Toll-like Receptor 4 mRNA Expression Research Articles

Effect of nicotinic acetylcholine receptor α7 subunit gene on liver inflammatory reaction in mice with nonalcoholic steatohepatitis and related mechanisms

Objective: To investigate the effect of nicotinic acetylcholine receptor α7 (α7nAChR) subunit gene on liver inflammation in mice with nonalcoholic steatohepatitis (NASH) and related mechanisms. Methods: C57BL/6J mice and α7nAChR gene knockout mice were fed for 24 weeks to establish the NASH model, and the mice were sacrificed to isolate and culture the primary liver macrophages. After the treatment with nicotine and endotoxin, ELISA was used to measure the levels of the inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in supernatant; indirect immunofluorescence assay and Western blot were used to observe the effect on the NF-κB signaling pathway, and quantitative PCR was used to measure the mRNA expression of Toll-like receptor-4 (TLR-4) in macrophages. An analysis of variance was used for comparison of means between multiple groups. Results: The results of ELISA showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher levels of IL-6 and TNF-α in supernatant (IL-6: 1 599±65 pg/ml vs 1 465±45 pg/ml, P < 0.05; TNF-α: 1 567±66 pg/ml vs 1 433±50 pg/ml, P < 0.05). The results of Western blot showed that compared with the endotoxin+nicotine group of C57 NASH mice, the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative protein expression of phosphorylated NF-κB and TLR-4 (NF-κB: 69 425±600 vs 51 133±200, P < 0.05; TLR-4: 93 387±684 vs 64 198±630, P < 0.05). The results of indirect immunofluorescence assay showed that the endotoxin+nicotine group of gene knockout NASH mice had a significantly higher fluorescence intensity of NF-κB than the endotoxin+nicotine group of C57 NASH mice. The results of PCR showed that the endotoxin+nicotine group of gene knockout NASH mice had significantly higher relative mRNA expression of TLR-4 than the endotoxin+nicotine group of C57 NASH mice (4.13±0.13 vs 2.93±0.14, P < 0.05). Conclusion: The α7nAChR gene knockout can aggravate the degree of inflammatory reaction in NASH, and its mechanism may be related to the fact that the NF-κB signaling pathway cannot be inhibited, which aggravates inflammatory reaction.

In vitro cow uterine response to Escherichia coli, leukotrienes and cytokines

The aim was to determine the dynamic profile of interactions between Escherichia coli (E. coli) and the actions of leukotrienes (LTs) and TNF and INFγ (cytokines) in the uterus in vitro. Uterine explants (N=6) were incubated for 2, 12 and 24h either as E. coli-treated (106CFU) or non-treated and/or with: LTB4 and C4 (10−6M, for both LTs), LTs receptors antagonists (aLTR; 10−6M) and/or cytokines (each 10ng/ml). Toll Like Receptor 4 (TLR4) mRNA expression increased in explants incubated with E. coli, cytokines and LTs after 2 and 12h and aLTR inhibited the effect of LTs in explants incubated with E. coli (P<0.05). IL-6 mRNA expression was up-regulated in E.coli-treated explants with cytokines after 2h and cytokines with LTs after 12h (P<0.05). E. coli increased prostaglandin (PG)E2 output after all examined time points, and PGF2α and IL-6 levels in E.coli-treated explants after 12 and 24h with cytokines, with LTs (P<0.05). aLTR inhibited LT stimulating action on PGs and IL-6 output in explants incubated with E. coli after 12 and 24h (P<0.05). LTs modify and enhance experimentally induced infection: TLR4 and IL-6 mRNA expression, IL-6 and PGs secretion, and cytokines participate in this process.

Ginkgolide A inhibits lipopolysaccharide-induced inflammatory response in human coronary artery endothelial cells via downregulation of TLR4-NF-κB signaling through PI3K/Akt pathway.

Ginkgolide A (GA) is a one of the active components of Ginkgo biloba. We aimed to detect the effects GA on the lipopolysaccharide (LPS)-induced inflammatory response in human coronary artery endothelial cells (HCAECs) and whether the effects are associated with the inhibition of toll-like receptor 4 (TLR4)-NF-κB signaling through PI3K/Akt pathway. HCAECs were stimulated with LPS and treated with GA or TLR4 inhibitor CLI-095. A PI3K/Akt inhibitor LY294002 was used to block the PI3K/Akt pathway. The toxic effects of GA, LPS and LY294002 on HCAEC were evaluated using MTT assay. Levels inflammatory mediators, TLR4 mRNA, NF-κB signaling activity were valuated. We found LPS stimulation significantly increased the release of IL-6, IL-8, MCP-1 and TNF-α from HCAECs, elevated the TLR4 mRNA expression and activated the NF-κB signaling. GA and CLI-095 abolished the LPS-induced inflammatory mediator release and NF-κB signaling activation, and GA reduced the TLR4 mRNA expression without affecting cell viability. However, PI3K/Akt blocking abolished the effects of GA on HCAECs. We conclude that GA could attenuate the LPS-induced inflammatory response in HCAECs and the anti-inflammatory activity might be associated with the inhibition of TLR4-NF-κB signaling through PI3K/AKT pathway. These findings suggest a therapeutic potential of GA in endothelial inflammation.

