Compound c17 alleviates inflammatory cardiomyopathy in streptozotocin-induced diabetic mice by targeting MyD88.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a common complication of diabetes mellitus and is associated with increased morbidity and mortality due to cardiac dysfunction. Chronic inflammation plays a significant role in the development of DCM, making it a promising target for novel pharmacological strategies. Our previous study has synthesized a novel compound, c17, which exhibited strong anti-inflammatory activity by specifically targeting to myeloid differentiation primary response 88 (MyD88). In this study, we evaluated the therapeutic effect of c17 in DCM. METHODS: The small molecular selective MyD88 inhibitor, c17, was used to evaluate the effect of MyD88 on DCM in both high concentration of glucose- and palmitic acid-stimulated macrophages and streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) mice. RESULTS: The treatment of c17 in T1DM mice resulted in improved heart function and reduced cardiac hypertrophy, inflammation and fibrogenesis. RNA sequencing analysis of the heart tissues revealed that c17 effectively suppressed the inflammatory response by regulating the MyD88-dependent pathway. Co-immunoprecipitation experiments further confirmed that c17 disrupted the interaction between MyD88 and Toll-like receptor 4 (TLR4), consequently inhibiting downstream NF-κB activation. In vitro studies demonstrated that c17 exhibited similar anti-inflammatory activity by targeting MyD88 in macrophages, which are the primary regulators of cardiac inflammation. Furthermore, conditioned medium derived from c17-treated macrophages showed reduced capacity to induce hypertrophy, pro-fibrotic reactions, and secondary inflammation in cardiomyocytes. CONCLUSIONS: In conclusion, the small-molecule MyD88 inhibitor, c17, effectively combated the inflammatory DCM, therefore could be a potential candidate for the treatment of this disease.

Caffeine Ameliorates AKT-Driven Nonalcoholic Steatohepatitis by Suppressing De Novo Lipogenesis and MyD88 Palmitoylation.

Dysregulated hepatic lipogenesis represents a promising druggable target for treating nonalcoholic steatohepatitis (NASH). This work aims to evaluate the therapeutic efficacy of caffeine in a NASH mouse model displaying increased hepatic lipogenesis driven by constitutive hepatic overexpression of the active v-akt murine thymoma viral oncogene homolog (AKT). Caffeine was administered in the AKT mice to study the efficacy in vivo. AKT-transfected and insulin-stimulated human hepatoma cells were used for in vitro experiments. The results demonstrated that caffeine ameliorated hepatic steatosis and inflammatory injury in vivo. Mechanistically, caffeine repressed the AKT/mTORC1 and SREBP-1/ACC/FASN signaling in mice and in vitro. Furthermore, caffeine impaired NF-κB activation by stabilizing IκBα, resulting in a reduction of proinflammatory mediators interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). Notably, caffeine abolished mTORC1/FASN-dependent MyD88 palmitoylation, which could be essential for its anti-inflammatory potential. Collectively, these results suggest that caffeine consumption could be advantageous in the prevention and therapy of NASH, especially in the subset accompanied by increased de novo lipogenesis.

DL0410 Alleviates Memory Impairment in D-Galactose-Induced Aging Rats by Suppressing Neuroinflammation via the TLR4/MyD88/NF-κB Pathway.

