Cryptoporus volvatus polysaccharides attenuate LPS-induced expression of pro-inflammatory factors via the TLR2 signaling pathway in human alveolar epithelial cells

Context: Cryptoporus volvatus (Peck) Hubb grows wild in China, and its fruiting bodies have been used traditionally to treat asthma and bronchitis.

Objectives: This study evaluates the anti-inflammatory effect of Cryptoporus polysaccharides (CP) extracted from fruiting bodies of C. volvatus on lipopolysaccharide (LPS)-induced pro-inflammatory factors and the signaling pathways involved in human alveolar epithelial cells.

Materials and methods: To evaluate the effects of CP on LPS-induced pro-inflammatory factors, A549 cells were pre-incubated with CP 1, 10, and 100?μg/ml for 1?h and then stimulated with LPS 10?μg/ml for 24?h. The expression of pro-inflammatory factors monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), Toll-like receptor 2 (TLR2), and phosphorylation of ERK1/2, p38, and NF-κB p65 were measured by q-PCR, ELISA, and western blotting.

Results: CP decreased LPS-induced mRNA expression of MCP-1, TNF-α, and IL-1β (IC₅₀?=?83.3, 85.2, and 91.6?μg/ml, respectively) and their correspondent protein expression (IC₅₀?=?88.6, 76.4, and 81.6?μg/ml, respectively). Investigation of potential mechanisms indicated that CP 100?μg/ml reduced LPS-induced expression of TLR2 mRNA (66.9%, p?<?0.01) and protein (63.2%, p?<?0.01) that was a result of the decreased pro-inflammatory factors. LPS induction increased the expression of TLR2 and the phosphorylation of p38 and ERK1/2, NF-kB p65 concomitantly. CP 100?μg/ml inhibited the LPS-induced phosphorylation of the signaling proteins (p?<?0.05).

Conclusions: This suggests that CP pretreatment down-regulates LPS-mediated inflammation in lung epithelial cells. This study further confirmed that CP is a potential anti-inflammatory drug for the treatment of airway inflammatory diseases.     

Thymoquinone attenuates liver fibrosis via PI3K and TLR4 signaling pathways in activated hepatic stellate cells

Thymoquinone (TQ) is the major active compound derived from the medicinal Nigella sativa. In the present study, we investigated the anti-fibrotic mechanism of TQ in lipopolysaccharide (LPS)-activated rat hepatic stellate cells line, T-HSC/Cl-6. T-HSC/Cl-6 cells were treated with TQ (3.125, 6.25 and 12.5 μM) prior to LPS (1 μg/ml). Our data demonstrated that TQ effectively decreased activated T-HSC/Cl-6 cell viability. TQ significantly attenuated the expression of CD14 and Toll-like receptor 4 (TLR4). TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinase-protein kinase B (Akt) phosphorylation. The expression of α-SMA and collagen-I were significantly decreased by TQ. Furthermore, TQ decreased X linked inhibitor of apoptosis (XIAP) and cellular FLIP (c-FLIP_L) expression, which are related with the regulation of apoptosis. Furthermore, TQ significantly increased the survival against LPS challenge in d-galactosamine (d-GlaN)-sensitized mice, and decreased the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were in line with in vitro results. Our data demonstrated that TQ attenuates liver fibrosis partially via blocking TLR4 expression and PI3K phosphorylation on the activated HSCs. Therefore, TQ may be a potential candidate for the therapy of hepatic fibrosis.     

Stimulation of pro-inflammatory responses by mebendazole in human monocytic THP-1 cells through an ERK signaling pathway

Oral helminthic mebendazole (MBZ) has been reported to cause liver injury with inflammatory responses. However, the underlying mechanism remains unknown. To examine the inflammatory reactions, we investigated whether MBZ and other helminthic drugs increase the release of pro-inflammatory cytokines and chemokines using human monocytic cells. The release of interleukin (IL)-8 and tumor necrosis factor (TNF) α from human monocytic THP-1 cells was significantly increased by treatment with MBZ, albendazole (ABZ), fenbendazole (FBZ), or oxibendazole (OBZ), but not by albendazole sulfoxide or praziquantel, suggesting that MBZ and structurally similar drugs can stimulate monocytes and increase the release of pro-inflammatory cytokines. MBZ also significantly increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2 in THP-1 cells. Pretreatment with the MAP kinase/ERK kinase 1/2 inhibitor U0126 significantly suppressed the increase of IL-8 and TNFα levels by MBZ, ABZ, FBZ, or OBZ treatment in THP-1 cells, but the p38 mitogen-activated protein kinase inhibitor SB203580 or JNK1/2 inhibitor SP600125 did not. These results suggested that an ERK1/2 pathway plays an important role in the release of IL-8 and TNFα in THP-1 cells treated with MBZ and structurally similar drugs. In conclusion, the release of inflammatory mediators by MBZ might be one of the mechanisms underlying immune-mediated liver injury. This in vitro method may be useful to predict adverse inflammatory reactions that lead to hepatotoxicity.     

