Protein Expression Levels Of TLR4 Research Articles

Polysaccharide of Atractylodes macrocephala Koidz regulates LPS-mediated mouse hepatitis through the TLR4-MyD88-NFκB signaling pathway

Feed corruption and poor breeding environment could cause widespread bacterial infection which could cause severe liver inflammation and lead to liver damage, even death. It has been proved that Polysaccharide of Atractylodes macrocephala Koidz (PAMK) could improve the immunity of animal, but the mechanism of its protective effect on hepatitis has been rarely reported. This study investigated the protective effect of PAMK on mouse liver through LPS-induced liver inflammatory. The results showed that LPS caused swelling of hepatocytes, disappearance of hepatic cord structure and infiltration of a large number of inflammatory cells, and LPS could up-regulated mRNA and protein expression levels of TLR4, MyD88, IKBα and NFκB, increased cytokines IL-1β, IL-4, IL-6 and TNF-α levels, enhance the levels of antioxidant enzymes CAT, GSH-PX, SOD, iNOs and MDA. PAMK pretreatment could relieved histopathological damage caused by LPS, and could activate the TLR4-MyD88-NFκB signalling pathway, reduce the levels of IL-1β, IL-6 and TNF-α, increase IL-4 levels, inhibit the levels of GSH-PX and MDA. These results indicate that PAMK could reduce inflammatory damage and oxidative stress in mice and play a protective role in the early stages of LPS invasion of the liver.

Genistein Attenuates Isoflurane-Induced Neuroinflammation by Inhibiting TLR4-Mediated Microglial-Polarization in vivo and in vitro.

BackgroundIsoflurane, a widely used anesthetic in surgery, has been found to induce neurotoxicity. In parallel, genistein is thought to attenuate isoflurane-induced neurotoxicity, although underlying molecular mechanisms are still unclear. In this study, we studied the protective effects of genistein on isoflurane-induced neuroinflammation in rats and BV2 cells.MethodsSprague-Dawley rat pups were exposed to 0.75% isoflurane for 6 hours at postnatal day 7 (P7), and genistein (20, 40, or 80 mg/kg/day) or saline administered from P3 to P15. Hippocampal single-cell suspensions were prepared and apoptosis analyzed by flow cytometry. mRNA expression was determined by RT-qPCR, while protein expression was assessed using Western blot, immunochemistry and immunofluorescence. TLR4 was knocked-out in BV2 cells through CRISPR-Cas9.ResultsGenistein treatment reduced isoflurane-induced apoptosis and inflammation in rat hippocampus. Importantly, genistein promoted M2 and suppressed M1 microglia polarization in rat hippocampus after stimulation with isoflurane. In addition, genistein reduced isoflurane-induced protein expression levels of TLR4, MyD88, TRAF6, p-TAK1, p-p38, p-ERK, p-IκBα and p-NF-κB in rat hippocampus. In BV2 cells exposed to isoflurane, genistein treatment decreased IL-1β, TNF-α, IL-6 and IL-8 mRNA expressions, promoted M2 and suppressed M1 microglia polarization. Similarly, genistein also decreased TLR4 protein levels in isoflurane-induced BV2 cells. However, genistein did not affect CD16, iNOS, CD206 and Arg1 protein levels in TLR4-KO BV2 cells exposed to isoflurane.ConclusionGenistein attenuates isoflurane-induced neurotoxicity by inhibiting TLR4-mediated microglial inflammation in vivo and in vitro.

MiR-489-3p inhibits TLR4/NF-κB signaling to prevent inflammation in psoriasis.

Psoriasis is a chronic inflammatory skin disease whose etiology has not yet been determined. MicroRNAs (miRs) regulate the early stages of psoriasis and are targets for therapeutic intervention. The present study aimed to investigate the functional role of miR-489-3p in psoriasis. The present study first assessed the expression levels of miR-489-3p and Toll-like receptor (TLR)4 mRNA using reverse transcription-quantitative PCR, and also detected the protein expression levels of TLR4 and NF-κB via western blot analysis. TargetScan and miRDB target gene prediction tools were used to confirm the regulation of Toll-like receptor (TLR)4 by miR-489-3p. Moreover, a Cell Counting Kit (CCK)-8 assay was conducted to evaluate cell viability, while cell cycle and colony formation assays were performed to evaluate cell proliferation. Human keratinocytes (HaCaT) were co-transfected with TLR4-small interfering RNA and miR-489-3p-inhibitor plasmids, and analysis of cell proliferation and inflammatory cytokine secretion was performed using CCK-8 assay and ELISA. It was found that miR-489-3p expression was downregulated in patients with psoriasis. Bioinformatics analysis identified that TLR4 was a direct target of miR-489-3p. This was confirmed via luciferase reporter assays in HaCaT cells. The overexpression of miR-489-3p inhibited the TLR4/NF-κB signaling pathway and reduced cell proliferation. TLR4 silencing alleviated the effects of miR-489-3p, and enhanced cell proliferation and inflammatory cytokine secretion. Taken together, these data suggested that miR-489-3p may be a key effector of psoriasis, which promotes inflammatory responses by direct targeting of TLR4. miR-489-3p therefore represents a promising prognostic biomarker and therapeutic target for psoriasis treatment.

