Expression Levels Of TLR4 Research Articles

The elevated expression of TLR4 and MMP9 in human abdominal aortic aneurysm tissues and its implication

BackgroundToll-like receptor 4 (TLR4) and matrix metalloproteinase 9 (MMP9) have been investigated to play significant roles in the formation of abdominal aortic aneurysm (AAA). But the reports on the expression pattern of TLR4 and MMP9 in human AAA specimens were relatively scant. The aim of this study was to make a detailed analysis of TLR4 and MMP9 expression in situ and their association with clinical parameters involved in human AAA.Methods40 AAA specimens were obtained from full-thickness aneurysmal tissues at the maximal dilation area during the open surgical repair, and 8 non-aneurysmal abdominal aortas from transplant donors served as controls. Expression of TLR4 and MMP9 protein was determined by immunohistochemistry.ResultsThere were increased levels of TLR4 and MMP9 expression in human AAA tissues. Compared with macrophages or SMCs, lymphocytes showed a higher positive rate of TLR4 and MMP9 staining, and an elevated ratio of high MMP9 expression (all P < 0.05). There existed a significant association between TLR4 and MMP9 expression (r = 0.767, P < 0.001), and both TLR4 and MMP9 levels were statistically related to circulating CRP. Moreover, TLR4 expression in situ indicated a positive correlation with its serum level (r = 0.654, P = 0.006). Multiple analysis revealed that high TLR4 expression in situ was associated with the risk of large AAA (OR = 6.211, 95%CI = 1.226–31.480, P = 0.027), while high MMP9 expression was correlated to the presence of thrombus within AAA (OR = 5.494, 95%CI = 1.181–25.562, P = 0.030), separately compared with their low expression.ConclusionsThis study confirmed the overexpression of TLR4 and MMP9 in human AAA tissues, and their close relationship implying in the pathogenesis of AAA. We further provided evidence that TLR4 had a potential effect on AAA size and MMP9 could influence the occurrence of thrombus within AAA.

Study on Anti-Inflammatory Activity of Niloticin by Targeting MD-2 Based on Computer-Aided Drug Design and Biolayer Interferometry

Niloticin is an active compound from Cortex phellodendri, but its antiinflammatory activity has not yet been explored. The aim of the present study was to assess the drug potential of niloticin and to study the MD-2-targeting mechanism of its anti-inflammatory activity. Niloticin’s drug potential was analyzed using the Traditional Chinese Medicine Systems Pharmacology Database. Molecular docking and biolayer interferometry technology were used to explore the anti-inflammatory mechanism of niloticin by targeting myeloid differentiation protein 2 (MD-2), which mediates a series of Toll-Like Receptor (TLR) 4-dependent inflammatory responses. The cytokines involved in the LPSTLR4/ MD-2-NF-κB pathway were evaluated by ELISA, RT-PCR, and western blot. The results showed that niloticin has drug potential and could bind to MD- 2. Niloticin had no impact on cell viability. Niloticin could significantly decrease the levels of NO, IL-6, TNF-a, and IL-1β (P&lt;0.01) induced by LPS. IL-1β, IL-6, iNOS, TNF-a, and COX-2 mRNA expression levels were decreased by niloticin (all P&lt;0.01). Compared with the control group, TLR4, p65, MyD88, p-p65, and iNOS expression levels induced by LPS were suppressed by niloticin (all P&lt;0.01). In conclusion, niloticin is a potential MD-2 antagonist. It might interact with MD-2 to play an anti-inflammatory role by suppressing the activation of the LPS-TLR4/MD-2-NF-κB signaling pathway.

The effects of guluronic acid (G2013), a new emerging treatment, on inflammatory factors in nonalcoholic steatohepatitis patients under in vitro conditions

Background Nonalcoholic Steatohepatitis (NASH) results from the accumulation of fatty acids in the liver. The elevated production of pro-inflammatory factors is the reason for the hyper inflammation in NASH. The α-L-Guluronic acid (G2013), a new member of NSAID family, is a plant-originated agent with immunomodulatory properties. The current study investigated the effects of G2013 on inflammatory factors in PBMCs of NASH patients. Methods PBMCs of 14 NASH patients and 14 healthy controls were isolated and cultured. The patient’s cells were treated with low (5 µg/mL) and moderate (25 µg/mL) doses of G2013 alongside the diclofenac optimum dose (3 µg/mL). The expression and secretion levels of variables were assessed by real-time PCR and ELISA, respectively. Results Findings indicated that the expression levels of TLR4 and NF-κB, as well as the secretion levels of TNF-α and IL-6 cytokines, were significantly elevated in NASH patients compared to healthy individuals. The expression levels of TLR4 and NF-κB were strikingly downregulated in treated cells of patients in both low and moderate doses of G2013. A considerable reduction was obtained in the secretion level of IL-6 using both low and moderate doses of G2013 and in the secretion level of TNF-α using the moderate dose of G2013. Conclusion The results indicated that G2013 could meaningfully decrease the expression and secretion levels of evaluated factors (TLR4, NF-κB, TNF-α, and IL-6) in PMBCs of NASH cases. Since there is no effective treatment for NASH patients, we hope that G2013 would be a promising immunomodulatory agent in reducing inflammation and improvement of patients.

