Gastrodin Attenuates Colitis and Prevents Tumorigenesis in Mice by Interrupting TLR4/MD2/NF-κB Signaling Transduction.

INTRODUCTION: Chronic inflammation is one of the causative factors for tumorigenesis. Gastrodin is a main active ingredient isolated from Gastrodia elata Blume, a famous medicinal herb with a long edible history. AIM: This study aimed to explore the effects of gastrodin on colitis-associated carcinogenesis (CRC) in mice and to elucidate its potential molecular mechanisms. METHODS: Balb/c mice were induced with azoxymethane (AOM) and dextran sulfate sodium (DSS) for 12 weeks. Gastrodin (50 mg/kg) was administered via oral gavage three times per week until the end of the experiment. Disease indexes, including body weight, bloody diarrhea, colon length, histopathological score, and tumor size, were measured. Tumor cell proliferation was evaluated by BrdU incorporation assay and tumor cell cytotoxicity was assessed by cell counting kit (CCK-8). The expression levels of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling molecules, NF-κB luciferase, and pro-inflammatory cytokines were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), immunoblotting, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), or reporter gene assays. The binding affinity between gastrodin and myeloid differentiation protein-2 (MD2) was analyzed by molecular docking and cellular thermal shift assay (CETSA). RESULTS: Gastrodin administration was demonstrated to mitigate various CRC-related symptoms in mice, including weight loss, diarrhea, and tissue abnormalities. Notably, gastrodin suppressed tumor cell growth during colitis- associated tumorigenesis, resulting in fewer and smaller adenomas in the colon. Unlike irinotecan, a broadspectrum antitumor drug, gastrodin did not exhibit apparent cytotoxicity in various colorectal adenocarcinoma cell lines. Additionally, gastrodin downregulated TLR4/NF-κB signaling molecules and pro-inflammatory mediators in mice and macrophages. Molecular docking and CETSA experiments suggested that gastrodin binds to the MD2 protein, potentially interfering with the recognition of lipopolysaccharide (LPS) by TLR4, leading to NF-κB pathway inhibition. CONCLUSION: This study provides evidence for the first time that gastrodin attenuated colitis and prevented colitisrelated carcinogenesis in mice, at least partially, by diminishing tumor-promoting cytokines through the interruption of TLR4/MD2/NF-κB signaling transduction.

Dried tangerine peel polysaccharide (DTPP) alleviates hepatic steatosis by suppressing TLR4/MD-2-mediated inflammation and endoplasmic reticulum stress.

Non-alcoholic fatty liver disease (NAFLD) is a complex pathogenic metabolic syndrome characterized by increased inflammation and endoplasmic reticulum stress. In recent years, natural polysaccharides derived from traditional Chinese medicine have shown significant anti-inflammatory effects, making them an attractive therapeutic option. However, little research has been conducted on the therapeutic potential of dried tangerine peel polysaccharide (DTPP) - one of the most important medicinal resources in China. The results of the present study showed that DTPP substantially reduced macrophage infiltration in vivo and suppressed the expression of pro-inflammatory factors and endoplasmic reticulum stress-related genes. Additionally, surface plasmon resonance analysis revealed that DTPP had a specific affinity to myeloid differentiation factor 2, which consequently suppressed lipopolysaccharide-induced inflammation via interaction with the toll-like receptor 4 signaling pathway. This study provides a potential molecular mechanism underlying the anti-inflammatory effects of DTPP on NAFLD and suggests DTPP as a promising therapeutic strategy for NAFLD treatment.

Dehydrocostus lactone alleviates irinotecan-induced intestinal mucositis by blocking TLR4/MD2 complex formation.