Xiaoyaosan Exerts Therapeutic Effects on the Colon of Chronic Restraint Stress Model Rats via the Regulation of Immunoinflammatory Activation Induced by the TLR4/NLRP3 Inflammasome Signaling Pathway.

Depression is the neurological manifestation most commonly associated with gastrointestinal diseases. The release of inflammatory cytokines mediated by TLR4/NLRP3 inflammasome signaling-induced immunoinflammatory activation may represent a common pathogenic process underlying the development of gastrointestinal diseases and depression. Clinical studies have indicated that Xiaoyaosan (XYS) can relieve depressive behavior by improving gastrointestinal symptoms. We previously demonstrated that XYS can reduce colonic inflammation in a rat model of chronic unpredictable mild stress; however, the precise anti-inflammatory mechanisms involved remain unclear. Here, we investigated whether XYS can ameliorate depressive behavior through regulating the TLR4/NLRP3 inflammasome signaling pathway, thereby inhibiting immunoinflammatory activation and reducing colonic proinflammatory cytokine levels. Fifty-two healthy male Sprague–Dawley rats were randomly divided into four groups (control, model, XYS, and fluoxetine). The latter three groups were subjected to 21 days of chronic restraint stress to generate a model of stress-induced depression. XYS and fluoxetine were administered intragastrically. Behavioral changes in the rats were assessed after 21 days. Serum and colon samples were collected, and the relative levels of the inflammation indicators IL-6, IL-1β, and TNF-α were determined by ELISA. Pathological changes in colon tissue were assessed by hematoxylin and eosin staining. The levels of TLR4, MyD88, NF-κB-p65, TAK1, IRAK1, and TRAF6 were detected by immunohistochemistry, while the gene and protein expression levels of TLR4, MyD88, NF-κB-p65, TAK1, IRAK1, TRAF6, NLRP3, ASC, and caspase-1 were detected by quantitative polymerase chain reaction (qPCR) and Western blotting. The results indicated that XYS could improve the depressive-like behavior and the weight loss of rats with stress-induced depression. Furthermore, depressed rats treated with XYS exhibited decreased expression levels of TLR4, MyD88, NF-κB-p65, TAK1, IRAK1, TRAF6, NLRP3, ASC, and caspase-1 in colonic tissue; reduced colon and serum concentrations of the inflammatory factors IL-6, IL-1β, and TNF-α; and lowered levels of colonic inflammation.

Wheat peptide protects against ethanol-induced gastric mucosal damage through downregulation of TLR4 and MAPK

• Excessive alcohol consumption can lead to gastric mucosal damage. • Wheat peptides repressed gastric inflammation through the reduction of inflammatory factor. • Wheat peptides prevented gastric oxidative stress through the inhibition of lipid peroxides. • Wheat peptides down-regulated protein expression levels of TLR4, MAPK and ERK. • Ethanol-induced gastric mucosal damage in rats can be prevented by wheat peptides. Excessive alcohol consumption can induce gastric mucosal damage. The present work aimed to examine the protective effect of wheat peptides against ethanol-induced gastric mucosal damage in rats. Wheat peptides (WP) were administered daily by gavage for 30 days before the rats were treated with anhydrous ethanol (1 mL). Histological observations were performed, as well as the anti-oxidation effect and anti-inflammatory action. WP increased the superoxide dismutase activity and prostaglandin E-2 (PGE2) levels and decreased the malondialdehyde and interleukin-8 (IL-8) levels. WP suppressed gastric mucosa inflammation through the reduction of TNF-α, MPO, IL-6, and IL-1β. WP repressed the expression of caspase-3, and enhanced the expression of anti-apoptotic protein EGFR. Furthermore, WP down-regulated protein expression levels of TLR4, MAPK and ERK, suggesting that WP have a potential effect to inhibit ethanol-induced inflammatory responses via blocking TLR4/MAPK/ERK signal transduction. The potential mechanism may be associated with enhancement of antioxidation, anti-inflammatory action and anti-apoptosis function.

PCSK9 and LRP5 in macrophage lipid internalization and inflammation

Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-density lipoprotein receptors (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between proprotein convertase subtilisin/kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation. Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 h of LDL incubation and lasts up to 24 h; however, in the absence of both LRP5 and PCSK9, there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally, PCSK9 participates in TLR4/NFkB signalling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB were detected in PCSK9 silenced cells after lipid loading, indicating a downregulation of the TLR4/NFκB pathway. Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5, there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favouring inflammation.

