Search Results (69)

Background: An acute angle-closure attack (AAC) is an ocular emergency that results from a rapid increase in intraocular pressure (IOP). Sustained IOP elevation induces severe degeneration of retinal ganglion cells (RGCs) without treatment. Overactivated microglia, key participants in innate immune responses, have critical roles in the pathogenesis of IOP-induced RGC death, although precise mechanisms remain unclear. In the present study, we used a rat ex vivo acute glaucoma model to investigate the role of microglial signaling in RGC death and examined whether pharmacological depletion of microglia using a CSF-1R inhibitor, PLX5622, exerts neuroprotection against pressure-induced retinal injury. Methods: Ex vivo rat retinas were exposed to hydrostatic pressure (10 mmHg or 75 mmHg) for 24 h. Pressure-dependent changes in retinal microglia and RGCs were detected by immunofluorescence. Morphological changes in the retina and RGC apoptosis were examined using light microscopy and TUNEL staining, respectively. The expression of NLRP3, active caspase-1, pro IL-1β, and IL-1β were examined using Western blotting. Effects of PLX5622, an agent that depletes microglia, were examined in morphology, apoptosis, and protein expression assays, while TAK-242, a TLR4 inhibitor, was examined against protein expression. Results: Pressure loading at 75 mmHg markedly increased activated microglia and apoptotic RGCs in the isolated retinas. Western blotting revealed increases in expression of NLRP3, active caspase-1, pro IL-1β, and IL-1β at 75 mmHg compared to 10 mmHg. Inhibition of pressure-induced increases in NLRP3 by TAK-242 indicates that pressure elevation induces RGC death via activation of the TLR4–NLRP3 inflammasome cascade. PLX5622 depleted microglia at 75 mmHg and significantly decreased expression of NLRP3, active caspase-1, pro IL-1β, and IL-1β at 75 mmHg, resulting in preservation of RGCs. Conclusions: These results indicate that pressure elevation induces proliferation of inflammatory microglia and promotes IL-1β production via activation of the TLR4–NLRP3 inflammasome cascade, resulting in RGC death. Pharmacological depletion of microglia with PLX5622 could be a potential neuroprotective approach to preserve RGCs from inflammatory cytokines in AAC eyes. Full article

Influenza A virus (IAV) is a major cause of respiratory illness in humans and animals. Secondary bacterial infections, especially those caused by Staphylococcus aureus (SA), significantly increase influenza-related morbidity and mortality. However, the mechanisms underlying these co-infections remain unclear. In this study, we examined how IAV infection influences SA-induced inflammation in lung epithelial cells. Our study was conducted based on in vitro experiments. First, we infected MLE-12 cells with IAV, confirming viral replication and the resulting cell damage. SA was then introduced 24 h or 36 h post-infection, and the cellular responses were measured. We assessed cell viability, cell-free DNA, Citrullinated histone H3, and the mRNA expression of TLR4 and proinflammatory cytokines. Our results showed that IAV+SA stimulation significantly increased upregulated TLR4 expression and inflammatory damage. To further explore TLR4’s role, we used the inhibitor TAK-242 and a TLR4 siRNA knockdown. Both approaches reduced the inflammatory response triggered by IAV and SA stimulation. These findings suggest that TLR4 is a key mediator in the enhanced inflammation observed during IAV and SA co-infection, offering a potential target for therapeutic intervention. Full article

Nickel oxide nanoparticles (NiONPs) can induce liver fibrosis, and their mechanism may be related to non-coding RNA, nuclear receptor signal transduction and ferroptosis, but the regulatory relationship between them is not clear. In this study, we aimed to investigate the role of hsa_circ_0001944 in regulating the Farnesol X receptor (FXR)/Toll-like receptor 4 (TLR4) pathway and ferroptosis in NiONPs-induced collagen deposition. We observed decreased FXR expression, increased TLR4 expression and alterations in ferroptosis features in both the rat liver fibrosis and the LX-2 cell collagen deposition model. To investigate the regulatory relationship among FXR, TLR4 and ferroptosis, we treated LX-2 cells with FXR agonist (GW4064), TLR4 inhibitor (TAK-242) and ferroptosis agonist (Erastin) combined with NiONPs. The results showed that TAK-242 alleviated collagen deposition by increasing ferroptosis features. Furthermore, GW4064 reduced the expression of TLR4, increased the ferroptosis features and alleviated collagen deposition. The results indicated that FXR inhibited the expression of TLR4 and enhanced the ferroptosis features, which were involved in the process of collagen deposition in LX-2 cells induced by NiONPs. Subsequently, we predicted that hsa_circ_0001944 might regulate FXR through bioinformatics analysis, and found NiONPs reduced the expression of hsa_circ_0001944 in LX-2 cells. Overexpression of hsa_circ_0001944 increased FXR level, reduced TLR4 level, increased the ferroptosis features and alleviated collagen deposition in LX-2 cells. In summary, we demonstrated that hsa_circ_0001944 regulates the FXR/TLR4 pathway and ferroptosis alleviate collagen formation induced by NiONPs. Full article

