mRNA Expression Levels Of Toll-like Receptor 4 Research Articles

Role of OPG/RANKL/RANK/TLR4 signaling pathway in sepsis-associated acute kidney injury

BackgroundSepsis-associated acute kidney injury (SA-AKI) has high mortality rates. The osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK)/Toll-like receptor 4 (TLR4) pathway and its potential role in SA-AKI pathogenesis remain to be fully understood. Herein, we addressed this issue using mouse models.MethodsAn SA-AKI mouse model was established using the cecal ligation and puncture method (CLP). Mice were grouped into sham, CLP model, CLP + recombinant RANKL, and CLP + anti-RANKL groups. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured to assess kidney function. ELISA was used to detect serum IL-1β, TNF-α, and IL-6 levels. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of OPG, RANKL, RANK, and TLR4 in kidney tissues. HE staining was performed to evaluate the pathological changes.ResultsThe CLP model group showed higher levels of Scr and BUN, indicating impaired kidney function in SA-AKI, compared to the sham group. Treatment with recombinant RANKL in the CLP + recombinant RANKL group reduced Scr and BUN levels, while anti-RANKL treatment in the CLP + anti-RANKL group elevated their levels. Moreover, the CLP model group had significantly increased IL-1β, TNF-α, and IL-6 than the sham group, indicating elevated inflammation in SA-AKI. The CLP + recombinant RANKL group demonstrated decreased cytokine levels, whereas the CLP + anti-RANKL group showed an increase. Additionally, the histopathological evaluation revealed distinct kidney tissue damage in the CLP model group. Recombinant RANKL treatment reduced this damage, while anti-RANKL treatment exacerbated it. Mechanically, the mRNA and protein expression of RANKL were significantly decreased, while those of OPG, RANK, and TLR4 were significantly increased in the CLP model group and the CLP + anti-RANKL group. Interestingly, treatment with recombinant RANKL reversed these changes, as evidenced by significantly increased RANKL but decreased OPG, RANK, and TLR4.ConclusionThe OPG/RANKL/RANK/TLR4 pathway is involved in SA-AKI pathogenesis. Recombinant RANKL treatment attenuates the inflammatory response and kidney tissue damage in SA-AKI, possibly via regulating this pathway. This pathway shows promise as a therapeutic target for SA-AKI.

Three Artemisia pollens trigger the onset of allergic rhinitis via TLR4/MyD88 signaling pathway.

The prevalence of allergic rhinitis is high, making it a relatively common chronic condition. Countless patients suffer from seasonal Allergic rhinitis (AR). The objective of this investigation is to examine the potential involvement of common pollen allergens in seasonal allergic rhinitis, and study the proposed mechanism of Toll-like receptor 4 (TLR4)/Myeloid differentiation primary response gene 88 (MyD88) signaling pathway in the induction of AR. A mouse AR model (sensitized group) was constructed with pollen extracts and ovalbumin(OVA) of Artemisia annua (An), Artemisia argyi (Ar) and Artemisia Sieversiana (Si), and thereafter, AR symptom score was performed. After successful modeling, mouse serum and nasal mucosa tissues were extracted for subsequent experiments. The expression levels of immunoglobulin E (IgE), Interleukin (IL)-4, IL-5, IL-13 and Tumor Necrosis Factor-α (TNF-α) in serum were detected using Enzyme-linked immunosorbent assay (ELISA); Hematoxylin-eosin (H&E) staining methods were used to observe the pathological changes of the nasal mucosal tissue; Utilizing immunohistochemistry (IHC) staining, the expression levels of TLR4, MyD88 and Nuclear factor kappa B (NF-κB) p65 in mouse nasal mucosa were quantified; The mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of sensitized mice were detected with Quantitative reverse transcription PCR (qRT-PCR) and Western Blot. Finally, the in vitro culture of Human nasal mucosal epithelial cells (HNEpC) cells was conducted, and cells were treated with 200µg/ml Artemisia annua pollen extractand OVA for 24h. Western Blot assay was used to detect the expression level of TLR4, MyD88 and NF-κB p65 proteins before and after HNEpC cells were treated with MyD88 inhibitor ST-2825. On the second day after AR stimulation, the mice showed obvious AR symptoms. H&E results showed that compared to the control group, the nasal mucosal tissue in the sensitized group was significantly more inflamed. Furthermore, ELISA assay showed increased expression levels of IgE, IL-4, IL-5, IL-13 and TNF-α in serum of mice induced by OVA and Artemisia annua pollen, Artemisia argyi pollen and Artemisia Sieversiana pollen than those of the control group. However, the expression level of IL-2 was lower than that of the control group (P < 0.05). Using Immunohistochemistry staining visually observed the expression levels of TLR4, MyD88 and NF-κB p65 in mouse nasal mucosa tissues and quantitatively analyzed. The expression levels of TLR4, MyD88 and NF-κB p65 in the sensitized group were higher than those in the control group, and the differences were statistically significant (P < 0.05). The results from qRT-PCR and Western Blot showed that the mRNA and protein expression levels of TLR4, MyD88 and NF-κB p65 in nasal mucosa of the sensitized group were significantly higher than those in the control group (P < 0.05). Finally, HNEpC cells were cultured in vitro and analyzed using Western Blot. The expression levels of TLR4, MyD88 and NF-κB p65 in OVA and An groups were significantly increased (P < 0.05). After ST-2825 treatment, TLR4 protein expression was significantly increased (P < 0.05) and MyD88 and NF-κB p65 protein expression were significantly decreased (P < 0.05). To sum up, the occurrence and development of AR induced by OVA and pollen of Artemisia annua, Artemisia argyi and Artemisia Sieversiana were related to TLR4/MyD88 signal pathway.