MiR-146a alleviates inflammation of acute gouty arthritis rats through TLR4/MyD88 signal transduction pathway.

The aim of this study was to explore the effect of micro-ribonucleic acid (miR)-146a on acute gouty arthritis rats through Toll-like receptor-4/myeloid differentiation factor 88 (TLR4/MyD88) signal transduction pathway. A total of 30 clean-grade Sprague-Dawley rats were divided into three groups, including agomiR-146a group (n=10), antagomiR-146a group (n=10) and negative control group (NC, n=10). The model was successfully established via a one-time injection of sodium urate into ankle joint cavity. Subsequently, agomiR-146a (10 μL), antagomiR-146a (10 μL) and normal saline (10 μL) were intrathecally injected into rats in the three groups at 1 h before injection and 12 h, 24 h, 48 h and 72 h after injection, respectively. The ankle joint swelling index, joint dysfunction index and joint inflammation index of rats in the three groups were closely monitored. After 72 h of observation, the rats were euthanized, and synovial tissues were collected from the knee joint. The expression and distribution of nuclear factor-κB (NF-κB) in synovial tissues were detected using the immunohistochemical method. Meanwhile, the expression levels of inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6) were detected via enzyme-linked immunosorbent assay. Furthermore, the mRNA and protein expression levels of TLR4 and MyD88 were detected via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blotting, respectively. No statistically significant differences in the joint swelling index, joint dysfunction index, joint inflammation index, TLR4 and MyD88 and related inflammatory factors were found between the NC group and antagomiR-146a group. Compared with the NC group, agomiR-146a group showed markedly reduced ankle joint swelling index (p<0.05). Meanwhile, joint landing behavior and inflammatory swelling were significantly relieved in the agomiR-146a group (p<0.05). The mRNA and protein expression levels of TLR4 and MyD88 were remarkably decreased as well (p<0.05). Furthermore, the expression and distribution of NF-κB in synovial tissues of agomiR-146a group was markedly reduced when compared with the NC group (p<0.05). In addition, agomiR-146a group exhibited significantly lower expression levels of inflammatory factors (TNF-α, IL-1 and IL-6) in synovial tissues (p<0.05). MiR-146a alleviates joint inflammation of acute arthritis in rats through the TLR4/MyD88/NF-κB signaling pathway, which may become a new therapeutic target.

1441LRP5 and PCSK9 in inflammatory cells immunomodulation

Abstract Background Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. The disruption of the hepatic interaction between Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) and Low-Density Lipoprotein Receptor (LDLR) downregulates blood cholesterol levels and reduces cardiovascular events. Recent data suggest that other members of the LDLR superfamily may be targets of PCSK9. Purpose The aim of this work is to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target, and to study the role of PCSK9 and LRP5 in foam cell formation and hence, in the mechanism of lipid accumulation and atherosclerotic plaque formation. Methods Intracellular protein and lipid localization, cholesteryl esters (CE) accumulation; quantification of structural and inflammatory proteins expression and immunoprecipitation analyses were performed in primary cultures of human inflammatory cells (monocytes and macrophages) silenced for LRP5 or PCSK9 and challenged with modified LDLs. Results We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages have less intracellular CE accumulation. In LDL treated macrophages internalization of LRP5-bound LDL starts after 30 minutes of incubation and lasts up to 24hours. The SREBP-2 promoter is not involved in LRP5 regulation but it does regulate macrophage PCSK9 expression. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally we studied the role of PCSK9 and LRP5 in the inflammatory response by TLR4/NFkB signaling pathway. We show decreased TLR4 protein expression levels and decreased nuclear translocation of NFκB in PCSK9 silenced-inflammatory cells after lipid loading indicating a downregulation of the proinflammatory pathway TLR4/NFκB. Increased gene expression is observed in TNF-α and IL1β after lipid-loading that is abolished in PCSK9-silenced macrophages. Furthermore release of the proinflammatory cytokines TNF-α and IL1β is decreased in PCSK9-silenced macrophages. LRP5 protein expression is increased in lipid-loaded macrophages independent of the presence or absence of PCSK9. Conclusions These results demonstrate that, in human macrophages, LRP5 is internalized with lipids. Furthermore, PCSK9 and LRP5 can form a complex in the cytoplasm of lipid-loaded macrophages opening the possibility that PCSK9 induces lysosomal LRP5 degradation in a similar manner than it does with LDLR. Finally we also show that PCSK9 gene interference decreases inflammation and supports a role for PCSK9 as an inflammatory mediator in atherosclerosis. Acknowledgement/Funding CN16/11/00411-LB; TERCEL RD16/0011/018-LB; FIS2016-02014 -MBP; SEC2015 to MBP

Screening of Antiviral Components of Ma Huang Tang and Investigation on the Ephedra Alkaloids Efficacy on Influenza Virus Type A.