IL-1β promotes tubulointerstitial injury in MPO-ANCA-associated glomerulonephritis .

It is widely accepted that tubulointerstitial injury (TII) is caused by glomerular injury (GI) in glomerular diseases. Glomerular endocapillary inflammation may result in crescent formation and exuded protein leakage, which may induce TII in antineutrophil cytoplasmic antibody-associated glomerulonephritis (ANCAGN). However, some reports have indicated a glomerulonephritis-independent mechanism of TII in ANCAGN. The aim of this study was to determine the principle cytokines correlated with TII severity and to elucidate a characteristic mechanism for TII in ANCAGN. 28 myeloperoxidase-ANCA-positive ANCAGN patients were enrolled, and their kidney biopsy specimens were histologically evaluated with regard to GI and TII. The mRNA expression of various cytokines was examined in 28 specimens. Interleukin (IL)-1β was significantly correlated with the severity of TII. The mRNA expression of Toll-like receptor 4 (TLR4) and Nod-like receptor family pyrin domain-containing-3 (NLRP3) also correlated with TII severity. Immunohistochemical analysis demonstrated that TLR4 protein was positively stained in the tubulointerstitial infiltrating cells. NRLP3 protein was detected in macrophages in the severe infiltrating area but was absent or only very faintly expressed in the glomeruli. These results indicated that NLRP3 inflammasome-dependent processing in macrophages releases the mature active form of IL-1β, which may lead to the development and deterioration of TII. Sterile inflammation leads to the formation of ANCA-mediated neutrophil extracellular traps (NETs), which may stimulate macrophages and dendritic cells via TLR4 and induce NF-κB-dependent mRNA expression and translation of pro-IL-1β. Simultaneously, damage-associated molecular pattern signals resulting from NETs promote NLRP3 inflammasome-dependent processing and release mature active IL-1β. Sterile inflammation utilizing the NLRP3 inflammasome might be a characteristic reaction limited to the tubulointerstitium. Thus, neutralizing IL-1β may be a promising strategy to suspend the progress of TII and improve the prognosis of chronic kidney disease resulting from ANCAGN. .

Puerarin exerts the protective effect against chemical induced dysmetabolism in rats

In this study, we aim to explore the potential benefits of puerarin on metabolic function of liver fibrosis (LF) rat induced by carbon tetrachloride (CCl4), and to investigate with the underlying molecular mechanism targeted on liver and pancreas tissues. In methodology, The LF rats were prepared through intragastrically giving CCl4 twice each week (2ml/kg, v/w) for 8weeks, and dosed puerarin (20, 40mg/kg) were given three times each week via intraperitoneal injection. After being conducted with oral glucose tolerance test (OGTT), the blood samples of rat were harvested for biochemical tests, as well as the liver and pancreas were isolated for histological examination and biochemical assays. The findings showed that puerarin-administered rats resulted in reduced glucose tolerance, blood insulin level, sero-enzymes of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and increased plasma level of high-density lipoprotein cholesterol (HDL-C) and reduced low-density lipoprotein cholesterol (LDL-C) content in serum. Further, the intrahepatic collagen deposits were lessened and positive cell of alpha smooth muscle actin (α-SMA) was lessened in puerarin treatment, while the pro-apoptotic cell numbers of Caspase 3, Bax in pancreatic islets were reduced dose-dependently. Moreover, the mRNA expressions of toll-like receptor 4 (TLR4) and tumor necrosis factor (TNF-α) in liver and pancreas were downregulated. In addition, TNF-α protein in the CCl4-lesioned liver and pancreas was reduced. Our findings demonstrate that puerarin contributes to attenuating the metabolic dysfunctions of CCl4-damaged liver and pancreas, in which the possible mechanisms may be linked to inhibition of inflammatory stress and normalization of metabolic homoeostasis in the liver and pancreas.

Quercetin modulates toll-like receptor-mediated protein kinase signaling pathways in oxLDL-challenged human PBMCs and regulates TLR-activated atherosclerotic inflammation in hypercholesterolemic rats.

