Liquiritin,a novel inhibitor of TRPV1 and TRPA1, protects against LPS-induced acute lung injury

TRPV1 and TRPA1 are cation channels that play key roles in inflammatory signaling pathways. They are co-expressed on airway C-fibers, where they exert synergistic effects on causing inflammation and cough. Licorice, the root of Glycyrrhiza uralensis, has been widely used in China as an anti-inflammatory and anti-coughing herb. To learn if TRPV1 and TRPA1 might be key targets of the anti-inflammatory and antitussive effects of licorice, we examined liquiritin, the main flavonoid compound and active ingredient of licorice, on agonist-evoked TRPV1 and TRPA1 activation. Liquiritin inhibited capsaicin- and allyl isothiocyanate-evoked TRPV1 and TRPA1 whole-cell currents, respectively, with a similar potency and maximal inhibition. In a mouse acute lung injury (ALI) model induced by the bacterial endotoxin lipopolysaccharide, which involves both TRPV1 and TRPA1, an oral gavage of liquiritin prevented tissue damage and suppressed inflammation and the activation of NF-κB signaling pathway in the lung tissue. Liquiritin also suppressed LPS-induced increase in TRPV1 and TRPA1 protein expression in the lung tissue, as well as TRPV1 and TRPA1 mRNA levels in cells contained in mouse bronchoalveolar lavage fluid. In cultured THP-1 monocytes, liguiritin, or TRPV1 and TRPA1 antagonists capsazepine and HC030031, respectively, diminished not only cytokine-induced upregulation of NF-κB function but also TRPV1 and TRPA1 expression at both protein and mRNA levels. We conclude that the anti-inflammatory and antitussive effects of liquiritin are mediated by the dual inhibition of TRPV1 and TRPA1 channels, which are upregulated in nonneuronal cells through the NF-κB pathway during airway inflammation via a positive feedback mechanism.     

Deficiency in TLR4 signal transduction ameliorates cardiac injury and cardiomyocyte contractile dysfunction during ischemia

Toll-like receptor 4 (TLR4), a proximal signalling receptor in innate immune responses to lipopolysaccharide of gram-negative pathogens, is expressed in the heart. Accumulating evidence have consolidated the notion that TLR4 plays an essential role in the pathogenesis of cardiac dysfunction. However, the molecular mechanisms of TLR4 responsible for ischemia-induced cardiac dysfunction remain unclear. To address the signalling mechanisms of TLR4-deficiency cardioprotection against ischemic injury, in vivo regional ischemia was induced by occlusion of the left anterior descending coronary artery in wild-type (WT) C3H/HeN and TLR4-mutated C3H/HeJ mice. The results demonstrated that blunted ischemic activation of p38 mitogen-activated protein kinase and JNK signalling occurred in C3H/HeJ hearts versus C3H/HeN hearts, while ERK and AMP-activated protein kinase (AMPK) signalling pathways were augmented during ischemia in C3H/HeJ hearts versus C3H/HeN hearts. Intriguingly, ischemia-stimulated endoplasmic reticulum stress was higher in C3H/HeN hearts than that in C3H/HeJ as demonstrated by up-regulation of Grp78/BiP, Gadd153/CHOP and IRE-1α. Myocardial infarct, caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining demonstrated that C3H/HeN hearts suffered more damage than those of C3H/HeJ hearts during ischemia. Moreover, isolated cardiomyocytes from C3H/HeJ hearts showed resistance to hypoxia-induced contractile dysfunction compared to those from C3H/HeN hearts, which are associated with greater hypoxic activation of AMPK and ERK signalling, better intracellular Ca²⁺ handling in C3H/HeJ versus C3H/HeN cardiomyocytes. These findings suggest that the cardioprotective effects against ischemic injury of hearts with deficiency in TLR4 signalling may be mediated through modulating AMPK and ERK signalling pathway during ischemia.     

Role of TLR1 and TLR6 in the host defense against disseminated candidiasis

Toll-like receptor-1 (TLR1) and TLR6 are receptors of the TLR family that form heterodimers with TLR2. The role of TLR1 and TLR6 for the recognition of the fungal pathogen Candida albicans was investigated. TLR1 is not involved in the recognition of C. albicans, and TLR1 knock-out (TLR1-/-) mice showed a normal susceptibility to disseminated candidiasis. In contrast, recognition of C. albicans by TLR6 modulated the balance between Th1 and Th2 cytokines, and TLR6 knock-out mice displayed a defective production of IL-10 and an increased IFN-gamma release. Production of the monocyte-derived cytokines tumor necrosis factor, IL-1, and IL-6 was normal in TLR6-/- mice, and this was accompanied by a normal susceptibility to disseminated candidiasis. In conclusion, TLR6 is involved in the recognition of C. albicans and modulates the Th1/Th2 cytokine balance, but this results in a mild phenotype with a normal susceptibility of TLR6-/- mice to Candida infection.     

