TLR4 Inhibitor Research Articles

CLEC12A sensitizes differentially responsive breast cancer cells to the anti-cancer effects of artemisinin by repressing autophagy and inflammation.

Enhanced inflammatory responses promote tumor progression by activating toll-like receptors (TLRs), which in turn are inhibited by C-type lectin like receptors (CTLRs), like CLEC12A. Although the presence of CLEC12A in acute myeloid leukemia is well established, its role in non-hematopoietic tumors is still obscure. In hematopoietic tumors, CLEC12A mostly inhibits TLRs and modulates inflammatory responses via NF-κB signaling. In this study, the fate of tumor progression was determined by modulating CLEC12A using artemisinin (ART), a FDA-approved anti-malarial drug, known for its anti-cancer and immunomodulatory properties with minimal adverse effects on normal cells. Effects of ART were primarily determined on hematological factors and primary metastatic organs, such as lungs, kidney and liver in normal and tumor-bearing BALB/c mice. Tumor-bearing mice were treated with different concentrations of ART and expressions of CLEC12A and associated downstream components were determined. CLEC12A was overexpressed in MDA-MB-231 and 4T1 cells, and the effects of ART were analyzed in the overexpressed cells. Silencing TLR4 using vivo morpholino was performed to elucidate its role in tumor progression in response to ART. Finally, CLEC12A modulation by ART was evaluated in the resident cancer stem cell (CSC) population. ART did not alter physiology of normal mice, in contrast to tumor-bearing mice, where ART led to tumor regression. In addition, ART reduced expression of CLEC12A. Expectedly, TLR4 expression increased, but surprisingly, that of NF-κB (RelA) and JNK/pJNK decreased, along with reduced inflammation, reduced autophagy and increased apoptosis. All the above observations reverted on overexpression of CLEC12A in MDA-MB-231 and 4T1 cells. Inhibition of TLR4, however, indicated no change in the expressions of CLEC12A, NF-κB, or apoptotic markers. The effect of ART showed a similar trend in the CSC population as in cancer cells. This study, for the first time, confirmed a differential role of CLEC12A in non-hematopoietic tumor and cancer stem cells in response to ART. Subsequent interaction and modulation of CLEC12A with ART induced tumor cell death and abrogation of CSCs, confirming a more comprehensive tumor therapy with reduced risk of recurrence. Therefore, ART may be repurposed as an effective drug for cancer treatment in future.

Effect of quercetin on acute appendicitis by reduction of Caspase-1 and inflammatory factors release through Toll-like receptor 4/nuclear factor kappa-B pathway

It has been reported that, quercetin can improve body’s inflammatory response through Toll-like receptor 4/nuclear factor kappa-B (TLR4/NF-κB) signaling, thereby playing a role in treating acute appendicitis. Firstly, acute appendicitis model was established in rats, where blank group, acute appendicitis model group, and quercetin intervention group (quercetin group) were set up to detect the effect of quercetin on acute appendicitis. In addition, the model, Toll-like receptor 4 inhibitor (TAK-242), Lipopolysaccharides (LPS), quercetin+ TAK-242, and quercetin+LPS groups were set to assess whether quercetin reduces Caspase-1 (CASP-1) through TLR4/NF-KB signaling and regulates the release of inflammatory factors to exert the effect on treating acute appendicitis. (1) Quercetin significantly inhibited the biological effects of tissue inflammation in rats with appendicitis and this process was related to decreased expression of TLR4 and NF-κB; (2) Quercetin also inhibited TLR4 and NF-κB expression and after using TLR4 inhibitors, the release of inflammatory factors in rat appendix tissue was controlled, especially in the quercetin+TAK-242 group. Quercetin can significantly block the development of inflammatory response in acute appendicitis, and this process is mainly achieved by reducing inflammatory factors. In addition, quercetin can effectively inhibit TLR4/NF-κB signaling related to anti-inflammatory effect, thereby down-regulating the expression of CASP-1, and finally inhibiting the inflammatory response of acute appendicitis.