Oxidative stress and neuroinflammation have been demonstrated to be linked with Alzheimer's disease (AD). In this study, we examined the protective effects of DL0410 in aging rats and explored the underlying mechanism against oxidative damage and neuroinflammation, which was then validated in LPS-stimulated BV2 microglia. We firstly investigated the improvement effects of DL0410 on learning and memory abilities and explored the potential mechanisms in D-gal-induced aging rats. An 8-week treatment with DL0410 significantly improved the learning and cognitive function of D-gal-stimulated Alzheimer's-like rats in the Morris water maze test, step-down test, and novel object recognition test, and the therapeutic effect of DL0410 at 10 mg/kg was even better than that of donepezil. What is more, the results showed that DL0410 alleviated neuron injury, increased the number of synapses, and improved the level of postsynaptic density protein 95 (PSD95) in the hippocampus and cortex. Next, we examined the protective effects of DL0410 against oxidative damage and neuroinflammation. Our observations indicated that DL0410 reduced the production of harmful oxidation products and promoted the antioxidative system, decreased the levels of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interleukin 6 (IL-6), and increased anti-inflammatory cytokines IL-10. Moreover, DL0410 inhibited the activation of astrocytes and microglia and suppressed the activation of the TLR4/MyD88/NF-κB signaling pathway. The anti-inflammation effect of DL0410 was further confirmed in LPS-stimulated BV2 cells, and the results showed that DL0410 reduced the level of inflammatory factors and inhibited the activation of the TLR4/MyD88/TRAF6/NF-κB signaling pathway in BV2 microglia. Molecular docking results indicated that DL0410 occupied the LPS recognition site in the TLR4/MD2 complex. Furthermore, the enhanced expression of claudin-1, claudin-5, occludin, CX43, and ZO-1 indicated that DL0410 protected the blood-brain barrier (BBB) integrity. Together, these results suggest that DL0410 exerts neuroprotective effects against hippocampus and cortex injury induced by D-galactose, and the possible mechanisms include antioxidative stress, antineuroinflammation, improving synaptic plasticity, and maintaining BBB integrity, which is mediated by the TLR4/MyD88/NF-κB signaling pathway inhibition. We suggest that DL0410 is a promising candidate for AD treatment.

Treadmill exercise alleviates neuronal damage by suppressing NLRP3 inflammasome and microglial activation in the MPTP mouse model of Parkinson's disease.

Treadmill exercise has been recognized as an effectively therapeutic strategy for Parkinson's disease (PD). However, its exact molecular mechanism of promoting PD remain unclear. Recently, the NLRP3 inflammasome is considered to play a critical role in the pathogenesis of PD. In this study, we investigated whether NLRP3 inflammasome was involved in treadmill exercise-induced neuroprotection and anti-inflammation effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. 8-week-old male mice (C57BL/6 strain) were divided into four groups: Control, MPTP, MPTP + EX and EX. MPTP was intraperitoneally injected into mice to establish chronic PD model. The open-field test and pole test were used to assess motor function. The results showed that treadmill exercise significantly alleviated motor dysfunction and dopaminergic neuron degeneration induced by MPTP. In addition, we also found that treadmill exercise suppressed MPTP-triggered microglia activation and the co-localization of NLRP3+/Iba-1+ cells in the substantia nigra. These effects were associated with suppression NLRP3 inflammasome via down-regulation of TLR4/MyD88/NF-κB pathway. Overall, our study demonstrated that treadmill exercise could effectively alleviates neuronal damage via inhibition of NLRP3 inflammasome and microglial activation in MPTP-induced PD mouse model.

Vitamin D3 Controls TLR4- and TLR2-Mediated Inflammatory Responses of Endometrial Cells.

OBJECTIVES: Vitamin D has potent immunoregulatory features and modulates innate and adaptive immune responses. There is a significant association between intrauterine infection-associated inflammatory responses and pregnancy complications such as abortion and preterm labor. Here, we investigated how 1,25 (OH)2 D3 could modulate inflammatory responses of endometrial cells. DESIGN: This is an in vitro experimental study. Endometrial stromal cells (ESCs) and whole endometrial cells (WECs) were collected from 15 apparently normal women, and the immunomodulatory effects of 1,25 (OH)2 D3 on lipopolysaccharide (LPS)- or lipoteichoic acid (LTA)-treated ESCs and WECs were investigated. Participants/Materials, Setting, and Methods: Women with no history of abortion, infertility, endometriosis, or sign of vaginal infection were enrolled in this study. Endometrial samples were collected by gynecologists using a Pipelle pipette in the proliferative phase of the menstrual cycle. WECs and ESCs were collected and treated with either LPS or LTA. The levels of IL-6, IL-8, and TNF-α in culture supernatants were quantified using the ELISA technique. TLR2, TLR4, and MyD88 expressions were assessed by RT-qPCR. TLR4 expression at the protein level was studied by the Western blot technique. RESULTS: 1,25 Dihydroxycholecalciferol (1,25 (OH)2 D3) significantly reduced TNF-α production in LPS-activated ESCs and TNF-α and IL-6 production by LTA-stimulated WECs. In contrast, 1,25 (OH)2 D3 pretreatment increased the production of IL-8 by LPS- and LTA-stimulated endometrial cells. 1,25 (OH)2 D3 pretreatment markedly reduced LPS-induced TLR4 protein expression by ESCs. LPS treatment of ESCs significantly induced MyD88 gene expression. This effect was reversed when these cells were pretreated with 1,25 (OH)2 D3 before stimulation with LPS. LIMITATIONS: Because of the small size of samples, doing experiments all together on some samples was not feasible. Confirmation of the results obtained here needs well-designed in vivo studies. CONCLUSIONS: 1,25 (OH)2 D3 is an immunomodulatory molecule essential for maintaining endometrial immune homeostasis by controlling potentially harmful inflammatory responses associated with female reproductive tract infections.

