Toll-like Receptor 4 mRNA Levels Research Articles

Anti-HSV-1 effect of dihydromyricetin from Ampelopsis grossedentata via the TLR9-dependent anti-inflammatory pathway

ObjectivesHerpes simplex virus 1 (HSV-1) is one of the most prevalent viruses in humans worldwide. Owing to limited therapeutic options mainly with acyclovir (ACV) and analogues and the emergence of ACV-resistant strains, new drugs with different modes of action and low toxicity are required. The aim of this study was to determine the anti-HSV-1 effect and mechanism of action of the flavonoid compound dihydromyricetin (DHM) from Ampelopsis grossedentata. MethodsThe HSV-1 inhibitory effect of DHM was evaluated by measuring plaque formation and generation of progeny virus as well as expression of HSV-1-related genes in Vero cells. The molecular mechanism of the antiviral activity of DHM against HSV-1 was explored by real-time quantitative PCR and ELISA. ResultsDHM presented a significant inhibitory effect on HSV-1 plaque formation and generation of progeny virus, with an EC50 (50% effective concentration) of 12.56 μM in Vero cells. Furthermore, expression of HSV-1 immediate-early genes (ICP4 and ICP22), early genes (ICP8 and UL42) and late genes (gB, VP1/2) was decreased by DHM at concentrations of 16 μM and 32 μM. DHM specifically suppressed mRNA levels of Toll-like receptor 9 (TLR9), leading to inhibition of the inflammatory transcriptional factor NFκB and a decrease in TNFα. ConclusionThese findings indicate that the effective inhibitory activity of DHM was achieved by suppressing TNFα production in a TLR9-dependent manner. Although further studies are needed to better characterise the activity of DHM in vivo, the results suggest this extract as a promising new anti-HSV-1 agent.

The role of TLR4/MyD88/NF-κB pathway in periodontitis-induced liver inflammation of rats.

ObjectivesThe aim of this study was to clarify the immune mechanism of hepatic injury induced by periodontitis using a rat model.MethodsTwenty‐four SPF male Wistar rats were randomly divided into two groups: control group (CG) and periodontitis group (PG). In order to induce experimental periodontitis, we tied the wire ligature around bilateral maxillary first molar of rats. After 8 weeks, the following indicators were valued: gingival index, tooth mobility, probing pocket depth; indexes about oxidative stress and circulating biomarkers; bone retraction by micro‐CT analysis; Toll‐like receptor 4 (TLR4), myeloid differential protein‐88 (MyD88), and nuclear factor kappa B (NF‐κB) by qRT‐PCR and Western blotting; tumor necrosis factor α (TNF‐α), and interleukin‐6 (IL‐6) by qRT‐PCR and immunohistochemical staining; inflammation of periodontal and hepatic tissues by histopathological observation.ResultsPeriodontal indicators and micro‐CT results showed the raised levels of inflammatory response and bone retraction in PG compared with CG. The mRNA and protein levels of TLR4, MyD88, NF‐κB, TNF‐α, and IL‐6 have indicated high values in PG versus CG. Histopathological analysis revealed a correlation between periodontitis and hepatic injury.ConclusionTLR4/MyD88/NF‐κB pathway may play a role in periodontitis‐induced liver inflammation of rats.

Ketamine alleviates HMGB1-induced acute lung injury through TLR4 signaling pathway.

Acute lung injury (ALI) is a common critical respiratory disease that seriously threatens human health. Ketamine has good anti-inflammatory and immune-regulating properties that can delay the lung injury process. High mobility group box protein 1 (HMGB1) plays an important role in the occurrence, development and treatment of ALI. Toll-like receptor 4 (TLR4) is the receptor for HMGB1. The aim of this study was to determine the role of the HMGB1 TLR4 signaling pathway in the treatment of ALI using ketamine. A total of 30 healthy, male, 8-week-old Sprague-Dawley rats were randomly, equally divided into a control group, an lipopolysaccharide (LPS) group and a ketamine group. In order to establish a rat ALI model, 15 mg/kg of LPS was injected into the femoral veins. Ketamine was intravenously injected (10 mg/kg) into the experimental group rats. The rats were euthanized 24 h after modeling and lung tissue samples were collected. Western blot was used to test TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 protein expression in the lung tissue. Real-time polymerase chain reaction (RT-PCR) was performed to detect TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA levels. Compared with the controls, the LPS group had significantly higher TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA and protein levels (p < 0.05). These levels were significantly lower after ketamine intervention in comparison with the LPS group (p < 0.05). A positive correlation was found between TLR4 and HMGB1 expression in the LPS and ketamine groups (r = 0.952, p < 0.001; r = 0.941, p < 0.001). Ketamine attenuates HMGB1-induced ALI, possibly by regulating the TLR4 signaling pathway.

