Search Results (27)

Stroke represents a significant public health burden, ranking as a leading cause of death and disability globally. The prevalence of stroke increases with age, with ischemic stroke accounting for nearly 87% of cases globally. The pathophysiology of stroke is characterized by neuronal injury, neuroinflammation, and oxidative stress, which exacerbate brain damage and hinder recovery. Myeloid Differentiation Factor 2 (MD2), an accessory protein of Toll-like receptor 4 (TLR4), has emerged as a key player in mediating inflammatory responses in stroke. This short review discusses the molecular mechanisms by which MD2 contributes to neuroinflammation and neuronal death following stroke and highlights MD2 as a promising therapeutic target for stroke treatment. Subsequently, we investigate the potential of MD2 inhibitors, their underlying mechanisms, and the therapeutic prospects of such inhibitors in reducing stroke-induced brain damage. Full article

Mesenchymal stem cells (MSCs) are one of the main residents in the bone marrow (BM) and have an essential role in the regulation of haematopoietic stem cell (HSC) differentiation and proliferation. Myelodysplastic syndromes (MDSs) are a group of myeloid disorders impacting haematopoietic stem and progenitor cells (HSCPs) that are characterised by BM failure, ineffective haematopoiesis, cytopenia, and a high risk of transformation through the expansion of MDS clones together with additional genetic defects. It has been indicated that MSCs play anti-tumorigenic roles such as in cell cycle arrest and pro-tumorigenic roles including the induction of metastasis in MDS and leukaemia. Growing evidence has shown that MSCs have impaired functions in MDS, such as decreased proliferation capacity, differentiation ability, haematopoiesis support, and immunomodulation function and increased inflammatory alterations within the BM through some intracellular pathways such as Notch and Wnt and extracellular modulators abnormally secreted by MSCs, including increased expression of inflammatory factors and decreased expression of haematopoietic factors, contributing to the development and progression of MDSs. Therefore, MSCs can be targeted for the treatment of MDSs and leukaemia. However, it remains unclear what drives MSCs to behave abnormally. In this review, dysregulations in MSCs and their contributions to myeloid haematological malignancies will be discussed. Full article

Serine/arginine-rich splicing factor 3 (SRSF3), the smallest member of the SR protein family, serves multiple roles in RNA processing, including splicing, translation, and stability. Recent studies have shown that SRSF3 is implicated in several inflammatory diseases. However, its impact on macrophage inflammation remains unclear. Herein, we determined the expression of SRSF3 in inflammatory macrophages and found that the level of SRSF3 was increased in macrophages within atherosclerotic plaques, as well as in RAW-264.7 macrophages stimulated by lipopolysaccharides. Moreover, the downregulation of SRSF3 suppressed the levels of inflammatory cytokines by deactivating the nuclear factor κB (NFκB) pathway. Furthermore, the alternative splicing of myeloid differentiation protein 2 (MD2), a co-receptor of toll-like receptor 4 (TLR4), is regulated by SRSF3. The depletion of SRSF3 increased the level of the shorter MD2B splicing variants, which contributed to inflammatory inhibition in macrophages. In conclusion, our findings imply that SRSF3 regulates lipopolysaccharide-stimulated inflammation, in part by controlling the alternative splicing of MD2 mRNA in macrophages. Full article

The SARS-CoV-2 Omicron variants have replaced all earlier variants, due to increased infectivity and effective evasion from infection- and vaccination-induced neutralizing antibodies. Compared to earlier variants of concern (VoCs), the Omicron variants show high TMPRSS2-independent replication in the upper airway organs, but lower replication in the lungs and lower mortality rates. The shift in cellular tropism and towards lower pathogenicity of Omicron was hypothesized to correlate with a lower toll-like receptor (TLR) activation, although the underlying molecular mechanisms remained undefined. In silico analyses presented here indicate that the Omicron spike protein has a lower potency to induce dimerization of TLR4/MD-2 compared to wild type virus despite a comparable binding activity to TLR4. A model illustrating the molecular consequences of the different potencies of the Omicron spike protein vs. wild-type spike protein for TLR4 activation is presented. Further analyses indicate a clear tendency for decreasing TLR4 dimerization potential during SARS-CoV-2 evolution via Alpha to Gamma to Delta to Omicron variants. Full article