Alogliptin Attenuates Lipopolysaccharide-Induced Neuroinflammation in Mice Through Modulation of TLR4/MYD88/NF-κB and miRNA-155/SOCS-1 Signaling Pathways.

BACKGROUND: Endotoxin-induced neuroinflammation plays a crucial role in the pathogenesis and progression of various neurodegenerative diseases. A growing body of evidence supports that incretin-acting drugs possess various neuroprotective effects that can improve learning and memory impairments in Alzheimer's disease models. Thus, the present study aimed to investigate whether alogliptin, a dipeptidyl peptidase-4 inhibitor, has neuroprotective effects against lipopolysaccharide (LPS)-induced neuroinflammation and cognitive impairment in mice as well as the potential mechanisms underlying these effects. METHODS: Mice were treated with alogliptin (20 mg/kg/d; p.o.) for 14 days, starting 1 day prior to intracerebroventricular LPS injection (8 µg/µL in 3 µL). RESULTS: Alogliptin treatment alleviated LPS-induced cognitive impairment as assessed by Morris water maze and novel object recognition tests. Moreover, alogliptin reversed LPS-induced increases in toll-like receptor 4 and myeloid differentiation primary response 88 protein expression, nuclear factor-κB p65 content, and microRNA-155 gene expression. It also rescued LPS-induced decreases in suppressor of cytokine signaling gene expression, cyclic adenosine monophosphate (cAMP) content, and phosphorylated cAMP response element binding protein expression in the brain. CONCLUSION: The present study sheds light on the potential neuroprotective effects of alogliptin against intracerebroventricular LPS-induced neuroinflammation and its associated memory impairment via inhibition of toll-like receptor 4/ myeloid differentiation primary response 88/ nuclear factor-κB signaling, modulation of microRNA-155/suppressor of cytokine signaling-1 expression, and enhancement of cAMP/phosphorylated cAMP response element binding protein signaling.

Quercetin alleviates neonatal hypoxic-ischemic brain injury by inhibiting microglia-derived oxidative stress and TLR4-mediated inflammation.

OBJECTIVE AND DESIGN: Microglia stimulated by oxygen glucose deprivation (OGD) were treated with quercetin to investigate the effect on oxidative stress and the inflammatory response and to explore whether toll-like receptor 4 (TLR4) signaling was involved. In addition, the effect of quercetin on the neurological functions of neonatal mice with hypoxic-ischemic brain injury (HIBI) was examined. MATERIALS AND SUBJECTS: Mouse BV2 microglial cells and postnatal day 7 neonatal mice were used. TREATMENT: A predetermined concentration of quercetin was used in cell experiments. Quercetin was injected i.p. (50 mg/kg) at three time points after HI insult: 0, 24, and 48 h. METHODS: Cell viability assay, Western blotting, qRT-RCR, ELISA, HIBI model construction and behavioral tests. RESULTS: This study first showed that quercetin protected BV2 cells from OGD-induced damage and reversed the changes in microglial oxidative stress-related molecules. Second, quercetin inhibited OGD-induced expression of inflammatory factors in BV2 cells and suppressed TLR4/MyD88/NF-κB signaling. Finally, quercetin was disclosed to be effective in mitigating cerebral infarct volume and cognitive and motor function deficits in HIBI mice. CONCLUSION: These results suggest that the neuroprotective effect of quercetin in HIBI mice is partially due to the inhibition of oxidative stress and TLR4-mediated inflammatory responses in activated microglia.