Tlr2 Gene Deletion Delays Retinal Degeneration in Two Genetically Distinct Mouse Models of Retinitis Pigmentosa.

Although considered a rare retinal dystrophy, retinitis pigmentosa (RP) is the primary cause of hereditary blindness. Given its diverse genetic etiology (>3000 mutations in >60 genes), there is an urgent need for novel treatments that target common features of the disease. TLR2 is a key activator of innate immune response. To examine its role in RP progression we characterized the expression profile of Tlr2 and its adaptor molecules and the consequences of Tlr2 deletion in two genetically distinct models of RP: Pde6brd10/rd10 (rd10) and RhoP23H/+ (P23H/+) mice. In both models, expression levels of Tlr2 and its adaptor molecules increased in parallel with those of the proinflammatory cytokine Il1b. In rd10 mice, deletion of a single Tlr2 allele had no effect on visual function, as evaluated by electroretinography. However, in both RP models, complete elimination of Tlr2 attenuated the loss of visual function and mitigated the loss of photoreceptor cell numbers. In Tlr2 null rd10 mice, we observed decreases in the total number of microglial cells, assessed by flow cytometry, and in the number of microglia infiltrating the photoreceptor layers. Together, these results point to TLR2 as a mutation-independent therapeutic target for RP.

Overexpression of TLR7 and TLR9 Occurs Before Onset Symptoms In First-Degree Relatives of Rheumatoid Arthritis Patients

Autoantibodies have a central role in the physiopathology of Rheumatoid Arthritis (RA). However, the responsible factors that trigger and perpetuate the autoantibodies production are unknown. Toll-like receptors (TLRs) have been considered as promotors of autoantibodies production to break down the immunotolerance in RA. Evaluate the expression levels of TLR7 and TLR9 as well as their correlation with autoantibodies in first-degree relatives (FDR) of RA patients (seropositive and seronegative to ACPA), respect to early RA (eRA) and chronic RA (cRA) patients. We selected 32 RA patients (16 as eRA and 16 as cRA) and 32 FDR of RA patients (16 seropositive and 16 seronegative to ACPA). Expression levels of TLR7 and TLR9 in whole blood samples from each group were measured by real-time PCR using total RNA extracted from each subject. Also, correlation analysis between TLRs expression and autoantibodies was performed. The expression of TLR7 and TLR9 was diminished in RA patients (p <0.01) but elevated in ACPA- FDR (p <0.0001) and ACPA+ FDR (p <0.05) with a positive correlation between them (r=0.749, p <0.000). Moreover, the expression levels of TLR7 correlate positively with ACPA levels in both seropositive ACPA+ FDR subjects (r=0.582, p=0.018) and eRA patients (r=0.593, p=0.020). Our results showed overexpression of TLR7 and TLR9 may occur in preclinical RA subjects. TLR7 overexpression correlated with ACPA levels' production, suggesting TLR7 may play a role in ACPA development.

Anti-inflammatory effects of microRNA-223 on sepsis-induced lung injury in rats by targeting the Toll-like receptor signaling pathway.