Hyaluronan ameliorates LPS-induced acute lung injury in mice via Toll-like receptor (TLR) 4-dependent signaling pathways

Toll-like receptor-4 (TLR4) signaling has been implicated in innate immunity and acute inflammation following acute lung injury (ALI). As such, modulating inflammatory response through TLR4 represents an attractive therapeutic approach to treat ALI. Increasing evidence demonstrates that hyaluronan (HA) can modulate TLR4 activation and has shown early promise as a therapeutic agent in ALI. However, the mechanism associated with HA has not been fully elucidated. In the current study, we sought to determine the effects of HA on lipopolysaccharide (LPS)-induced inflammatory response and gain insights into the mechanism of action in mice with intratracheal instillation of LPS. Our results demonstrate that in contrast to mice challenged with LPS, pretreatment with HA significantly inhibited inflammatory cell recruitment, attenuated lung injury and suppressed the level of cytokine/chemokine in bronchial alveolar lavage fluid (BALF). Investigation of the mechanism responsible for inhibition of LPS activation showed HA treatment significantly inhibited the nuclear translocation of NF-κB p65 and protein expression of myeloid differentiation primary response protein (MyD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) and p38 MAPK, JNK and ERK activation in lung tissue. Furthermore, we compared the protection effect of HA in TLR4-deficient mice with those of genetically matched wild type (WT) mice in an acute model of lung injury. However, in TLR4-deficient mice, HA pretreatment before LPS instillation fail to affect the LPS response. Therefore, our findings suggest that HA pretreatment attenuated LPS-induced ALI and the anti-inflammatory function of HA was partial dependent on TLR4, which shed new light on potential elements that regulate the lung injury response.     

Toll样受体4介导炎症及凋亡信号通路在新生儿小肠结肠炎动物模型中的作用探讨

目的探讨Toll样受体(TLR)4炎症及凋亡信号通路对于新生儿坏死性小肠结肠炎(NEC)炎症及肠道细胞凋亡的影响。方法通过多因素联合作用对新生TLR4基因缺失小鼠和相应野生型小鼠进行诱导NEC动物模型。采用实时荧光定量聚合酶链反应(RTPCR)检测肠组织TLR4、Re LA/p65、胱天蛋白酶(caspases)8,caspase 9,caspase3的m RNA表达,并检测肠道细胞的凋亡水平变化。结果被成功诱导NEC后细胞凋亡水平有明显变化,缺失株处理组Re LA/p65与Caspase 8表达下降,Caspase 9表达上调,野生株处理组Re LA/p65与Caspase 8表达上调,Caspase 9表达下降。结论 TLR4介导的炎症-凋亡信号通路对NEC病程中的细胞凋亡起到了抑制作用。     

雷公藤次碱通过调控TLR4/MyD88/TRAF6信号通路抑制LPS诱导的RAW264.7细胞炎症反应

目的 探讨雷公藤次碱抗炎活性及其作用机制。方法 用细胞计数盒-8 (CCK-8)法考察雷公藤次碱对小鼠单核巨噬细胞白血病细胞RAW 264.7 增殖活性的影响,用酶联免疫吸附法（ELISA）检测雷公藤次碱对脂多糖（LPS）诱导的RAW264.7细胞分泌细胞因子一氧化氮（NO）、白细胞介素-1β（IL-1β）、 肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）的影响,用免疫印迹法考察雷公藤次碱对白介素受体相关激酶（IRAK）、肿瘤坏死因子受体相关蛋白6（TRAF6）、核因子κB抑制因子α（IκBα）、核因子κB（NF-κB p65）、分裂原激活的蛋白激酶p38（p38）、c-Jun氨基末端激酶（JNK）、细胞外调节蛋白激酶（ERK）在LPS刺激的RAW264.7细胞中的表达及其磷酸化的影响。结果 雷公藤次碱在25、50、100 μmol/L浓度下对RAW264.7细胞无显著毒性,并可显著抑制细胞因子NO、IL-1β、TNF-α和IL-6含量。免疫印迹法检测结果显示雷公藤次碱可显著抑制IRAK及TRAF6的表达;显著抑制ERK、P38和JNK的磷酸化;抑制IκBα的降解,降低NF-κB p65的核转运水平。结论 雷公藤次碱具有体外抗炎活性,其作用机制可能介导TLR4/MyD88/TRAF6信号通路。     