LPS suppresses expression of asialoglycoprotein-binding protein through TLR4 in thioglycolate-elicited peritoneal macrophages

Macrophages are known to express various types of endocytosis receptors that mediate the removal of foreign pathogens. Macrophage asialoglycoprotein-binding protein (M-ASGP-BP) is a Gal/GalNAc-specific lectin, which functions as an endocytosis receptor. We found here that LPS is able to down-regulate the mRNA expression of M-ASGP-BP in a time-dependent manner using thioglycolate-elicited rat and mouse peritoneal macrophages. However, LPS does not modulate the mRNA expression of M-ASGP-BP from macrophages of C3H/HeN mice, which have a point mutation of TLR4, the primary LPS receptor. Furthermore, an inhibitor of NF-κB was observed to efficiently block the suppressive effect of LPS on M-ASGP-BP as well as to inhibit the phosphorylated IκB. These results demonstrate that the mRNA expression of M-ASGP-BP is down-regulated by the LPS-mediated TLR4 pathway involving NF-κB activation, suggesting that engagement of M-ASGP-BP by LPS may yield a negative signal that interferes with the LPS-induced positive signals mediated by proinflammatory cytokines.     

Role of TLR4 in lipopolysaccharide-induced acute kidney injury: Protection by blueberry

Inflammation has been implicated in the pathophysiology of kidney disorders. Previous studies have documented the contributions of various inflammatory cascades in the development of kidney and other organ dysfunctions. The Toll-like receptor 4 (TLR4) inflammatory pathway is a major contributor of inflammation in the kidney. Interestingly, lipopolysaccharide (LPS), a specific ligand for TLR4, has been shown to induce acute kidney injury (AKI) in animal models. We have previously studied the beneficial effects of nonpharmacological agents, particularly blueberries (BB), in attenuating inflammation and oxidative stress. We hypothesize that BB protect against the LPS-induced AKI by inhibiting TLR4 activation and kidney injury markers. Twelve-week-old male Sprague-Dawley rats received a BB solution or saline intragastric gavage for 2 days. One group of BB and saline-gavaged animals was injected with LPS (10 mg/kg bw). Another group of rats was injected with VIPER (0.1 mg/kg iv), a TLR4-specific inhibitory peptide, 2 h before LPS administration. Compared to LPS-administered rats, the BB-pretreated animals exhibited improved glomerular filtration rate, elevated renal blood flow, and a reduced renal vascular resistance. In addition, a reduction in the rate of production of free radicals, namely total reactive oxygen species (ROS) and superoxide, was observed in the BB-supplemented LPS group. Gene and protein expressions for TLR4, proinflammatory cytokine, and acute kidney injury markers were also attenuated in animals that were pretreated with BB as measured by real time RT-PCR and Western blotting, respectively. These results in the BB-pretreated group were consistent with those in the VIPER-treated rats, and indicate that BB protects against AKI by inhibiting TLR4 and its subsequent effect on inflammatory and oxidative stress pathways.     

Decursin negatively regulates LPS-induced upregulation of the TLR4 and JNK signaling stimulated by the expression of PRP4 in vitro

ABSTRACT

The current investigation was carried out to analyze the correlation of bacterial lipopolysaccharide (LPS) and pre-mRNA processing factor 4B (PRP4) in inducing inflammatory response and cell actin cytoskeleton rearrangement in macrophages (Raw 264.7) and colorectal (HCT116) as well as skin cancer (B16-F10) cells. Cell lines were stimulated with LPS, and the expression of PRP4 as well as pro-inflammatory cytokines and proteins like IL-6, IL-1β, TLR4, and NF-κB were assayed. The results demonstrated that LPS markedly increased the expression of PRP4, IL-6, IL-1β, TLR4, and NF-κB in the cells. LPS and PRP4 concomitantly altered the morphology of cells from an aggregated, flattened shape to a round shape. Decursin, a pyranocoumarin from Angelica gigas, inhibited the LPS and PRP4-induced inflammatory response, and reversed the induction of morphological changes. Finally, we established a possible link of LPS with TLR4 and JNK signaling, through which it activated PRP4. Our study provides molecular insights for LPS and PRP4-related pathogenesis and a basis for developing new strategies against metastasis in colorectal cancer and skin melanoma. Our study emphasizes that decursin may be an effective treatment strategy for various cancers in which LPS and PRP4 perform a critical role in inducing inflammatory response and morphological changes leading to cell survival and protection against anti-cancer drugs.     