The aim of the present study was to investigate the mediation of micro RNA (miR)-223 on the anti-inflammatory effect of the Toll-like receptor (TLR) signaling pathway on sepsis-induced lung injury in rats via negatively regulating the expression of interleukin (IL)-6. Sprague-Dawley rats were used in the present study. It was determined whether miR-223 is differentially expressed in the lung using reverse transcription-quantitative PCR techniques and the content of cytokines in bronchoalveolar lavage (BAL) fluid was detected. The protein expression levels of TLR4 and nuclear factor (NF)-κB p65 were examined by western blotting and the pathological changes in the lung tissues of the sepsis group were observed. Hematoxylin and eosin was used to stain the lung tissues. The alveoli in the sham group exhibited a normal structure and morphology. In the sepsis group, the alveoli of the lung tissues were surrounded by numerous neutrophils, the mesenchyme was swollen, regions of the alveolar wall exhibited fibrosis and the alveolar wall was thickened. Furthermore, in the sepsis group, miR-223 expression was increased in the lung tissues when compared with that in the sham group. The content of cytokines, IL-6 and IL-1β in the BAL fluid was significantly increased when compared with that of the sham group and TLR4 and NF-κB were also highly expressed. In addition, when compared with RAW264.7 cells that were overexpressing miR-223, the content of IL-6 and IL-1β in the supernatant and protein expression of TLR and NF-κB in cells were markedly decreased. Thus, it was demonstrated that miR-223 negatively regulated the expression of IL-6, mediating the TLR4/NF-κB signaling pathway and exerting an anti-inflammatory effect in sepsis-induced lung injury.

Ischemic postconditioning ameliorates acute kidney injury induced by limb ischemia/reperfusion via transforming TLR4 and NF-\u03baB signaling in rats

BackgroundThe present study investigated the influence of ischemic postconditioning (I-postC) on the adjustment of renal injury after limb ischemia-reperfusion (I/R) injury, to elucidate the mechanisms of the Toll-like receptor 4 (TLR 4)/NF-κB signaling pathway using histopathological and immunohistochemical methods.MethodsMale Sprague-Dawley rats were randomly assigned to five groups (numbered from 1 to 5): the sham group (Group 1, only the anesthesia procedure was conducted without limb I/R), the I/R group (Group 2, 4 h of reperfusion was conducted following 4 h limb ischemia under anesthesia), the I/R + I-postC group (Group 3, 4 h of ischemia and 4 h of reperfusion was conducted; before perfusion, 5 min of limb ischemia and 5 min of reperfusion were performed in the rats and repeated 3 times), the I/R + TAK group (Group 4, rats were injected with TLR4 antagonist TAK through the caudal vein before limb ischemia and reperfusion under anesthesia), the TAK group (Group 5, rats were injected with TAK, and the anesthesia procedure was conducted without limb I/R). Histological changes in the kidney in different groups were observed, and the extent of tubular injury was assessed. Changes in biochemical indexes and the expression of inflammatory factors, TLR4, and NF-κB were also evaluated.ResultsCompared with rats in the I/R group, the secretion of inflammatory factors and the expression levels of TLR4 and NF-κB were decreased in rats in the I/R + I-postC group. Histological analysis revealed renal injury, including inflammatory cell infiltration, dilatation of the tubuli lumen, congestion in glomerular capillaries, degeneration of tubuli epithelial cells, and necrosis was ameliorated by I-postC. Immunohistochemical studies showed that I/R-induced elevation in TLR4 and NF-κB expression was reduced by I-postC treatment. Moreover, the expression levels of TLR4, NF-κB, and inflammatory factors in rats in the I/R + TAK group were also decreased, and the renal pathological lesion was alleviated, which was similar to that in rats in the I/R + I-postC group.ConclusionsThe present findings suggest that I-postC can reduce tissue injury and kidney inflammation induced by limb I/R injury, possibly via inhibition of the TLR4 and NF-κB pathways.

Safflower extract improves depression in mice by inhibiting the TLR4-NLRP3 inflammation signaling pathway.

Safflower extract (SE) improves depression in mice by inhibiting the TLR4-NLRP3 inflammatory signaling pathway. Chronic unpredictable mild stress (CUMS) was used to establish a mouse model of depression. A total of 60 adult male ICR mice were randomly divided into 6 groups: control group, depression group (CUMS only), SE (10 mg/kg) + depression group (CUMS+SE, 10 mg/kg), SE (30 mg/kg) + depression group (CUMS+SE, 30 mg/kg), Cli-095 + depression group (CUMS+Cli-95), and fluoxetine hydrochloride (FLU) + depression group (CUMS+FLU). We assessed the depressive behaviors of these mice using the sucrose preference test (SPT), the open field test (OFT), the forced swim test (FST), and the tail suspension test (TST). We measured the expression levels of SOD, MDA, GSH-Px, 5-HT, NE, TNF-α, IL-1β, and IL-6 using ELISA kits. Western blot was used to determine the relative expression levels of TLR4, p38, NF-κB, NLRP3, and caspase-1. SE significantly improved the results of the SPT, OFT, FST, and TST. SE also increased the expression levels of 5-HT and NE in the prefrontal cortex while decreased the expression levels of TNF-α, IL-1β, and IL-6 compared with CUMS. SE (10 or 30 mg/kg) or FLU (10 mg/kg) significantly inhibited the expression of TLR4 and p-p38 induced by CUMS. SE significantly inhibited the expression of p-NF-κB in the prefrontal cortex and hippocampus induced by CUMS. The decrease of NLRP3 and caspase-1 were obviously reversed after administration of SE (10 or 30 mg/kg) or FLU (10 mg/kg). The results showed that SE has potential antidepressant effects in CUMS mice, and its underlying mechanism may be related to its effects on inflammation and the TLR4-NF-κB-NLRP3 signaling pathway in the brain.