Inhibitory effects of amantadine on the production of pro-inflammatory cytokines by stimulated in vitro human blood

Treatment with amantadine (AMA), an N-methyl-D-aspartate (NMDA) receptor antagonist and antidepressant drug, increased the antidepressant activity of subsequent drugs in experimental studies and in patients suffering from treatment-resistant depression (TRD). Recent evidence indicates that depression may be accompanied by activation of an inflammatory response. These data indicate that pro-inflammatory cytokines may play a role in the etiology of depression, particularly in TRD. The present in vitro study shows the ability of AMA, used at concentrations between 10^?7 to 10^?5 M, to reduce the production of the pro-inflammatory cytokines, specifically interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). In addition, AMA treatment increased the production of the negative immunoregulator, interleukin-10 (IL-10). Furthermore, the combined treatment of AMA with fluoxetine (FLU), but not imipramine (IMI), had a stronger immunomodulatory effect on cytokine production than AMA alone. The above data provide additional rationale for the treatment of patients suffering from depression with a combination of AMA and a selective serotonin reuptake inhibitor.     

Effect of environmental pollutants on the production of pro-inflammatory cytokines by normal human dermal keratinocytes

The effect of the environmental pollutants, diesel exhaust particles (DEP) and formaldehyde (FA), on the production of pro-inflammatory cytokines (interleukin (IL)-1alpha, IL-1beta, tumor necrosis factor (TNF)-alpha and IL-8) by normal human dermal keratinocytes (hKCs) was investigated. Normal hKCs were incubated with various concentrations of DEP (0.4, 0.8, 4, or 20 microg/ml) or FA (0.25, 0.5, 1, or 5 microg/ml), and cytokine production was then determined by enzyme-linked immunosorbent assay (ELISA). DEP (20 microg/ml) induced IL-1beta production without altering cell growth. The increased production of IL-1beta induced by this concentration of DEP was further enhanced by the presence of phorbol 12-myristate 13-acetate (PMA), although PMA alone did not affect the levels of IL-1beta. IL-8 production was also increased by DEP (0.4 and 0.8 microg/ml), which is consistent with the results that these concentrations of DEP increased the number of cells significantly after 72 h incubation. Although FA alone did not stimulate the production of IL-1beta or IL-8 by keratinocytes, FA (0.5 microg/ml and 5 microg/ml) significantly increased IL-8 and IL-1beta production, respectively, in cells stimulated with PMA. IL-1alpha production was not modulated by FA or DEP even in the presence of PMA. TNF-alpha was produced by unstimulated keratinocytes at barely detectable levels after 48 h incubation. Although basal levels of TNF-alpha in the culture supernatants were increased after stimulation with PMA, neither pollutant alone nor combination with PMA affected the levels of TNF-alpha. These in vitro findings suggest that environmental pollutants may act as modulating factors of cutaneous inflammation by affecting the ability of keratinocytes to release pro-inflammatory cytokines.     

Icariside II potentiates paclitaxel-induced apoptosis in human melanoma A375 cells by inhibiting TLR4 signaling pathway