BACKGROUND: Irinotecan (CPT-11) is used as chemotherapeutic drug for treatment of colorectal cancer. However, without satisfactory treatments, its gastrointestinal toxicities such as diarrhea and intestinal inflammation severely restrained its clinical application. Roots of Aucklandia lappa Decne. are used as traditional Chinese medicine to relieve gastrointestinal dysfunction and dehydrocostus lactone (DHL) is one of its main active components. Nevertheless, the efficacy and mechanism of DHL against intestinal mucositis remains unclear. PURPOSE: The present study aimed to investigate the protective effects of DHL on CPT-11-induced intestinal mucositis and its underlying mechanisms. METHODS: The protective effect of DHL was investigated in CPT-11-induced mice and lipopolysaccharide (LPS)+CPT-11 induced THP-1 macrophages. Body weight, diarrhea score, survival rate, colon length, and histopathological changes in mice colon and jejunum were analyzed to evaluate the protective effect of DHL in vivo. And DHL on reducing inflammatory response and regulating TLR4/NF-κB/NLRP3 pathway in vivo and in vitro were explored. Moreover, DHL on the interaction between TLR4 and MD2 was investigated. And silencing TLR4 targeted by siRNA was performed to validate the mechanisms of DHL on regulating the inflammation. RESULTS: DHL prevented CPT-11-induced intestinal damage, represented by reducing weight loss, diarrhea score, mortality rate and the shortening of the colon. Histological analysis confirmed that DHL prevented intestinal epithelial injury and improved the intestinal barrier function in CPT-11 induced mice. Besides, DHL significantly downregulated the level of inflammatory cytokines by inhibiting TLR4/NF-κB/NLRP3 signaling pathway in CPT-11-induced mice and LPS+CPT-11-induced THP-1 macrophages. In addition, DHL blocked TLR4/MD2 complex formation. Molecular docking combined with SIP and DARTS assay showed that DHL could bind to TLR4/MD2 and occludes the hydrophobic pocket of MD2. Furthermore, Silencing TLR4 abrogated the effect of DHL on LPS+CPT-11 induced inflammatory response in THP-1 macrophages. Additionally, DHL ameliorate the CPT-11-induced intestinal mucositis without affecting the anti-tumor efficacy of CPT-11 in the tumor xenograft mice. CONCLUSION: This study found that DHL exhibited the anti-inflammatory effects in CPT-11-induced intestinal mucositis by inhibiting the formation of TLR4/MD2 complex and then regulation of NF-κB/NLRP3 signaling pathway. DHL is potentially served as a novel strategy of combined medication with CPT-11.

Radix Glycyrrhizae extract and licochalcone a exert an anti-inflammatory action by direct suppression of toll like receptor 4.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Glycyrrhizae (GL), a herbal medicine that is widely available, has shown advantages for a variety of inflammatory diseases. Toll like receptor 4 (TLR4) pathway has been shown to play a key role in the progression of inflammation. AIM OF THE STUDY: The purpose of this study was to investigate the involvement of TLR4 in the anti-inflammatory mechanism of GL extract and its active constituent on acute lung injury (ALI). MATERIALS AND METHODS: A model of inflammation produced by lipopolysaccharide (LPS) was established in C57BL/6 mice and macrophages derived from THP-1. To screen the active components of GL, molecular docking was used. Molecular dynamics and surface plasmon resonance imaging (SPRi) were used to study the interaction of a specific drug with the TLR4-MD2 complex. TLR4 was overexpressed by adenovirus to confirm TLR4 involvement in the anti-inflammatory activities of GL and the chosen chemical. RESULTS: We observed that GL extract significantly reduced both LPS-induced ALI and the production of pro-inflammatory factors including TNF-α, IL-6 and IL-1ß. Additionally, GL inhibited the binding of Alexa 488-labeled LPS (LPS-488) to the membrane of THP-1 derived macrophages. GL drastically reduce on the expression of TLR4 and the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-B (NF-κB). Furthermore, molecular docking revealed that Licochalcone A (LicoA) docked into the LPS binding site of TLR4-MD2 complex. MD2-LicoA binding conformation was found to be stable using molecular dynamic simulations. SPRi indicated that LicoA bound to TLR4-MD2 recombinant protein with a KD of 3.87 × 10-7 M. LicoA dose-dependently reduced LPS-488 binding to the cell membrane. LicoA was found to significantly inhibit LPS-induced lung damage and inflammation. Furthermore, LicoA inhibited TLR4 expression, MAPK and NF-κB activation in a dose-dependent manner. The inhibitory effects of GL and LicoA on LPS-induced inflammation and TLR4 signaling activation were partly eliminated by TLR4 overexpression. CONCLUSION: Our findings imply that GL and LicoA exert inhibitory effects on inflammation by targeting the TLR4 directly.