Baicalin suppresses renal fibrosis through microRNA-124/TLR4/NF-κB axis in streptozotocin-induced diabetic nephropathy mice and high glucose-treated human proximal tubule epithelial cells.

Renal fibrosis is a major pathological event in the development of diabetic nephropathy (DN). Baicalin is a flavonoid glycoside that possesses multiple pharmacological properties including anti-fibrotic activity. In the present study, the effects of baicalin on renal fibrosis along with related molecular basis were investigated in streptozotocin (STZ)-induced DN mouse model and high glucose (HG)-treated HK-2 human proximal tubule epithelial cell model. Renal injury was evaluated through blood urea nitrogen (BUN) and serum creatinine (Scr) levels and urine albumin creatine ratio (ACR). Renal fibrosis was assessed by type IV collagen (COLIV) and fibronectin (FN) protein expression and histopathologic analysis via Masson trichrome staining. Protein levels of COLIV, FN, NF-κB inhibitor alpha (IκBα), phosphorylated IκBα (p-IκBα), p65, phosphorylated p65 (p-p65), and toll-like receptor 4 (TLR4) were measured by western blot assay. MicroRNA-124 (miR-124) and TLR4 mRNA levels were detected by RT-qPCR assay. The interaction of miR-124 and TLR4 was examined by bioinformatics analysis, luciferase reporter assay, and RIP assay. Baicalin or miR-124 attenuated renal injury and fibrosis in STZ-induced DN mice. Baicalin inhibited the increase of COLIV and FN expression induced by HG through upregulating miR-124 in HK-2 cells. TLR4 was a target of miR-124. MiR-124 inhibited TLR4/NF-κB pathway activation and the inactivation of the NF-κB pathway hindered COLIV and FN expression in HG-stimulated HK-2 cells. Baicalin prevented renal fibrosis by increasing miR-124 and inactivating downstream TLR4/NF-κB pathway in DN, hinting the pivotal values of baicalin and miR-124 in the management of DN and renal fibrosis.

(-)-Epicatechin Mitigates High Fat Diet-induced Inflammation in the Hippocampus and Altered Recognition Memory in Mice

Abstract Objectives This project investigated the capacity of dietary (-)-epicatechin (EC) to mitigate hippocampal inflammation and impaired memory in high fat diet (HFD)-fed mice. Methods Healthy 6 weeks old male C57BL/6J mice (10 mice/group) were fed for 13 weeks either: a control diet (10% total calories from fat), a high fat diet (60% total calories from lard fat), or the control and high fat diets supplemented with 20 mg EC/kg body weight. Between weeks 10 and 12 of the dietary intervention, object recognition memory was evaluated by the novel object recognition task and short-term spatial memory by the object location memory task, and the Morris Water Maze. After 13 weeks on the dietary treatments, mice were euthanized, and brain tissues and blood were collected. Hippocampus was isolated, flash-frozen in liquid nitrogen, and stored at −80°C. Metabolic endotoxemia was assessed by measuring plasma lipopolysaccharide (LPS) levels. Gene expressions related to inflammation (Toll-like receptor 4 (TLR4) and tumor necrosis factor-α (TNF-α)), activation of microglia (ionized calcium-binding adapter molecule 1 (Iba-1)), and oxidative stress (NADPH oxidase 4 (NOX4)) were analyzed in the hippocampus with RT-qPCR. Results After 13 weeks on the dietary treatments, HFD-fed mice developed obesity, endotoxemia, and showed increased parameters of hippocampal inflammation, i.e., high mRNA levels of TLR4, Iba-1, and NOX4. While not affecting body weight gain, EC supplementation prevented all other HFD-induced changes. Impaired recognition memory was observed in HFD-fed mice, which was prevented by EC supplementation. Neither HFD consumption nor EC supplementation affected mouse spatial memory. Conclusions EC supplementation prevented short-term recognition memory in HFD-induced obese mice, which could be in part due to the capacity of EC to mitigate metabolic endotoxemia and associated hippocampal inflammation and oxidative stress. Funding Sources HA Jastro Shields Award.