Total glucosides of peony regulate toll-like receptor to inhibit inflammatory response in diabetic nephropathy

Diabetic nephropathy (DN) is a common chronic disease affecting people’s life. Total glucosides of peony (TGP) regulates TLR4/NF-κB activation. This study aims at investigating the underlying mechanism of TGP in DN treatment. A retrospective analysis of clinical data was conducted to observe periodic acid-silver metheramine (PASM) staining and detect the expression of TLR4 and VEGF in DN. Additionally, rats were injected with Streptozocin (STZ) (30–60 mg) according to the ratio of 45–65 mg/kg to establish DN model. 8 weeks later, rats of experimental group were intraperitoneally injected with TGP. Kidney was collected for HE staining to observe morphological changes. Fibronectin expression was detected by histochemistry and the level of TLR4/NF-κB pathway-related proteins and apoptosis were detected by Western blot. PASM staining revealed focal tubular atrophy and interstitial fibrosis in DN patients. The renal medulla gradually disappeared and infiltration increased in control group, while inflammatory cells decreased in experimental group. The expression of fibronectin, TLR4 and p-p65 was decreased after TGP treatment. TGP treatment significantly decreased fibronectin expression and TGP treatment-induced inhibition of NF-κB signaling enhanced apoptotic activation of HK-2 cells, which was partially reversed by TLR4 and NF-κB inhibitors. TGP can inhibit inflammatory response of DN via regulating TLR4/NF-κB.

Sex differences in the biomechanical and biochemical responses of caudal rat intervertebral discs to injury.

Intervertebral disc degeneration (IDD) is a major cause of low back pain (LBP) worldwide. Sexual dimorphism, or sex-based differences, appear to exist in the severity of LBP. However, it is unknown if there are sex-based differences in the inflammatory, biomechanical, biochemical, and histological responses of intervertebral discs (IVDs). Caudal (Coccygeal/Co) bone-disc-bone motion segments were isolated from multiple spinal levels (Co8 to Co14) of male and female Sprague-Dawley rats. Changes in motion segment biomechanics and extracellular matrix (ECM) biochemistry (glycosaminoglycan [GAG], collagen [COL], water, and DNA content) were evaluated at baseline and in response to chemical insult (lipopolysaccharide [LPS]) or puncture injury ex vivo. We also investigated the contributions of Toll-like receptor (TLR4) signaling on responses to LPS or puncture injury ex vivo, using a small molecule TLR4 inhibitor, TAK-242. Findings indicate that IVD motion segments from female donors had greater nitric oxide (NO) release in LPS groups compared to male donors. HMGB1 release was increased in punctured discs, but not LPS injured discs, with no sex effect. Although both male and female discs exhibited reductions in dynamic moduli in response to LPS and puncture injuries, dynamic moduli from female donors were higher than male donors across all groups. In uninjured (baseline) samples, a significant sex effect was observed in nucleus pulposus (NP) DNA and water content. Female annulus fibrosus (AF) also had higher DNA, GAG, and COL content (normalized bydry weight), but lower water content than male AF. Additional injury- and sex-dependent effects were observed in AF GAG/DNA and COL/DNA content. Finally, TAK-242 improved the dynamic modulus of female but not male punctured discs. Our findings demonstrate that there are differences in rat IVD motion segments based on sex, and that the response to injury in inflammatory, biomechanical, biochemical, and histological outcomes also exhibit sex differences. TLR4 inhibition protected against loss of mechanical integrity of puncture-injured IVD motion segments, with differences responses based on donor sex.

Design, synthesis and anti-rheumatoid arthritis activity of target TLR4 inhibitors

A series of 1-(2-oxocyclohexyl)butane-1, 3-dione derivatives were designed and synthesized as TLR4 inhibitors by modifying the core structure of the lead compound ((6, 8-dioxo-1, 2, 3, 4, 6, 7, 8, 8a-octahydronaphthalen-2-yl) carbamate)). In vitro, compound 3p significantly inhibited the proliferation of rat synovial cells, inhibited the proliferation of LPS-induced RAW264.7 cells, and inhibited TLR4 activity, with IC50 values of 1.21 ± 0.09 μM, 0.73 ± 0.05 μM and 0.43 ± 0.03 μM, respectively, which was superior to the positive control methotrexate. In vivo anti-rheumatoid arthritis evaluation, compound 3p can significantly inhibit the inflammatory factors TNF-α, IL-1β and IL-6, so as to achieve the therapeutic purpose. In the preliminary mechanism study, compound 3p has obvious regulatory effects on the abnormal increase of TLR4, JAK2 and STAT3 protein and gene expression related to inflammatory signaling pathway in RAW264.7 cells. In summary, this study aims to develop more effective candidates for rheumatoid arthritis.