Paeoniflorin exerts antidepressant-like effects through enhancing neuronal FGF-2 by microglial inactivation.

Ethnopharmacological relevance Paeonia lactiflora is a famous Traditional Chinese medicine widely used for immunological regulation. Paeoniflorin, the main component of Paeonia lactiflora, exerts neuroprotective and antidepressant-like effects in rodents. AIM OF THE STUDY: Fibroblast growth factor 2 (FGF-2) is essentially required in the central nervous system as it acts as both a neurotrophic factor and an anti-inflammatory factor participating in the regulation of proliferation, differentiation and apoptosis of neurons in the brain. However, it is unclear whether paeoniflorin could exert antidepressant effects via regulating FGF-2. MATERIALS AND METHODS: In the present study, the effects of paeoniflorin were evaluated in depressive mice induced by the endotoxin lipopolysaccharide (LPS) injection. RESULTS: The results showed that paeoniflorin markedly increased sucrose preference and reduced immobility time in LPS mice, indicating antidepressant effects. Consistent with the results from molecular docking showing paeoniflorin antagonizes TLR4, NF-κB and NLRP3, the biochemical analysis also indicated paeoniflorin inhibited TLR4/NF-κB/NLRP3 signaling, decreased proinflammatory cytokine levels and microglial activation in the hippocampus of LPS induced mice. In addition, the levels of neuronal FGF-2 and the density of dendritic spine were improved by paeoniflorin. More importantly, the FGFR1 inhibitor SU5402 prevented the antidepressant effects of paeoniflorin and blocked the neuroinflammatory and neurogenic regulatory effects of paeoniflorin, indicating that FGF-2/FGFR1 activation was required for the effects of paeoniflorin. CONCLUSION: Taken together, the results demonstrate that paeoniflorin exhibits neuroprotective and antidepressant effects in mice, which may be mediated by activating neuronal FGF-2/FGFR1 signaling via the inhibition of microglial activation in the hippocampus.

N-palmitoyl-D-glucosamine, a Natural Monosaccharide-Based Glycolipid, Inhibits TLR4 and Prevents LPS-Induced Inflammation and Neuropathic Pain in Mice.

Toll-like receptors (TLRs) are key receptors through which infectious and non-infectious challenges act with consequent activation of the inflammatory cascade that plays a critical function in various acute and chronic diseases, behaving as amplification and chronicization factors of the inflammatory response. Previous studies have shown that synthetic analogues of lipid A based on glucosamine with few chains of unsaturated and saturated fatty acids, bind MD-2 and inhibit TLR4 receptors. These synthetic compounds showed antagonistic activity against TLR4 activation in vitro by LPS, but little or no activity in vivo. This study aimed to show the potential use of N-palmitoyl-D-glucosamine (PGA), a bacterial molecule with structural similarity to the lipid A component of LPS, which could be useful for preventing LPS-induced tissue damage or even peripheral neuropathies. Molecular docking and molecular dynamics simulations showed that PGA stably binds MD-2 with a MD-2/(PGA)3 stoichiometry. Treatment with PGA resulted in the following effects: (i) it prevented the NF-kB activation in LPS stimulated RAW264.7 cells; (ii) it decreased LPS-induced keratitis and corneal pro-inflammatory cytokines, whilst increasing anti-inflammatory cytokines; (iii) it normalized LPS-induced miR-20a-5p and miR-106a-5p upregulation and increased miR-27a-3p levels in the inflamed corneas; (iv) it decreased allodynia in peripheral neuropathy induced by oxaliplatin or formalin, but not following spared nerve injury of the sciatic nerve (SNI); (v) it prevented the formalin- or oxaliplatin-induced myelino-axonal degeneration of sciatic nerve. SIGNIFICANCE STATEMENT We report that PGA acts as a TLR4 antagonist and this may be the basis of its potent anti-inflammatory activity. Being unique because of its potency and stability, as compared to other similar congeners, PGA can represent a tool for the optimization of new TLR4 modulating drugs directed against the cytokine storm and the chronization of inflammation.

Astilbin prevents osteoarthritis development through the TLR4/MD-2 pathway.