Acupoint application therapy alleviates pain by regulating immune function through inhibiting TLR4/MyD88/NF-κB p65 signaling in a primary dysmenorrhea rat model.

To investigate the effects of graphene-based warm uterus acupoint paste on uterine Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear transcription factor-kappa B p65 (NF-κB p65) signaling pathway and Th1/Th2 immune balance in primary dysmenorrhea ( PD ) model rats, so as to reveal its immunological mechanisms of relieving dysmenorrhea. Thirty SD female rats were randomly divided into 3 groups：normal group, model group and acupoint paste group, with 10 rats in each group. PD rat model was established by subcutaneous injection of estradiol benzoate for 10 consecutive days. At the same time of modeling, graphene-based warm uterus acupoint paste was applied to the acupoints of "Guanyuan" (CV4), bilateral "Zigong" (EX-CA1) and "Sanyinjiao" (SP6) of rats in the acupoint paste group. The application was continuously applied once daily for 10 d, 5 h each time. On the 11th day, oxytocin was injected intraperitoneally to observe the writhing latency, writhing times within 30 min and writhing score of rats in each group. The spleen and thymus indexes were calculated. The pathological changes of spleen and thymus tissue were observed after HE staining. The contents of serum immunoglobulin (Ig) A, IgG, tumor necrosis factor-α (TNF-α), interleukin (IL)-2, interferon-γ (IFN-γ), IL-4 and IL-10 were detected by ELISA . The protein and mRNA expression levels of TLR4, MyD88 and NF-κB p65 in rat uterine tissue were detected by Western blot and real-time quantitative PCR, respectively. Compared with the normal group, the writhing times and writhing scores within 30 min of rats in the model group were significantly increased(P<0.001), and the rats showed writhing reaction (P<0.01). The spleen index and thymus index were significantly decreased(P<0.01, P<0.05). The spleen and thymus had obvious pathological changes. The contents of IgA, IgG, TNF-α, IL-2 and IFN-γ in serum were significantly increased, while the contents of serum IL-4 and IL-10 were significantly decreased(P<0.001, P<0.01). The expression levels of TLR4, MyD88, NF-κB p65 protein and corresponding mRNA in uterine tissue were significantly increased(P<0.001). Following intervention, compared with the model group, the writhing latency time of rats in the acupoint paste group was prolonged, and the writhing times and writhing scores within 30 min were significantly decreased (P<0.001). The spleen index and thymus index were significantly increased(P<0.01, P<0.05). The pathological changes of spleen and thymus were improved. The contents of serum IgA, IgG, TNF-α, IL-2 and IFN-γ were significantly decreased, while the contents of IL-4 and IL-10 were significantly increased(P<0.001, P<0.05, P<0.01). The expression of TLR4, MyD88, NF-κB p65 protein and the corresponding mRNA levels in uterine tissue were decreased(P<0.001, P<0.01). Graphene-based warm uterus acupoint paste can regulate the immune balance of Th1/ Th2 by regulating TLR4/ MyD88/ NF-κB p65 signaling pathway, repair the pathological damage of immune tissue, improve immune function, and effectively relieve the pain symptoms of PD rats.