Involvement of the Hsp70/TLR4/IL-6 and TNF-α pathways in delayed-onset muscle soreness.

Delayed-onset muscle soreness (DOMS) is a very common condition in athletes and individuals not accustomed to physical activity that occurs after moderate/high-intensity exercise sessions. The activation of microglial Toll-like receptor 4 (TLR4) in the spinal cord has been described to be important for the induction and maintenance of persistent pain. Based on that, we hypothesize that 70 kilodalton heat-shock protein (Hsp70), a mediator released by exercise, could activate microglial TLR4 in the spinal cord, releasing proinflammatory cytokines and contributing to the start of DOMS. In fact, we found that the knockout of TLR4, myeloid differentiation primary response 88 (MyD88), interleukin-6 (IL-6), or both tumor necrosis factor-α (TNF-α) receptor 1 and TNF-α receptor 2 in mice prevented the development of DOMS following acute aerobic exercise in contrast to the findings in male C57BL/6 wild-type mice. Furthermore, DOMS in exercised wild-type mice was also prevented after pre-treatment with microglia inhibitor, TLR4 antagonist, and anti-Hsp70 antibody. During exercise-induced DOMS, Hsp70 mRNA, TLR4 mRNA, and protein levels, as well as Iba-1 (a microglial marker), IL-6, and TNF-α protein levels, were increased in the muscle and/or spinal cord. Together, these findings suggest that Hsp70 released during exercise-induced DOMS activates the microglial TLR4/IL-6/TNF-α pathway in the spinal cord. Thus, the blockade of TLR4 activation may be a new strategy to prevent the development of DOMS before intense exercise.

Protective Effects of Clostridium Butyricum in a Murine Model of Dextran Sodium Sulfate-Induced Colitis That Involve Inhibition of the TLR2 Signaling Pathway and T Helper 17 Cells

This study aimed to investigate the role of Clostridium butyricum (C. butyricum) in conjunction with the Toll-like receptor2 (TLR2) signaling pathway and T helper 17 (Th17) cells in dextran sodium sulfate (DSS)-induced colitis in mice. Forty 8-week-old BALB/c mice were randomly divided into 5 groups of 8 mice for 7 days: control, DSS (5% DSS), DSS+C. butyricum (1×109 CFU), DSS+C. butyricum (1×108 CFU) and DSS+C. butyricum (1×107 CFU) groups. We assessed the disease activity index (DAI) and histological damage scores. The expression levels of TLR2, myeloid differentiation factor 88 (MyD88), nuclear factor kappa-B p65 (NF-κBp65), interleukin (IL) 17 (IL17), IL23 and retineic acid receptor related orphan nuclear receptor gamma t (RORγt) were determined through immunohistochemical staining, western blot and quantitative real-time PCR (qRT-PCR). The expression levels of CD3+CD4+IL17+ cells in peripheral blood were measured by flow cytometry. C. butyricum dose-dependently decreased DAI and histological damage scores in DSS mice and down-regulated the mRNA and protein levels of TLR2, MyD88 and NF-κBp65 in mouse colon tissue (all P < 0.05). In addition, C. butyricum dose-dependently decreased the levels of CD3+CD4+IL17+ cells in peripheral blood and down-regulated the mRNA and protein levels of IL17, IL23 and RORγt in mouse colon tissue (all P < 0.05). Moreover, the effect of C. butyricum on TLR2 was positively correlated with IL17, IL23 and RORγt. C. butyricum exerts a dose-dependently protective effect on acute intestinal inflammation induced by DSS in mice, by inhibiting the TLR2 signaling pathway, down-regulating the expression of IL23 and RORγt, and inhibiting the secretion of IL17.

Immune Response to Salmonella Enteritidis Infection in Broilers Immunized Orally With Chitosan-Based Salmonella Subunit Nanoparticle Vaccine.