Contribution of toll-like receptor 2 and nicotinamide adenine dinucleotide phosphate oxidase to the trimethylamine N-oxide-induced inflammatory reactions in U937-derived macrophages.

BACKGROUNDTrimethylamine N-oxide (TMAO) is emerging as a new generation of metabolites related to the activation of inflammatory reactions in the macrophages during atherosclerosis. Stress-activation of cell surface toll-like receptors (TLRs) as well as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) is also assumed to be involved in TMAO-induced inflammatory reaction in the macrophages. To elucidate the possible contribution of TLRs and NOX to the mentioned signaling pathway, we aimed to simultaneously evaluate the expression level of TLR2, TLR6, and NOX2 in TMAO-treated macrophages.METHODS2.5 × 106 cells of U937-derived macrophages were treated in triplicates with different concentrations (37.5, 75, 150, and 300 μM) of TMAO for 24 hours. The cells were also treated with tunicamycin (TUN), as a positive control of stress. Normal control group (CTR) cells received no treatment. The viability of treated cells was checked by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was also used to evaluate the relative expression (fold change) of TLR2, TLR6, and NOX2 at messenger ribonucleic acid (mRNA) levels. One-way analysis of variance (ANOVA) with post-hoc Dunnett’s test was performed to compare every mean with that of the control.RESULTSNo cell death occurred because of treatments. Dose of 300 μM of TMAO significantly increased the relative expression of both TLR2 and NOX2 compared to the CTR cells (P < 0.001 for both). The elevation of TLR6 was not statistically significant in all groups of TMAO-treated cells (P > 0.050).CONCLUSIONOur results provide documentation supporting contribution of TLR2 and NOX2 to previously described inflammatory reactions induced by TMAO in macrophages. In addition, they may clarify the proatherogenic role of TMAO in foam cell formation as well as abnormal activation of macrophages during atherosclerosis.

Expression of TLR4 and major inflammatory cytokines in patients with bladder cancer of different grade and stage.

The bladder cancer is immunogenic, and neoantigens generated by tumor cells trigger a notable immune response in the host. On the other hand, multiple immune escape mechanisms allow for avoiding the recognition by the host immune system. Toll-like receptor type 4and inflammatory cytokines play major role in the immune response to bladder cancer. To assess the expression of TLR4and the genes of major inflammatory cytokines in tumor cells and in unaffected tissue of the bladder. The pairs of samples from the urinary bladder tumor and unaffected adjacent tissue were obtained from 50surgically treated patients with bladder cancer. The level of expression of TLR4, TGF-β1, INF-γ, TNF-α genes was evaluated by real-time polymerase chain reaction. Bladder cancer cells are characterized by lower expression levels of TLR4, TGF-β1, INF-γ, TNF-α as compared to unaffected tissue. In patients with recurrent cancer, expression of TLR-4and cytokines does not change both in tumor and in unaffected tissue of the bladder. Expression of TLR4is identically low both in low- and high-grade cancer. Expression levels of the INF-γ and TNF-α are remarkably low in muscle-invasive cancer compared to the unaffected bladder tissue. The level of TGF-β1in bladder cancer is comparable to the unaffected tissue of the bladder, while in the intact and metastatic lymph nodes it is significantly upregulated. Bladder cancer tissue differs from the unaffected part of the bladder wall in the level of TLR4, TGF-β1, INF-γ, TNF-α expression.

Aureusidin derivative CNQX inhibits chronic colitis inflammation and mucosal barrier damage by targeting myeloid differentiation 2 protein.