Escherichia coli (E. coli) is a frequent gram-negative bacterium that causes nosocomial infections, affecting more than 100 million patients annually worldwide. Bacterial lipopolysaccharide (LPS) from E. coli binds to toll-like receptor 4 (TLR4) and its co-receptor’s cluster of differentiation protein 14 (CD14) and myeloid differentiation factor 2 (MD2), collectively known as the LPS receptor complex. LPCAT2 participates in lipid-raft assembly by phospholipid remodelling. Previous research has proven that LPCAT2 co-localises in lipid rafts with TLR4 and regulates macrophage inflammatory response. However, no published evidence exists of the influence of LPCAT2 on the gene expression of the LPS receptor complex induced by smooth or rough bacterial serotypes. We used RAW264.7—a commonly used experimental murine macrophage model—to study the effects of LPCAT2 on the LPS receptor complex by transiently silencing the LPCAT2 gene, infecting the macrophages with either smooth or rough LPS, and quantifying gene expression. LPCAT2 only significantly affected the gene expression of the LPS receptor complex in macrophages infected with smooth LPS. This study provides novel evidence that the influence of LPCAT2 on macrophage inflammatory response to bacterial infection depends on the LPS serotype, and it supports previous evidence that LPCAT2 regulates inflammatory response by modulating protein translocation to lipid rafts. Full article

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex is a key receptor of the innate immune system and a major driver of inflammation that is responsible for the multifaceted defense response to Gram-negative infections. However, dysfunction in the tightly regulated mechanisms of TLR4-mediated signaling leads to the uncontrolled upregulation of local and systemic inflammation, often resulting in acute or chronic disease. Therefore, the TLR4/MD-2 receptor complex is an attractive target for the design and development of anti-inflammatory therapies which aim to control the unrestrained activation of TLR4-mediated signaling. Complex structure–activity relationships and species-specificity behind ligand recognition by the TLR4/MD-2 complex complicate the development of MD-2-specific TLR4 antagonists. The restriction of the conformational flexibility of the disaccharide polar head group is one of the key structural features of the newly developed lipid A—mimicking glycophospholipids, which are potential inhibitors of TLR4-mediated inflammation. Since phosphorylation has a crucial influence on MD-2–ligand interaction, glycolipids with variable numbers and positioning of phosphate groups were synthesized and evaluated for their ability to inhibit TLR4-mediated pro-inflammatory signaling in human and murine immune cells. A bis-phosphorylated glycolipid was found to have nanomolar antagonist activity on human TLR4 while acting as a partial agonist on murine TLR4. The glycolipid inhibited mTLR4/MD-2-mediated cytokine release, acting as an antagonist in the presence of lipopolysaccharide (LPS), but at the same time induced low-level cytokine production. Full article

Acute kidney injury (AKI) is described as the abrupt decrease in kidney function always accompanied by inflammation. The roots of Oxybaphus himalaicus Edgew. have long been used in Tibetan folk medicine for the treatment of nephritis. Nevertheless, modern pharmacological studies, especially about the underlying mechanism of O. himalaicus medications, are still lacking. Here, in lipopolysaccharide (LPS)-induced RAW264.7 macrophages, the O. himalaicus extract (OE) showed significant anti-inflammatory activity with the dose dependently reducing the LPS-stimulated release of nitric oxide and the mRNA level and protein expression of inflammatory cytokines and reversed the activation of nuclear factor kappa B (NF-κB). Co-immunoprecipitation assay indicated that OE inhibited Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD2) complex formation and further suppressed both myeloid differentiation factor 88 (MyD88)-dependent and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent cascades activation. In addition, OE could restrain NADPH oxidase 2 (NOX2) endocytosis by blocking TLR4/MD2 complex formation to prevent reactive oxygen species production. In LPS-induced AKI mice, OE treatment mitigated renal injury and inflammatory infiltration by inhibiting TLR4/MD2 complex formation. UPLC-MS/MS analysis tentatively identified 41 components in OE. Our results indicated that OE presented significant anti-inflammatory activity by inhibiting TLR4/MD2 complex formation, which alleviated LPS-induced AKI in mice. Full article

Molecular interactions between respiratory syncytial virus (RSV) fusion protein (F protein) and the cellular receptor Toll-like receptor 4 (TLR4) and myeloid differentiation factor-2 (MD-2) protein complex are unknown. Thus, to reveal the detailed molecular interactions between them, in silico analyses were performed using various bioinformatics techniques. The present simulation data showed that the neutralizing antibody (NT-Ab) binding sites in both prefusion and postfusion proteins at sites II and IV were involved in the interactions between them and the TLR4 molecule. Moreover, the binding affinity between postfusion proteins and the TLR4/MD-2 complex was higher than that between prefusion proteins and the TLR4/MD-2 complex. This increased binding affinity due to conformational changes in the F protein may be able to form syncytium in RSV-infected cells. These results may contribute to better understand the infectivity and pathogenicity (syncytium formation) of RSV. Full article