Derangements of TLR2 Likely Play a Significant Role in the Thrombosis of Ph(-) Myeloproliferative Neoplasm

Introduction . We had previously reported that TLR 2 levels were significantly elevated in patients with polycythemia vera and MPN ( PV , ET, MF grouped together ) and TLR2 levels were related to JAK2 mutation and leukocytosis ( Blood (2022) 140 (Supplement 1): 12185-12186.). Since MPN has increased incidence of thrombosis in patients with JAK2V617F mutation and leukocytosis , therefore , current studies were done to test whether TLR-2 is related to thrombosis in MPN . In vitro platelet activation ( an pre-thrombotic marker) and clinical episodes of thrombosis were then investigated. . Material and Methods. 119 patients ( 51ET, 37 PV, 31MF, 21 CTR) were studied . 1) platelet activation. : The diluted blood was incubated with platelet agonists Pam3CSK4 (TLR2 agonist) or lipopolysaccharide (LPS) (TLR4 agonist) for 15 minutes at room temperature , then platelet activation was measured by Flow cytometry assaying CD40L, P-selectin, PAC-1 and LPI ( leukocyte-platelet-interaction). We obtained more consistent results from the LPI methods . Therefore, LPI methods were used to measure the platelet activation. LPI method was measured by CD45 and CD41 antibodies (Miltenyi Biotec, Auburn, CA), and platelet activation were measured as the percentage of CD45 cells with aggregated CD41. To test whether TLR2 or TLR4 were responsible for the LPI response ,TLR2 inhibitor C29 (InvivoGen, San Diego, CA) or the TLR4 inhibitor C34 ((Sigma-Aldrich, St. Louis, MO) were added to the LPI assay. Effects of inhibitions were measured by fold-changes which were calculated by dividing the LPI values after activation by either Pam3CSK4 (TLR2 agonist) or LPS (TLR4 agonist), with or without added inhibitors, by the LPI values before activation or inhibition. Results . 1) As shown in Fig A, a) un-activated MPN (U-MPN) patients had more LPI than un-activated controls (u-CTR), with mean ± SE of 13.01 ± 1.12 and 5.01 ± 0.97 in U-MPN and U-CTR, respectively (P<0.01). This is consistent with previous reports that un-activated MPN patients have more intrinsically activated platelets than un-activated controls .b) Greater LPI response by activation by Pam3CSK4, (TLR2 agonist ) than by LPS ( lipopolysaccharide)) (TLR4 agonist) . MPN and controls ( mean ± SE ) were of 22.25 ± 0.99 and 10.46 ± 0.44 ( by Pam3CSK4) and 12.12 ± 2.16 and 4.34 ± 0.32 ( by LPS) (P<0.0005) respectively . 2). Fig B showed the TLR2 inhibitor C29 significantly suppressed LPI ( measured by fold-changes ) , in un-activated MPN (0.38 ± 0.05), activated controls (0.42 ± 0.11), and activated MPN (0.42 ± 0.05). Conversely, the TLR4 inhibitor C34 had no inhibitory effect on un-activated MPN (0.98 ± 0.03), activated MPN (1.02 ± 0.02), or activated controls (0.84 ± 0.06) . 3) As shown in Table 2 , TLR 2 levels elevated Patients ( TLR2-E) ( defined as more than mean plus 2 standard deviation of controls (> 310 MFI) , have more thrombosis episode (29%) than TLR2 -normal patients (11%) . Conclusion . TLR2 likely plays a significant role in thrombosis in MPN patients on the basis : 1) JAK2 mutated patients have more TLR2 levels 2) TLR2-E patients have more leukocytosis than TLR-N patients 3) LPI was significantly elevated with TLR2 agonist ( Pam3SK4) but not by TLR4 agonist ( LPS) , and LPI was significantly decreased by TLR2 inhibitor and not by TLR4 inhibitor. 4) clinical evidence of more thrombosis in TLR -E patients than TLR-N patients.

Trichinella spiralis galectin binding to toll-like receptor 4 induces intestinal inflammation and mediates larval invasion of gut mucosa

Previous studies showed that Trichinella spiralis galectin (Tsgal) facilitates larval invasion of intestinal epithelium cells (IECs). However, IEC proteins binding with Tsgal were not identified, and the mechanism by which Tsgal promotes larval invasion is not clear. Toll-like receptors (TLRs) are protein receptors responsible for recognition of pathogens. The aim of this study was to investigate whether recombinant Tsgal (rTsgal) binds to TLR-4, activates inflammatory pathway in gut epithelium and mediates T. spiralis invasion. Indirect immunofluorescence (IIF), GST pull-down and co-immunoprecipitation (Co-IP) assays confirmed specific binding between rTsgal and TLR-4 in Caco-2 cells. qPCR and Western blotting showed that binding of rTsgal with TLR-4 up-regulated the TLR-4 transcription and expression in Caco-2 cells, and activated p-NF-κB p65 and p-ERK1/2. Activation of inflammatory pathway TLR-4/MAPK-NF-κB by rTsgal up-regulated pro-inflammatory cytokines (IL-1β and IL-6) and down-regulated anti-inflammatory cytokine TGF-β in Caco-2 cells, and induced intestinal inflammation. TAK-242 (TLR-4 inhibitor) and PDTC (NF-κB inhibitor) significantly inhibited the activation of TLR-4 and MAPK-NF-κB pathway. Moreover, the two inhibitors also inhibited IL-1β and IL-6 expression, and increased TGF-β expression in Caco-2 cells. In T. spiralis infected mice, the two inhibitors also inhibited the activation of TLR-4/MAPK-NF-κB pathway, ameliorated intestinal inflammation, impeded larval invasion of gut mucosa and reduced intestinal adult burdens. The results showed that rTsgal binding to TLR-4 in gut epithelium activated MAPK-NF-κB signaling pathway, induced the expression of TLR-4 and pro-inflammatory cytokines, and mediated larval invasion. Tsgal might be regarded as a candidate molecular target of vaccine against T. spiralis enteral invasive stage.