Although Ma Huang Tang (MHT) has long been considered as a classical formula for respiratory infections like influenza, bronchitis and asthma, its chemical ingredients that really exert the main efficacy are still obscure. In this study we aimed to screen its antiviral components and investigate the potential mechanisms. The MDCK cellular research results showed that, among nine predominant ingredients of MHT, L-methylephedrin (LMEP), L-ephedrine (LEP) and D-pseudo- ephedrine (DPEP) significantly inhibited the proliferation of influenza A virus in vitro, and the inhibitory effect at 24 h after the treatment was more obvious than that at 48 h. They also significantly inhibited the mRNA expression levels of related genes in the TLR3, TLR4 and TLR7 signaling pathways, which were accompanied with the down-regulation of TNF-α level and the up-regulation of IFN-β level in the cell supernatant. Therefore, three Ephedra alkaloids exert an antiviral effect in vitro which may be closely related to the inhibition of viral replication and the modulation of inflammatory response. Animal research further indicated, at the 3rd and 7th days after infection, LEP and DPEP significantly attenuated lung injury, decreased lung index, virus load in the lung and the level of IL-1β in serum, inhibited the mRNA expression levels of TNF-α, TLR3, TLR4, TLR7, MyD88, NF-κB p65 and RIG-1 as well as the protein expression levels of TLR4, TLR7, MyD88 and NF-κB p65 and markedly increased thymus index, the level of IL-10 in serum and the mRNA expression level of IFN-γ. LEP and DPEP have certain protective effects on the influenza virus-infected mice, which may be associated with their abilities of effectively alleviating lung injury, improving the immunologic function of infected mice and adjusting the host’s TLRs and RIG-1 pathways. The overall findings demonstrate that, as effective and inexpensive natural substances, Ephedra alkaloids and MHT may have potential utility in clinical management.

Effect of Bufei Yishen Granules Combined with Electroacupuncture in Rats with Chronic Obstructive Pulmonary Disease via the Regulation of TLR-4/NF-κB Signaling.

Background The combined therapy of Bufei Yishen granules (BY) and electroacupuncture (EA) has shown good effects clinically in treating chronic obstructive pulmonary disease (COPD). The present study aimed to observe the effects of the BY + EA combination in a COPD rat model and dissect the potential mechanisms via Toll-like receptor (TLR) 4/nuclear factor kappa B (NF-κB) signaling. Methods The COPD rats were treated with normal saline, aminophylline, Bufei Yishen granules, electroacupuncture, or Bufei Yishen granules combined with electroacupuncture. The pulmonary function; lung tissue histology; levels of inflammatory factors; expression levels of TLR-4, inhibitor of nuclear factor kappa B (IκB), and NF-κB; and activation of NF-κB in the lung tissues were evaluated. Results Pulmonary function was markedly decreased in the COPD rats, and the lung tissue histology of the COPD rats showed severe pathological changes. The pulmonary function and lung tissue morphology in the treatment groups (APL, BY, EA, and BY + EA) were improved. The increased levels of the inflammatory cytokines interleukin (IL)-1β and IL-6 indicated a chronic inflammatory state in the COPD rats. In the BY, EA, and BY + EA groups, the levels of IL-1β and IL-6 were decreased, especially in the BY + EA group. In addition, the mRNA and protein expression levels of TLR-4, IκB, and NF-κB were obviously downregulated in the BY and BY + EA groups; and the NF-κB p65 activation was significantly decreased in the BY, EA, and BY + EA groups. Conclusions Bufei Yishen granules and electroacupuncture have curative effects in COPD rats, and the combination therapy of Bufei Yishen granules and electroacupuncture is superior. The TLR-4/NF-κB pathway may be involved in the potential mechanisms by which Bufei Yishen granules and electroacupuncture reduce inflammation.

Pomegranate peel polyphenols inhibits inflammation in LPS-induced RAW264.7 macrophages via the suppression of TLR4/NF-κB pathway activation.