Our previous study has found that aureusidin can inhibit inflammation by targeting myeloid differentiation 2 (MD2) protein. Structural optimization of aureusidin gave rise to a derivative named CNQX. LPS was used to induce inflammation in intestinal macrophages; flow cytometry, PI staining and Hoechst 33342 staining were used to detect the apoptotic level of macrophages; enzyme‐linked immunosorbent assay (ELISA) was utilized to detect the expression level of inflammatory factors (including IL‐1β, IL‐18 and TNF‐α); immunofluorescence staining was used to investigate the expression of MD2; Western blot was employed to measure the protein level of TLR4, MD2, MyD88 and p‐P65. As a result, CNQX with IC50 of 2.5 μM can significantly inhibit the inflammatory damage of macrophages, decrease apoptotic level, reduce the expression level of inflammatory factors and simultaneously decrease the expression level of TLR4, MD2, MyD88 as well as p‐P65. Caco‐2 cell line was used to simulate the intestinal mucosal barrier in vitro, LPS was employed to induce cell injury in Caco‐2 (to up‐regulate barrier permeability), and CNQX with IC50 of 2.5 μl was used for intervention. Flow cytometry was used to detect the apoptotic level of Caco‐2 cells, trans‐epithelial electric resistance (TEER) was measured, FITC‐D was used to detect the permeability of the intestinal mucosa, and Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin‐1, MyD88, TLR4 and MD2). As a result, CNQX decreased the apoptotic level of Caco‐2 cells, increased TEER value, decreased the expression levels of MyD88, TLR4 and MD2, and increased the protein levels of tight junction proteins (including occludin and claudin‐1). C57BL/6 wild‐type mice were treated with drinking water containing Dextran sulphate sodium (DSS) to establish murine chronic colitis model. After CQNX intervention, we detected the bodyweight, DAI score and H&E tissue staining to evaluate the life status and pathological changes. Immunohistochemistry (IHC) staining was used to detect the expression of MD2 protein, tight junction protein (including occludin and claudin‐1). Transmission electron microscopy and FITC‐D were used to detect intestinal mucosal permeability. Western blot was used to detect the expression levels of tight junction proteins (including occludin, claudin‐1, MyD88, TLR4 and MD2) in the intestinal mucosa tissue. Consequently, CNQX can inhibit the intestinal inflammatory response in mice with colitis, inhibit the mucosal barrier injury, increase the expression of tight junction proteins (including occludin and claudin‐1) and decrease the expression levels of MyD88, TLR4 and MD2. Mechanistically, pull‐down and immunoprecipitation assays showed that CNQX can inhibit the activation of TLR4/MD2‐NF‐κB by binding to MD2 protein.Collectively, in this study, we found that CNQX can suppress the activation of TLR4 signals by targeting MD2 protein, thereby inhibiting inflammation and mucosal barrier damage of chronic colitis.

TLR2 and TLR4 mRNA expression levels in liver transplant patients with acute rejection

Toll-like receptors (TLRs) have important role in transplant outcomes by activating the innate immune system and production of pro-inflammatory cytokines, leading to graft rejection. We assessed the expression level of TL2 and TLR4 in acute rejection (AR) on the 1st and 7th-day post-transplantation. TLR2 and TLR4 expressions were evaluated by real-time PCR in both the AR group (n = 50) and non-AR (n = 50), compared with the control group. Also, the correlation of the expression levels of TLRs between both the 1st and 7th day was analyzed. ROC curve analysis was used to determine the cut-off value for TLRs expression. TLR4 mRNA expression was significantly up-regulated in AR patients vs. the controls on the 1st day (p ≤ 0.05) and it was down-regulated in non-AR vs. controls on the 1st day (p ≤ 0.05). Also, TLR4 expression had decreased in both AR and non-AR groups vs. control on the 7th day (p ≤ 0.05). Both TLR2 and TLR4 expression in comparison to non-AR had increased in the AR group on the 7th day (p ≤ 0.05). TLR2 expression positively correlated between 1st and 7th day in AR (r = 0.3, (p ≤ 0.05) and non-AR group (r = 0.2, p ≤ 0.05). ROC curve analysis showed a cut-off value of TLR2 up to 0.98 with sensitivity 71.05 (95%CI = 54.1–84.6) and specificity 63.27 (95%CI = 48.3–76.6) that could distinguish between AR and non-AR group (p ≤ 0.05). The data support that both TLR2 and TLR4 expression have an effective role in AR after liver transplantation and could be used as possible biomarkers for AR to choose better therapeutic strategies based on immunological aspects.

YAP/TEAD1 Complex Is a Default Repressor of Cardiac Toll-Like Receptor Genes.