Salmonella enterica serovar Enteritidis (S. Enteritidis, SE) infection in broilers causes a huge economic loss and public health risk. We previously demonstrated that orally delivered chitosan based (CS) Salmonella subunit nanoparticle (NP) vaccine containing immunogenic outer membrane proteins (OMP) and flagellin (FLA) of SE [CS-NP(OMP+FLA)] induces immune response in broilers. The objective of this study was to evaluate the dose- and age-dependent response and efficacy of CS-NP(OMP+FLA) vaccine in broilers. Three-day old birds were vaccinated and boosted once or twice. Additional groups were vaccinated at three weeks with no booster or boosted once a week later. Each dose of CS-NP vaccine had either 10 or 50 μg of OMP+FLA antigens. Our data revealed that two doses of vaccine were required to induce substantial immune response. Birds received 2 doses of CS-NP(OMP+FLA) vaccine at 3 days and 3 weeks of age with 10 μg antigens, and birds inoculated twice at 3 and 4 weeks of age with 50 μg antigens had lowest challenged bacterial load in the cecal contents with over 0.5 log10 reduction. In CS-NP(OMP+FLA) vaccinated birds, antigen-specific splenocyte proliferation, mucosal and systemic antibody response and the frequency of IFNγ-producing T cells were increased compared to control groups. At the molecular level, in the cecal tonsils of CS-NP(OMP+FLA) immunized birds, mRNA levels of toll-like receptor (TLR) 2 and TLR 4, and cytokines IL-4 and IL-10 were upregulated. The CS-NP(OMP+FLA) vaccine given orally has the potential to induce a protective immune response against SE infection in broilers.

The role of TLR4/NF-κB signaling pathway in activated microglia of rats with chronic high intraocular pressure and vitro scratch injury-induced microglia

Glaucoma is a kind of blind-causing disease with structural damages of optic nerve and defection of visual field. It is believed that the death of retinal ganglion cell (RGC) is a consequential event of over-reactive immune orchestral cells such as microglia. Previous evidences in animal and clinical studies show the innate immunity plays a pivotal role in neuro-inflammation of glaucoma. Toll-like receptor 4 (TLR4) is expressed on microglia and mediates many neuroinflammatory diseases. We aimed to explore the impacts of high intraocular pressure (IOP) on rat microglia in retina and the regulation of TLR4/NF-κB signaling pathway in scratched microglia cells. In our study, we successfully established chronic high IOP rat model by episcleral vein cauterization (EVC) which behaved like the chronic glaucoma. Besides, we set up an in vitro scratch-induced injury model in rat microglia cells. We found the level of activated microglia cells were significantly increased in the retina of chronic high IOP groups. Moreover, the inhibition of TLR4/NF-κB signaling pathway suppressed the expression of TLR4 protein and mRNA levels of P50, IL-6 and TNF-α. Our original study provided a theoretical basis on targeting TLR4/NF-κB to suppress pro-inflammatory factors releasing in activated microglia and it might be a good treatment target to prevent glaucoma from progressing.

The efficacy of lactic acid bacteria usage in turbot Scophthalmus maximus on intestinal microbiota and expression of the immune related genes

To understand the efficacy of lactic acid bacteria (LAB) as probiotics on the growth, immune response and intestinal microbiota of turbot Scophthalmus maximus, in this study, the Leuconostoc mesenteroides HY2 strain screened from wide caught fish was bath administrated for juvenile turbot with no bacteria administrated as control. The mRNA levels of toll-like receptors 3 (TLR3), interleukin 8 (IL-8) and interferon induced with helicase C domain 1 (IFIH1) in different organs (i.e. intestine, liver, spleen, kidney, brain and skin) were analyzed using RT-PCR technology. The intestinal microbiota was analyzed by 16S rRNA sequencing, in which principal co-ordinates analysis (PCoA) as well as cluster analysis was performed. The results showed that the specific growth rate of turbot in the LAB treatment was significantly higher than those of the control group (P < 0.05). The expression levels of TLR3, IL-8 and IFIH1 were significantly up-regulated in the organs of LAB treatment, except that IL-8 was slightly down-regulated in kidney. A total of 42 phyla in intestinal microbiota were identified. The composition of intestinal microbiota showed significant differences between LAB treatment and the control group. Shannon index in the LAB treatment was significantly increased while Simpson index significantly declined. The PCoA and cluster analysis exhibited significant differences in the composition and abundance between the two groups. Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria acted as biomarkers which may have effects to promote absorption and/or trigger the immune function. In conclusion, the administration of HY2 strain was capable of improving growth performance of turbot by enhancing immune response and optimizing structure and diversity of intestinal microbiota.