MCPIP1 alleviates depressive‑like behaviors in mice by inhibiting the TLR4/TRAF6/NF‑κB pathway to suppress neuroinflammation.

Lipopolysaccharide‑induced (LPS) neuroinflammation serves an important role in the development of depression. Monocyte chemotactic protein‑1‑induced protein1 (MCPIP1, also known as ZC3H12A and Regnase‑1) possesses endoribonuclease and deubiquitinase activities. In the present study, the effects of MCPIP1 on LPS‑induced depression were assessed. A mouse model of hippocampal neuroinflammation was established by intraperitoneal injection of LPS. Microglia were treated with LPS, MCPIP1 overexpression vector, MCPIP1 knockdown vector or TLR4 inhibitor. MCPIP1 alleviated LPS‑induced depressive‑like behaviors. MCPIP1 facilitated M2 polarization of microglia. MCPIP1 attenuated the inflammatory response in microglia via inhibition of the TLR4/TNF receptor associated factor 6 (TRAF6)/NF‑κB signaling pathway. The results indicated that MCPIP1 accelerated M2‑polarization of microglia and alleviated depressive‑like behaviors of mice via the inhibition of the TLR4/TRAF6/NF‑κB signaling pathway.

Improvement on renal interstitial fibrosis in rats with renal failure via TLR4/NF-κB signaling pathway by means of panax notoginseng saponins

Panax notoginseng saponins (PNS) have excellent effects on treating cardiovascular and cerebrovascular diseases, tumors and organ interstitial diseases. This study assessed the effect of PNS on renal interstitial fibrosis (RIF). Four groups of models were firstly created, which included; renal failure model, healthy group, model group, PNS group, and then established TLR4 inhibitor group (TAK-242 group), TLR4 activator group (LPS group), combined PNS and TLR4 inhibitor group (PNS+ TAK-242 group) and combined PNS and TLR4 agonist (PNS+LPS group). TLR4/NF-κB signaling pathway and regulatory mechanism for PNS were observed along with analysis of TGF-β1, α-SMA, Collagen I and FN expressions in kidney tissues. The PNS significantly inhibited changes of renal interstitial fibrosis in rats with renal failure, and this process was related to decreased expressions of TLR4 and NF-κB. The PNS also inhibited the expression of TLR4, and expressions of TGF-β1, α-SMA, Collagen I and FN were down-regulated in kidney cells after using TAK-242, especially in the PNS+TAK-242 group. The PNS can thus significantly inhibit the process of renal fibrosis, and this process is due to the fact that, PNS inhibits the activity of TLR4/NF-κB signaling pathway to down-regulate the expressions of fibrosis-related factors TGF-β1, α-SMA, etc., in exerting its effects.

Lactobacillus casei combined with Lactobacillus reuteri alleviate pancreatic cancer by inhibiting TLR4 to promote macrophage M1 polarization and regulate gut microbial homeostasis