Combination therapy of paclitaxel (taxol) with natural anti-tumor agents that are capable of inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Our previous study showed that icariside II (IS), derived from Herba Epimedii, inhibited the proliferation of melanoma cells in vivo and in vitro through the regulation of apoptosis. In this report, the combination effects of paclitaxel and IS were investigated in human melanoma A375 cells. As compared to the treatment with paclitaxel alone, the co-administration of IS and paclitaxel resulted in an enhancement of apoptosis as revealed by WST-8 and PI assays. Meanwhile, Western blot analysis showed that the co-administration of IS and paclitaxel resulted in increases of cleaved caspase-3, one of the terminal pro-apoptotic proteins. In melanoma, IL-8 and VEGF are positively correlated with disease stage and a high probability of progression. We demonstrated that treatment of A375 cells with IS in combination with paclitaxel resulted in a significant decrease in the production of IL-8 and VEGF, compared with paclitaxel alone. Recent studies suggest that TLR4–MyD88–ERK signaling may be a novel target for reversing chemoresistance to paclitaxel. Our flow cytometry and Western blot data showed that paclitaxel activated TLR4–MyD88–ERK signaling and that IS treatment could effectively inhibit this paclitaxel-induced activation of TLR4–MyD88–ERK signaling. In conclusion, this study demonstrated for the first time that IS could potentiate paclitaxel-induced apoptosis in melanoma cells. These effects were mediated, at least in part, by inhibiting the activation of the TLR4 signal transduction pathways. These findings support further preclinical evaluation of IS as a new potential anti-tumor agent.     

Lead alters intracellular protein signaling and suppresses pro-inflammatory activation in TLR4 and IFNR-stimulated murine RAW 264.7 cells,in vitro

Lead (Pb) is a persistent environmental pollutant that has a structure and charge similar to many ions, such as calcium, that are essential for normal cellular function. Pb may compete with calcium for protein binding sites and inhibit signaling pathways within the cell affecting many organ systems including the immune system. The aim of the current study was to assess whether the calcium/calmodulin pathway is a principal target of environmentally relevant Pb during pro-inflammatory activation in a RAW 264.7 macrophage cell line. RAW 264.7 cells were cultured with 5 μM Pb(NO₃)₂, LPS, rIFNγ, or LPS+rIFNγ for 12, 24, or 48 hr. Intracellular protein signaling and multiple functional endpoints were investigated to determine Pb-mediated effects on macrophage function. Western blot analysis revealed that Pb initially modulated nuclear localization of NFκB p65 and cytoplasmic phosphorylation of CaMKIV accompanied by increased phosphorylation of STAT1β at 24 hr. Macrophage proliferation was significantly decreased at 12 hr in the presence of Pb, while nitric oxide (NO) was significantly reduced at 12 and 24 hr. Cells cultured with Pb for 12, 24, or 48 hr exhibited altered cytokine levels after specific stimuli activation. Our findings are in agreement with previous reports suggesting that macrophage pro-inflammatory responses are significantly modulated by Pb. Further, Pb-induced phosphorylation of CaMKIV (pCaMKIV), observed in the present study, may be a contributing factor in metal-induced autophagy noted in our previous study with this same cell line.     

Silica nanoparticles activate purinergic signaling via P2X7 receptor in dendritic cells,leading to production of pro-inflammatory cytokines

We examined the mechanism of SNP-mediated stimulation of IL-1β and IL-18 production via P2R-mediated pathways in mouse bone marrow dendritic cells (mBMDCs). Examination of uptake of SNPs with diameters of 30, 70, and 300 nm (SNP30, SNP70, and SNP300, respectively) by lipopolysaccharide-matured mBMDCs revealed that significant uptake of SNP30 occurred within as short a time as 1 h. Production of IL-1β and IL-18 by cells exposed to SNPs increased dose-dependently, and was highest in cells exposed to SNP30. The SNP30-induced cytokine production was significantly inhibited by ATPase (apyrase) and by P2X₇ receptor antagonist (A438079). ATP release was also highest in SNP30-exposed cells. Treatment of mBMDCs with exogenous ATP induced release of high levels of IL-1β and IL-18, and this release was also significantly inhibited by apyrase and A438079. The order of effectiveness of the three SNPs for inducing intracellular reactive oxygen species (ROS) production accorded well with those of cytokine production and ATP release. ROS production was inhibited by diphenyleneiodonium chloride (DPI). SNPs, especially SNP30, activate purinergic signaling in matured mBMDCs by inducing ATP release via P2X₇ receptor. ATP induces ROS production via NADPH oxidase, and ROS activate inflammasomes, leading to caspase-1-dependent processing of pro-cytokines and release of IL-1β and IL-18.     