Oxidative stress, ion concentration change and immune response in gills of common carp (Cyprinus carpio) under long-term exposure to bisphenol A

Bisphenol A (BPA) is a well-known phenolic environmental estrogen, widely distributed in the aquatic environment, which poses a toxic risk to the health of aquatic organisms. This study aimed to assess the effect of BPA on common carp gills by analyzing oxidative stress, ion equilibrium and immune response. Fish were exposed to five concentrations of BPA (0, 0.01, 0.1, 0.5, and 2 mg/L) for 30 days. Then gills were collected to assay biochemical parameters and gene expression. The results showed that BPA could decrease the levels of total antioxidant capacity (T-AOC), catalase (CAT), glutathione (GSH) and glutathione S-transferase (GST) and increase the levels of superoxide dismutase (SOD), malondialdehyde (MDA) and 8-hydroxy-2 deoxyguanosine (8-OHdG). The gene expression showed that BPA (2 mg/L) could affect the nuclear erythroid 2-related factor 2 (nrf2) signaling pathway, upregulate the gene expression of nrf2 and heme oxygenase 1 (ho-1). Meanwhile, BPA was found to change the activity of Na+/K+ ATPase, and increased the concentrations of Na+ and Ca2+ in gills of common carp. Also, high BPA concentration (0.5 or 2 mg/L) exposure increased the activity of alkaline phosphatase (AKP), blocked mRNA level of lysozyme-c (c-lyz), activated Toll-like receptors (TLRs) signaling pathway, enhanced the mRNA levels of toll-like receptor 2 (tlr2), receptor 4 (tlr4), myeloid differentiation factor 88 (myd88), interferon regulatory factor 3 (irf3), interleukin 1β (il-1β), interleukin 6 (il-6) and interleukin 10 (il-10). Overall, these results suggested that high BPA could induce oxidative damage, ion imbalance, immunosuppression and inflammatory response in gills of common carp.

Polysaccharides from Enteromorpha prolifera protect against carbon tetrachloride-induced acute liver injury in mice via activation of Nrf2/HO-1 signaling, and suppression of oxidative stress, inflammation and apoptosis.

To investigate whether polysaccharides from Enteromorpha prolifera (EPP) could protect against acute hepatic injury induced by CCl4, ICR mice were pretreated with EPP (150, 300, and 450 mg kg-1) and silymarin (100 mg kg-1) for 28 days before CCl4 induction. Pretreatment with EPP attenuated CCl4-induced elevated serum transaminase activities and histopathological alterations in the liver. In addition, EPP prevented CCl4-induced reduction of protein levels of phosphorylated nuclear factor E2-related factor 2 (p-Nrf2)/Nrf2, heme oxygenase-1 (HO-1), and mRNA levels of NADPH quinineoxidoreductase-1 (NQO-1), which, in turn, reduced hepatic oxidative stress injury. Furthermore, the hepatic protein levels of inflammatory mediators and the phosphorylation of nuclear factor-kappaB p65 (NF-κB p65) and I kappaB alpha (IκBα), and the mRNA levels of Toll-like receptor 2 (TLR2), TLR4, and prolyl-isomerase-1 (Pin-1) in the inflammatory signaling pathway were recovered in the EPP pretreated groups. Moreover, EPP prevented the hepatocellular apoptotic changes with inhibition of B-cell lymphoma 2 (Bcl-2), and the induction of Bcl-2-associated X (Bax) and Cleaved caspase-3 caused by CCl4. Taken together, these results indicated that EPP protected against hepatic injury induced by CCl4-derived reactive intermediates through the activation of Nrf2/HO-1 signaling, and suppression of oxidative stress, inflammation and apoptosis.

Resveratrol inhibits LPS-induced inflammation through suppressing the signaling cascades of TLR4-NF-κB/MAPKs/IRF3.

Resveratrol (Res) is a natural compound that possesses anti-inflammatory properties. However, the protective molecular mechanisms of Res against lipopolysaccharide (LPS)-induced inflammation have not been fully studied. In the present study, RAW264.7 cells were stimulated with LPS in the presence or absence of Res, and the subsequent modifications to the LPS-induced signaling pathways caused by Res treatment were examined. It was identified that Res decreased the mRNA levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response protein MyD88, TIR domain-containing adapter molecule 2, which suggested that Res may inhibit the activation of the TLR4 signaling pathway. It suppressed the expression levels of total and phosphorylated TLR4, NF-κB inhibitor, p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase, extracellular signal-regulated kinase 1/2 and interferon (IFN) regulatory factor 3 (IRF3) proteins. Following treatment with Res or specific inhibitors, the production of pro-inflammatory mediators including tumor necrosis factor-α, interleukin (IL)-6, IL-8 and IFN-β were decreased and the expression of anti-inflammatory mediator IL-10 was increased. These results suggested that Res may inhibit the signaling cascades of NF-κB, MAPKs and IRF3, which modulate pro-inflammatory cytokines. In conclusion, Res exhibited a therapeutic effect on LPS-induced inflammation through suppression of the TLR4-NF-κB/MAPKs/IRF3 signaling cascades.