BackgroundPancreatic cancer is a highly lethal disease with no effective treatments. Lactobacillus casei (L. casei) and Lactobacillus reuteri (L. reuteri) exhibited therapeutic effects on several cancers, but their roles in pancreatic cancer are unknown. This study aims to explore how L. casei & L. reuteri influence pancreatic cancer and the underlying mechanisms.MethodsPancreatic cancer cells were treated with L. casei & L. reuteri and co-cultured with macrophages in a transwell system in vitro. Pancreatic cancer xenograft model was established and L. casei & L. reuteri was used to treat mice in vivo. MTT, CCK-8 assay or immunohistochemical staining were used to determine the proliferation of pancreatic cancer cells or tumor tissues. Transwell assay was applied to test the migration and invasion of pancreatic cells. RT-qPCR was utilized to assess TLR4 and MyD88 expressions in pancreatic cells or tumor tissues. WB, immunofluorescence staining, or flow cytometry was used to evaluate the M1/M2 polarization of macrophages. Besides, the composition of gut microbiota of tumor-bearing mice was determined by 16 S rRNA sequencing, and ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) untargeted metabolomics was used to evaluate the metabolic profiles of feces.ResultsL. casei & L. reuteri inhibited the proliferation, migration, invasion of pancreatic cancer cells and pancreatic cancer cell-induced M2 polarization of macrophages by suppressing TLR4. Meanwhile, L. casei & L. reuteri repressed pancreatic cancer growth and promoted M1 macrophage polarization. Besides, L. casei & L. reuteri reduced fecal Alloprevotella and increased fecal azelate and glutamate in nude mice, while TLR4 inhibitor TAK-242 increased Clostridia UCG-014, azelate, uridine, methionine sulfoxide, oxypurinol, and decreased glyceryl monoester in the feces of pancreatic tumor-bearing mice. Fecal oxypurinol and glyceryl monoester levels were positively or negatively associated with gut Clostridia UCG-014 abundance, respectively.ConclusionL. casei & L. reuteri alleviate pancreatic cancer by inhibiting TLR4 to promote macrophage M1 polarization and regulate gut microbial homeostasis.

Characteristic chromanone acids from Calophyllum membranaceum: Determination of C-3 configuration and anti-inflammatory activity

One undescribed homologous furanochromanone (1) featuring a 6/6/5/3 tetracyclic skeleton and four highly oxidized pyranochromanones (2−5), along with a set of four pyranochromanone stereoisomers [(±)-6a and (±)-6b], were isolated from the leaves of Calophyllum membranaceum Gardn. Et Champ. Their structures were elucidated by using spectroscopic data, Snatzke's method, quantum-chemical calculations, and X-ray crystallographic analysis. The correlation of characteristic Cotton effects and specific chemical shifts with C-3 configuration provided a convenient approach to assign the C-3 configuration of 2,3-dimethylchromanones. The stereochemical assignments of 3-OH substituted pyranochromanones by quantum-based NMR methods following single/double MTPA derivatization were consistent with the ECD/NMR prediction, which verified the feasibility and reliability of the proposed empirical rule. The underlying mechanism was further clarified by conformational and molecular orbital analyses. Moreover, biological evaluation and binding assays demonstrated that compound 3 (KD = 0.45 μM) tightly binds to the TLR4–MD2 target, thereby inhibiting the TLR4/MyD88-dependent and -independent signal pathways. This study provides the first evidence that Calophyllum chromanones are a novel structural type of TLR4 inhibitors, exerting their anti-inflammatory effects by disrupting the binding between TLR4 and MD2.

Estrogen/estrogen receptor activation protects against DEN-induced liver fibrosis in female rats via modulating TLR-4/NF-kβ signaling

AimMen are more susceptible to liver fibrosis (LF) than women. However, the underlying molecular mechanism, especially the role of estrogen/estrogen receptor (ER) activation in this sexual dimorphism is unclear. Therefore, the aim of the current study was to investigate the impact and the underlying molecular mechanisms of estrogen/ER activation on diethyl nitrosamine (DEN)-induced LF. Main methodsThirty ovariectomized (OVX) female rats were randomly allocated into five groups (n = 6), and received no treatment, diethyl nitrosamine (DEN), DEN/fulvestrant, DEN/silymarin or DEN/estradiol benzoate (EB). In addition, three sham groups received no treatment, DEN or DEN/fulvestrant, and one control group that neither ovariectomized nor treated. Directly after treatment, liver injury biomarkers were measured. In addition, hepatic tissue hydroxyproline, TNF- α, TGF- β, and IL-10 were evaluated. Expression of NF-kβ, CD68 (a marker for macrophage infiltration), ER-β and TLR-4 were measured. Finally, liver tissue histopathology was assessed. Key findingsOvariectomy aggravates DEN-induced LF, as it significantly elevated all liver tissue injury biomarkers. This effect has become even worse after blocking ER by fulvestrant, indicating a protective role of estrogen/ER activation against DEN-induced LF. Inhibition of TLR-4/NF-kβ signaling pathway contributed to this protective effect, as estrogen deprivation or blocking of ER significantly activates this pathway during the onset of LF. While administration of EB or silymarin (selective ER-β activator) improved LF indices and deactivated this pathway. SignificanceThese results provide new insight into the pivotal role of estrogen/ER activation via modulation of TLR-4/NF-kβ, in the alleviation of LF pathogenesis.