MyD88-dependent and -independent signalling via TLR3 and TLR4 are differentially modulated by Δ9-tetrahydrocannabinol and cannabidiol in human macrophages.

Toll-like receptors (TLRs) are sensors of pathogen-associated molecules that trigger inflammatory signalling in innate immune cells including macrophages. All TLRs, with the exception of TLR3, promote intracellular signalling via recruitment of the myeloid differentiation factor 88 (MyD88) adaptor, while TLR3 signals via Toll-Interleukin-1 Receptor (TIR)-domain-containing adaptor-inducing interferon (IFN)-ß (TRIF) adaptor to induce MyD88-independent signalling. Furthermore, TLR4 can activate both MyD88-dependent and -independent signalling (via TRIF). The study aim was to decipher the impact of the highly purified plant-derived (phyto) cannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), when delivered in isolation and in combination (1:1), on MyD88-dependent and -independent signalling in macrophages. We employed the use of the viral dsRNA mimetic poly(I:C) and endotoxin lipopolysaccharide (LPS), to induce viral TLR3 and bacterial TLR4 signalling in human Tamm-Horsfall protein-1 (THP-1)-derived macrophages, respectively. TLR3/TLR4 stimulation promoted the activation of interferon (IFN) regulatory factor 3 (IRF3) and TLR4 promoted the activation of nuclear factor (NF)-κB signalling, with downstream production of the type I IFN-ß, the chemokines CXCL10 and CXCL8, and cytokine TNF-α. THC and CBD (both at 10 µM) attenuated TLR3/4-induced IRF3 activation and induction of CXCL10/IFN-ß, while both phytocannabinoids failed to impact TLR4-induced IκB-α degradation and TNF-α/CXCL8 expression. The role of CB1, CB2 and PPARγ receptors in mediating the effect of THC and CBD on MyD88-independent signalling was investigated. TLRs are attractive therapeutic targets given their role in inflammation and initiation of adaptive immunity, and data herein indicate that both CBD and THC preferentially modulate TLR3 and TLR4 signalling via MyD88-independent mechanisms in macrophages. This offers mechanistic insight into the role of phytocannabinoids in modulating cellular inflammation.

R848 Is Involved in the Antibacterial Immune Response of Golden Pompano (Trachinotus ovatus) Through TLR7/8-MyD88-NF-κB-Signaling Pathway.

R848 is an imidazoquinoline compound that is a specific activator of toll-like receptor (TLR) 7/8 and is often used in immunological research in mammals and teleosts. However, the immune responses initiated by R848 through the TLR7/8 pathway in response to bacterial infection remain largely unexplored in teleosts. In the current study, we investigated the antibacterial response and the participating signaling pathway initiated by R848 in golden pompano (Trachinotus ovatus). We found that R848 could stimulate the proliferation of head kidney lymphocytes (HKLs) in a dose-dependent manner, enhance the survival rate of HKLs, and inhibit the replication of bacteria in vivo. However, these effects induced by R848 were significantly reduced when chloroquine (CQ) was used to blocked endosomal acidification. Additionally, an in vivo study showed that R848 strengthened the antibacterial immunity of fish through a TLR7/8 and Myd88-dependent signaling pathway. A cellular experiment showed that Pepinh-MYD (a Myd88 inhibitor) significantly reduced the R848-mediated proliferation and survival of HKLs. Luciferase activity analysis showed that R848 enhanced the nuclear factor kappa B (NF-κB) activity, whereas this activity was reduced when CQ and Pepinh-MYD were present. Additionally, when an NF-κB inhibitor was present, the R848-mediated pro-proliferative and pro-survival effects on HKLs were significantly diminished. An in vivo study showed that knockdown of TLR7, TLR8, and Myd88 expression in golden pompano via siRNA following injection of R848 resulted in increased bacterial dissemination and colonization in fish tissues compared to that of fish injection of R848 alone, suggesting that R848-induced antibacterial immunity was significantly reduced. In conclusion, these results indicate that R848 plays an essential role in the antibacterial immunity of golden pompano via the TLR7/8-Myd88-NF-κB- signaling pathway.