Yeast cultures alleviate gossypol induced inflammatory response in liver tissue of Ussuri catfish (Pseudobagrus ussuriensis)

The present study explored the mechanisms of gossypol induced inflammatory response in liver tissue of Ussuri catfish (Pseudobagrus ussuriensis), and investigated whether the yeast culture could relieve the inflammatory response of fish. For this purpose, Ussuri catfish were fed two diets with or without yeast culture for 8 weeks, and then fish were treated with 300 mg gossypol/kg body weight by intragastric administration for 15 days. Fish pre-fed diet without yeast culture supplementation and treated with gossypol oral administration (Gos group) had higher interleukin-1β (IL-1β) and interleukin 8 (IL-8) mRNA levels compared with fish pre-fed diet without yeast culture supplementation and treated with sodium carboxymethylcellulose solution oral administration (control group) and pre-fed yeast culture diet and treated with gossypol oral administration (Gos/YC group), but had lower interleukin 10 mRNA level than the control group (P < .05). Simultaneously, hepatic toll-like receptor 2 (TLR2), myeloid differentiation factor 88 (MyD88) and nuclear transcription factor kappa B p65 (NF-κB p65) mRNA levels of fish in Gos group were also up-regulated. However, dietary yeast culture supplementation remarkably prevented the increase of IL-1β and IL-8 mRNA levels and the decrease of IL-10 mRNA level by inhibiting the excessive activation of TLR2-MyD88-NF-κB signaling pathway through down-regulating TLR2, NF-κB p65 (P < .05) and MyD88 (P > .05) mRNA levels. Besides, the lowest serum lysozyme and alkaline phosphatase activities were observed in fish of Gos group. There was an increased trend in immunoglobulin M content among the groups. Fish in Gos group had significantly lower hepatic catalase, superoxide dismutase and total antioxidant capacity compared with those fish in the control group (P < .05). Higher occurrences of the dilatation in sinusoids, karyolysis, hydropic degenerations and nuclei shifting to the cellular periphery were detected in Gos group compared with those in the control group, coupled with increased serum aspartate aminotransferase activity. Lower occurrences of these histopathological symptoms were observed in the Gos/YC group compared with those of fish in the Gos group. Overall, the results mentioned above indicated that gossypol could result in immune and oxidative damage, liver injury and inflammatory response and dietary yeast culture supplementation could alleviate these negative responses for Ussuri catfish.

Beneficial effects of a host gut-derived probiotic, Bacillus pumilus, on the growth, non-specific immune response and disease resistance of juvenile golden pompano, Trachinotus ovatus

A potential host-derived probiotic, Bacillus pumilus A97, was successfully screened from 434 isolates from the intestines of healthy adult Trachinotus ovatus based on in vitro assays of non-haemolytic activity, extracellular enzyme activity, inhibitory activity against pathogens (Vibrio or Streptococcus), tolerance to gastrointestinal stress and bile salts, cell surface hydrophobicity, autoaggregation, biofilm formation and antibiotic susceptibility. Dietary supplementation with B. pumilus A97 (1.0 × 108 CFU·g−1) for 56 days significantly improved the weight gain, specific growth rate, feed efficiency and non-specific immune responses of T. ovatus (P < .05). The mRNA levels of toll-like receptor 8 (TLR8) in the intestines and toll-like receptor 9 (TLR9) in the kidneys of T. ovatus were significantly upregulated (P < .05). Higher survival rates post-Vibrio ponticus-challenge was found in T. ovatus fed an A97 diet than in the control fish. All the results suggest that B. pumilus A97 has great potential as an effective dietary probiotic for improving the growth, immune status and disease resistance of T. ovatus.