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a novel target for reducing low-density lipoprotein cholesterol. PCSK9 activates the atherosclerosis process through pro-inflammation signaling. Furthermore, the serum level of PCSK9 is positively correlated with mortality in patients with heart failure (HF). Cardiac fibrosis plays a crucial role in the pathophysiology of HF. In this study, we intended to examine whether PCSK9 can increase fibroblast activities and explore what its underlying mechanisms are. Migration, proliferation analyses, and Western blotting were used on human cardiac fibroblasts with and without PCSK9. Alirocumab (a PCSK9 inhibitor, 10 mg/kg/week intra-peritoneally for 28 consecutive days) was treated in isoproterenol (100 mg/kg, subcutaneous injection)-induced HF rats. PCSK9 (50, 100 ng/mL) increased proliferation, myofibroblast differentiation capability, and collagen type I production. Compared with control cells, PCSK9 (100 ng/mL)-treated cardiac fibroblasts showed higher nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3), interleukin (IL)-1, myofibroblast differentiation, and collagen production capabilities, which were attenuated by MCC950 (an NLRP3 inhibitor, 100 μmol/L). PCSK9 upregulated Myd88 and NF-κB signaling, which were reduced by TAK242 (a toll-like receptor (TLR) 4 inhibitor, 10 μmol/L). Moreover, alirocumab significantly improved left ventricular systolic function and attenuated fibrosis in HF rats. In conclusion, PCSK9 upregulates NLRP3 signaling and the profibrotic activities of cardiac fibroblasts through the activation of TLR4/Myd88/NF-κB signaling. Full article

Toll-like receptor 4 (TLR4) has been implicated in the production of uncontrolled inflammation within the body, known as the cytokine storm. Studies that employ machine learning (ML) in the prediction of potential inhibitors of TLR4 are limited. This study introduces AICpred, a robust, free, user-friendly, and easily accessible machine learning-based web application for predicting inhibitors against TLR4 by targeting the TLR4-myeloid differentiation primary response 88 (MyD88) interaction. MyD88 is a crucial adaptor protein in the TLR4-induced hyper-inflammation pathway. Predictive models were trained using random forest, adaptive boosting (AdaBoost), eXtreme gradient boosting (XGBoost), k-nearest neighbours (KNN), and decision tree models. To handle imbalance within the training data, resampling techniques such as random under-sampling, synthetic minority oversampling technique, and the random selection of 5000 instances of the majority class were employed. A 10-fold cross-validation strategy was used to evaluate model performance based on metrics including accuracy, balanced accuracy, and recall. The XGBoost model demonstrated superior performance with accuracy, balanced accuracy, and recall scores of 0.994, 0.958, and 0.917, respectively, on the test. The AdaBoost and decision tree models also excelled with accuracies ranging from 0.981 to 0.992, balanced accuracies between 0.921 and 0.944, and recall scores between 0.845 and 0.891 on both training and test datasets. The XGBoost model was deployed as AICpred and was used to screen compounds that have been reported to have positive effects on mitigating the hyperinflammation-associated cytokine storm, which is a key factor in COVID-19. The models predicted Baricitinib, Ibrutinib, Nezulcitinib, MCC950, and Acalabrutinib as anti-TLR4 compounds with prediction probability above 0.90. Additionally, compounds known to inhibit TLR4, including TAK-242 (Resatorvid) and benzisothiazole derivative (M62812), were predicted as bioactive agents within the applicability domain with probabilities above 0.80. Computationally inferred compounds using AICpred can be explored as potential starting skeletons for therapeutic agents against hyperinflammation. These predictions must be consolidated with experimental screening to enhance further optimisation of the compounds. AICpred is the first of its kind targeting the inhibition of TLR4-MyD88 binding and is freely available at http://197.255.126.13:8080. Full article