Juzentaihoto, a Kampo medicine, enhances IL-12 production by modulating Toll-like receptor 4 signaling pathways in murine peritoneal exudate macrophages

Juzentaihoto (TJ-48), a Kampo medicine, has been reported to affect the immune system. Although toll-like receptors (TLRs) have been identified as receptors of innate immunity, the effects of TJ-48 on TLR signaling pathways have not been thoroughly investigated. Here we evaluated the effects of TJ-48 on TLR4 signaling pathways. Peritoneal exudate macrophages (PEMs) isolated from mice orally administered TJ-48 for 11 days were stimulated with lipopolysaccharide (LPS), a ligand of TLR4, in vitro. Production of IL-12 p40 was significantly augmented in TJ-48-treated PEMs compared with that in vehicle PEMs, without affecting the surface expression of TLR4. Treatment with chemical inhibitors of NF-kappa B and p38 mitogen-activated protein kinases (MAPKs) in vitro inhibited LPS-induced IL-12 production, whereas JNK and ERK inhibitors increased IL-12 production. Immunoblotting with phosphorylation-state specific antibodies demonstrated that TJ-48 differentially affected LPS-induced phosphorylation of NF-kappa B and MAPKs. In PEMs treated with TJ-48, LPS-induced phosphorylation of p65 NF-kappa B and p38 MAPK was augmented, while that of JNK and ERK was attenuated compared with those in vehicle PEMs. These results suggest that selective modulation of the TLR4 signaling pathways by TJ-48 is involved in enhanced production of IL-12 in PEMs.     

Vitamin D receptor signaling of monocytic differentiation in human leukemia cells: role of MAPK pathways in transcription factor activation

Among the many important physiological functions of the activated vitamin D receptor (VDR) is the signaling of monocytic differentiation, first demonstrated by conversion of malignant myeloid leukemia cells to nonproliferating cells with mature monocyte/macrophage appearance. However, the understanding of how 1, 25-dihydroxyvitamin D3 (1,25D) signals monocytic differentiation is still developing. Recent advances summarized here include the role of the principal "mitogen-activated protein kinase" (MAPK) pathways, their potential downstream target the CCAAT/enhancer binding protein beta (C/EBP beta), cell cycle related proteins, and cyclin-dependent kinase 5 (Cdk5) in 1,25D-induced differentiation. The precise steps by which activated VDR signals differentiation are incompletely understood in any of the cell types known to respond to 1,25D. We have focused our studies on HL60 cells, a widely available cell line derived from a patient with promyeloblastic leukemia, with the goal of achieving as clear a picture as possible with the currently available tools. In this model, outlined in Fig. 1, a plausible sequence of events is presented, with the caveats that these are not the only pathways activated by liganded VDR, and that several other pathways, also operative, remain to be convincingly demonstrated. The details of the scheme will be discussed in the sections below.     

Involvement of mitogen-activated protein kinases and NFkappaB in LPS-induced CD40 expression on human monocytic cells

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFkappaB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFkappaB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFkappaB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFkappaB activation, and CD40 expression. Moreover, blockage of MAPK and NFkappaB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFkappaB.     

Okra polysaccharide-2 plays a vital role on the activation of RAW264.7 cells by TLR2/4-mediated signal transduction pathways

Polysaccharide is the main active component of okra (Abelmoschus esculentus L.) and it can effectively stimulate the activation of macrophages. However, the immune regulatory mechanism is still not clear. Therefore, the present study aimed to reveal the possible mechanism by investigating the effect of okra polysaccharide-2 (RPS-2) on Toll-like receptor (TLR) 2/4-mediated signal transduction pathways in RAW264.7 murine macrophage cells. In order to confirm whether RPS-2 stimulated macrophages activation via TLR2 or TLR4, RAW264.7 murine macrophage cells were pretreated with TLR2/4 inhibitors for 1 h before RPS-2 treatment, and then the NO, IL-10, TNF-α levels were tested. The results indicated that both TLR2 and TLR4 were the keys of immune regulatory effect of RPS-2. Afterwards, the effect of RPS-2 on NF-κB and MAPKs signaling pathways were studied by western blot analysis. It showed RPS-2 induced the phosphorylation of p65, IκBα, p38, ERK1/2 and JNK. At the same time, the specific inhibitors reduced these phosphorylation levels as well as NO, IL-10 and TNF-α amounts. In a word, RPS-2 activated macrophages by NF-κB and MAPKs signal transduction pathways.