Targeting IRAK4 disrupts inflammatory pathways and delays tumor development in chronic lymphocytic leukemia.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and -unmutated cases. In the Eµ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

Effect of Toll-Like Receptor 4/Myeloid Differentiation Factor 88 Inhibition by Salvianolic Acid B on Neuropathic Pain After Spinal Cord Injury in Mice.

BACKGROUND: Spinal cord injury (SCI) is a common type of injury, and about half of patients affected by SCI will suffer from neuropathic pain within a year after injury. However, the treatment effect of neuropathic pain is far from satisfactory. Our study attempted to reveal whether salvianolic acid B (SalB) could relieve the neuropathic pain caused by SCI in mice by inhibiting the Toll-like receptor 4 (TLR4)/Myeloid differentiation factor 88 (MyD88) pathway. METHODS: The mice were randomly divided into a sham group, model group, high-dose treatment group, and low-dose treatment group. The high- and low-dose groups received varying doses of SalB after modeling. RESULTS: The increase of pain sensitivity was evaluated by detecting paw withdrawal mechanical threshold and withdrawal thermal latency. Messenger RNA and protein expression levels of TLR4 and myD88 were detected by using quantitative reverse-transcription polymerase chain reaction and western blot, respectively. Compared with the model group, there was a significant reduction in paw withdrawal mechanical threshold and withdrawal thermal latency after SalB treatment. CONCLUSIONS: SalB reduced the release of tumor necrosis factor-α and substance P by inhibiting the TLR4/MyD88 pathway in the SCI mouse model. This not only resulted in lower pain, but also contributed to long-term relief of mechanical hyperalgesia.

[The effects of silica dust on the expression of MyD88 and TRAF6 mRNA of lung macrophages in rats].

Objective: To investigate the effects of silica dust on the expression of Myeloid differentiation factor 88 (MyD88) mRNA and tumor necrosis factor receptor-associated factor (TRAF6) mRNA of lung macrophages in rats. Methods: Selecting 40 SPF-class Wistar rats with average weight (200±20) g randomly divided into control group and 30 d, 60 d, 120 d experimental groups with 10 rats in each group according to body weight. The experimental groups rats were injected with 1 ml of SiO(2) (100 mg/ml) suspension through the trachea into lung only once, then they were respectively killed after 30, 60, 120 days. The control group rats were injected with 1 ml of saline into lung, and killed after 120 days. The lungs of the rats were taken for pathological observation. Lung macrophages were extracted and counted, and their activity was detected by MTT. RT-qPCR was used to assess the relative contents of MyD88 mRNA and TRAF6 mRNA. Results: Silica dust inhalation led to infiltration of lung tissue cells, thickening the alveolar wall and destruction of alveolar structure. The longer the exposure to dust, the more obvious the results were. The number of macrophages in all experimental groups and activity in the 30 d, 60 d groups were significantly higher than that in the control group (P<0.05) . Among them, 30 d group had the largest number and the highest activity. Compared with the control group, the expression of MyD88 mRNA and TRAF6 mRNA of lung macrophages in rats increased in the experimental groups (P<0.05) , especially in the 60 d group. Conclusion: Silica dust inhalation can increase the expression of MyD88 and TRAF6 in macrophages, suggesting that silica dust can induce silicosis fibrosis by activating TLR/NF-κB signal pathway.

Allicin Improves Lung Injury Induced by Sepsis via Regulation of the Toll-Like Receptor 4 (TLR4)/Myeloid Differentiation Primary Response 88 (MYD88)/Nuclear Factor kappa B (NF-κB) Pathway.