Toll-like receptors (TLRs) are pattern recognition receptors that have a unique and essential function in innate immunity. The effect of quercetin on TLR-mediated downstream signaling mechanism and its effect on TLR-mediated MAP kinase and Akt pathways were studied in oxLDL-stimulated hPBMCs using specific inhibitors. The pretreatment of hPBMCs with specific TLR inhibitor, CLI-095, decreased the NF-κB nuclear translocation and TNF-α release by oxLDL. When the cells treated with inhibitor and quercetin together, the inhibition was more effective. The specific inhibitor for p38 MAPK, SB203580, reduced the phosphorylated p38 level and decreased the NF-κB activation and TNF-α release by oxLDL-challenged hPBMCs. This inhibitor showed enhanced inhibition when treated with quercetin together. The inhibitors for ERK1/2, PD98059, and for JNK, SP606125, also showed inhibitory effect on NF-κB activation and TNF-α release by oxLDL-simulated hPBMCs. Quercetin supplementation enhanced the inhibition of nuclear translocation of NF-κB and the release of cytokines. TLR4 inhibition study confirmed the downstream signaling mechanism mediated by NF-κB which is involved in the oxLDL-induced inflammatory response, and quercetin suppresses the cytokine, TNF-α release by modulating TLR-NF-κB signaling pathway. In addition to NF-κB signaling pathway, inflammation induced by oxLDL was also related to the activation of p38MAPK, ERK1/2 and JNK, and Akt pathways, and the protective effect of quercetin may be also related to the inhibition of activation of these pathways. Quercetin significantly downregulated the elevated mRNA expression of TLRs and cytokine TNF-α in HCD-fed atherosclerotic rats in vivo. As quercetin possesses inhibition on both TLR-NF-κB signaling pathway and TLR-mediated MAPK pathway, it is evident that it can be used as a therapeutic agent to ameliorate atherosclerotic inflammation. Since quercetin is the major flavonoid and forms the backbone of many other flavonoids and this study provides strong evidence that it has potent anti-inflammatory effect, quercetin may be a promising agent for the prevention and treatment of atherosclerosis and promote health by reducing harmful vascular inflammation.

Fungal cell wall agents and bacterial lipopolysaccharide in organic dust as possible risk factors for pulmonary sarcoidosis.

BackgroundComposition of organic dust is very complex, involving particles of microbial, animal and plant origin. Several environmental exposure studies associate microbial cell wall agents in organic dust with various respiratory symptoms and diseases. The aim of the present study was to investigate the in vitro effects of the co-exposure of fungal cell wall agents (FCWAs) and bacterial lipopolysaccharide (LPS) on inflammatory immune responses of peripheral blood mononuclear cells (PBMCs) from patients with pulmonary sarcoidosis.MethodsPBMCs from 22 patients with pulmonary sarcoidosis and 20 healthy subjects were isolated and stimulated in vitro with FCWAs (soluble and particulate (1 → 3)-β-D-glucan, zymosan and chitosan) and/or LPS. Subsequently, cytokines were measured by ELISA and the mRNA expression of dectin-1, toll-like receptor 2 (TLR2), TLR4 and mannose receptor (MR) was analysed by real-time RT-PCR.ResultsPatients with sarcoidosis had a significantly higher secretion of inflammatory cytokines tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-10 and IL-12 (1.7-fold, 2.0-fold, 2.2-fold, and 2.8-fold, respectively; all p < 0.05) after in vitro co-stimulation of PBMCs with FCWAs and LPS. We showed that PBMCs from patients with sarcoidosis had a higher baseline mRNA expression of dectin-1, TLR2, TLR4 and MR (6-fold, 11-fold, 18-fold, and 4-fold, respectively). Furthermore, we found a reduced expression of dectin-1, TLR2 and TLR4 after stimulation with FCWAs and/or LPS, although the reduction was significantly weaker in patients than in healthy subjects.ConclusionsIn conclusion, co-stimulation with FCWAs and LPS of PBMC from patients with sarcoidosis caused a weaker reduction of dectin-1, TLR2, TLR4 receptors expression, which could increase the sensitivity of PBMCs, leading to excessive inflammatory cytokine responses and result in the development or progression of pulmonary sarcoidosis.