The innate immune receptor toll-like receptor 4 (TLR4) is known as a sensor for the gram-negative bacterial cell wall component lipopolysaccharide (LPS). TLR4 activation leads to a strong pro-inflammatory response in macrophages; however, it is also recognised to play a key role in cancer. Recent studies of the opioid receptor (OR)-independent actions of opioids have identified that TLR4 can respond to opioids. Opioids are reported to weakly activate TLR4, but to significantly inhibit LPS-induced TLR4 activation. The action of opioids at TLR4 is suggested to be non-stereoselective, this is because OR-inactive (+)-isomers of opioids have been shown to activate or to inhibit TLR4 signalling, although there is some controversy in the literature. While some opioids can bind to the lipopolysaccharide (LPS)-binding cleft of the Myeloid Differentiation factor 2 (MD-2) co-receptor, pharmacological characterisation of the inhibition of opioids on LPS activation of TLR4 indicates a noncompetitive mechanism. In addition to a direct interaction at the receptor, opioids affect NF-κB activation downstream of both TLR4 and opioid receptors and modulate TLR4 expression, leading to a range of in vivo outcomes. Here, we review the literature reporting the activity of opioids at TLR4, its proposed mechanism(s), and the complex functional consequences of this interaction. Full article

Artemisinin (also known as Qinghaosu), an active component of the Qinghao extract, is widely used as antimalarial drug. Previous studies reveal that artemisinin and its derivatives also have effective anti-inflammatory and immunomodulatory properties, but the direct molecular target remains unknown. Recently, several reports mentioned that myeloid differentiation factor 2 (MD-2, also known as lymphocyte antigen 96) may be the endogenous target of artemisinin in the inhibition of lipopolysaccharide signaling. However, the exact interaction between artemisinin and MD-2 is still not fully understood. Here, experimental and computational methods were employed to elucidate the relationship between the artemisinin and its inhibition mechanism. Experimental results showed that artemether exhibit higher anti-inflammatory activity performance than artemisinin and artesunate. Molecular docking results showed that artemisinin, artesunate, and artemether had similar binding poses, and all complexes remained stable throughout the whole molecular dynamics simulations, whereas the binding of artemisinin and its derivatives to MD-2 decreased the TLR4(Toll-Like Receptor 4)/MD-2 stability. Moreover, artemether exhibited lower binding energy as compared to artemisinin and artesunate, which is in good agreement with the experimental results. Leu61, Leu78, and Ile117 are indeed key residues that contribute to the binding free energy. Binding free energy analysis further confirmed that hydrophobic interactions were critical to maintain the binding mode of artemisinin and its derivatives with MD-2. Full article

Ergosta-7, 9 (11), 22-trien-3β-ol (EK100) was isolated from Cordyceps militaris, which has been used as a traditional anti-inflammatory medicine. EK100 has been reported to attenuate inflammatory diseases, but its anti-inflammatory mechanism is still unclear. We were the first to investigate the effect of EK100 on the Toll-like receptor 4 (TLR4)/nuclear factor of the κ light chain enhancer of B cells (NF-κB) signaling in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells and the green fluorescent protein (GFP)-labeled NF-κB reporter gene of Drosophila. EK100 suppressed the release of the cytokine and attenuated the mRNA and protein expression of pro-inflammatory mediators. EK100 inhibited the inhibitor kappa B (IκB)/NF-κB signaling pathway. EK100 also inhibited phosphatidylinositol-3-kinase (PI3K)/Protein kinase B (Akt) signal transduction. Moreover, EK100 interfered with LPS docking to the LPS-binding protein (LBP), transferred to the cluster of differentiation 14 (CD14), and bonded to TLR4/myeloid differentiation-2 (MD-2) co-receptors. Compared with the TLR4 antagonist, resatorvid (CLI-095), and dexamethasone (Dexa), EK100 suppressed the TLR4/AKT signaling pathway. In addition, we also confirmed that EK100 attenuated the GFP-labeled NF-κB reporter gene expression in Drosophila. In summary, EK100 might alter LPS docking to LBP, CD14, and TLR4/MD-2 co-receptors, and then it suppresses the TLR4/NF-κB inflammatory pathway in LPS-stimulated RAW264.7 cells and Drosophila. Full article