BACKGROUND The aim of this study was to assess the effects and mechanisms of allicin in a sepsis-induced lung injury in vivo study. MATERIAL AND METHODS The rats (n=54) were divided into 6 groups: Normal, DMSO, LPS, LPS+LD, LPS+MD, and LPS+HD groups. After being treated by different methods, we collected the lung tissues of different groups and evaluated the pathology by HE staining and positive apoptosis cells by TUNEL. We assessed the W/D ratio, inflammatory cytokines (TNF-alpha, IL-6 and IL-1ß), and relative protein expressions (TLR4, MyD88, NF-kappaB, caspase-3, and caspase-9) by IHC assay. RESULTS Compared with LPS group, the lung injury and positive cell number of allicin treated groups were significantly improved with dose-dependent (P<0.05, respectively) and the W/D ratio and TNF-alpha, IL-6 and IL-1ß concentration were significantly down-regulation compared with those of LPS group with dose-dependent (P<0.05, respectively). By IHC, the TLR4, MyD88, NF-kappaB, caspase-3 and caspase-9 protein activities of allicin treated groups were significantly suppressed compared with those of LPS group (P<0.05, respectively) in lung tissues. CONCLUSIONS This in vivo study shows that allicin improved sepsis-induced lung injury by regulation of TLR4/MyD88/NF-kappaB.

Prolyl hydroxylases positively regulated LPS-induced inflammation in human gingival fibroblasts via TLR4/MyD88-mediated AKT/NF-κB and MAPK pathways.

OBJECTIVES: Prolyl hydroxylases (PHDs) play essential roles in oxygen-sensing system, whereas the effects of PHDs on inflammation have not been totally uncovered. Our study aimed to investigate the role of PHDs in lipopolysaccharide (LPS)-induced inflammation of human gingival fibroblasts (HGFs) and clarify the potential mechanisms. MATERIALS AND METHODS: A pan hydroxylase inhibitor, dimethyloxallyl glycine (DMOG), and RNA interference were used to explore the role of PHDs in inflammation. Cytotoxic effect of DMOG was determined by cell-counting kit-8 and flow cytometry respectively. The secretion levels of IL-6 and IL-8 were assessed by ELISA. The mRNA levels of inflammatory cytokines, Toll-like receptor (TLR) 4 and MyD88 were evaluated by quantitative real-time PCR. The activation of NF-κB, mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways were detected by western blot and the nuclear translocation of NF-κB p65 was examined by immunofluorescence. Downregulation of PHD1 and PHD2 was performed with siRNA transfection. RESULTS: Dimethyloxallyl glycine inhibited LPS-induced inflammatory cytokine, TLR4 and MyD88 expression in gene level and the elevated secretion of IL-6 and IL-8 was also downregulated. Additionally, LPS-induced activation of NF-κB, MAPK and AKT pathways was abolished by DMOG treatment. Importantly, LPS-induced inflammatory cytokine expression was merely suppressed by PHD2 knockdown. CONCLUSIONS: Prolyl hydroxylases acted as a positive regulator in LPS-induced inflammation of HGFs via TLR4/MyD88-mediated NF-κB, MAPK and AKT signalling pathways and PHD2 among three isoforms was principally responsible for the effects.

Biochanin A Reduces Inflammatory Injury and Neuronal Apoptosis following Subarachnoid Hemorrhage via Suppression of the TLRs/TIRAP/MyD88/NF-κB Pathway.

Inflammatory injury and neuronal apoptosis participate in the period of early brain injury (EBI) after subarachnoid hemorrhage (SAH). Suppression of inflammation has recently been shown to reduce neuronal death and neurobehavioral dysfunction post SAH. Biochanin A (BCA), a natural bioactive isoflavonoid, has been confirmed to emerge the anti-inflammatory pharmacological function. This original study was aimed at evaluating and identifying the neuroprotective role of BCA and the underlying molecular mechanism in an experimental Sprague-Dawley rat SAH model. Neurobehavioral function was evaluated via the modified water maze test and modified Garcia neurologic score system. Thus, we confirmed that BCA markedly decreased the activated level of TLRs/TIRAP/MyD88/NF-κB pathway and the production of cytokines. BCA also significantly ameliorated neuronal apoptosis which correlated with the improvement of neurobehavioral dysfunction post SAH. These results indicated that BCA may provide neuroprotection against EBI through the inhibition of inflammatory injury and neuronal apoptosis partially via the TLRs/TIRAP/MyD88/NF-κB signal pathway.