Arsenic, a well-known environmental endocrine disruptor, exerts interference on the body’s endocrine system. Our previous investigations have demonstrated that chronic exposure to sodium arsenite (NaAsO₂) can induce thyroid damage and dysfunction in Sprague–Dawley (SD) rats. Vitamin D (VD) is an indispensable fat-soluble vitamin that plays a crucial role in maintaining thyroid health. In recent years, numerous studies have demonstrated the association between VD deficiency and the development of various thyroid disorders. However, the precise intervention roles and mechanisms of VD in arsenic-induced thyroid injury remain elusive. This study aimed to investigate the intervention effect of VD on NaAsO₂-induced thyroid dysfunction in SD rats. The results demonstrated that exposure to NaAsO₂ activates the TLR4/NF-κB signaling pathway in thyroid tissue of rats, leading to apoptosis of thyroid cells and subsequent inflammatory damage and disruption of serum thyroid hormone secretion. Supplementation with TAK-242 (a TLR4 inhibitor) and VD effectively inhibits the activation of the TLR4/NF-κB signaling pathway in rat thyroid tissue exposed to NaAsO₂, thereby reducing the inflammatory damage and dysfunction caused by arsenic exposure. In conclusion, the findings of this study offer innovative insights into the application of VD in the prevention and treatment of thyroid dysfunction caused by arsenic exposure. Full article

(1) Background: Small ubiquitin-like modifiers (SUMOs) are pivotal in post-translational modifications, influencing various cellular processes, such as protein localization, stability, and genome integrity. (2) Methods: This review explores the SUMO family, including its isoforms and catalytic cycle, highlighting their significance in regulating key biological functions in thyroid cancer. We discuss the multifaceted roles of SUMOylation in DNA repair mechanisms, protein stability, and the modulation of receptor activities, particularly in the context of thyroid cancer. (3) Results: The aberrant SUMOylation machinery contributes to tumorigenesis through altered gene expression and immune evasion mechanisms. Furthermore, we examine the therapeutic potential of targeting SUMOylation pathways in thyroid cancer treatment, emphasizing the need for further research to develop effective SUMOylation inhibitors. (4) Conclusions: By understanding the intricate roles of SUMOylation in cancer biology, we can pave the way for innovative therapeutic strategies to improve outcomes for patients with advanced tumors. Full article

Background: The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial to viral entry and can cause cardiac injuries. Toll-like receptor 4 (TLR4) and NOD-, LPR-, and pyrin-domain-containing 3 (NLRP3) inflammasome are critical immune system components implicated in cardiac fibrosis. The spike protein activates NLRP3 inflammasome through TLR4 or angiotensin-converting enzyme 2 (ACE2) receptors, damaging various organs. However, the role of spike protein in cardiac fibrosis in humans, as well as its interactions with NLRP3 inflammasomes and TLR4, remain poorly understood. Methods: We utilized scratch assays, Western blotting, and immunofluorescence to evaluate the migration, fibrosis signaling, mitochondrial calcium levels, reactive oxygen species (ROS) production, and cell morphology of cultured human cardiac fibroblasts (CFs) treated with spike (S1) protein for 24 h with or without an anti-ACE2 neutralizing antibody, a TLR4 blocker, or an NLRP3 inhibitor. Results: S1 protein enhanced CFs migration and the expressions of collagen 1, α-smooth muscle actin, transforming growth factor β1 (TGF-β1), phosphorylated SMAD2/3, interleukin 1β (IL-1β), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). S1 protein increased ROS production but did not affect mitochondrial calcium content and cell morphology. Treatment with an anti-ACE2 neutralizing antibody attenuated the effects of S1 protein on collagen 1 and TGF-β1 expressions. Moreover, NLRP3 (MCC950) and NF-kB inhibitors, but not the TLR4 inhibitor TAK-242, prevented the S1 protein-enhanced CFs migration and overexpression of collagen 1, TGF-β1, and IL-1β. Conclusion: S1 protein activates human CFs by priming NLRP3 inflammasomes through NF-κB signaling in an ACE2-dependent manner. Full article

ApTOLL, a TLR4 modulator aptamer, has demonstrated cerebroprotective effects in a permanent ischemic stroke mouse model, as well as safety and efficacy in early phase clinical trials. We carried out reverse translation research according to STAIR recommendations to further characterize the effects and mechanisms of ApTOLL after transient ischemic stroke in rats and to better inform the design of pivotal clinical trials. Adult male rats subjected to transient middle cerebral artery occlusion were treated either with ApTOLL or the vehicle intravenously at different doses and time-points. ApTOLL was compared with TAK-242 (a TLR4 inhibitor). Female rats were also studied. After neurofunctional evaluation, brains were removed for infarct/edema volume, hemorrhagic transformation, and histologic determinations. Peripheral leukocyte populations were assessed via flow cytometry. ApTOLL showed U-shaped dose-dependent cerebroprotective effects. The maximum effective dose (0.45 mg/kg) was cerebroprotective when given both before reperfusion and up to 12 h after reperfusion and reduced the hemorrhagic risk. Similar effects occurred in female rats. Both research and clinical ApTOLL batches induced slightly superior cerebroprotection when compared with TAK-242. Finally, ApTOLL modulated circulating leukocyte levels, reached the brain ischemic tissue to bind resident and infiltrated cell types, and reduced the neutrophil density. These results show the cerebroprotective effects of ApTOLL in ischemic stroke by reducing the infarct/edema volume, neurofunctional impairment, and hemorrhagic risk, as well as the peripheral and local immune response. They provide information about ApTOLL dose effects and its therapeutic time window and target population, as well as its mode of action, which should be considered in the design of pivotal clinical trials. Full article