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS. Full article

Inflammatory Bowel Diseases (IBD) are chronic inflammations associated with progressive degradation of intestinal epithelium and impairment of the local innate immune response. Restoring of epithelial integrity and of the mucosal barrier function, together with modulation of inflammatory and innate immune markers, represent targets for alternative strategies in IBD. The aim of our study was to evaluate the effects of a diet including 8% grape seed meal (GSM), rich in bioactive compounds (polyphenols, polyunsaturated fatty acids (PUFAs), fiber) on the markers of colonic epithelial integrity, mucosal barrier function, pro-inflammatory, and innate immunity in DSS-treated piglets used as animal models of intestinal inflammation. Our results have demonstrated the beneficial effects of bioactive compounds from dietary GSM, exerted at three complementary levels: (a) restoration of the epithelial integrity and mucosal barrier reinforcement by modulation of claudins, Occludin (OCCL) and Zonula-1 (ZO-1) tight junction genes and proteins, myosin IXB (MYO9B) and protein tyrosine phosphatase (PTPN) tight junction regulators and mucin-2 (MUC2) gene; (b) reduction of pro-inflammatory MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) genes and activities; and (c) suppression of the innate immune TLR-2 (Toll-like receptor-2) and TLR-4 (Toll-like receptor-4) genes and attenuation of the expression of MyD88 (Myeloid Differentiation Primary Response 88)/MD-2 (Myeloid differentiation factor-2) signaling molecules. These beneficial effects of GSM could further attenuate the transition of chronic colitis to carcinogenesis, by modulating the in-depth signaling mediators belonging to the Wnt pathway. Full article

Imperatorin (IMP) could downregulate several inflammatory transcription factor signaling pathways. Some studies have pointed out that IMP could interfere with toll-like receptor 4 (TLR4) signaling. This study evaluates how IMP interferes with the TLR4 co-receptors signaling through the protein-ligand docking model, Western blotting, immunofluorescence (IF), and atomic force microscopy (AFM) assays in lipopolysaccharide (LPS) stimulated macrophage-like RAW264.7 cells in vitro. The results of the protein-ligand docking demonstrate that IMP interferes with LPS binding to the LPS-binding protein (LBP), the cluster of differentiation 14 (CD14), and the toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) co-receptors in LPS-stimulated RAW264.7 cells. Compared with TLR4 antagonist CLI-095 or dexamethasone, IMP could suppress the protein expressions of LBP, CD14, and TLR4/MD-2 in LPS-stimulated cells. Furthermore, the three-dimensional (3D) image assay of the AFM showed IMP could prevent the LPS-induced morphological change in RAW264.7 cells. Additionally, IMP could activate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, and it increased the antioxidative protein expression of heme oxygenase-1 (HO-1), superoxidase dismutase (SOD), and catalase (CAT). Our results are the first to reveal that the anti-inflammatory effect of IMP interferes with LPS binding to TLR4 co-receptor signaling and activates the antioxidative Nrf2 signaling pathway. Full article

Toll-like receptors (TLRs) are transmembrane proteins that are key regulators of innate and adaptive immune responses, particularly TLR4, and they have been identified as potential drug targets for the treatment of disease. Several low-molecular-weight compounds are being considered as new drug targets for various applications, including as immune modulators. Mygalin, a 417 Da synthetic bis-acylpolyamine, is an analog of spermidine that has microbicidal activity. In this study, we investigated the effect of mygalin on the innate immune response based on a virtual screening (VS) and molecular docking analysis. Bone marrow-derived macrophages and the cell lines J774A.1 and RAW 264.7 stimulated with lipopolysaccharide (LPS) were used to confirm the data obtained in silico. Virtual screening and molecular docking suggested that mygalin binds to TLR4 via the protein myeloid differentiation factor 2 (MD-2) and LPS. Macrophages stimulated by mygalin plus LPS showed suppressed gene expression of tumor necrosis factor (TNF-α), interleukine 6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as inhibition of signaling protein p65 of the nuclear factor κB (NF-κB), resulting in decreased production of nitric oxide (NO) and TNF-α. These results indicate that mygalin has anti-inflammatory potential, being an attractive option to be explored. In addition, we reinforce the importance of virtual screening analysis to assist in the discovery of new drugs. Full article