MUC1 attenuates neutrophilic airway inflammation in asthma by reducing NLRP3 inflammasome-mediated pyroptosis through the inhibition of the TLR4/MyD88/NF-κB pathway

BackgroundNeutrophilic airway inflammation is a challenge in asthma management and is associated with poor patient prognosis. Mucin 1 (MUC1), which contains a cytoplasmic tail (MUC1-CT), has been found to mediate glucocorticoid sensitivity in asthma; however, its role in modulating neutrophilic airway inflammation in asthma remains unknown.MethodsHuman-induced sputum cells were collected from healthy participants (n = 12), patients with mild-to-moderate asthma (n = 34), and those with severe asthma (n = 18). In vitro human lung bronchial 1 epithelial cell line (BEAS-2B) was transfected with small interfering RNA against MUC1 (MUC1-siRNA) and then stimulated by lipopolysaccharide (LPS), where some cells were pretreated with a TLR4 inhibitor (TAK-242). In vivo mouse model of asthmatic neutrophil airway inflammation was induced by ovalbumin (OVA)/LPS. Some groups were intraperitoneally injected with MUC1-CT inhibitor (GO-203) and/or TAK-242 .ResultsThe mRNA expression of MUC1 was downregulated in the induced sputum of patients with asthma and correlated with asthmatic neutrophilic airway inflammation. The mRNA expressions of TLR4, MyD88, nucleotide-binding oligomerization domain-like pyrin domain-containing protein 3 (NLRP3), caspase-1, interleukin (IL)-18, and IL-1β in induced sputum cells of patients with asthma were upregulated and related to the mRNA expression of MUC1. LPS activated the TLR4 pathway and NLRP3-mediated pyroptosis in BEAS-2B cells in vitro, which were significantly aggravated after MUC1-siRNA transfection. Furthermore, MUCl-CT interacted with TLR4, and the interaction between TLR4 and MyD88 was significantly increased after MUCl-siRNA transfection. Moreover, TAK-242 ameliorated TLR4/MyD88/nuclear factor kappa B (NF-κB) pathway activation, NLRP3 inflammasome-mediated pyroptosis, and neutrophilic inflammation exacerbated by MUC1 downregulation. GO-203 exacerbated TLR4/MyD88/NF-κB pathway activation in vivo, and NLRP3 inflammasome-mediated pyroptosis reduced in a mouse model of asthmatic neutrophil airway inflammation induced by OVA/LPS; these pathological changes were partially alleviated after TAK-242 application.ConclusionThis study revealed that MUC1 downregulation plays an important role in asthmatic neutrophilic airway inflammation. MUC1-CT reduces NLRP3 inflammasome-mediated pyroptosis by inhibiting the activation of the TLR4/MyD88/NF-κB pathway, thereby attenuating neutrophil airway inflammation in patients with asthma.

Azomethine functionalized platform for the selective detection of Zr(IV) ion, biological evaluation and potent TLR-4 inhibitor

In this study, a rapid-sensing material featuring azomethine functionalized Schiff base has been developed to address the pressing need for zirconium detection. The sensor demonstrates remarkable selectivity for zirconium (IV) ions, surpassing its performance with respect to other relevant metal ions. The elucidation of compound structures was accomplished through X-ray crystallography, unveiling a new approach to zirconium (IV) ion detection with a high binding constant of 8.01 × 104 M−1 and an exceptionally low limit of detection (LOD) of 31.5 nM. Moreover, the binding stoichiometry between Probe 4a and Zr(IV) was determined to be 1:1, as confirmed by both Job's plot analysis and spectroscopy analysis. Successful Zr(IV) ion detection in real samples was achieved using synthesized compound 4a. Furthermore, compound 4a exhibits notable antibacterial and antioxidant properties. Molecular docking analysis with the TLR-4 receptor protein supports the antibacterial efficacy of compound 4a, revealing a robust binding energy of -8.23 kcal mol−1.

Nanoplastics activate a TLR4/p38-mediated pro-inflammatory response in human intestinal and mouse microglia cells

The crescent presence of nanoplastics in the environment raises concerns regarding their potential impact on health. This study exposed human colon adenocarcinoma cells (HT29) and microglia cells (N9) to nanoplastics (25 nm, 50 nm, and 100 nm Polystyrene) to investigate their inflammatory responses, which are vital for body's defence. Although cytotoxicity remained generally low, HT29 cells exhibited a notable upregulation of p50 and p38 expression, concomitant with elevated TLR4 expression, in contrast with N9 cells that showed a less pronounced upregulation of these proteins. Additionally, nanoplastic exposure increased IL-1ß levels, partially attenuated by pre-exposure to TLR4 or p38 inhibitors. Intriguingly, N9 cells exposed to nanoplastics exhibited substantial increases in iNOS mRNA. This effect was entirely prevented by pre-exposure to TLR4 or p38 inhibitors, while TNF-α mRNA levels remained relatively stable. These findings underscore the potential of nanoplastics to activate inflammatory pathways, with response kinetics varying depending on the cell type.

