junction dysfunction as well as macrophage infiltration in WT mice, but not in FXR KO mice. Interesting, GW4064 suppresses NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome mediates tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1ß, as well as mitochondrial respiratory complexes mRNA expression in WT and FXR KO mice treated with LPS. This results demonstrated that central roles of FXR in coordinating regulation of both inflammation and mitochondrial dysfunction. We propose that GW4064 is promising therapeutic agent for treatment of ileocolitis.

Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways.

Obstructive sleep apnea (OSA) is associated with nonalcoholic fatty liver disease (NAFLD), and causes chronic intermittent hypoxia (CIH) during sleep. Inflammation is associated with the development of metabolic complications induced by CIH. Research suggests that innate immune mechanisms are involved in the pro-inflammatory pathways of liver fibrosis. The purpose of this study was to investigate whether innate immune responses induce liver fibrosis, and to evaluate mechanisms underlying hepatic inflammation related to CIH in a murine diet-induced obesity (DIO) model. Inflammatory and oxidative stress markers, TLR4, MyD88, Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-ß (TRIF), I-κB, NF-κB, p38 MAPK, c-JNK, and ERK activation, were measured in the serum and liver. As a result, α1(I)-collagen mRNA was significantly higher in DIO mice exposed to CIH than in the control groups. CIH mice exhibited liver fibrosis and significantly higher protein expression of TLR4, MyD88, phosphorylated (phospho-) I-κB, and phospho-ERK1/2 activation in the liver, and higher expression of NF-κB than that in the controls. TRIF, p38 MAPK, and JNK activation did not differ significantly between groups. We conclude that CIH in DIO mice leads to liver fibrosis via TLR4/MyD88/MAPK/NF-kB signaling pathways.

Coronary neutrophil extracellular trap burden and deoxyribonuclease activity in ST-elevation acute coronary syndrome are predictors of ST-segment resolution and infarct size.

RATIONALE: Mechanisms of coronary occlusion in ST-elevation acute coronary syndrome are poorly understood. We have previously reported that neutrophil (polymorphonuclear cells [PMNs]) accumulation in culprit lesion site (CLS) thrombus is a predictor of cardiovascular outcomes. OBJECTIVE: The goal of this study was to characterize PMN activation at the CLS. We examined the relationships between CLS neutrophil extracellular traps (NETs), bacterial components as triggers of NETosis, activity of endogenous deoxyribonuclease, ST-segment resolution, and infarct size. METHODS AND RESULTS: We analyzed coronary thrombectomies from 111 patients with ST-elevation acute coronary syndrome undergoing primary percutaneous coronary intervention. Thrombi were characterized by immunostaining, flow cytometry, bacterial profiling, and immunometric and enzymatic assays. Compared with femoral PMNs, CLS PMNs were highly activated and formed aggregates with platelets. Nucleosomes, double-stranded DNA, neutrophil elastase, myeloperoxidase, and myeloid-related protein 8/14 were increased in CLS plasma, and NETs contributed to the scaffolds of particulate coronary thrombi. Copy numbers of Streptococcus species correlated positively with dsDNA. Thrombus NET burden correlated positively with infarct size and negatively with ST-segment resolution, whereas CLS deoxyribonuclease activity correlated negatively with infarct size and positively with ST-segment resolution. Recombinant deoxyribonuclease accelerated the lysis of coronary thrombi ex vivo. CONCLUSIONS: PMNs are highly activated in ST-elevation acute coronary syndrome and undergo NETosis at the CLS. Coronary NET burden and deoxyribonuclease activity are predictors of ST-segment resolution and myocardial infarct size.

Interleukin 17A and Toll-like Receptor 4 in Patients with Arterial Hypertension.

BACKGROUND/AIMS: Immune responses are involved in arterial hypertension. An observational cross-sectional case control study was conducted to estimate the association between Toll-like receptor 4 (TLR4) expression and interleukin (IL)-17A serum levels in patients with controlled and non-controlled hypertension. METHODS: We have enrolled 105 non-complicated otherwise healthy hypertensive patients: 53 with well-controlled blood pressure and 52 non-controlled. TLR4 peripheral monocytes expression and serum IL-17A levels were determined by flow cytometry and ELISA, respectively. RESULTS: Non-controlled patients exhibited higher TLR4 expression than well-controlled (25.60 vs. 21.99, P=0.011). TLR4 expression was lower in well-controlled patients who were prescribed beta blockers (18.9 vs. 22.6, P=0.005) and IL-17A concentration was higher in patients using diuretics in either group (1.41 vs. 2.01 pg/ml, P<0.001; well-controlled 1.3 vs. 1.8 pg/ml, P= 0.023; non-controlled 1.6 vs. 2.3 pg/ml, P=0.001). Correlation between IL-17A concentration and hypertension duration was observed in non-controlled patients (Spearman correlation coefficient . ρ=0.566, P<0.001) whereas in well-controlled patients a correlation was found between hypertension duration and TLR4 expression (ρ=0.322, P=0.020). CONCLUSIONS: Arterial hypertension stimulates the immune response regardless of blood pressure regulation status. Prolonged hypertension influences peripheral monocyte TLR4 expression and IL-17A serum levels. Anti-hypertensive drugs have different immunomodulatory effects: diuretics are associated with higher IL-17A concentration and beta-blockers with lower TLR4 expression.