BackgroundsInflammatory response mediated by activated immune cells is a vital process in host defense system while responding to various stresses. Our previous studies have indicated that pomegranate peel polyphenols (PPPs) and their main components punicalagin (PC) and ellagic acid (EA) decreased pro-inflammatory cytokines and inflammatory mediators by regulating the mitogen-activated protein kinases (MAPKs) pathway, but whether these tested polyphenols play an important role in NF-κB signaling pathway, another crucial pathway of inflammation, remains unclear.ObjectiveIn this study, we analyzed the anti-inflammatory effect of these polyphenols via TLR4-NF-κB pathway in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.MethodsDifferent concentrations of PPPs, PC, and EA were pre-incubated with RAW264.7 macrophages and then stimulated with LPS (1 μg/mL), and the effects of reactive oxygen species and TLR4 were investigated. Moreover, NF-κB p65 nuclear translocation and phosphorylation, and degradation of IκB were measured by Western blot. Furthermore, the influence of pro-inflammatory cytokines was detected by enzyme-linked immunosorbent assay (ELISA).ResultsOur data showed that PPPs, PC, and EA inhibited LPS-induced intracellular ROS production and suppressed the mRNA and protein expression levels of TLR4 in a dose-dependent manner. Moreover, the anti-inflammatory mechanism was involved in blocking LPS-induced phosphorylation, degradation of IκB, and nuclear translocation of p65. Additionally, PPPs and PC exhibited a stronger anti-inflammatory effect than that of EA.ConclusionThe results indicated that PPPs possess potent anti-inflammatory effect, and PC was the main effective component in PPPs, which provided new insights into the utilization of PPPs to prevent inflammation-associated disorders.

Atorvastatin alleviates early hypertensive renal damage in spontaneously hypertensive rats

This study aimed to examine the effects of atorvastatin on early hypertensive renal damage and explored the underlying mechanisms. 12-week-old salt-loaded spontaneous hypertensive rats (SHRs) were divided into four groups: atorvastatin (AVT), losartan potassium (LP), atorvastatin combined with peroxisome proliferators-activated receptor γ (PPAR-γ) inhibitor (AVT + GW9662), and saline. During 10 weeks administration blood pressure and urea albumin creatinine ratio were determined. We also examined the renal function, pathological changes of kidney, inflammatory cytokines in the serum and the association of the change of inflammatory factors in the kidney tissue. AVT did not reduce the mortality of the SHRs. AVT reduced the blood pressure of SHRs, but the effect was not comparable to that of LP. AVT significantly decreased urine protein. AVT and LP displayed comparable effects by significantly decreasing inflammatory cytokines (hs-CRP, IL-1β, IL-6, TNF-α, and TGF-β) levels in serum. AVT and LP both apparently improved renal pathological changes and significantly reduced the infiltration of macrophage in renal tubular interstitial. Both mRNA and protein expression levels of TLR4, NFκB, MCP-1 were significantly down regulated in AVT and LP groups. There was no significant change in macrophage polarity. The addition of PPAR-γ inhibitor partially reduced the anti-inflammatory effect of AVT. These results mean that Atorvastatin can alleviates the pathology of hypertensive renal damage. Atorvastatin protects the kidney by reducing the apparent inflammation in salt-loaded SHRs. Atorvastatin alleviates inflammation partially by augmenting expression of PPAR-γ.

TLR4/MyD88 signaling determines the metastatic potential of breast cancer cells

The influence of Toll-like receptor (TLR)4/myeloid differentiation factor (MyD)88 signaling on the invasion and metastasis of cancer cells has been previously reported. The purpose of the present study was to determine the role of TLR4/MyD88 in breast cancer cell migration and invasion, and to discover novel therapeutic targets for breast cancer treatment. TLR4, MyD88 and high mobility group box 1 (HMGB1) mRNA expression levels were assessed in highly invasive human MDA-MB-231 breast cancer cells, breast cancer cells with a low rate of invasion (MCF-7) and normal human MDA-Kb2 mammary gland cells by reverse transcription-quantitative polymerase chain reaction. The protein expression levels of these markers were detected by western blotting and immunofluorescence. Randomly selected breast cancer and paracarcinoma tissues were used to measure TLR4 and MyD88 protein expression levels by immunohistochemistry. The mRNA and protein expression levels of TLR4 and MyD88 were significantly higher in MDA-MB-231 cells compared with either MCF-7 cells or MDA-Kb2 cells. The mRNA and protein expression levels of HMGB1 were comparable in the two breast cancer cell lines, with no statistical difference (P>0.05). TLR4 and MyD88 protein expression levels were also significantly higher in breast cancer tissues compared with paracarcinoma tissues (P<0.05). TLR4 and MyD88 protein expression levels were positively correlated with axillary lymph node metastasis and histological grade (P<0.05). TLR4/MyD88 expression levels were positively correlated with the metastasis of breast cancer cells. TLR4/MyD88 may be useful as a novel biomarker to evaluate the prognosis and treatment of patients with breast cancer.