Substantial heterogeneity of inflammatory cytokine production and its inhibition by a triple cocktail of toll-like receptor blockers in early sepsis.

Early sepsis is a life-threatening immune dysregulation believed to feature a "cytokine storm" due to activation of pattern recognition receptors by pathogen and danger associated molecular patterns. However, treatments with single toll-like receptor (TLR) blockers have shown no clinical benefit. We speculated that sepsis patients at the time of diagnosis are heterogeneous in relation to their cytokine production and its potential inhibition by a triple cocktail of TLR blockers. Accordingly, we analyzed inflammatory cytokine production in whole blood assays from early sepsis patients and determined the effects of triple TLR-blockade. Whole blood of 51 intensive care patients sampled within 24h of meeting Sepsis-3 criteria was incubated for 6h without or with specific TLR2, 4, and 7/8 stimuli or suspensions of heat-killed S. aureus or E. coli bacteria as pan-TLR challenges, and also with a combination of monoclonal antibodies against TLR2 and 4 and chloroquine (endosomal TLR inhibition), subsequent to dose optimization. Concentrations of tumor necrosis factor (TNF), Interleukin(IL)-6, IL-8, IL-10, IL-1α and IL-1β were measured (multiplex ELISA) before and after incubation. Samples from 11 sex and age-matched healthy volunteers served as controls and for dose-finding studies. Only a fraction of sepsis patient samples revealed ongoing cytokine production ex vivo despite sampling within 24h of first meeting Sepsis-3 criteria. In dose finding studies, inhibition of TLR2, 4 and endosomal TLRs reliably suppressed cytokine production to specific TLR agonists and added bacteria. However, inflammatory cytokine production ex vivo was only suppressed in the high cytokine producing samples but not in the majority. The suppressive response to TLR-blockade correlated both with intraassay inflammatory cytokine production (r=0.29-0.68; p<0.0001-0.04) and cytokine baseline concentrations (r=0.55; p<0.0001). Upon meeting Sepsis-3 criteria for less than 24h, a mere quarter of patient samples exhibits a strong inflammatory phenotype, as characterized by increased baseline inflammatory cytokine concentrations and a stark TLR-dependent increase upon further ex vivo incubation. Thus, early sepsis patient cohorts as defined by Sepsis-3 criteria are very heterogeneous in regard to inflammation. Accordingly, proper ex vivo assays may be useful in septic individuals before embarking on immunomodulatory treatments.

Anti-influenza drug screening and inhibition of apigetrin on influenza A virus replication via TLR4 and autophagy pathways

Activation of Toll-like receptor (TLR) 4 plays important roles in the influenzaA virus (IAV) infection. To explore TLR4 inhibitors, 161 traditional Chinese medicines (TCMs) were screened. Further, we screened out Ixeris sonchifolia Hance, and its active compound, Apigetrin (apigenin-7-O-glucoside). Antiviral activity of Apigetrin was determined by plaque assay. We also further investigated the influence of Apigetrin on immune signaling pathways including TLRs, MAPK, NF-κB and autophagy pathways. The in-vitro results showed that the extract and its several ingredients could significantly inhibit IAV replication. Apigetrin significantly improved IAV-induced oxidative stress, inhibited the IAV-induced cytokine storm by suppressing the excessive activation of TLR3/4/7, JNK/p38 MAPK and NF-κB. Apigetrin decreased autophagosome accumulation and promoted degradation of IAV protein. Interestingly, Apigetrin antiviral activity was reversed by using H2O2 and the agonists of TLR4, JNK/p38, NF-κB and autophagy. Most important, the in-vitro effective concentration is higher than the reported plasma concentration. The in-vivo test showed that Apigetrin significantly increased the average survival time, reduced the lung edema and IAV replication. In conclusion, we have found that Ixeris sonchifolia Hance and its several ingredients can inhibit IAV infection, and the mechanisms of action of Apigetrin against IAV is by regulating TLR4 and autophagy signaling pathways.