Placenta-on-a-chip model for assessing the transport and toxicity of xenobiotics in vitro

Glaucoma is a kind of blind-causing disease with structural damages of optic nerve and defection of visual field. It is believed that the death of retinal ganglion cell (RGC) is a consequential event of over-reactive immune orchestral cells such as microglia. Previous evidences in animal and clinical studies show the innate immunity plays a pivotal role in neuro-inflammation of glaucoma. Toll-like receptor 4 (TLR4) is expressed on microglia and mediates many neuroinflammatory diseases. We aimed to explore the impacts of high intraocular pressure (IOP) on rat microglia in retina and the regulation of TLR4/NF-κB signaling pathway in scratched microglia cells. In our study, we successfully established chronic high IOP rat model by episcleral vein cauterization (EVC) which behaved like the chronic glaucoma. Besides, we set up an in vitro scratch-induced injury model in rat microglia cells. We found the level of activated microglia cells were significantly increased in the retina of chronic high IOP groups. Moreover, the inhibition of TLR4/NF-κB signaling pathway suppressed the expression of TLR4 protein and mRNA levels of P50, IL-6 and TNF-α. Our original study provided a theoretical basis on targeting TLR4/NF-κB to suppress pro-inflammatory factors releasing in activated microglia and it might be a good treatment target to prevent glaucoma from progressing.

Interaction of notch signaling pathway with toll-like receptor 4 on the function of CD14+ monocytes in chronic hepatitis C patients

Objective: To observe the expressional changes in Notch signaling pathway and toll-like receptor 4 (TLR4) and their interactions on the functions of CD14(+) monocytes in chronic hepatitis C patients. Methods: A total of 24 treatment-naïve chronic hepatitis C cases and 10 healthy individuals, who visited Shaanxi Provincial People's Hospital from August to October 2017, were enrolled. Selected CD14(+) monocytes were stimulated by the Notch signaling pathway inhibitor DAPT or transfected with TLR4 siRNA, and the levels of Notch1, Notch2, Hes1 and Hes5 mRNA were detected by real-time quantitative PCR. TLR4 protein levels and phosphorylation of NF-κB was detected by Western blot. ELISA was used to detect the level of cytokines secreted from CD14(+) monocytes. A t-test or paired t-test was used for comparison between groups. Results: The relative expression of Notch1 mRNA (3.97 ± 2.03 vs. 0.91 ± 0.76, P < 0.01) and downstream of Notch signaling pathway (5.96 ± 2.31 vs. 0.99 ± 0.45, P < 0.01), Hes1 mRNA and Hes5 mRNA (4.31 ± 1.05 vs. 0.84 ± 0.20, P < 0.01) in CD14+ monocytes of chronic hepatitis C patients was significantly higher than that of healthy individuals. The relative expression of TLR4 mRNA (5.14 ± 1.09 vs. 1.27 ± 0.39) and protein level in CD14(+) monocytes of chronic hepatitis C patients were significantly higher than those of healthy individuals (P < 0.01). An inhibition of Notch signaling pathway with DAPT had reduced the relative expression level of TLR4 mRNA (2.58 ± 1.36 vs. 4.34 ± 1.88, P < 0.05), protein expression and phosphorylation of NF-B in CD14(+) monocytes of chronic hepatitis C patients. Furthermore, the secretion level of MCP-1 [(94.32 ± 23.59) pg/ml vs. (64.07 ± 9.39) pg/ml, P < 0.01] and IL-8 [(12.54 ± 4.89) pg/ml vs. (7.92 ± 3.01) pg/ml, P < 0.05] was significantly reduced. TLR4 siRNA transfection reduced the expressions of Notch1 mRNA (2.09 ± 1.72 vs. 3.73 ± 1.75, P < 0.05), Hes1 (2.87 ± 0.84 vs. 5.54 ± 0.97, P < 0.01), and Hes5 (2.89 ± 0.93 vs. 4.51 ± 1.54, P < 0.01) in CD14(+) monocytes of chronic hepatitis C patients. Conclusion: Interaction of Notch signaling pathway with TLR4 can promote the function of CD14(+) monocytes in chronic hepatitis C patients.

Improved metal allergen reactivity of artificial skin models by integration of Toll-like receptor 4-positive cells.