Study on the effects of total flavonoids from litchi nucleus on nuclear translocation of nuclear factor-kappa B and related protein expression in rat hepatic stellate cell

Objective: The effect of total flavonoids of litchi (TFL) on nuclear translocation of nuclear factor-kappa B (NF- kappa B) in rat hepatic stellate cell line (HSC-T6) induced by transforming growth factor - beta 1 (TGF- beta 1) in vitro was studied to explore the mechanism of action of anti-hepatic fibrosis drugs. Methods: HSC-T6 was cultured in vitro, induced by TGFβ1 for 24 h, and then treated with TFL at 125, 250 and 500 μg/ml for 48 h. The effect of TFL on NF-κB nuclear translocation in HSC-T6 was observed by confocal laser microscopy. The effects of TFL on the expression of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and Collagen I protein were detected by western blot. The expressions of TLR4 and p-NF-κB p65 were detected by immunofluorescence. Data were presented as mean±SEM. Homogeneity test of variance was performed and then followed by one-way analysis of variance (ANOVA). The multiple comparisons between groups were performed by LSD test. P < 0.05 was considered statistically significant. Results: Confocal laser scanning microscopy showed TFL inhibited the nuclear translocation of NF-κB in activated HSC-T6 in a concentration-dependent manner and TFL down regulated the protein expression levels of TLR4, p-IκB ɑ, p-NF-κB p65, NF-κB and collagen I protein in HSC-T6 in a concentration-dependent manner. Conclusion: The mechanism of TFL against hepatic fibrosis may be related to the inhibition of nuclear translocation of NF-κb in the activated HSC-T6 and the expression of TLR4, P-iκbɑ, P-nf-κb p65, NF-κb and collagen I protein in HSC-T6.

Upregulation of HMGB1-TLR4 inflammatory pathway in focal cortical dysplasia type II

BackgroundWe attempted to determine whether the inflammatory pathway HMGB1-TLR4 and the downstream pro-inflammatory cytokines is upregulated in focal cortical dysplasia (FCD) type II and whether there is a correlation between the TLR4 upregulation and disease duration or frequency of epileptic seizures.MethodsFCD type II and peri-FCD paired tissues resected from eight children with refractory epilepsy were collected. Through real-time qPCR, Western blot, and co-immunoprecipitation, we examined the differences between FCD lesions and peri-FCD tissues with respect to mRNA expression, protein expression, and protein interaction in HMGB1-TLR4 pathway biomarker and downstream pro-inflammatory factors in whole brain tissue. Then, we used immunofluorescence to examine the difference between FCD lesions and peri-FCD tissues with respect to protein expression and intracellular distribution of HMGB1-TLR4 pathway biomarker in neurons, astrocytes, and oligodendrocytes. Correlation between level of TLR4 expression and disease duration or frequency of epileptic seizures in patients was also analyzed.ResultsThe protein expression levels of TLR4, cytoplasm HMGB1, TLR4/MyD88 complex, ubiquitination of TRAF6, p-IKK, p-IκB-α, p-NF-κB p65, and IL-1β and TNF-α in lesion tissues were significantly higher than those in peri-FCD controls. Total mRNA expression levels of TLR4, IL-1β, and TNF-α in lesion tissues were significantly higher than those in peri-FCD controls, but HMGB1 had no significant change. In neurons and astrocytes inside the lesions, the expression of TLR4 protein was significantly higher than that in peri-FCD tissues, and HMGB1 was mainly expressed in the cytoplasm, while expressed in the nuclei in peri-FCD tissues. But in oligodendrocytes, there was no upregulation of HMGB1-TLR4 pathway in both lesions and peri-FCD tissues. We did not identify the correlation between the level of TLR4 activation and disease duration or frequency of epileptic seizures.ConclusionThe HMGB1-TLR4 pathway was upregulated in the neurons and astrocytes inside FCD type II lesions, which led to an increase in the release of downstream pro-inflammatory cytokines. Correlation between the level of TLR4 activation and duration or frequency of epileptic seizures was not identified.

Effects of Kudingcha Nanoparticles in Hyperlipidaemic Rats Induced by a High Fat Diet