Effects of toll like receptor 4 overexpression on blood parameters, body composition, and gastrointestinal microbial diversity in sheep

Genetic modifications can improve the health status and disease resistance of farm animals. In the previous study, genetically modified (GM) sheep overexpressing toll like receptor 4 (TLR4) were produced. The GM sheep had stronger ability to eliminate invasive microbes compared to the wild-type (WT) sheep. Physiological status and immune homeostasis, blood parameters, body composition, and gastrointestinal microbial diversity were evaluated in this study to elucidate the effects of TLR4 overexpression. In a set of 10 GM rams, the TLR4 mRNA and protein expression levels in the peripheral blood mononuclear cells, muscle, kidney, and spleen were higher than those of the WT (n = 10; P < 0.05). No significant differences in the parameters of red blood cells, white blood cells, platelets and in the composition of circulating T lymphocyte subsets were observed between the GM and WT sheep (P > 0.05). Of the biochemical parameters, only total protein levels differed (were higher) in the GM sheep than in the WT sheep (P < 0.05) while no significant differences were observed for the other indices (P > 0.05). GM and WT sheep showed similar offal weights and muscle fatty acid and amino acid compositions (P > 0.05). Furthermore, TLR4 overexpression did not affect the community structure of the gastrointestinal tract microbiota. Eleven KEGG pathways associated with the TLR4 gene, physiology and biochemistry, growth and metabolism, fatty acids, and amino acids did not demonstrate significant differences between the GM and WT sheep (P > 0.05). In conclusion, TLR4 overexpression had no principal or adverse effects on the physiological development and health of sheep, except for the general mRNA and protein expression levels.

Expression of HMGB1-TLR4 in Placentas from Preeclamptic Pregnancies and Its Effect on Proliferation and Invasion of HTR-8/SVneo Cells

Objectives: Placental inflammation possibly underlies preeclampsia (PE) pathogenesis. This study aimed to investigate the expression of the high mobility box group 1 (HMGB1)-toll-like receptor 4 (TLR4) signalling pathway in preeclamptic placentas and determine whether HMGB1 regulates the biological behaviour of trophoblasts in vitro. Design: Placental biopsies were taken from 30 preeclamptic patients and 30 normotensive controls. In vitro experiments were carried out in HTR-8/SVneo human trophoblast cells. Participants/Materials, Setting, Methods: HMGB1, TLR4, and nuclear factor kappa B (NF-κB) mRNA and protein were quantified to compare their expression in human placentas from preeclamptic and normotensive pregnancies. HTR-8/SVneo cells were stimulated with HMGB1 (50–400 μg/L) for 6–48 h, and proliferation and invasion of HTR-8/SVneo cells were measured via Cell Counting Kit-8 and transwell assays. HTR-8/SVneo cells were also transfected with HMGB1 and TLR4 small interfering RNA (siRNA) to investigate the effect of knocking down these proteins. The mRNA and protein expression of TLR4, NF-κB, and matrix metalloproteinase 9 (MMP-9) were determined using quantitative real-time PCR and Western blotting, respectively. Data were analysed with either a t-test or one-way analysis of variance. Results: The mRNA and protein levels of HMGB1, TLR4, and NF-κB were significantly higher in the placentas from preeclamptic pregnancies than from normal pregnancies (p < 0.05). HMGB1 stimulation (at concentrations up to 200 μg/L) of HTR-8/SVneo cells significantly increased invasion and proliferation over time. However, at an HMGB1 stimulation concentration of 400 μg/L, the invasion and proliferation ability of HTR-8/SVneo cells decreased. Compared to controls, mRNA and protein expression levels of TLR4, NF-κB, and MMP-9 increased (mRNA level fold change: 1.460, 1.921, 1.667; protein level fold change: 1.600, 1.750, 2.047) when stimulated with HMGB1 (p < 0.05) but decreased when HMGB1 was knocked down (p < 0.05). TLR4 siRNA transfection combined with HMGB1 stimulation reduced the mRNA (fold change: 0.451) and protein (fold change: 0.289) expression of TLR4 (p < 0.05), while NF-κB and MMP-9 were unaffected (p > 0.05). Limitations: Only one trophoblast cell line was used in this study, and the findings were not confirmed in animal studies. Conclusions: This study explored the pathogenesis of PE from two aspects: inflammation and trophoblast invasion. The overexpression of HMGB1 in placentas from preeclamptic pregnancies suggests this protein may be involved in PE pathogenesis. In vitro, HMGB1 was found to regulate the proliferation and invasion of HTR-8/SVneo cells by activating the TLR4-NF-κB-MMP-9 pathway. These findings have implications for targeting HMGB1 could be a therapeutic strategy for treating PE. In the future, we will further verify this in vivo and in other trophoblast cell lines, further exploring the molecular interactions of the pathway.