Chronic inflammation causes muscle wasting. Because most inflammatory cytokine signals are mediated via TGF-β-activated kinase-1 (TAK1) activation, inflammatory cytokine-induced muscle wasting may be ameliorated by the inhibition of TAK1 activity. The present study was undertaken to clarify whether TAK1 inhibition can ameliorate inflammation-induced muscle wasting. SKG/Jcl mice as an autoimmune arthritis animal model were treated with a small amount of mannan as an adjuvant to enhance the production of TNF-α and IL-1β. The increase in these inflammatory cytokines caused a reduction in muscle mass and strength along with an induction of arthritis in SKG/Jcl mice. Those changes in muscle fibers were mediated via the phosphorylation of TAK1, which activated the downstream signaling cascade via NF-κB, p38 MAPK, and ERK pathways, resulting in an increase in myostatin expression. Myostatin then reduced the expression of muscle proteins not only via a reduction in MyoD1 expression but also via an enhancement of Atrogin-1 and Murf1 expression. TAK1 inhibitor, LL-Z1640-2, prevented all the cytokine-induced changes in muscle wasting. Thus, TAK1 inhibition can be a new therapeutic target of not only joint destruction but also muscle wasting induced by inflammatory cytokines. Full article

Increased low-density lipoprotein levels are risk factors for diabetic neuropathy. Diabetes mellitus is associated with elevated metabolic stress, leading to oxidised low-density lipoprotein formation. Therefore, it is important to investigate the mechanisms underlying the pathogenesis of diabetic neuropathy in diabetes complicated by dyslipidaemia with increased levels of oxidised low-density lipoprotein. Here, we examined the effects of hyperglycaemia and oxidised low-density lipoprotein treatment on Schwann cell death and its underlying mechanisms. Immortalised mouse Schwann cells were treated with oxidised low-density lipoprotein under normo- or hyperglycaemic conditions. We observed that oxidised low-density lipoprotein-induced cell death increased under hyperglycaemic conditions compared with normoglycaemic conditions. Moreover, hyperglycaemia and oxidised low-density lipoprotein treatment synergistically upregulated the gene and protein expression of toll-like receptor 4. Pre-treatment with TAK-242, a selective toll-like receptor 4 signalling inhibitor, attenuated hyperglycaemia- and oxidised low-density lipoprotein-induced cell death and apoptotic caspase-3 pathway. Our findings suggest that the hyperactivation of toll-like receptor 4 signalling by hyperglycaemia and elevated oxidised low-density lipoprotein levels synergistically exacerbated diabetic neuropathy; thus, it can be a potential therapeutic target for diabetic neuropathy. Full article

Necroptosis, a form of necrosis, and alterations in mitochondrial dynamics, a coordinated process of mitochondrial fission and fusion, have been implicated in the pathogenesis of cardiovascular diseases. This study aimed to determine the role of mitochondrial morphology in canonical necroptosis induced by a combination of TNFα and zVAD (TNF/zVAD) in H9c2 cells, rat cardiomyoblasts. Time-course analyses of mitochondrial morphology showed that mitochondria were initially shortened after the addition of TNF/zVAD and then their length was restored, and the proportion of cells with elongated mitochondria at 12 h was larger in TNF/zVAD-treated cells than in non-treated cells (16.3 ± 0.9% vs. 8.0 ± 1.2%). The knockdown of dynamin-related protein 1 (Drp1) and fission 1, fission promoters, and treatment with Mdivi-1, a Drp-1 inhibitor, had no effect on TNF/zVAD-induced necroptosis. In contrast, TNF/zVAD-induced necroptosis was attenuated by the knockdown of mitofusin 1/2 (Mfn1/2) and optic atrophy-1 (Opa1), proteins that are indispensable for mitochondrial fusion, and the attenuation of necroptosis was not canceled by treatment with Mdivi-1. The expression of TGFβ-activated kinase (TAK1), a negative regulator of RIP1 activity, was upregulated and the TNF/zVAD-induced RIP1-Ser166 phosphorylation, an index of RIP1 activity, was mitigated by the knockdown of Mfn1/2 or Opa1. Pharmacological TAK1 inhibition attenuated the protection afforded by Mfn1/2 and Opa1 knockdown. In conclusion, the inhibition of mitochondrial fusion increases TAK1 expression, leading to the attenuation of canonical necroptosis through the suppression of RIP1 activity. Full article