Osteoarthritis has become one of the main diseases affecting the life of many elderly people with high incidence of disability, and local chronic inflammation in the joint cavity is the most crucial pathological feature of osteoarthritis. Astilbin is the main active component in a variety of natural plants such as Hypericum perforatum and Sarcandra glabra, which possess antioxidant and anti-inflammatory effects. At present, there is no study about the protective effect of Astilbin for osteoarthritis. The purpose of this study was to investigate the effect of Astilbin in human OA chondrocytes and mouse OA model, which was established by surgery-mediated destabilization of the medial meniscus (DMM). In vitro, we found that Astilbin pre-treatment inhibited lipopolysaccharide (LPS)-induced overproduction of inflammation-correlated cytokines such as nitric oxide (NO), prostaglandin E2 (PGE2), tumour necrosis factor α (TNF-α) and interleukin 6 (IL-6), and suppressed overexpression of inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Astilbin, on the other hand, prevented the LPS-induced degradation of extracellular matrix (ECM) by down-regulating MMP13 (matrix metalloproteinases 13) and ADAMTS5 (a disintegrin and metalloproteinase with thrombospondin motifs 5). Moreover, by inhibiting the formation of the TLR4/MD-2/LPS complex, Astilbin blocked LPS-induced activation of TLR4/NF-κB signalling cascade. In vivo, Astilbin showed the chondro-protective effect in the surgical-induced OA mouse models. In conclusion, our findings provided evidence that develops Astilbin as a potential therapeutic drug for OA patients.

Melatonin-sulforaphane hybrid ITH12674 attenuates glial response in vivo by blocking LPS binding to MD2 and receptor oligomerization.

Neuroinflammation is increasingly associated to the onset and progression of neurodegenerative diseases. Furthermore, several lines of evidence have demonstrated the capacity of aberrant protein aggregates to activate the immune response, accelerating the advance of the disease. Compound ITH12674 is a melatonin-sulforaphane hybrid designed to exert a dual drug-prodrug mechanism of action that combines potent NRF2 induction and free radical scavenger activity. ITH12674 also showed neuroprotective properties in oxidative stress related models, that were dependant on its NRF2 inducing properties. Given the high impact of neuroinflammation in the pathogenesis of neurodegeneration, we foresaw to study the anti-inflammatory properties of ITH12674. ITH12674 reduced inflammatory markers in glial cell cultures and hippocampal tissue after LPS administration. The anti-inflammatory effect was related to inhibition of TLR4 receptors due to a direct interaction with the TLR4/MD2 complex at the hydrophobic cavity of MD2. ITH12674 is endowed with anti-inflammatory properties, that are complementary to the NRF2 inducing activity and neuroprotective properties. Thus, ITH12674 could be of potential interest for the treatment of diseases with chronic neuroinflammation.

Inhibition of myeloid differentiation factor 2 by baicalein protects against acute lung injury.

BACKGROUND: ALI/ARDS is characterized by severe hypoxemic respiratory failure attributed to inflammatory tissue injury. There are no treatment modalities able to prevent/reverse the dire pathological sequelae in these patients. Evidence links the inflammatory lung injury to uncontrolled activation of the immune signaling complex, TLR4-MD2 (Toll-like receptor-myeloid differentiation factor 2). Baicalein, a natural flavonoid, is reported to have robust anti-inflammatory properties, but its inhibition mechanism remains unclear. HYPOTHESIS/PURPOSE: This study investigated the protective mechanisms of baicalein on ALI/ARDS. METHODS: We used two experimental mouse models of LPS-induced ALI, pulmonary infection model (intratracheal LPS), and systemic infection model (intravenous LPS). Blood, BALF, lung and liver tissues were analyzed using routine methods. In vitro studies using peritoneal mouse macrophages or recombinant proteins were designed to elucidate inhibition mechanisms of baicalein. RESULTS: Our critical new findings revealed that Baicalein was an MD2 inhibitor, directly bound to MD2, effectively suppressing TLR4-MD2 activation and the subsequent MAPK and NF-κB signaling. The inhibited MD2 prevented development of inflammatory tissue injury and improved survival. The importance of MD2 in the inflammatory injury in ALI was corroborated by data obtained from MD2-/- mice, which did not develop the characteristic LPS-induced lung tissue damage. Thus, the findings indicated that MD2 was critical for development of ALI, functioning as an early upstream signal driving the progression of inflammatory injury. CONCLUSION: Baicalein, as a direct and selective MD2 inhibitor, inhibited the early upstream TLR4-MD2 signaling and is a promising therapeutic agent for the treatment of ALI/ARDS.