Toll-like receptor 4 signaling pathway in sensory neurons mediates remifentanil-induced postoperative hyperalgesia via transient receptor potential ankyrin 1.

Background: Remifentanil-induced postoperative hyperalgesia (RIH) refers to a state of hyperalgesia or aggravated pre-existing pain after remifentanil exposure. There has been considerable interest in understanding and preventing RIH. However, the mechanisms responsible for RIH are still not completely understood. Toll-like receptor 4 (TLR4), a classic innate immune receptor, has been detected in sensory neurons and participates in various nociceptive conditions, whereas its role in RIH remains unclear. Transient receptor potential ankyrin 1 (TRPA1) always serves as a nociceptive channel, whereas its role in RIH has not yet been investigated. This study aimed to determine whether the TLR4 signaling pathway in sensory neurons engaged in the development of RIH and the possible involvement of TRPA1 during this process. Methods: A rat model of remifentanil-induced postoperative hyperalgesia (RIH) was established, which presented decreased paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL). The mRNA and protein expression levels of TLR4, phosphorylated NF-κB, and TRPA1 in the dorsal root ganglion (DRG) from RIH model were analyzed by real-time PCR, western blot, and immunofluorescence. The TLR4 antagonist TAK-242 and the TRPA1 antagonist HC-030031 were applied to determine the role of sensory neuron TLR4 signaling and TRPA1 in RIH. Results: Compared with control, PWMT and PWTL were significantly decreased in RIH model. Moreover, the mRNA and protein expression of TLR4 and TRPA1 in DRG were upregulated after remifentanil exposure together with increased NF-κB phosphorylation. TLR4 antagonist TAK-242 mitigated mechanical pain in RIH together with downregulated expression of TLR4, phosphorylated NF-κB, and TRPA1 in DRG neurons. In addition, TRPA1 antagonist HC-030031 also alleviated mechanical pain and decreased TRPA1 expression in RIH without affecting TLR4 signaling in DRG. Conclusions: Taken together, these results suggested that activation of TLR4 signaling pathway engaged in the development of RIH by regulating TRPA1 in DRG neurons. Blocking TLR4 and TRPA1 might serve as a promising therapeutic strategy for RIH.

Association of the Toll-like receptor 4 and NOX4 gene and protein levels in asthmatic patients with metabolic syndrome: A case-control study.

Understanding the contributing of influence inflammatory biomarkers in asthmatic patients with metabolic syndrome is more important. Whereby, the present study considering the important association of NADPH oxidase4 (NOX4) and Toll- like receptor4 (TLR4) in the respiratory inflammatory responses in asthmatic patients with metabolic syndrome (AS-MetS) and asthmatic (AS) patients. In this case-control study, 30 AS and 34 AS-MetS patients were enrolled. The Peripheral blood mononuclear cells (PBMCs) mRNA and protein levels of TLR4 and NOX4 were measured by qRT-PCR and western blot, respectively. Then their correlation was evaluated. The significant down-regulation of mRNA and protein PBMCs expression levels of TLR4 were observed in the AS-MetS group in comparison to AS one (P=0.03), but the NOX4 expression was non-significant. Additionally, the significant correlation was exhibited between mRNA expression levels of NOX4 and TLR4 in both AS-MetS (r= 0.440, P=0.009) and AS groups (r=0.909, P=0.0001). The association between TLR4 mRNA level and triglyceride in AS-MetS group (r=0.454, P=0.008,) and also white blood cells (WBC) in AS group (r= -0.507, P=0.006,) were significant. The metabolic syndrome can significantly influence the expressions of TLR4 in AS-MetS. This study indicated that TLR4 and NOX4 altogether may provide valuable molecular knowledge of their relation with metabolic syndrome criteria for finding major pathways in different phenotype of asthma.