An increased concentration of palmitate in circulation is one of the most harmful factors in obesity. The von Willebrand factor (vWF), a protein involved in haemostasis, is produced and secreted by the vascular endothelium. An increased level of vWF in obese patients is associated with thrombosis and cardiovascular disease. The aim of this study was to investigate a palmitate effect on vWF in endothelial cells and understand the mechanisms of palmitate-activated signalling. Human umbilical vein endothelial cells (HUVECs) incubated in the presence of palmitate, exhibited an increased VWF gene expression, vWF protein maturation, and stimulated vWF secretion. Cardamonin, a Nuclear Factor kappa B (NF-κB) inhibitor, abolished the palmitate effect on VWF expression. The inhibition of Toll-like receptor (TLR) 2 with C29 resulted in the TLR4 overactivation in palmitate-treated cells. Palmitate, in the presence of TLR4 inhibitor TAK-242, leads to a higher expression of TLR6, CD36, and TIRAP. The silencing of TLR4 resulted in an increase in TLR2 level and vice versa. The obtained results indicate a potential mechanism of obesity-induced thrombotic complication caused by fatty acid activation of NF-κB signalling and vWF upregulation and help to identify various compensatory mechanisms related to TLR4 signal transduction. Full article

TAK1 (transforming growth factor-beta-activated kinase 1) is a crucial therapeutic target in inflammation-related diseases. This study investigated the inhibitory potential of cannflavin A, a flavonoid found in Cannabis sativa, against TAK1. Through in silico approaches, including drug-likeness analysis, ADMET assessment, molecular docking, and molecular dynamics simulation, the binding affinity and stability of cannflavin A were evaluated. The results demonstrate that cannflavin A exhibits excellent ADMET properties and displays superior binding affinity and stability at the ATP binding site of TAK1 when compared to the known inhibitor takinib. Notably, the decomposition of binding free energy unveils critical amino acid residues involved in TAK1 binding, underscoring the inhibitory effect of cannflavin A through TAK1 inhibition. These findings highlight the potential of cannflavin A as a TAK1 inhibitor and its significant implications for the development of targeted therapies in inflammation-related diseases. Through modulating inflammatory signaling pathways, cannflavin A holds promise for more effective and tailored treatment strategies, particularly in rheumatoid arthritis. This study contributes to the current understanding of cannflavin A’s application and provides a foundation for further research and innovative approaches in targeted therapies for inflammatory conditions. Full article

Chronic widespread pain (CWP) is associated with a high rate of disability and decreased quality of life in people with HIV-1 (PWH). We previously showed that PWH with CWP have increased hemolysis and elevated plasma levels of cell-free heme, which correlate with low endogenous opioid levels in leukocytes. Further, we demonstrated that cell-free heme impairs β-endorphin synthesis/release from leukocytes. However, the cellular mechanisms by which heme dampens β-endorphin production are inconclusive. The current hypothesis is that heme-dependent TLR4 activation and macrophage polarization to the M1 phenotype mediate this phenomenon. Our novel findings showed that PWH with CWP have elevated M1-specific macrophage chemokines (ENA-78, GRO-α, and IP-10) in plasma. In vitro, hemin-induced polarization of M0 and M2 macrophages to the M1 phenotype with low β-endorphins was mitigated by treating cells with the TLR4 inhibitor, TAK-242. Similarly, in vivo phenylhydrazine hydrochloride (PHZ), an inducer of hemolysis, injected into C57Bl/6 mice increased the M1/M2 cell ratio and reduced β-endorphin levels. However, treating these animals with the heme-scavenging protein hemopexin (Hx) or TAK-242 reduced the M1/M2 ratio and increased β-endorphins. Furthermore, Hx attenuated heme-induced mechanical, heat, and cold hypersensitivity, while TAK-242 abrogated hypersensitivity to mechanical and heat stimuli. Overall, these results suggest that heme-mediated TLR4 activation and M1 polarization of macrophages correlate with impaired endogenous opioid homeostasis and hypersensitivity in people with HIV. Full article