Epimedium sagittatum inhibits TLR4/MD-2 mediated NF-κB signaling pathway with anti-inflammatory activity.

BACKGROUND: Epimedium sagittatum (Sieb.et Zucc.) Maxim., Ying-Yang-Huo in Chinese has been used as a traditional Chinese medicine and is deemed to "reinforce the kidney Yang". Previous studies showed that E. sagittatum could modulate the immune system and treat some chronic disease such as rheumatic arthritis, cardiovascular diseases and osteoporosis. The aim of this study is to evaluate the anti-inflammatory effects of ethyl acetate extracts (YYHs) of E. sagittatum and its mechanisms of action. METHODS: In order to explore the composition of YYHs, YYHs was analyzed using high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS/MS) and in comparison with reference standards. Anti-inflammatory model was established in LPS-induced RAW264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Production of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were measured by enzyme-linked immunosorbent assays (ELISA). In addition, expression of p-p65 protein and TLR4/MD-2 complex was detected by western blots and flow cytometric, respectively. Nuclear factor kappa B (NF-κB) nuclear translocation was observed by fluorescence microscope. RESULTS: A total of eight compounds were identified, of which icariside II was the most abundant compound. YYHs (12.5-50 µg/mL) had no obvious cytotoxic effect on cells, and remarkably inhibited LPS-induced production of NO, TNF-α and IL-2 with a dose-dependent manner. Additionally, YYHs up-regulated expression of p-p65 and TLR4/MD-2 complex. Further research showed that YYHs significantly suppressed NF-κB p65 nuclear translocation. CONCLUSION: In brief, YYHs contributed to the inhibition of LPS-induced inflammatory response through the TLR4/MD-2-mediated NF-κB pathway and may be a potential choice to combat inflammation diseases. It includes a schema of pathways at the end of the paper.

Amphiphilic Guanidinocalixarenes Inhibit Lipopolysaccharide (LPS)- and Lectin-Stimulated Toll-like Receptor 4 (TLR4) Signaling.

We recently reported on the activity of cationic amphiphiles in inhibiting TLR4 activation and subsequent production of inflammatory cytokines in cells and in animal models. Starting from the assumption that opportunely designed cationic amphiphiles can behave as CD14/MD-2 ligands and therefore modulate the TLR4 signaling, we present here a panel of amphiphilic guanidinocalixarenes whose structure was computationally optimized to dock into MD-2 and CD14 binding sites. Some of these calixarenes were active in inhibiting, in a dose-dependent way, the LPS-stimulated TLR4 activation and TLR4-dependent cytokine production in human and mouse cells. Moreover, guanidinocalixarenes also inhibited TLR4 signaling when TLR4 was activated by a non-LPS stimulus, the plant lectin PHA. While the activity of guanidinocalixarenes in inhibiting LPS toxic action has previously been related to their capacity to bind LPS, we suggest a direct antagonist effect of calixarenes on TLR4/MD-2 dimerization, pointing at the calixarene moiety as a potential scaffold for the development of new TLR4-directed therapeutics.

The acute exposure of tetrachloro-p-benzoquinone (a.k.a. chloranil) triggers inflammation and neurological dysfunction via Toll-like receptor 4 signaling: The protective role of melatonin preconditioning.

This study is aimed to investigate the inflammation and neurological dysfunction induced by tetrachloro-p-benzoquinone (TCBQ) through Toll-like receptor 4 (TLR4) signaling. We also investigated the protective role of melatonin as an antioxidant and anti-inflammatory agent. In vitro model was established by rat pheochromocytoma PC12 cells, meanwhile, TLR4 wild-type (C57BL/6) and knockout mice (C57BL/10ScNJ TLR4-/-) were used as in vivo model. In vitro study showed TCBQ exposure enhanced the expression of TLR4, myeloid differentiation factor 88 (MyD88) at both transcriptional and post-transcriptional levels. By contrast, melatonin decreased TLR4 and MyD88 expressions. Moreover, our result indicated that melatonin disrupted the formation of TLR4/MyD88/MD2/CD14 complex. In addition, melatonin terminated TCBQ-mediated phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular regulated protein kinase (ERK) signaling and hampered its downstream pro-inflammatory cytokine releases. In vivo result also indicated TLR4 deficiency partially protected against TCBQ-induced morphological and neuropathological changes in mice brain, suggested the role of TLR4. In conclusion, melatonin modulates TCBQ-mediated inflammatory genes through TLR4/MyD88-dependent signaling pathway. Our current study, to the best of our knowledge, is the first time show melatonin not only disrupt the binding of TLR4 and MyD88, but also restricted the formation of TLR4/MD2/CD14 complex, suggesting that melatonin supplementary may represent a valuable therapeutic strategy for inflammatory neurological dysfunction.