Impact of Toll-like Receptor 4 Expression on Inflammatory Responses Related to Premature Membrane Rupture Induced by Lipopolysaccharide.

This study aimed to determine the mechanism through which the expression of Toll-like receptor 4 (TLR4) influences the lipopolysaccharide (LPS)-induced inflammatory response, a condition that is associated with premature rupture of membranes (PROM). Human myeloid leukemia mononuclear cells (THP-1) were employed as the experimental model. These cells were treated with LPS and the TLR4 inhibitor CLI-095 and subsequently divided into three groups. A range of assays were utilized, including methyl thiazolyl tetrazole (MTT) assay, real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) for measuring TLR4 and tumor necrosis factor α (TNF-α) mRNA levels, double antibody sandwich enzyme-linked immunosorbent assay (ELISA) for assessing monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase 9 (MMP-9), as well as secretion levels of interleukin (IL)-6 and IL-1β. And western blotting was used to detect the expression of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB) p65, which are components of the TLR4 downstream signaling pathway. The LPS-induced proliferation of THP-1 cells was significantly inhibited (p < 0.05) when compared with normal THP-1 cells. Moreover, LPS also promoted TLR4 mRNA and protein expression levels, TNF-α mRNA expression, secretion of inflammatory factors, and phosphorylation of ERK and NF-κB p65 proteins (p < 0.05). On the other hand, administration of the TLR4 inhibitor CLI-095 significantly inhibited the expression of TLR4 mRNA and protein. It also effectively increased the proliferative activity of THP-1 cells and inhibited the secretion of TNF-α and inflammatory factors, as well as the phosphorylation of ERK and NF-κB p65 proteins (p < 0.05). In summary, suppressing TLR4 expression can mitigate inflammatory responses, thereby reducing the likelihood of premature rupture of membranes during pregnancy, which is often triggered by such inflammation.

The Role of TLR4-Induced Macrophage Pyroptosis in the Pathogenesis of Severe Aplastic Anemia

Objective: By studying the level of macrophage pyroptosis in patients with severe aplastic anemia (SAA) and its effect on CD8+ T cells, we investigated the mechanisms by which Toll-like receptor 4 (TLR4) induces macrophage pyroptosis and regulates the immune status in SAA. Methods: We derived macrophages in vitro from bone marrow samples collected from SAA treatment-naive patients, remission patients, and healthy controls and cultured the macrophages. Flow cytometry, quantitative reverse transcription polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay were used to detect the levels of pyroptosis and the expression levels of TLR4. Knockdown of TLR4 or the addition of TLR4 inhibitors in bone marrow-derived macrophages of SAA treatment-naive patients was performed to explore the effects of changes in TLR4 levels on the pyroptosis level of macrophages in SAA patients and on the killing effect of CD8+ T cells. Results: 1. The relative mRNA and protein levels of interleukin (IL)-1β, IL-18, NLRP3, caspase-1, and gasdermin D (GSDMD) in macrophages in the SAA treatment-naive group were significantly higher than those in the healthy control group, and the concentrations of IL-1β and IL-18 were significantly increased in the supernatants of macrophage cultures. IL-1β, IL-18, NLRP3, caspase-1, and GSDMD were significantly negatively correlated with the severity of the disease(Fig.A). 2. The relative mRNA and protein expression levels of TLR4 in macrophages were significantly higher in the SAA treatment-naive group than in the remission group or the control group. In addition, the relative mRNA expression level of TLR4 was positively correlated with the expression levels of IL-1β, IL-18, NLRP3, caspase-1, and GSDMD. After TLR4 knockdown or the addition of TLR4 inhibitors, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, caspase-1, and GSDMD were significantly lower than those in the control group, and the concentrations of IL-1β and IL-18 in the culture supernatant were significantly lower than those in the control group. 3. After CD8+ T cells were cocultured with macrophages with TLR4 knockdown or with the addition of inhibitors, the expression of perforin and granzyme B in CD8+ T cells was significantly reduced. Further coculture of CD8+ T cells with K562 reduced the apoptosis level of K562. Conclusion: In SAA patients, the level of macrophage pyroptosis was increased, and the expression levels of TLR4 in macrophages were increased and positively correlated with the level of macrophage pyroptosis. Inhibiting TLR4 expression reduced the level of macrophage pyroptosis. TLR4 might induce pyroptosis of macrophages in SAA patients and enhance the killing effect of CD8+ T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical significance of high mobility group box 1/toll-like receptor 4 in obese diabetic patients.