Effect of dexmedetomidine on liver injury in rats with obstructive jaundice

Objective To evaluate the effect of dexmedetomidine on liver injury in the rats with obstructive jaundice. Methods Forty-five healthy male Sprague Dawley rats, weighing 250-300 g, aged 8-9 weeks, were divided into 3 groups (n=15 each) using a random number table: sham operation group (S group), obstructive jaundice group (OJ group) and dexmedetomidine group (D group). Obstructive jaundice was induced in rats by division and double ligation of the common bile duct in OJ and D groups.Dexmedetomidine 100 μg/kg was injected intraperitoneally at 72 h after establishment of the model in group D. At 3, 5 and 24 h after administration, blood samples were collected from hearts for determination of the plasma alanine aminotransferase (ALT) and C-reactive protein (CRP) levels.After blood sampling at each time point, the specimens from the external right lobe of the liver were obtained for detection of the expression of Toll-like receptor 4 (TLR4) mRNA (by real-time polymerase chain reaction) and TLR4 content (by enzyme-linked immunosorbent assay) in liver tissues and for pathological examination of liver tissues (with light microscope). Results Compared with group S, the plasma ALT and CRP levels were significantly increased at each time point after administration, and the expression of TLR4 mRNA in liver tissues was significantly up-regulated, and TLR4 content in liver tissues was significantly increased in OJ and D groups (P<0.05). Compared with group OJ, the plasma ALT and CRP levels were significantly decreased at each time point after administration, and the expression of TLR4 mRNA in liver tissues was significantly down-regulated, and TLR4 content in liver tissues was significantly decreased in group D (P<0.05). The degree of damage to liver tissues was significantly attenuated in group D compared with group OJ, and was aggravated in group D compared with group S. Conclusion Dexmedetomidine can reduce liver injury in the rats with obstructive jaundice. Key words: Dexmedetomidine; Jaundice, obstructive; Liver diseases

Molecular characterization and expression analysis of toll-like receptor 2 in response to bacteria in silvery pomfret intestinal epithelial cells

Toll-like receptor 2 (TLR2) has been shown to play a crucial role in the host defense of pathogenic microbes in innate immunity. In this study, the full-length cDNA of TLR2 in silvery pomfret (Pampus argenteus) was cloned by homology cloning and the rapid amplification of cDNA ends (RACE) technique. The complete cDNA sequence of TLR2 was 2932 bp, containing an open reading frame (ORF) of 2469 bp encoding 822 amino acids. A multiple alignment analysis of the silvery pomfret TLR2 protein-coding sequence with other known TLR2 sequences from Oplegnathus fasciatus, Epinephelus coioides, Larimichthys crocea, Miichthys miiuy, Oreochromis niloticus, Paralichthys olivaceus, Trematomus bernacchii, Sparus aurata, and Chionodraco hamatus exhibited a high degree of homology of 78.83%, 75.91%, 74.21%, 74.94%, 71.95%, 72.57%, 73.68%, 75%, and 72.52 respectively, between these fish. Analysis of the TLR2 domain structures indicated that TLR2 from the silvery pomfret has the typical structural features of proteins that belong to the TLR family, including one transmembrane domain, eleven leucine-rich repeats (LRRs), and one Toll/IL-1 receptor homology domain (TIR). In vitro immunostimulation experiments revealed that Lactobacillus plantarum and Clostridium butyricum induce high levels of TLR2 mRNA and protein expression, but they induce only moderate levels of IL-8 and TNF-α production compared to Vibrio anguillarum. This suggests that TLR2 might play a vital role in the L. plantarum and C. butyricum-mediated immune response. In contrast, V. anguillarum significantly increased the secretion of IL-8 and TNF-α and induced cell apoptosis and necrosis. Due to the lower expression of TLR2 and higher levels of IL-8 and TNF-α induced by V. anguillarum, we hypothesize that a V. anguillarum infection is independent of the TLR2-induced production of pro-inflammatory cytokines. These results indicate that TLR2 may be involved in molecular interactions between the host and commensal bacteria, that exist in the silvery pomfret intestinal tract.

Exercise intensity and the expression of toll-like receptor 4 and nuclear transcription factor-κB in the diabetic liver

Objective To investigate the changes in toll-like receptor 4 (TLR4) and nuclear transcription factor-κB (NF-κB) expression in the livers of diabetic rats after exercise at different intensities. Methods Fifty Sprague-Dawley rats were randomly divided into a control group (NC), a diabetic group (DM), a low intensity exercise group (LIE), a medium intensity exercise group (MIE) and a high intensity exercise group (HIE), each of 10. The control group was given a normal diet. Type 2 diabetes was induced in the others through feeding a high-fat and high-sugar diet, as well as injecting a small dose of streptozotocin intraperitoneally. The three exercising groups swam to 40%, 70% and 100% of their exhaustion times once a day, 6 d/w for 6 weeks. Due to infection, drowning and other reasons, only 8 rats in each of the DM, LIE and MIE groups, as well as 7 in the HIE group survived. After the intervention, body weight, liver weight and fasting blood glucose (FBG) were tested. The expression of TLR4 and NF-κB mRNA in the livers was tested using RT-PCR technology, and the expression of TLR4 and NF-κB protein in the liver was tested using immunohistochemistry. Results Compared with the NC group, the average body weight and liver weight of the other four groups decreased significantly, while the FBG and the expression of TLR4 and NF-κB mRNA and protein increased significantly. There were significant differences among the exercise groups with the lowest values in the MIE group. Conclusion Six weeks of aerobic exercise can reduce FBG and the level of expression of TLR4 and NF-κB mRNA and protein in the liver, at least in rats. The effect of exercise at medium intensity is best. Key words: Exercise; Diabetes mellitus; Liver; Toll-like receptor 4; Nuclear transcription factor-κB

Oral citrulline supplementation protects female mice from the development of non-alcoholic fatty liver disease (NAFLD).