Flavonoids from Radix Tetrastigmae improve LPS-induced acute lung injury via the TLR4/MD-2-mediated pathway.

Total flavonoids isolated from Radix Tetrastigmae (RTFs) possess immunomodulatory activity, particularly on inflammation. In mice with lipopolysaccharide (LPS)­induced acute lung injury (ALI), treatment with RTFs at 40, 80 and 160 mg/kg significantly reduced leukocyte infiltration, improved histopathological changes in lung tissues and decreased the LPS­induced production of several inflammatory mediators in the bronchoalveolar lavage fluid (BALF), which included the chemotatic factors, granulocyte colony­stimulating factor, monocyte inflammatory protein­1α and B­lymphocyte colony inflammatory cytokines, including interleukin (IL)­1ß, IL­6, IL­12p40 and tumor necrosis factor­α, in a dose­dependent manner. In addition, the expression of the Toll­like receptor 4 (TLR4)/myeloid differentiation factor­2 (MD­2) compound, the phosphorylation of p38 mitogen­activated protein kinase (p38MAPK), c­Jun N­terminal kinase (JNK) and nuclear transcription factor­κB (NF­κB), in addition to the DNA binding activity of NF­κB p65 in lung tissues, were all attenuated following RTF treatment. However, RTF treatment had no effect on extracellular signal­regulated kinase (ERK). In conclusion, RTFs contributed to the regulation of LPS­induced ALI through the TLR4/MD-2-mediated NF­κB, JNK and p38MAPK pathways. This may be a potential therapeutic option for the treatment of inflammatory diseases.

Celastrol blocks binding of lipopolysaccharides to a Toll-like receptor4/myeloid differentiation factor2 complex in a thiol-dependent manner.

ETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii (lei gong teng; Thunder of God Vine), which belongs to the Celastraceae family, has long been used in traditional Chinese medicine to treat inflammation and rheumatoid arthritis. Celastrol is a bioactive compound isolated from T. wilfordii. AIM OF THE STUDY: We investigated whether celastrol suppressed binding of lipopolysaccharides (LPS) to myeloid differentiation factor 2 (MD2), thereby downregulating Toll-like receptor4 (TLR4) activation in mouse primary macrophages. MATERIALS AND METHODS: Cytokine expression was determined by polymerase chain reaction analysis and enzyme-linked immunosorbent assay in bone marrow-derived primary macrophages (BMDMs). The kinase activity of tank-binding kinase 1 (TBK1) was examined by a luciferase reporter assay and an in vitro kinase assay. LPS binding to MD2 was examined by an in vitro binding assay and confocal microscopy analysis. RESULTS: Celastrol reduced LPS-induced expression of inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12, and IL-1ß, at both the mRNA and protein levels in BMDMs. Celastrol suppressed LPS binding to MD2, as shown by the in vitro binding assay, whereas it did not inhibit TBK1. In addition, co-localization of LPS with MD2 in BMDMs was blocked by celastrol. The inhibitory effects of celastrol on LPS binding to MD2 were reversed by thiol donors (N-acetyl-L-cysteine and dithiothreitol), suggesting that the thiol reactivity of celastrol contributes to its inhibitory effects on TLR4 activation in macrophages. CONCLUSION: Our results demonstrate that celastrol suppresses TLR4 activation through the inhibition of LPS binding to the TLR4/MD2 complex. These results provide a novel mechanism of action by which celastrol contributes to the anti-inflammatory activity of T. wilfordii.

Vizantin inhibits endotoxin-mediated immune responses via the TLR 4/MD-2 complex.