Background/Aims: The herbal medicine Kudingcha has a bitter taste and low bioavailability for lipid reduction. To improve the bioavailability and ameliorate the compliance, we prepared Kudingcha nanoparticles and investigated their effect in hyperlipidaemic rats. In addition, the safety and lipid-lowering mechanism of the Kudingcha nanoparticles were examined. Methods: Kudingcha nanoparticles were prepared by ionotropic gelation and spray-drying. Seventy rats were randomly assigned into eight groups: a normal fat diet group (NF), a high-fat group (HF), a spontaneous recovery group (SR), a Kudingcha group (KDC), a blank nanoparticle group (B-N), and a Kudingcha nanoparticle groups (low, medium and high doses). All groups (except for the normal fat diet group) were fed a high-fat diet to establish hyperlipidaemia. Different interventions were administered to the treatment groups for four weeks. Serum lipids were measured using commercially available kits according to the recommended protocols. Liver morphology and histopathology were examined by a light microscope. The mRNA and protein levels of TLR4 and NF-κB were determined by RT-PCR and Western blotting, respectively. In addition, acute toxicity was evaluated by the LD50 test. Results: The Kudingcha nanoparticles were spherical and had a smooth surface. The size distribution of the nanoparticles was 100-600 nm. Acute toxicity results revealed that the Kudingcha nanoparticles were a non-toxic substance. Compared with regular Kudingcha, TG and TC decreased distinctly in the Kudingcha nanoparticles, especially for the moderate and high dose groups (p<0.05). Moreover, the Kudingcha nanoparticles were superior in lowering body, liver and adipose tissue weights compared to Kudingcha (p<0.05). With respect to antioxidant properties, the nanoparticles also revealed an outstanding impact on serum SOD and MDA. In addition, liver morphology and histology in the moderate and high dose nanoparticle groups were similar to those in the normal group. Finally, the mRNA and protein expression levels of TLR4 and NF-κB in the liver tissue of the nanoparticle groups were significantly upregulated compared with the KDC group (p<0.05). Conclusions: Based on these results, we conclude that Kudingcha nanoparticles are a potent lipid-lowering agent and may have a potential role in the treatment of hyperlipidaemia and fatty liver disease.

Intermittent High Glucose Exacerbates A-FABP Activation and Inflammatory Response through TLR4-JNK Signaling in THP-1 Cells

Background Glucose fluctuation confers additional risks on diabetes-related vascular diseases, but the underlying mechanisms are unknown. Macrophage activation mediated by TLR4-JNK signaling plays an important role during the progress of diabetes. In the present study, we hypothesize that glucose fluctuation results in macrophage inflammation through TLR4-JNK signaling pathways. Methods THP-1 cells were treated with normal glucose (5 mM), constant high glucose (25 mM), and intermittent high glucose (rotation per 6 h in 5 mM or 25 mM) for 24 h. The mRNA and protein expression levels of TLR4, p-JNK, and adipocyte fatty acid-binding protein (A-FABP) were determined, and the proinflammatory cytokines TNF-α and IL-1β were quantified. Results In constant high glucose, TLR4 expression and JNK phosphorylation levels increased, and this effect was more pronounced in intermittent high glucose. Accordingly, the expression of A-FABP and the release of the proinflammatory cytokines TNF-α and IL-1β also increased in response to constant high glucose, an effect that also was more evident in intermittent high glucose. The inhibition of p-JNK by SP600125 did not attenuate TLR4 expression, but totally inhibited both A-FABP expression and the production of the proinflammatory cytokines TNF-α and IL-1β in both constant and intermittent high glucose. Conclusions Intermittent high glucose potentiates A-FABP activation and inflammatory responses via TLR4/p-JNK signaling in THP-1 cells. These findings suggest a more detrimental impact of glucose fluctuation on macrophage inflammation in diabetes-related vascular diseases than thus far generally assumed.

Angiotensin 1-7 ameliorates caerulein-induced inflammation in pancreatic acinar cells by downregulating Toll-like receptor 4/nuclear factor-κB expression.

The present study aimed to investigate the effects of angiotensin (Ang) 1–7 on caerulein (CAE)-stimulated nuclear factor (NF)-κB, Toll-like receptor (TLR4) and cytokine expression using pancreatic acinar AR42J cells. AR42J cells were treated with 10 nmol/l CAE for various durations. In addition, cells were pretreated with various concentrations of Ang 1–7 or A779, a specific antagonist of Ang 1–7, and were stimulated with CAE for 12 h. Control cells were treated with vehicle (F-12K complete medium with 2% fetal bovine serum, 10 U/ml penicillin and 100 mg/ml streptomycin) alone. The mRNA and protein expression levels of TLR4, NF-κB, interleukin (IL)-6, IL-8, IL-10 and tumor necrosis factor-α (TNF-α) were determined by western blotting, immunofluorescence and reverse transcription-quantitative polymerase chain reaction. CAE treatment stimulated TLR4 and NF-κB expression within AR42J cells. Immunofluorescence indicated that TLR4 was expressed on the membranes and in the cytoplasm of AR42J cells, whereas NF-κB expression accumulated in the cytoplasm and nuclei. CAE-induced expression of TLR4 and NF-κB within AR42J cells was abrogated by 10−5 mmol/l Ang 1–7; however, TLR4 and NF-κB expression was enhanced with the addition of A779, particularly 10−5 mmol/l. In addition, treatment with 10−6 and 10−5 mmol/l Ang 1–7 significantly mitigated CAE-induced expression of IL-6, IL-8 and TNF-α, whereas it enhanced IL-10 expression. Conversely, A779 treatment enhanced the CAE-induced expression of IL-6, IL-8 and TNF-α, and reduced IL-10 expression in AR42J cells. In conclusion, these results suggested that Ang 1–7 may attenuate CAE-induced inflammation by downregulating TLR4, NF-κB and proinflammatory cytokine expression within AR42J cells. Therefore, Ang 1–7 may exert protective effects against the pathological progression of AP in a cell model of AP induced by CAE and may be considered in the development of treatments for this disease.