Impairments of intestinal barrier function are discussed as risk factors for the development and progression of non-alcoholic fatty liver disease (NAFLD). Studies suggest an association between arginine/citrulline homeostasis and the development of liver damages. Here, the effect of an oral L-citrulline (Cit) supplement on the development of a Western-style diet (WSD)-induced NAFLD was determined in mice. Female 6- to 8-week-old C57BL/6J mice were either pair-fed a liquid Western-style or control diet (C)±2.5g/kg bodyweight Cit for 6weeks (C+Cit or WSD+Cit). Indices of liver damage, glucose metabolism, intestinal barrier function and NO synthesis were measured. While bodyweight gain was similar between groups, markers of glucose metabolism like fasting blood glucose and HOMA index and markers of liver damage like hepatic triglyceride levels, number of neutrophils and plasminogen activator inhibitor-1 protein levels were significantly lower in WSD+Cit-fed mice when compared to WSD-fed mice only. Protein levels of the tight junction proteins occludin and zonula occludens-1 in duodenum were significantly lower in mice fed a WSD when compared to those fed a WSD+Cit (-~70 and -~60%, respectively, P<0.05), whereas portal endotoxin levels, concentration of 3-nitrotyrosine protein adducts in duodenum and toll-like receptor-4 mRNA expression in livers of WSD+Cit-fed mice were markedly lower than in WSD-fed mice (-~43%, P=0.056; -~80 and -~48%, respectively, P<0.05). Our data suggest that the protective effects of supplementing Cit on the development of NAFLD in mice are associated with a decreased translocation of endotoxin into the portal vein.

Ischemic post-conditioning attenuates renal ischemic reperfusion injury via down-regulation of toll-like receptor 4 in diabetic rats

Background: Ischemia/reperfusion (I/R) injury, which is commonly seen in the field of renal surgery or transplantation, is a major cause of acute renal failure (ARF). The ischemic ARF in diabetic rats is much more severe than that in the normal rats exposed to as same ischemic time. Ischemic post-conditioning (IPO) is a phenomenon by which intermittent interruptions of blood flow in the early phase of reperfusion can protect organs from I/R injury. To determine whether the renal protection effect of IPO mediates by toll-like receptor 4 (TLR4) signaling pathway in diabetic rats.Methods: Streptozotocin-induced diabetic rats were randomly divided into three groups: sham operation group, I/R group, and IPO group. Except sham operation group, rats were subjected to 30 min of renal ischemia, both with and without treatment with IPO, then reperfusion 24 h. Light microscope and transmission electronic microscope were used to observe structural changes of renal tubule. RT-PCR was used to measure TLR4 and tumor necrosis factor-alpha (TNF-α) mRNA expression level, renal TLR4 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) protein expression was detected by Western blot.Results: The results demonstrated that IPO markedly decreased renal ischemic injury caused by I/R and inhibited the proinflammatory expression levels of TLR4, TNF-α, and NF-κB, all of which up-regulated by I/R in diabetic rats.Conclusion: Taken together, our results suggest that proper IPO may have protective effect on the ischemic injury mediated by renal I/R, which might be associated with inhibition of TLR4 signaling pathway in diabetic rats.

Peripheral blood toll-like receptor 4 correlates with rapid virological response to pegylated-interferon and ribavirin therapy in hepatitis C genotype 1 patients.

BackgroundToll-like receptors (TLRs) are effectors of the innate immune system that are able to recognize hepatitis C virus (HCV) and give rise to an immune response. Failure of interferon (IFN)-α-based treatment is related to host immunity. Therefore, we sought to study the clinical importance of TLRs in HCV genotype 1 patients who received pegylated IFN (PEG-IFN) plus ribavirin (RBV) therapy.MethodsWe enrolled 79 treatment-naïve patients with HCV genotype 1. Patients completed a 24- to 48-week course of response-guided therapy. Peripheral blood monocyte (PBMC) expression of mRNA for TLRs 2, 3, 4, 7, and 9 was quantified by real-time PCR before therapy. TLR mRNA expression is shown as a log ratio relative to GAPDH mRNA (log 2 -(∆Ct)).ResultsForty-five patients (57.0 %) showed a rapid virological response (RVR). Univariate analysis revealed that TLR 2, 3, 4, 7, and 9 were significantly lower in the RVR group than in the non-RVR group (P = 0.001, 0.014, < 0.001, 0.008, and 0.001, respectively). Multivariate analysis revealed that TLR 4 < −2 log (OR: 7.17, 95 % CI: 1.70–30.34, P = 0.007) was an independent predictor for RVR. In addition, levels of TLR 2, 3, 4, 7, and 9 were positively correlated with HCV viral load (P = 0.009, 0.013, < 0.001, 0.007, and 0.001, respectively).ConclusionsA low level of TLR 4 mRNA in PMBCs was correlated with RVR, which indicates that TLR4 may play a critical role in HCV recognition and activation of innate immunity. TLR expression levels were correlated with HCV viral load, indicating that TLR activation upon exposure to HCV may subsequently limit HCV replication.Electronic supplementary materialThe online version of this article (doi:10.1186/s12876-016-0492-6) contains supplementary material, which is available to authorized users.