Vizantin has immunostimulating properties and anticancer activity. In this study, we investigated the molecular mechanism of immune activation by vizantin. THP-1 cells treated with small interfering RNA for TLR-4 abolished vizantin-induced macrophage activation processes such as chemokine release. In addition, compared with wild-type mice, the release of MIP-1ß induced by vizantin in vivo was significantly decreased in TLR-4 knockout mice, but not in TLR-2 knockout mice. Vizantin induced the release of IL-8 when HEK293T cells were transiently cotransfected with TLR-4 and MD-2, but not when they were transfected with TLR-4 or MD-2 alone or with TLR-2 or TLR-2/MD-2. A dipyrromethene boron difluoride-conjugated vizantin colocalized with TLR-4/MD-2, but not with TLR-4 or MD-2 alone. A pull-down assay with vizantin-coated magnetic beads showed that vizantin bound to TLR-4/MD-2 in extracts from HEK293T cells expressing both TLR-4 and MD-2. Furthermore, vizantin blocked the LPS-induced release of TNF-α and IL-1ß and inhibited death in mice. We also performed in silico docking simulation analysis of vizantin and MD-2 based on the structure of MD-2 complexed with the LPS antagonist E5564; the results suggested that vizantin could bind to the active pocket of MD-2. Our observations show that vizantin specifically binds to the TLR-4/MD-2 complex and that the vizantin receptor is identical to the LPS receptor. We conclude that vizantin could be an effective adjuvant and a therapeutic agent in the treatment of infectious diseases and the endotoxin shock caused by LPS.

Role of berberine in anti-bacterial as a high-affinity LPS antagonist binding to TLR4/MD-2 receptor.

BACKGROUND: Berberine is an isoquinoline alkaloid mainly extracted from Rhizoma Coptidis and has been shown to possess a potent inhibitory activity against bacterial. However, the role of berberine in anti-bacterial action has not been extensively studied. METHODS: The animal model was established to investigate the effects of berberine on bacterial and LPS infection. Docking analysis, Molecular dynamics simulations and Real-time RT-PCR analysis was adopted to investigate the molecular mechanism. RESULTS: Treatment with 40 mg/kg berberine significantly increased the survival rate of mice challenged with Salmonella typhimurium (LT2), but berberine show no effects in bacteriostasis. Further study indicated that treatment with 0.20 g/kg berberine markedly increased the survival rate of mice challenged with 2 EU/ml bacterial endotoxin (LPS) and postpone the death time of the dead mice. Moreover, pretreatment with 0.05 g/kg berberine significantly lower the increasing temperature of rabbits challenged with LPS. The studies of molecular mechanism demonstrated that Berberine was able to bind to the TLR4/MD-2 receptor, and presented higher affinity in comparison with LPS. Furthermore, berberine could significantly suppressed the increasing expression of NF-κB, IL-6, TNFα, and IFNß in the RAW264.7 challenged with LPS. CONCLUSION: Berberine can act as a LPS antagonist and block the LPS/TLR4 signaling from the sourse, resulting in the anti-bacterial action.

Zhankuic acid A isolated from Taiwanofungus camphoratus is a novel selective TLR4/MD-2 antagonist with anti-inflammatory properties.

TLR4, a membrane receptor that functions in complex with its accessory protein myeloid differentiation factor-2 (MD-2), is a therapeutic target for bacterial infections. Taiwanofungus camphoratus is highly valued as a medicinal mushroom for cancer, hypertension, and inflammation in traditional medicine. Zhankuic acid A (ZAA) is the major pharmacologically active compound of T. camphoratus. The mechanism of action of T. camphoratus or ZAA has not been fully elucidated. We analyzed the structure of human TLR4/MD-2 complex with ZAA by X-score and HotLig modeling approaches. Two Abs against MD-2 were used to verify the MD-2/ZAA interaction. The inflammation and survival of the mice pretreated with ZAA and injected with LPS were monitored. The modeling structure shows that ZAA binds the MD-2 hydrophobic pocket exclusively via specific molecular recognition; the contact interface is dominated by hydrophobic interactions. Binding of ZAA to MD-2 reduced Ab recognition to native MD-2, similar to the effect of LPS binding. Furthermore, ZAA significantly ameliorated LPS-induced endotoxemia and Salmonella-induced diarrhea in mice. Our results suggest that ZAA, which can compete with LPS for binding to MD-2 as a TLR4/MD-2 antagonist, may be a potential therapeutic agent for gram-negative bacterial infections.

[Effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-kappaB-TNF-alpha].

OBJECTIVE: To study the effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-kappaB-TNF-alpha in and its pharmacological mechanism of antiendotoxin. METHOD: RAW264. 7 macrophages were cultured and divided into 5 groups, namely the blank control group, the LPS model group, the sophoridine + LPS group, the TLR4/MD-2 blocking agent + LPS group and the anti-TLR4/MD-2 + sophoridine + LPS group. Cells and cell culture fluids were collected at 120 min after the each group was processed. The expression of TLR4 protein was measured by western blot, the distribution and expression of NF-kappaB protein were measured by immunocytochemistry, and the expression of NF-kappaB and TNF-alpha mRNA were measured by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The content of TNF-alpha in the cell supernatant was detected by using radioimmunoassay. RESULT: Compared with the LPS group, the expression of TLR4 protein, NF-kappaB mRNA, the rate of NF-kappaB entry the nucleus, TNF-alpha mRNA and TNF-alpha content in the cell supernatant were significantly decreased in the sophoridine + LPS group (P < 0.01). The rate of NF-kappaB entry the nucleus and TNF-alpha in the TLR4/MD-2 blocking agent + LPS group and the TLR4/MD-2 blocking agent + sophoridine + LPS group were notablly lower than that of the LPS model group (P < 0.01), close to that of the blank control group. However, there was no statistical significance between the two groups. CONCLUSION: TLR4/MD-2 may be one of sophoridine's targets. Sophordine's inhibitory effect on pathway activity of TLR4-NF-kappaB-TNF-alpha may be one of its antiendotoxin mechanisms.

Xanthohumol and related prenylated flavonoids inhibit inflammatory cytokine production in LPS-activated THP-1 monocytes: structure-activity relationships and in silico binding to myeloid differentiation protein-2 (MD-2).

Xanthohumol (XN) is a prenylated chalcone-type flavonoid found in hops and beer. Our objective of this study was to determine the anti-inflammatory activities of XN, isoxanthohumol (IX), and 15 related prenylated chalcones and flavanones, as well as their structure-activity relationships. The anti-inflammatory activities of the flavonoids were measured by their ability to inhibit lipopolysaccharide (LPS)-induced cytokine production in human monocytic THP-1 cells. The position, number, and length of the prenyl groups had a marked influence on the inhibitory activity of the prenylfavonoids towards MCP-1 and IL-6 production. The α,ß-unsaturated carbonyl moiety present in chalcones such as XN was not an absolute requirement for inhibitory activity, as the saturated XN derivative, tetrahydroxanthohumol (TX), showed inhibitory activity comparable to XN. With the aim to determine the mechanism of the observed anti-inflammatory effects, cellular protein levels of Toll-like receptor 4 (TLR4) were measured by Western blot 24 h following coexposure of THP-1 cells to LPS and either XN, TX, or IX. Only XN reduced the cellular TLR4 protein content. Therefore, an additional hypothesis was developed for an anti-inflammatory mechanism that involves the TLR4 coreceptor myeloid differentiation protein-2 (MD-2), which provides the actual binding site for LPS. Molecular docking studies showed that the complementarity of prenylated flavonoids with the hydrophobic MD-2 pocket (indicating goodness of fit) directly predicted their relative ability to inhibit MCP-1 and IL-6 production. In conclusion, prenylated flavonoids may suppress LPS-induced TLR4 activation at least partly by interfering with LPS binding to the TLR4 coreceptor MD-2, and XN (but not other prenylflavonoids) exerts an additional anti-inflammatory effect by downregulating the cellular TLR4 protein content.

Oxidized phospholipid inhibition of toll-like receptor (TLR) signaling is restricted to TLR2 and TLR4: roles for CD14, LPS-binding protein, and MD2 as targets for specificity of inhibition.

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysaccharide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-alpha production, IkappaBalpha degradation, p38 MAPK phosphorylation, and NF-kappaB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I.C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from approximately 30 microm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.