Requirement of Rab21 in LPS-induced TLR4 signaling and pro-inflammatory responses in macrophages and monocytes

Lipopolysaccharide (LPS) induces macrophage/monocyte activation and pro-inflammatory cytokines production by activating Toll-like receptor 4 (TLR-4) signaling. Rab GTPase 21 (Rab21) is a member of the Rab GTPase subfamily. In the present study, we show that LPS induced TLR4 and Rab21 association and endosomal translocation in murine bone marrow–derived macrophages (BMDMs) and primary human peripheral blood mononuclear cells (PBMCs). In BMDMs, shRNA-mediated stable knockdown of Rab21 inhibited LPS-induced expression and production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). Conversely, forced overexpression of Rab21 by an adenovirus construct potentiated LPS-induced IL-1β, IL-6 and TNF-α production in BMDMs. Further studies show that LPS-induced TLR4 endosomal traffic and downstream c-Jun and NFκB (nuclear factor-kappa B) activation were significantly inhibited by Rab21 shRNA, but intensified with Rab21 overexpression in BMDMs. Finally, in the primary human PBMCs, siRNA-induced knockdown of Rab21 significantly inhibited LPS-induced IL-1β, IL-6 and TNF-α production. Taken together, we suggest that Rab21 regulates LPS-induced pro-inflammatory responses by promoting TLR4 endosomal traffic and downstream signaling activation.     

TLR4-independent and PKR-dependent interleukin 1 receptor antagonist expression upon LPS stimulation

Dendritic cells (DCs) induce innate immune responses by recognizing bacterial LPS through TLR4 receptor complexes. In this study, we compared gene expression profiles of TLR4 knockout (TLR4^neg) DCs and wild type (TLR4^pos) DCs after stimulating with LPS. We found that the expression of various inflammatory genes by LPS were TLR4-independent. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since IL-1rn is a potent natural inhibitor of proinflammatory IL-1. Using RT-PCR, real-time PCR, immunoblotting and ELISA, we demonstrated that IL-1rn was induced by DCs stimulated with LPS in the absence of TLR4. 2-Aminopurine, a pharmacological PKR inhibitor, completely abrogated LPS-induced expression of IL-1rn in TLR4^neg DCs, suggesting that LPS-induced TLR4-independent expression of IL-1rn might be mediated by PKR pathways. Considering that IL-1rn is a physiological inhibitor of IL-1, TLR4-independent and PKR-dependent pathways might be crucial in counter-balancing proinflammatory effector functions of DCs resulted from TLR4-dependent activation by LPS.     

miR-146b overexpression ameliorates lipopolysaccharide-induced acute lung injury in vivo and in vitro

Acute respiratory distress syndrome (ARDS) is a type of acute lung injury (ALI), which causes high morbidity and mortality. So far, effective clinical treatment of ARDS is still limited. Recently, miR-146b has been reported to play a key role in inflammation. In the present study, we evaluated the functional role of miR-146b in ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. The miR-146b expression could be induced by LPS stimulation, and miR-146b overexpression was required in the maintenance of body weight and survival of ALI mice; after miR-146b overexpression, LPS-induced lung injury, pulmonary inflammation, total cell and neutrophil counts, proinflammatory cytokines, and chemokines in bronchial alveolar lavage (BAL) fluid were significantly reduced. The promotive effect of LPS on lung permeability through increasing total protein, albumin and IgM in BAL fluid could be partially reversed by miR-146b overexpression. Moreover, in murine alveolar macrophages, miR-146b overexpression reduced LPS-induced TNF-α and interleukin (IL)-1β releasing. Taken together, we demonstrated that miR-146b overexpression could effectively improve the LPS-induced ALI; miR-146b is a promising target in ARDS treatment.     