Identification and expression analysis of TLR2 in mucosal tissues of turbot (Scophthalmus maximus L.) following bacterial challenge.

The pathogen recognition receptors (PRRs), which can recognize the conserved pathogen-associated molecular patterns (PAMPs) of the bacteria, play key roles in the mucosal surfaces for pathogen recognition and activation of immune signaling pathways. However, our understanding of the PRRs and their activities in mucosal surfaces in the critical early time points during pathogen infection is still limited. Towards to this end, here, we sought to identify the Toll-like receptor 2 (TLR2) in turbot as well as its expression profiles in mucosal barriers following bacterial infection in the early time points. The full-length TLR2 transcript consists of open reading frame (ORF) of 2451 bp encoding the putative peptide of 816 amino acids. The phylogenetic analysis revealed the turbot TLR2 showed the closest relationship to Paralichthys olivaceus. The TLR2 mRNA expression could be detected in all examined tissues, with the most abundant expression level in liver, and the lowest expression level in skin. In addition, TLR2 showed different expression patterns following Vibrio anguillarum and Streptococcus iniae infection, but was up-regulated following both challenge, especially post S.iniae challenge. Characterization of TLR2 will probably contribute to understanding of a number of infectious diseases and broaden the knowledge of interactions between host and pathogen, which will eventually help in the development of novel intervention strategies for farming turbot.

Effect of pulse high-volume hemofiltration on Toll-like receptor in patients with severe sepsis.

The expression level and prognosis of Toll-like receptor 2 (TLR2) mRNA in peripheral blood mononuclear cells of patients with severe sepsis after applying pulse high-volume hemofiltration (PHVHF) were investigated. Sustained PHVHF treatment was carried out on 40 patients on the basis of conventional treatment for up to 72 h. Acute physiology and chronic health evaluation (APACHE) II scores of patients were compared before and after the treatment. CD4+, CD8+ lymphocyte counts and ratios in the peripheral blood were detected using FASort before and 24 and 48 h of PHVHF treatment. Enzyme-linked immunosorbent assay was adopted to detect tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) concentrations in plasma at different time points before and after 24, 48 and 72 h of treatment, while semi-quantitative reverse transcription-polymerase chain reaction technology was used to test TLR2 mRNA expression. After PHVHF treatment, APACHE II, Sequential Organ Failure Assessment scores were decreased (P<0.05). After 72 h of PHVHF treat-ment, TNF-α, IL-10, TLR2 mRNA expression levels in the plasma of patients were significantly decreased compared to before treatment (P<0.05), and the IL-10 / TNF-α ratio was much higher than before treatment (p<0.05). In conclusion, PHVHF can restore the pro-inflammatory/anti-inflammatory balance of the body, thereby improving the overall condition of the patients by removing inflammatory mediators and lowering TLR2 expression of mononuclear cell surface in peripheral blood.

Effect of Omega-3 fish oil fat emulsion on the expression of Toll-like receptor 4 and nuclear factor-κB in the neonatal rats hippocampus after hypoxic-ischemic

Objective To discuss whether Omega-3 fish oil fat emulsion has the potential protective mechanism for 7-day-old rats with hypoxic - ischemic brain damage (HIBD). Methods One hundred and sixty-eight 7-day-old SD rats were randomly divided into 4 groups: group A(sham group), group B(Omega-3 fish oil fat emulsion group), group C(normal fat emulsion group), group D(model group), and there were 42 cases in each group.Neonatal HIBD was induced by the method of Rice.Rats were sacrificed at 1 d, 3 d and 7 d after the surgery.Hippocampus was removed for Real-time PCR and Western blot test to detect Toll-like receptor 4 (TLR4 )and nuclear factor-κB (NF-κB )mRNA and protein expression.TUNEL staining comparison was done among different groups to observe the number of cellular apoptosis. Results HE staining of hippocampus CA1 area in 3 d showed that brain tissues in group A maintained normal structures; those in group D had much more brain cells with severe edema than other groups; TLR4 and NF-κB mRNA and protein expression levels in group D were higher than those in group A in 1 d (all P 0.05); TLR4 and NF-κB mRNA and protein expression levels in group B were lower than those in group C and group D in 7 d(all P 0.05). The apoptotic cell number of brain tissues in 3 d: group B (13.67±2.52) were lower than those in group C (27.67±2.52) and group D (41.00±3.61) (all P 0.05). Conclusions Omega-3 fish oil fat emulsion plays an important role in protecting neonatal rats with HIBD.The mechanisms were likely to reduce TLR4, NF-κB and cell apoptosis levels. Key words: Rat, newborn; Omega - 3 fish oil emulsion; Hypoxic-ischemia brain damage; Toll-like receptor 4; Nuclear factor-κB