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that modulate innate immune responses and play essential roles in the pathogenesis of heart diseases. Although important, the molecular mechanisms controlling cardiac TLR genes expression have not been clearly addressed. This study examined the expression pattern of Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, and Tlr9 in normal and disease-stressed mouse hearts. Our results demonstrated that the expression levels of cardiac Tlr3, Tlr7, Tlr8, and Tlr9 increased with age between neonatal and adult developmental stages, whereas the expression of Tlr5 decreased with age. Furthermore, pathological stress increased the expression levels of Tlr2, Tlr4, Tlr5, Tlr7, Tlr8, and Tlr9. Hippo-YAP signaling is essential for heart development and homeostasis maintenance, and YAP/TEAD1 complex is the terminal effector of this pathway. Here we found that TEAD1 directly bound genomic regions adjacent to Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, and Tlr9. In vitro, luciferase reporter data suggest that YAP/TEAD1 repression of Tlr4 depends on a conserved TEAD1 binding motif near Tlr4 transcription start site. In vivo, cardiomyocyte-specific YAP depletion increased the expression of most examined TLR genes, activated the synthesis of pro-inflammatory cytokines, and predisposed the heart to lipopolysaccharide stress. In conclusion, our data indicate that the expression of cardiac TLR genes is associated with age and activated by pathological stress and suggest that YAP/TEAD1 complex is a default repressor of cardiac TLR genes.

Sparstolonin B Exerts Therapeutic Effects on Collagen-Induced Arthritis by Inhibiting the NLRP3 Inflammasome and Reducing the Activity of α1,3-Fucosyltransferase

Objective. To explore the role of α1,3-fucosyltransferase in the mediation of rheumatoid arthritic inflammation, the protective effect of Sparstolonin B on rheumatoid arthritis (RA), and the mechanisms that regulate the NLRP3 inflammasome. Methods. Forty, weighing from 260-300 g, male Sprague-Dawley rats were randomly divided into the following groups: a sham operation group (Sham group), a rheumatoid arthritis model group (RA group), an RA+Sparstolonin B treatment group (RAS group), an RA+Iguratimod group (RAI group), and an RA+SsnB+NLRP3 inflammasome activator (Nigericin) group (RASN group); ten animals were allocated to each group. We determined the arthritis index for each group of rats, and pathological changes were evaluated by hematoxylin-eosin staining. We also used ELISAs to determine the serum levels of IL-17, IL-6, TNF-α, TGF-β, IL-18, and IL-1β. TUNEL staining was used to investigate apoptosis in synovial cells. IF was used to detect the release of ROS, ASC formation, and the expression levels of FucT-V and NLRP3. Western blotting was used to detect the protein expression levels of Bc1-2, Bax, TLR4, MYD88, NF-κB, pro-caspase-1, NLRP3, FucT-V, E-Selectin, and P-Selectin. We also performed in vitro experiments with Sparstolonin B and detected changes in 1,3-fucosyltransferase activity by ELISA. The pyroptosis-related phenotype, including ASC, was identified by immunofluorescence, while levels of NLRP-3, pro-IL-1, and pro-caspase-1 were detected by western blotting. Results. Sparstolonin B was showed to alleviate joint swelling in RA rats, inhibited inflammatory cell infiltration and the release of ROS, reduced damage caused by oxidative stress, and suppressed the rate of apoptosis in synovial cells. The administration of Sparstolonin B inhibited the secretion of IL-17 from Th17 cells and triggered the secretion of TGF-β from Treg cells, thus leading to the reduced expression of TLR4, MyD88, and NF-κB, and the suppression of TNF-α secretion. Moreover, Sparstolonin B downregulated the expression of NLRP3, inhibited ASC formation in vivo and in vitro, and reduced the levels of IL-18 and IL-1β. The expression levels of FucT-V, E-Selectin, and P-Selectin were also inhibited. Interestingly, these protective effects of Sparstolonin B could be blocked in RA rats by inhibiting the activation of the NLRP3 inflammasome. Conclusion. Sparstolonin B improved inflammatory responses and oxidative stress by inhibiting the NLRP3 inflammasome, inhibiting the expression of FucT-V and downregulating the TLR4/MYD88/NF-𝜅B signaling pathway in order to rescue RA.