Ursodeoxycholic acid protects against lung injury induced by fat embolism syndrome

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a life‐threatening disease with a high mortality rate, which was a common complication of fat embolism syndrome (FES). Ursodeoxycholic acid (UDCA) has been reported to exert potent anti‐inflammatory effects under various conditions. In vivo, perinephric fat was injected via tail vein to establish a rat FES model, the anti‐inflammatory effects of UDCA on FES‐induced lung injury were investigated through histological examination, ELISA, qRT‐PCR, Western blot and immunofluorescence. In vitro, human lung microvascular endothelial cells (HPMECs) were employed to understand the protective effects of UDCA. The extent of ALI/ARDS was evaluated and validated by reduced PaO₂/FiO₂ ratios, increased lung wet/dry (W/D) ratios and impaired alveolar‐capillary barrier, up‐regulation of ALI‐related proteins in lung tissues (including myeloperoxidase [MPO], vascular cell adhesion molecule 1 [VCAM‐1], intercellular cell adhesion molecule‐1 [ICAM‐1]), elevated protein concentration and increased proinflammatory cytokines levels (TNF‐α and IL‐1β) in bronchoalveolar lavage fluid (BALF). Pre‐treatment with UDCA remarkably alleviated these pathologic and biochemical changes of FES‐induced ALI/ARDS; our data demonstrated that pre‐treatment with UDCA attenuated the pathologic and biochemical changes of FES‐induced ARDS, which provided a possible preventive therapy for lung injury caused by FES.     

Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A^−/−) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and interferon (IFN)-β were significantly increased in SR-A^−/− mice compared to wild-type mice, and elevated nuclear factor kappa B (NFκB) activation was detected in SR-A^−/− macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NFκB in vitro. SR-A deletion also promoted the nuclear translocation of NFκB and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A^−/− macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.     

Cellular differentiation-induced attenuation of LPS response in HT-29 cells is related to the down-regulation of TLR4 expression

Intestinal epithelial cells not only present a physical barrier to bacteria but also participate actively in immune and inflammatory responses. The migration of epithelial cells from the crypt base to the surface is accompanied by a cellular differentiation that leads to important morphological and functional changes. It has been reported that the differentiation of colonic epithelial cells is associated with reduced interleukin (IL)-8 responses to IL-1beta. Although toll-like receptor 4 (TLR4) has been previously identified to be an important component of mucosal immunity to lipopolysaccharide (LPS) in the colon, little is known about the regulation of TLR4 in colonic epithelial cells during cellular differentiation. We investigated the effects of differentiation on LPS-induced IL-8 secretion and on the expression of TLR4. Differentiation was induced in colon cancer cell line HT-29 cells by butyrate treatment or by post-confluence culture and assessed by measuring alkaline phosphatase (AP) activity. IL-8 secretion was measured by ELISA, and TLR4 protein and mRNA expressions were followed by Western blot and RT-PCR, respectively. HT-29 cells were found to be dose-dependently responsive to LPS. AP activity increased in HT-29 cells by differentiation induced by treatment with butyrate or post-confluence culture. We found that IL-8 secretion induced by LPS was strongly attenuated in differentiated cells versus undifferentiated cells, and that cellular differentiation also attenuated TLR4 mRNA and protein expressions. Pretreating HT-29 cells with tumor necrosis factor (TNF)-alpha or interferon (INF)-gamma augmented LPS-induced IL-8 secretion and TLR4 expression. These TNF-alpha- or INF-gamma-induced augmentations of LPS response and TLR4 expression were all down-regulated by differentiation. Collectively, we conclude that cellular differentiation attenuates IL-8 secretion induced by LPS in HT-29 cells, and this attenuation is related with the down-regulation of TLR4 expression.     

The immunomodulation of endotoxin-induced acute lung injury by hesperidin in vivo and in vitro