Modulation of inflammation by toll-like receptor 4/nuclear factor-kappa B in diarrhea-predominant irritable bowel syndrome.

In order to investigate the function of toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signal pathways in the pathogenesis of diarrhea-predominant irritable bowel syndrome (IBS-D), IBS-D animal models were established in wistar rats challenged with acute and chronic stresses (29 days). Wistar rats without stress-challenged were used as controls. IBS-D models were randomly divided into two groups: one was treated with normal saline, another group was treated with TLR4/NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC) (50mg/kg/week) for continuous four times. Our results demonstrate that continuous stresses can induce the characteristic symptoms of IBS-D, including high wet stool rate and intestinal flora imbalance. Further examinations of colon tissues show that the protein expression levels of TLR4 and NF-κB in IBS-D groups are higher than that in control group. The secretory levels of interleukin (IL-8), tumor necrosis factor α (TNFα), and myeloid differentiation factor 88 (MyD88) are significantly increased in IBS-D group. Administration with PDTC effectively downregulates levels of these inflammatory factors. In contrast, interleukin-10 (IL-10) is in an opposite alteration with lower levels in IBS-D groups and the PDTC treatment increases it to the levels as in control group. Moreover, inhibition of the TLR4/NF-κB by PDTC improves the microstructure of intestinal mucosa mainly by increasing the height of villi. Our results suggest that TLR4/NF-κB signal pathway plays an important role in the modulation of inflammatory responses in IBS-D, which might be a therapeutic target for the IBS-D. All of these findings also provide the evidence concerning an inherent linkage between the axis of stress/NF-κB/inflammation and IBS-D.

Monoclonal antibody against Toll-like receptor4 attenuates ventilator-induced lung injury in rats by inhibiting MyD88- and NF-κB-dependent signaling.

The mechanisms through which mechanical ventilation causes non-infectious inflammatory diseases and lung injury are poorly understood. Animals models of this type of injury suggest that it involves signaling mediated by Toll‑like receptor (TLR)4and9. In this study, in order to gain further insight into the involvement of TLR4 in this type of injury, we performed invivo and invitro experiments to determine the mechanisms through which TLR4 triggers inflammation. We also examined whether the use of TLR4 monoclonal antibody(mAb) can alleviate this type of injury. For this purpose, rats were tracheotomized and administered intratracheal injections of anti‑TLR4 mAb or saline, and then ventilated for 4 h at a high tidal volume(HTV) of 40 ml/ kg or allowed to breathe spontaneously for the same period of time (controls). Alveolar macrophages(AMs) were isolated from the bronchoalveolar lavage fluid(BALF) of the rats and stimulated for 16h with tumor necrosis factor(TNF)‑α in the presence or absence of anti‑TLR4 mAb. Lung injury was assessed by examining lung histopathology, lung wet/dry weight ratio, BALF total protein and cytokine levels in BALF and plasma. The mRNA and protein expression levels of TLR4, TLR9, myeloid differentiation factor88(Myd88) and nuclear factor(NF)‑κB were measured in cultured macrophages. Compared to the controls (spontaneous breathing), the ventilated rats exhibited greater pulmonary permeability, more severe inflammatory cell infiltration/lung edema, and higher levels of interleukin(IL)‑1β, IL‑6 and TNF‑α in BALF and plasma. The AMs from the ventilated rats expressed higher mRNA and protein levels of TLR4, TLR9, Myd88 and NF‑κB compared with the macrophages from the spontaneously breathing rats. The ventilated rats pre‑treated with anti‑TLR4 mAb exhibited markedly attenuated signs of ventilation‑induced injury, such as less lung inflammation and pulmonary edema, fewer cells in BALF, and lower levels of ILs and TNF‑α in BALF and plasma. Similarly, the TNF‑α‑dependent increases in the mRNA and protein expression of TLR4, Myd88 and NF‑κB in AMs were attenuated when TNF‑α was co‑administered with anti‑TLR4 mAb than when TNF-α was administered alone. Co‑administering anti-TLR4 mAb also reduced the TNF‑α‑dependent secretion of ILs. On the whole, our data demonstrate that TLR4 contributes significantly to ventilation‑induced lung injury by activating the Myd88/NF‑κB pathway, and pre‑treating rats with anti‑TLR4 mAb partially protects them against this type of injury by inhibiting Myd88/NF-κB signaling.