Targeting HMGB1-TLR4 signaling by miR-216a-5p elevation alleviates the inflammatory behavioral hypersensitivity

Neuroinflammation induced by microglial activation has a critical role in inflammatory pain. In this study, we detected the function of miR-216a-5p in the progression of inflammatory behavioral hypersensitivity. Here, decreases of miR-216a-5p and up-regulation of high-mobility group box1 (HMGB1) were observed in complete freund’s adjuvant (CFA)-induced inflammatory pain model in mice and LSP-activated BV2 microglia. HMGB1 was identified as a target of miR-216a-5p by luciferase reporter system. Ectopic expression of miR-216a-5p suppressed microglial marker IBA-1 expression and subsequent pro-inflammatory cytokine releases (IL-1β, IL-6 and TNF-α) from LPS-activated microglia. Additionally, LPS exposure enhanced the protein expression levels of HMGB1, TLR4 and p-p65 NF-kB in microglia, which were abrogated following miR-216a-5p overexpression. Intriguingly, transfection of HMGN1 cDNA into BV2 microglial cells reversed the inhibitory effects of miR-216a-5p elevation on microglial activation-triggered inflammatory response. Intrathecal delivery of LV-miR-216a-5-p ameliorated CFA-evoked mechanical and thermal hyperalgesia in mice. Concomitantly, overexpressing miR-216a-5p also restrained the inflammatory response and microglia activation in CFA-induced inflammatory mouse models, concomitant with the decreases in the expression of HMGB1, TLR4 and p-p65 NF-kB in spinal cord. Thus, these findings highlight that miR-216a-5p may alleviate inflammatory behavioral hypersensitivity by blocking microglia-mediated neuroinflammation via targeting the HMGB1-TLR4-NF-kB pathway, supporting miR-216a-5p as a potential therapeutic avenue for inflammatory pain.

MiRNA-146a and miRNA-202-3p Attenuate Inflammatory Response by Inhibiting TLR4, IRAK1, and TRAF6 Expressions in Rats following Spinal Cord Injury

Spinal cord injury (SCI) is a catastrophic disease that induces a complex cascade of cellular reactions at the local lesion area, including secondary cell death and inflammatory reactions. Accumulating evidence has showed pro- and anti-inflammatory roles of microRNAs (miRNAs), a class of small RNAs, in SCI. The present study is aimed at investigating the effects of two miRNAs, miRNA-146a and miRNA-202-3p, on inflammatory response after SCI. Initially, we found that the expression levels of miRNA-146a and miRNA-202-3p were increased in the plasma samples of 32 SCI patients at days 3 and 7 after admission and the rat spinal cord at days 3 and 7 after SCI modeling compared with healthy controls and sham-operated rats, respectively. The expression levels of TLR4, IRAK1, and TRAF6 were declined in the rat spinal cord at days 1, 3, and 7 after SCI modeling compared with sham-operated rats. Injection of miRNA-146a mimic or miRNA-202-3p mimic decreased TLR4, IRAK1, and TRAF6 expressions in the rat spinal cord at days 1, 3, and 7 after SCI modeling, while injection of miRNA-146a antagomir or miRNA-202-3p antagomir produced opposed results. Subsequent results showed that the expression levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-8 were upregulated in the rat serum at days 1, 3, and 7 after SCI modeling compared with sham-operated rats. Injection of miRNA-146a mimic or miRNA-202-3p mimic decreased TNF-α, IL-1β, IL-6, and IL-8 expression levels in the rat serum at days 1, 3, and 7 after SCI modeling, while injection of miRNA-146a antagomir or miRNA-202-3p antagomir yielded opposed results. The expression levels of TNF-α, IL-1β, IL-6, and IL-8 were higher in the supernatants of PC12 cells transfected with anti-miRNA-146a or anti-miRNA-202-3p than in those transfected with si-TLR4, si-IRAK1, or si-TRAF6. These findings support the notion that miRNA-146a/miRNA-202-3p exerts anti-inflammatory functions after SCI.

Intrahepatic TLR3 and IFNL3 Expressions Are Associated with Stages of Fibrosis in Chronic Hepatitis C.