To investigate the modulation of lung local immune responses of hesperidin (HES) on the acute lung inflammation induced by LPS in vivo. Mice were challenged with intratracheal lipopolysaccharide (100 μg) 30 min before with treatment hesperidin (200 mg/kg oral administration) or vehicle. After 4 and 24 h, bronchoalveolar lavage fluid was obtained to measure proinflammatory (TNF-α, IL-1β, IL-6), anti-inflammatory (IL-10, IL-4, IL-12) cytokines, chemokines (KC, MCP-1 and MIP-2), total cell counts, nitric oxide production, and proteins. Lung histology was performed in inflated-fixed lungs. Hesperidin downregulate the LPS-induced expression of TNF-α, IL-1β, IL-6, KC, MIP-2, MCP-1, and IL-12. It also enhanced the production of IL-4, IL-10. Total leukocyte counts; nitric oxide production, iNOS expression, and proteins were significantly decreased by hesperidin. In vitro, HES suppressed the expression of IL-8 on A549 cells and THP-1 cells, the expression of TNF-α, IL-1β, and IL-6 on THP-1 cells, the expression of ICAM-1 and VCAM-1 on A549 cells which effect cell adhesion function. The suppression of those molecules is controlled by NF-κB and AP-1, which are activated by IκB and MAPK pathways. HES inhibits those pathways, thereby suppressing the expression of IL-8, TNFα, IL-1β, IL-6, IL-12, ICAM-1 and VCAM-1. This study indicates that HES had a markedly immunomodulatory effect in a clinically relevant model of ARDS. Nevertheless, further investigations are required to determine the potential clinical usefulness of HES in the adjunctive therapy of ARDS.     

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.     

Retracted: microRNA-542-5p protects against acute lung injury in mice with severe acute pancreatitis by suppressing the mitogen-activated protein kinase signaling pathway through the negative regulation of P21-activated kinase 1

Severe acute pancreatitis (SAP) is a condition associated with high rates of mortality and lengthy hospital stays. In the current study, SAP mouse models were established in BALB/c wild-type and P21-activated kinase 1 (PAK1) knockdown mice with the objective of determining the expression of microRNA-542-5p (miR-542-5p) and the subsequent elucidation of the mechanism by which it influences acute lung injury (ALI) by mediating mitogen-activated protein kinase (MAPK) signaling and binding to PAK1. The targeting relationship between miR-542-5p and PAK1 was verified using the bioinformatics prediction website and by the means of a dual-luciferase reporter assay. Following the SAP model establishment, the mice were assigned into various groups with the introduction of different mimic and inhibitors in an attempt to investigate the effects involved with miR-542-5p on inflammatory reactions among mice with SAP-associated ALI. Our results indicated that PAK1 was targeted and negatively mediated by miR-542-5p. Mice with SAP-associated ALI exhibited an increased wet-to-dry weight ratio, myeloperoxidase activity, serum amylase activity, TNF-α, interleukin-1 beta (IL-1β), and intercellular adhesion molecule-1 (ICAM-1) contents, p-p38MAPK, p-ERK1/2, and p-JNK protein levels as well as PAK1 positive expression, while decreased miR-542-5p levels were observed. Functionally, overexpression of miR-542-5p improves ALI in mice with SAP via inhibition of the MAPK signaling pathway by binding to PAK1.Based on the evidence from experimental models, miR-542-5p was shown to improve ALI among mice with SAP, while suggesting that the effect may be related to the inactivation of the MAPK signaling pathway and downregulation of PAK1 gene. Thus, miR-542-5p could serve as a promising target for ALI treatment.     

Hydrogen sulfide protects against acetaminophen-induced acute liver injury by inhibiting apoptosis via the JNK/MAPK signaling pathway

Acetaminophen (APAP) is a widely used over-the-counter analgesic and antipyretic. It can cause hepatotoxicity. Recent studies demonstrated that hydrogen sulfide (H₂S) exhibits cell protection in several cell types. This study was designed to investigate whether H ₂S ameliorated APAP-induced acute liver injury and to elucidate its mechanisms. In this study, we analyzed the detailed biological and molecular processes of APAP-induced hepatotoxicity using a bioinformatics analysis, which showed that apoptosis and the c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase pathway were confirmed to play critical roles in these processes. We further investigated the protective effects of H ₂S on APAP-induced hepatotoxicity. In vivo, we observed that the exogenous supplement of H ₂S ameliorated APAP-induced liver injury. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) systems were the endogenous pathway of H ₂S. The expression of CBS/CSE was decreased in APAP-treated mice, while H ₂S could significantly restore it. In addition, APAP-induced JNK activation was inhibited by H ₂S in vivo. In vitro, H ₂S abolished the active effects of APAP on caspase3, Bax, and Bcl-2 expressions as well as JNK phosphorylation in hepatocytes. It was found through flow cytometry that the amount of APAP-induced apoptotic hepatocytes was decreased in the presence of H ₂S. In conclusion, our results suggested that H ₂S attenuated APAP-induced apoptosis in hepatocytes through JNK/MAPK siganaling pathway.