Grape seed proanthocyanidin extract ameliorates murine autoimmune arthritis through regulation of TLR4/MyD88/NF-κB signaling pathway.

BACKGROUND/AIMS: Grape seed proanthocyanidin extract (GSPE) has been reported to have a beneficial effect on regulating inf lammation. However, the anti-inflammatory mechanism of GSPE remains unclear. The aim of this study was to verify the influence of GSPE on the Toll-like receptor 4 (TLR4)-mediated signaling pathway in the regulation of murine autoimmune arthritis. METHODS: Collagen-induced arthritis (CIA) was induced in dilute brown non-agouti (DBA)/1J mice. The mice were treated with GSPE (0 or 100 mg/kg) intraperitoneally. The severity of arthritis was assessed clinically, biochemically, and histologically. Immunostaining for TLR4 was performed. The expressions of TLR4 and downstream signaling molecules were analyzed by Western blot. The effect of GSPE on lipopolysaccharide (LPS)-induced TLR4 activation was also evaluated using RAW264.7 cells and fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis and from those with osteoarthritis. RESULTS: GSPE attenuated the clinical severity of arthritis and decreased histological damage. GSPE treatment reduced the number of TLR4-stained cells in the synovium of mice with CIA. GSPE also downregulated the expression of TLR4, myeloid differentiation factor 88 (MyD88) and phosphorylated IκBα synovial protein in CIA mice. Concurrently, GSPE inhibited the nuclear translocation of nuclear factor-κB (NF-κB) subunits (p65 and p50). LPS-induced TLR4 activation was suppressed by GSPE in human FLS as well as in murine macrophages in vitro. CONCLUSIONS: Our results demonstrated that GSPE ameliorated CIA by regulating the TLR4-MyD88-NF-κB signaling pathway.

Prophylactic supplement with melatonin successfully suppresses the pathogenesis of periodontitis through normalizing RANKL/OPG ratio and depressing the TLR4/MyD88 signaling pathway.

Periodontitis (PD) is an inflammatory disease characterized by gingival inflammation and resorption of alveolar bone. Impaired receptor activator of nuclear factor-kappa B ligand/osteoprotegerin (RANKL/OPG) signaling caused by enhanced production of pro-inflammatory cytokines plays an essential role in the pathogenesis of PD. Considering melatonin possesses significant anti-inflammatory property, this study aimed to determine whether prophylactic treatment with melatonin would effectively normalize RANKL/OPG signaling, depress toll-like receptor 4/myeloid differentiation factor 88 (TLR4/MyD88)-mediated pro-inflammatory cytokine activation, and successfully suppress the pathogenesis of PD. PD was induced in adult rats by placing the ligature at molar subgingival regions. Fourteen days before PD induction, 10, 50, or 100 mg/kg of melatonin was intraperitoneally injected for consecutive 28 days. Biochemical and enzyme-linked immunosorbent assay were used to detect TLR4/MyD88 activity, RANKL, OPG, interleukin 1ß, interleukin 6, and tumor necrosis factor-α levels, respectively. The extent of bone loss, bone mineral intensity, and calcium intensity was further evaluated by scanning electron microscopy, micro-computed tomography, and energy-dispersive X-ray spectroscopy. Results indicated that high RANKL/OPG ratio, TLR4/MyD88 activity, and pro-inflammatory cytokine levels were detected following PD. Impaired biochemical findings paralleled well with severe bone loss and reduced calcium intensity. However, in rats pretreated with melatonin, all above parameters were successfully returned to nearly normal levels with maximal change observed in rats receiving 100 mg/kg. As prophylactic treatment with melatonin effectively normalizes RANKL/OPG signaling by depressing TLR4/MyD88-mediated pro-inflammatory cytokine production, dietary supplement with melatonin may serve as an advanced strategy to strengthen oral health to counteract PD-induced destructive damage.