An inefficient immune response against the hepatitis C virus (HCV), combined with viral evasion mechanisms, is responsible for the chronicity of infection. The need to evaluate the innate mechanisms of the immune response, such as TLR3 and IFN-λ3, and their relationship with the virus–host interaction is important for understanding the pathogenesis of chronic hepatitis C. The present study aimed to investigate the gene expressions of TRL3 and IFNL3 in liver tissue, seeking to evaluate whether these could be potential biomarkers of HCV infection. A total of 23 liver biopsy samples were collected from patients with chronic HCV, and 8 biopsies were collected from healthy control patients. RNA extraction, reverse transcription and qPCR were performed to quantify the relative gene expressions of TLR3 and IFNL3. Data on the viral load; AST, ALT, GGT and AFP levels; and the viral genotype were collected from the patients′ medical records. The intrahepatic expression of TLR3 (p = 0.0326) was higher in chronic HCV carriers than in the control group, and the expression of IFNL3 (p = 0.0037) was lower in chronic HCV carriers than in the healthy control group. The expression levels of TLR3 (p = 0.0030) and IFNL3 (p = 0.0036) were higher in the early stages of fibrosis and of necroinflammatory activity in the liver; in contrast, TLR3 and IFNL3 expressions were lower in the more advanced stages of fibrosis and inflammation. There was no correlation between the gene expression and the serum viral load. Regarding the initial METAVIR scale scores, liver transaminase levels were lower in patients with advanced fibrosis when correlated with TLR3 and IFNL3 gene expressions. The results suggest that in the early stages of the development of hepatic fibrosis, TLR3 and IFN-λ3 play important roles in the antiviral response and in the modulation of the tolerogenic liver environment because there is a decrease in the intrahepatic expressions of TLR3 and IFNL3 in the advanced stages of fibrosis, probably due to viral evasion mechanisms.

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.

Baicalin relieves Mycoplasmapneumoniae infection‑induced lung injury through regulating microRNA‑221 to inhibit the TLR4/NF‑κB signaling pathway.

Mycoplasma pneumoniae (MP) is a common pathogen that can cause respiratory infections. MP pneumonia (MPP) leads to numerous complications, including lung injury and even death. The present study aimed to investigate the protective effects of Baicalin treatment on MP infection-induced lung injury and the molecular mechanism underlying these effects. Briefly, after mice were infected intranasally by MP and treated with Baicalin (80 mg/kg), serum levels of MP-immunoglobulin M (IgM) were detected by ELISA. The expression levels of C-reactive protein (CRP) in lung tissue were detected by immunohistochemistry and the bronchoalveolar lavage fluid (BALF) was examined by ELISA. Inflammatory factors and inflammatory cells in the BALF were assessed. The expression levels of microRNA (miR)-221 in lung tissue were examined by reverse transcription-quantitative PCR and pathological changes in lung tissue were detected by H&E staining. Cell apoptosis was evaluated by TUNEL assay and the protein expression levels of TLR4, MyD88 and NF-κB were detected by western blotting. Baicalin treatment significantly reduced serum levels of MP-IgM and CRP expression in lung tissue during MP infection. In addition, Baicalin decreased the levels of IL-1β, IL-6, IL-18 and TNF-α in the BALF, and the number of inflammatory cells. Baicalin also reduced the inflammatory infiltration in lung tissue induced by MP infection, improved the pathological changes detected in lung tissue, reduced apoptosis, and downregulated the protein expression levels of TLR4, MyD88 and NF-κB. Furthermore, Baicalin treatment downregulated the expression of miR-221 and the protective effects of Baicalin were attenuated by miR-221 overexpression. In conclusion, Baicalin has a therapeutic effect on mice with MP infection-induced lung injury, which may be related to inhibition of miR-221 expression and regulation of the TLR4/NF-κB signaling pathway.

Inhibition effect of Hsp90 on TLR2, TLR4, and MAPK signaling pathway in melanoma in-vitro

IntroductionThe Hsp90 is a member of heat shock proteins (HSPs) involved in protein folding and protecting cells from stress. Hsp90 is implicated in melanoma carcinogenesis in which its suppression could have beneficial applicability. This study aimed to investigate the effect of Hsp90 inhibition on TLR2 and TLR4 expression along with the MAPK signaling pathway in melanoma cancer cell line (A-375). Materials & methodsMelanoma cell line was exposed to Hsp90 inhibitor. After 48 h, the expression of Hsp90 was determined by Real-Time PCR and Western blotting. Then, TLR2 and TLR4 expression levels along with phosphorylated p38, ERK, JNK (MAPK signaling pathway) were assessed by qRT-PCR and Western blotting. MTT assay was used to determine the effect of Hsp90 inhibition on cell proliferation. ResultsThe Hsp90 inhibitor decreased the expression of Hsp90 after 48 h. This decrement led to decreased expression of TLR2 and TLR4 through the MAPK signaling pathway in melanoma cells. MTT assay revealed that anti- Hsp90 treatment caused a reduction of cell viability in a time-dependent manner. ConclusionThe results of this study determine the potential therapeutic ability of Hsp90 inhibitor in melanoma skin cancer cells by regulating TLR2 and TLR4 through MAPK signaling pathway in-vitro.