High mobility group box 1 (HMGB1) is an alarmin that may link to obesity and type 2 diabetes mellitus (T2DM). The present study analyzed the correlation between HMGB1/ Toll-like receptor 4 (TLR4) and certain biochemical parameters in obese (OB) diabetic patients. 40 normal glucose tolerant subjects (NGT) and 40 patients with newly diagnosed T2DM were enrolled. All patients were further divided into non-obese NGT (NGT-NOB), obese NGT (NGT-OB), non-obese T2DM (T2DM-NOB) and obese T2DM (T2DM-OB) groups according to body mass index (BMI).The levels of HMGB1 in serum were quantified using ELISA, whereas the mRNA expression levels of TLR4 in peripheral blood mononuclear cells were assessed using reverse transcription-quantitative PCR. The results suggested that the levels of HMGB1 and TLR4 were higher in NGT-OB and T2DM-NOB groups compared with those in NGT-NOB group. Similarly, the levels of these two markers were higher in T2DM-OB group compared with those in NGT-OB group. Correlation analysis indicated that the levels of HMGB1 and TLR4 were positively correlated with triglyceride (TG), fasting plasma glucose (FPG) levels and BMI, whereas a negative correlation between HMGB1 and high density lipoprotein (HDL) was noted. Linear regression analysis suggested that HMGB1 was associated with FPG and TG levels, whereas TLR4 was strongly associated with TG levels and BMI. The results demonstrated that the expression levels of HMGB1 and TLR4 in patients with T2DM or obesity were increased, which were associated with glycolipid metabolism disorders. Therefore, the HMGB1/TLR4 may serve a role in inflammatory process associated with obesity and T2DM.

KDM2B overexpression prevents myocardial ischemia-reperfusion injury in rats through regulating inflammatory response via the TLR4/NF-κB p65 axis.

Histone modifier lysine-specific demethylase 2B (KDM2B) has been previously reported to activate the inflammatory response by transcription initiation of the IL-6 gene. However, the effects of KDM2B on the inflammatory response during myocardial ischemia-reperfusion (I/R) injury and corresponding mechanisms remain poorly understood. The present study aimed to investigate the role and mechanism of KDM2B in myocardial I/R injury. Therefore, a myocardial I/R injury model was established in rats through coronary artery ligation. Adeno-associated virus-encoding KDM2B and small interfering RNA-KDM2B were designed to determine the effects of KDM2B on myocardial I/R injury using H&E staining and a TUNEL assay in the myocardial tissues. Reverse transcription-quantitative PCR and western blotting were performed to detect the mRNA and protein expression levels of KDM2B, toll-like receptor 4 (TLR4), NF-κB p65 and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3). ELISA was used to detect the levels of TNF-α, IL-6 and IL-1β in the peripheral blood samples. Pathological analysis demonstrated that the cells in the model group were disordered, with a large area of necrosis and neutrophil infiltration. Knocking down KDM2B expression significantly upregulated the mRNA and protein expression levels of TLR4, NLRP3, NF-κB p65 and the ratio of phosphorylated (p)-p65 to p65. KDM2B knockdown also significantly increased the levels of IL-1β, IL-6 and TNF-α in the peripheral blood, which aggravated myocardial injury and promoted the apoptosis of myocardial cells. However, overexpression of KDM2B downregulated the mRNA and protein expression levels of TLR4, NLRP3, NF-κB P65, the ratio of p-p65 to p65 whilst reducing the levels of IL-1β, IL-6 and TNF-α in the peripheral blood, which markedly improved myocardial injury and significantly inhibited the apoptosis of cells in myocardial tissue. In conclusion, the results indicated that overexpression of KDM2B may prevent myocardial I/R injury in rats by reducing the inflammatory response through regulation of the TLR4/NF-κB p65 axis.

MicroRNA-216a-5p in lipopolysaccharide-induced endothelial injury.