Reconstructed human epidermis (RhE) is widely used to replace animal models in order to assess the proinflammatory and allergenic effects of chemicals. Unfortunately, RhE lacks proinflammatory responsiveness for metal haptens, which are the most prevalent human contact allergens, raising concerns about its reliability for predicting skin allergens. To investigate whether this limitation of RhE might be attributable to a lack of functional expression of Toll-like receptor 4 (TLR4), which governs proinflammatory sensitivity to nickel and cobalt. RhE, dendritic cell (DC)-containing RhE and full-thickness skin equivalent (FTSE) were compared regarding their proinflammatory responsiveness to metal allergens. The incorporation of dermal fibroblasts was sufficient to confer metal sensitivity to RhE. Unlike keratinocytes, normal human fibroblasts expressed high levels of TLR4 mRNA and induced interleukin-8 expression upon stimulation with nickel or cobalt. Consistently, dermal isolates from FTSE expressed considerable amounts of TLR4 mRNA, whereas RhE or epidermis isolated from FTSE, normal human epidermis or inflamed human epidermis failed to express TLR4. Similarly, co-culture with TLR4-positive DCs bestowed RhE with proinflammatory responsiveness to metals. Our data suggest that FTSE or DC/RhE co-culture models can circumvent the shortcomings of RhE assays, and combine the benefits of complex and monoculture-based test systems in a single assay.

Fetal Membrane Inflammation Induces Preterm Birth Via Toll-Like Receptor 2 in Mice With Chronic Gingivitis

Inflammation is associated with preterm birth. We previously described a mouse model of chronic inflammation-induced preterm birth after dental Porphyromonas gingivalis infection. The aim of this study was to employ this model system to investigate the mechanisms through which enhanced uterine contractility induces preterm birth. Messenger RNA (mRNA) encoding contraction-associated proteins, such as oxytocin receptors, was measured at various gestational time points by real-time polymerase chain reaction (PCR). Spontaneous and oxytocin-induced uterine contractile activity at gestational day 18 was assessed using a tissue organ bath. The expression levels of Toll-like receptor 2 (TLR2), TLR4, cyclooxygenase (COX)-2, nuclear factor-kappa B (NF-κB) p65, and p38 mitogen-activated protein kinase (MAPK) on gestational day 18 were also determined by real-time PCR or Western blotting. Messenger RNA encoding contraction-associated proteins was increased at gestational day 18, and the spontaneous contractile activity (1.6-fold greater area under the contraction curve) and sensitivity to oxytocin (EC50: 8.8 nM vs 2.2 nM) were enhanced in the P gingivalis group compared to those in the control group. In the P gingivalis group, COX-2 mRNA expression was not elevated in the placenta or myometrium but was upregulated 2.3-fold in the fetal membrane. The TLR2 mRNA levels in the fetal membrane were 2.7-fold higher in the P gingivalis group, whereas TLR4 levels were not elevated. Activation of the NF-κB p65 and p38 MAPK pathways was enhanced in the fetal membrane of the P gingivalis group. Thus, in mice with chronic dental P gingivalis infection, TLR2-induced inflammation in the fetal membrane leads to upregulation of uterine contractility, leading to preterm birth.

Effects of MiR-146a on repair and inflammation in rats with spinal cord injury through the TLR/NF-κB signaling pathway.

This study aims to investigate the effects of micro ribonucleic acid-34a (miR-34a) on repair and inflammation of rats with spinal cord injury (SCI) through the toll-like receptor (TLR)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. In this study, 12 healthy rats (control group (CG)) and 24 SCI rats (experimental group (EG-1)) were selected as subjects. A total of 12 experimental rats randomly selected from EG-1 were injected with 5 µL agomiR-146 as EG-2 group. Serum levels of miR-146a, TLR, NF-κB, interleukin-8 (IL-8) and IL-6 of rats in CG and EG-1 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Furthermore, the protein levels of miR-146a, TLR, NF-κB, IL-8 and IL-6 in rats of CG and EG were detected via Western blotting. Spinal cord tissue sections of SCI rats after treatment with agomiR-146 were observed by hematoxylin and eosin staining (H&E) staining. The mRNA level of miR-146a in SCI rats was significantly lower than that in healthy rats, and the difference was statistically significant (p < 0.05). The mRNA levels of TLR, NF-κB, IL-8 and IL-6 in SCI rats were markedly higher than those in healthy rats, showing significant differences (p < 0.05). However, the relative mRNA level of miR-146a in EG-2 group was significantly higher than that in EG-1 group, with a significant difference (p < 0.05). Relative level of miR-146a was not significantly different between EG-2 group and CG group (p > 0.05). Meanwhile, the mRNA levels of TLR, NF-κB, IL-8 and IL-6 in EG-2 group were evidently lower than those in EG-1 group, displaying significant differences (p < 0.05). MiR-146a can promote the repair of SCI and reduce inflammatory responses in rats through the TLR/NF-κB signaling pathway.