Effect of lipopolysaccharide on toll-like receptor-4 signals in mouse cancer cells.

BACKGROUND: Recent findings showed that activated TLR signals on cancer cells might promote cancer progression. This study was designed to explore the influence of Toll-like receptor 4 (TLR4) agonist lipopolysaccharides (LPS) on mouse melanoma and breast cancer cell proliferation and their TLR4 signalling. METHODS: Mouse melanoma cell line (B16F10) and breast cancer cell line (4T1) were taken as models. They were treated with LPS (0, 1.25, 2.5, 5, 7.5, 10 µg/ml) for 4, 16, 24, 48 h and MTT assay was done. The expression of TLR4, MyD88, NF-κB mRNA was detected by quantitative real time-polymerase chain reaction method quantitatively. RESULTS: Ultra-pure LPS at 5 µg/ml concentration increased significantly B16F10 cell viability 24 hour after stimulation, but not in 4T1 cell. The mRNA levels of TLR4, MyD88 and NF-κB were significantly up-regulated in both cell lines by stimulating the cells at 5 µg/ml LPS. CONCLUSIONS: Our data demonstrated that B16F10 and 4T1 cells are responsive to LPS, but their responses are time and dose dependent. These results provide new ways to understand the TLR4 signalling in tumour cells (Fig. 2, Ref. 24).

A preliminary study on the proinflammatory mechanisms of Treponema pallidum outer membrane protein Tp92 in human macrophages and HMEC-1 cells.

AIMS: To determine proinflammatory mechanisms of Treponema pallidum outer membrane protein Tp92 in the early syphilis infection in human macrophages and HMEC-1 cells. METHODS: Recombinant Tp92 protein was used to stimulate target human macrophages and HMEC-1 cells. PDTC (Pyrrolidinedithiocarbamic acid), SB202190 and Z-YVAD-FMK were used to block the MyD88/NF-κB, MAPKs/p38 and NLRP3/Caspase-1 pathway, respectively. TNF-α, IL-1ß, IL-6, IL-8,NLRP3, casepase-1 were detected by ELISA or Western blot. Lactate dehydrogenase (LDH) activity was measured. RESULTS: Tp92 protein could significantly induced the secretion of proinflammatory cytokines TNF-α, IL-1ß, IL-6 and IL-8 in HMEC-1 cells, but not in macrophages except IL-8. When MyD88/NF-κB pathway was blocked, differences in the secretion of TNF-α, IL-6 and IL-1ß levels and LDH enzyme activity between Tp92 group and Tp92 + PDTC group were not significant (P > 0.05) in HMEC-1 cells and macrophages except IL-8(P < 0.05). When MAPKs/p38 pathway was blocked, differences in the secretion of TNF-α, IL-1ß, IL-6 and IL-8 and LDH enzyme activity both Tp92 group and Tp92 + SB2010190 group were not significant (P > 0.05) in HMEC-1 cells and macrophages. In contrast, when NLRP3/Caspase-1 pathway was blocked with Z-YVAD-FMK, TNF-α, IL-6 and IL-1ß levels, LDH enzyme activity, and Caspase-1 and NLRP3 protein levels were significantly declined (P < 0.05) in HMEC-1 cells except IL-8(P > 0.05). The LDH enzyme activity in macrophages was decreased before and after Z-YVAD-FMK blocking (P < 0.05),however, differences in the secretion of TNF-α, IL-1ß, IL-6 and IL-8 between Tp92 group and Tp92+Z-YVAD-FMK group in macrophages were not significant (P > 0.05). CONCLUSIONS: Tp92 protein may promote proinflammatory cytokines TNF-α, IL-1ß, IL-6 secretion of HMEC-1 cells, but not in macrophages, and increase the LDH enzyme activity of HMEC-1 cells and macrophages through NLRP3/Caspase-1 pathway. However, Tp92 protein may promote IL-8 secretion of HMEC-1 cells and macrophages through MyD88/NF-κB pathway.