Artesunate ameliorates severe acute pancreatitis (SAP) in rats by inhibiting expression of pro-inflammatory cytokines and Toll-like receptor 4

Severe acute pancreatitis (SAP) is a severe clinical condition with significant morbidity and mortality. Multiple organs dysfunction (MOD) is the leading cause of SAP-related death. The over-release of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α is the underlying mechanism of MOD; however, there is no effective agent against the inflammation. Herein, artesunate (AS) was found to increase the survival of SAP rats significantly when injected with 3.5% sodium taurocholate into the biliopancreatic duct in a retrograde direction, improving their pancreatic pathology and decreasing serum amylase and pancreatic lipase activities along with substantially reduced pancreatic IL-1β and IL-6 release. In vitro, AS-pretreatment strongly inhibited IL-1β and IL-6 release and their mRNA expressions in the pancreatic acinar cells treated with lipopolysaccharide (LPS) but exerted little effect on TNF-α release. Additionally, AS reduced the mRNA expressions of Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) p65 as well as their protein expressions in the pancreatic acinar cells. In conclusion, our results demonstrated that AS could significantly protect SAP rats, and this protection was related to the reduction of digestive enzyme activities and pro-inflammatory cytokine expressions via inhibition of TLR4/NF-κB signaling pathway. Therefore, AS may be considered as a potential therapeutic agent against SAP.

Lowering of Messenger Ribonucleic Acid Toll-Like Receptors 2-4,9 in Peripheral Blood Mononuclear Cells in Kidney Allograft Recipients, Relationships With Immunosuppressive Treatment, and Delayed Graft Function Occurrence

BackgroundBoth tacrolimus (Tac) and cyclosporine (CsA) inhibit control peripheral blood mononuclear cells (PBMC) after stimulation of various Toll-like receptors (TLR) at supra-pharmacological concentrations. Earlier studies demonstrated that 24 hours after kidney transplantation (KT), the expression of the TLR4 messenger RNA (mRNA) in PBMC from patients with subsequent delayed graft function (DGF+) was lower than in patients without DGF (DGF−). An assessment was made of the interaction of immunosuppression with TLR mRNA in PBMC and to verify whether the reduced expression of TLR-2,3,4,9 mRNA in PBMC is permanent in DGF+. MethodsWe investigated mRNA expression of TLR in non-stimulated PBMC. All patients were transplanted more than 1 month before PBMC acquisition. Patients were divided into groups with respect to positive or negative history of delayed graft function (DGF+/−). ResultsThe expression of TLR2, TLR3, and TLR9 in patients was lower than that in the control group. We found an association of Tac C0 with expression of TLR4 only and CsA dose per 1 kg body weight with TLR2 or up to 6 months after KT with TLR9. Mofetil mycophenolate (MMF)contributed to the change of TLR4 expression in the CsA group but not in the Tac group. TLR3 and TLR9 were nearly equally sensitive to both Tac and CsA, with a decrease of expression with respect to control. DGF+ was associated with variable degree of reduction of TLR2, TLR3, TLR4, and TLR 9 expression. ConclusionsWe showed the importance of immunosuppression and delayed graft function as factors that modify the overall expression of mRNA-TLR PBMC for a period of time after KT. Patients with a history of DGF have chronically decreased expression of mRNA TLR2, TLR3, TLR4, and TLR9. This fact is associated with poorer graft function. Measuring the expression of the TLR in the upper range of therapeutic doses of calcineurin inhibitors and MMF gives the opportunity to assess the strength, effectiveness, and toxicity of immunosuppression.

Resolvin D1 Protects Lipopolysaccharide-induced Acute Kidney Injury by Down-regulating Nuclear Factor-kappa B Signal and Inhibiting Apoptosis.