Neurotropin alleviates cognitive impairment by inhibiting TLR4/MyD88/NF-κB inflammation signaling pathway in mice with vascular dementia

Vascular dementia (VD) is the second most common cause of dementia after Alzheimer's disease. Neuroinflammation contributes to pathogenesis of VD. Neurotropin (NTP) is an analgesic that has been shown to suppress inflammation and neural repair. But its effects on VD are still unclear. Therefore, this study aimed to investigate the therapeutic effects and potential mechanisms of NTP in the VD model mice established by bilateral common carotid artery stenosis method. In VD mice, we found that NTP treatment increased cerebral blood flow by Laser speckle imaging, reduced neuron loss by Nissl, HE and immunochemistry staining, attenuated white matter damage by magnetic resonance imaging and ultrastructural damage by transmission electron microscope, improved cognitive functions by new object recognition test and three-chamber test, Y maze test and Morris water maze test, inhibited significantly glial activation by immunofluorescence methods, reduced the expression of TLR4, down-regulated expression of MyD88 and phosphorylation of NF-κB P65, decreased the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNFα. Further, we showed that administration of a TLR4 inhibitor TAK242 had a similar effect to NTP, while the TLR4 agonist CRX-527 attenuated the effect of NTP in the VD mice. Collectively, our study suggested that NTP alleviates cognitive impairment by inhibiting TLR4/MyD88/NF-κB inflammation signaling pathway in the VD mice. Thus, NTP may be a promising therapeutic approach and a potential TLR4 inhibitor for VD.

Ultrasound-assisted extraction and characterization of Penthorum chinense polysaccharide with anti-inflammatory effects

Penthorum chinense has been used in both food and medication for many years, and polysaccharide of which was considered as one of the bioactive compounds. However, the extraction process of polysaccharide from P. chinense (PCP) was not well optimized. Ultrasound-assisted extractionhas been widely employed in the extraction of natural products for its compliance with the concept of green and economic chemistry. To better investigate the structure and biology activity of PCP, response surface methodology was employed to optimize the ultrasound-assisted extraction conditions of PCP. The optimum extraction for the ultrasound-assisted extraction of PCP were obtained as ratio of solvent to material 40 mL/g, ultrasonic power 380 W, and extraction time of 50 min. The yield of PCP reached 8.71% under these optimized conditions. PCP was further purified by using anion exchange chromatography and gel filtration, an acidic fraction PCP-AP-1 was hereby obtained. The results of structural elucidation indicated that PCP-AP-1 was a typical pectic polysaccharide with a molecular weight of 66360 Da, mainly composed of galacturonic acid (68.5 mol%), followed by arabinose (9.8 mol%), rhamnose (9.4 mol%), glucose (7.7 mol%), with homogalacturonan region and rhamnogalacturonan I regions. In vitro study showed that PCP-AP-1 could improve the inflammation induced by lipopolysaccharide in intestinal epithelial cells, which was probably performed through the inhibition of multiple signaling pathways including the inhibition of TLR4, NOD1/2 and NF-κB pathway, as well as the reduction of NLRP3 inflammasome. This study defined the type of polysaccharide present in P. chinense and revealed a potential of application this plant in the prevention of intestinal inflammatory diseases.

Evodiamine alleviates DEHP-induced hepatocyte pyroptosis, necroptosis and immunosuppression in grass carp through ROS-regulated TLR4 / MyD88 / NF-κB pathway.

Di (2-ethylhexyl) phthalate (DEHP) is a neuroendocrine disruptor that can cause multi-tissue organ damage by inducing oxidative stress. Evodiamine (EVO) is an indole alkaloid with anti-inflammatory, antitumor, and antioxidant pharmacological activity. In this manuscript, the effects of DEHP and EVO on the pyroptosis, necroptosis and immunology of grass carp hepatocytes (L8824) were investigated using DCFH-DA staining, PI staining, IF staining, AO/EB staining, LDH kit, qRT-PCR and protein Western blot. The results showed that DEHP exposure upregulated reactive oxygen species (ROS) levels, promoted the expression of TLR4/MyD88/NF-κB pathway, increased the expression of genes involved in cell pyroptosis pathway (LDH, NLRP3, ASC, caspase1, IL-1β, IL-18 and GSDMD) and necroptosis-related genes (RIPK1, RIPK3 and MLKL). The expression of DEHP can also affect immune function, which can be demonstrated by variationsin the activation of antimicrobial peptides (LEAP2, HEPC, and β-defensin) and inflammatory cytokines (TNF-α, IL-2, IL-6 and IL-10). EVO regulates cellular antioxidant capacity by inhibiting ROS burst, reduces DEHP-induced cell pyroptosis and necroptosis to some extent, and restores cellular immune function, after co-exposure with EVO. The TLR4 pathway was inhibited by the co-treatment of TLR4 inhibitor TLR-IN-C34 and DEHP, which attenuated the expression of cell pyroptosis, necroptosis, and immunosuppression. Thus, DEHP induced pyroptosis, necroptosis and abnormal immune function in L8824cells by activating TLR4/MyD88/NF-κB pathway. In addition, EVO has a therapeutic effect on DEHP-induced toxic injury. This study further provides a theoretical basis for the risk assessment of plasticizer DEHP on aquatic organisms.