Background:Resolvin D1 (RvD1) is a newly found anti-inflammatory bioactive compound derived from polyunsaturated fatty acids. The current study aimed to explore the protective effect of RvD1 on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) and its possible mechanism.Methods:Both in vivo and in vitro studies were conducted. Male BALB/c mice were randomly divided into control group (saline), LPS group (LPS 5 mg/kg), RvD1 group (RvD1 5 μg/kg + LPS 5 mg/kg), and blockage group (Boc-MLP 5 μg/kg + RvD1 5 μg/kg + LPS 5 mg/kg). Boc-MLP is a RvD1 receptor blocker. The mice were intraperitoneally injected with these drugs and recorded for general condition for 48 h, while the blood and kidneys were harvested at 2, 6, 12, 24, and 48 h time points, respectively (n = 6 in each group at each time point). Human proximal tubule epithelial cells (HK-2) were randomly divided into control group (medium only), LPS group (LPS 5 μg/ml), RvD1 group (RvD1 10 ng/ml + LPS 5 μg/ml), and blockage group (Boc-MLP 10 ng/ml + RvD1 10 ng/ml + LPS 5 μg/ml). The cells were harvested for RNA at 2, 4, 6, 12, and 24 h time points, respectively (n = 6 in each group at each time point). Blood creatinine was tested by using an Abbott i-STAT portable blood gas analyzer. Tumor necrosis factor-α (TNF-α) level was detected by ELISA. Kidney pathology was observed under hematoxylin and eosin (HE) staining and transmission electron microscope (TEM). We hired immune-histological staining, Western blotting, and fluorescence quantitative polymerase chain reaction to detect the expression of RvD1 receptor ALX, nuclear factor-kappa B (NF-κB) signaling pathway as well as caspase-3. Kidney apoptosis was evaluated by TUNEL staining.Results:RvD1 receptor ALX was detected on renal tubular epithelials. Kaplan–Meier analysis indicated that RvD1 improved 48 h animal survival (80%) compared with LPS group (40%) and RvD1 blockage group (60%), while RvD1 also ameliorated kidney pathological injury in HE staining and TEM scan. After LPS stimulation, the mRNA expression of toll-like receptor 4, myeloid differentiation factor 88, and TNF-α in both mice kidneys and HK-2 cells were all up-regulated, while RvD1 substantially inhibited the up-regulation of these genes. Western blotting showed that the phosphorylated-IκB/IκB ratio in LPS group was significantly higher than that in the control group, which was inhibited in the RvD1 group. RvD1 could inhibit the up-regulation of cleaved-caspase-3 protein stimulated by LPS, which was prohibited in RvD1 blockage group. RvD1 group also had a lower proportion of apoptotic nuclei in mice kidney by TUNEL staining compared with LPS group.Conclusion:In LPS-induced AKI, RvD1 could decrease TNF-α level, ameliorate kidney pathological injury, protect kidney function, and improve animal survival by down-regulating NF-κB inflammatory signal as well as inhibiting renal cell apoptosis.

Effects of budesonide on toll-like receptor expression in alveolar macrophages from smokers with and without COPD.

IntroductionAlveolar macrophages (AMs) are equipped with innate immune receptors such as toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4). In primary bronchial epithelial cells, exposure of toll-like receptor (TLR) ligands or tumor necrosis factor-alpha (TNF-α) increased TLR2 mRNA expression and reduced interleukin-8 (IL-8) release when coincubated with glucocorticosteroids. The aim of this study was to compare TLR2 and TLR4 expression levels and the effect of a glucocorticosteroid after stimulation with TLR ligands on AMs from smokers with and without COPD compared with the healthy controls.Subjects and methodsBronchoalveolar lavage was performed, and AMs were isolated from smokers with (n=10) and without COPD (n=11) and healthy controls (n=10) and stimulated ex vivo with peptidoglycan (PGN), lipopolysaccharide (LPS), or TNF-α ± budesonide (Bud). Blocking antibodies to TLR2 or TLR4 were added before stimulation with LPS or PGN ± Bud, respectively. The release of proinflammatory cytokine (TNF-α), chemoattractant (CXCL8), and TLR expression was analyzed by enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction.ResultsLPS, PGN, and TNF-α induced an increased release of IL-8 and TNF-α in the AMs in all the groups independent of smoking or disease. These responses were inhibited by a glucocorticosteroid (Bud) in all the three groups, except PGN-induced IL-8 secretion in smokers without COPD. Bud increased TLR2 expression in the healthy controls and smokers without COPD. Costimulation of TLR ligands and Bud significantly enhanced TLR2 mRNA expression in both groups of smokers compared with TLR ligands alone. In smokers, costimulation with PGN and Bud significantly increased TLR2 expression when compared with Bud alone. On stimulation with the TLR4 agonist, LPS downregulated TLR4 mRNA expression in all the three groups.ConclusionThe combination of glucocorticosteroids with TLR ligands can increase TLR2 expression, thereby improving host defense in smokers. Also this combination can decrease the secretion of proinflammatory cytokines and chemokines as an anti-inflammatory response. Our findings indicate that glucocorticosteroid therapy strengthens immune defense pathways, which may have implication during exacerbation caused by microorganisms.