MicroRNAs (miRNAs/miRs) are a type of non-coding RNA that are closely associated with disease development and treatment. The present study aimed to investigate the role of miR-216a-5p in lipopolysaccharide (LPS)-induced endothelial injury in vitro. The EdU assay was performed to detect EdU-positive cells, while flow cytometric analysis was performed to detect apoptotic cells. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the expression levels of miR-216a-5p, Toll-like receptor 4 (TLR4), MyD88 and nuclear factor (NF)-κB(p65) and phosphorylated (p)-NF-κB(p65). Furthermore, p-NF-κB(p65) nuclear expression level was detected via cellular immunofluorescence. The dual-luciferase reporter assay was performed to verify the association between miR-216a-5p and TLR4. The results demonstrated that the number of EdU-positive cells significantly decreased, the apoptotic rate significantly increased, and TLR4, MyD88 and NF-κB(p65) mRNA expression levels were significantly upregulated.TLR4, MyD88 and p-NF-κB(p65) protein expression levels were significantly upregulated and p-NF-κB(p65) nuclear concentration was significantly enhanced in the small interfering RNA-miR-216a-5p and LPS groups (P<0.001, respectively) compared with the negative control group. However, the addition of miR-216a-5p significantly increased the number of EdU-positive cells, significantly decreased the apoptotic rate and significantly downregulated the mRNA expression levels of TLR4, MyD88 and NF-κB(p65), as well as the protein expression levels of TLR4, MyD88 and p-NF-κB(p65). In addition, the p-NF-κB(p65) nuclear concentration was significantly decreased in the miR-216a-5p group (P<0.001, respectively) compared with the LPS group. Taken together, the results suggest that overexpression of miR-216a-5p suppresses the effects of LPS induced endothelial injury.

Protective effect of total triterpenoids from Chaenomeles speciosa against Helicobacter pylori-induced gastritis in mice

This study investigated the protective effect of total triterpenoids from Chaenomeles speciosa against Helicobacter pylori(Hp)-induced gastritis in mice and explored its possible mechanism. The chronic atrophic gastritis(CAG) model mice were randomly divided into four groups of model, total triterpenoids from C. speciosa(50 and 100 mg·kg~(-1)) and triple therapy, with C57 BL/6 J mice without Hp infection taken as the normal group. Mice in the treatment groups were given corresponding drugs once a day for 4 weeks. Then the following indexes were detected: the contents of reactive oxygen species(ROS), monocyte chemotactic protein 1(MCP-1), keratinocyte chemokines(KC), TNF-α, IL-1β, IL-6, IL-18, IL-4 and IL-10 in blood and gastric tissue, the activities and contents of LDH, MPO, SOD, GSH-Px, CAT and MDA in gastric tissue and the activities of β-glucuronidase, β-galactosidase, cathepsins B and D in blood, gastric tissue and lysosome. Besides, the mRNA expression levels of Toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), Bcl-2, Bcl-xl, Bax and Bad in gastric tissue were determined by quantitative real-time PCR. Western blot was employed to detect the protein expression levels of TLR4, MyD88, p-IKKβ, p-IκBα, NOD-like receptor 3(NLRP3), apoptosis-associated speck-like protein(ASC), pro-caspase-1, caspase-1, thioredoxin-interacting protein(TXNIP), pro-IL-1β, pro-IL-18, Bcl-2, Bcl-xl, Bax, Bad, cytochrome C, apoptotic protease-activating factor-1(Apaf-1), pro-caspase-9, pro-caspase-3, cleaved-caspase-9, cleaved-caspase-3, poly(ADP-ribose) polymerase 1(PARP-1), cleaved-PARP-1 and cytosol and nucleus NF-κB p65 in gastric tissue. The results indicated that the total triterpenoids from C. speciosa significantly suppressed Hp proliferation, alleviated the damage to gastric mucosa and improved lymphocyte infiltration and gland atrophy. They were also effective in reducing the activities of β-glucuronidase, β-galactosidase, cathepsins B and D in blood and gastric tissue, elevating the activities of β-glucuronidase and cathepsin D in lysosomal organelles, decreasing the contents of ROS, MCP-1, KC, TNF-α, IL-1β, IL-6, IL-18 in blood, MDA content and MPO and LDH activities in gastric tissue and increasing the contents of IL-4 and IL-10 in blood and activities of SOD, CAT and GSH-Px in gastric tissue. Other phenomena were also observed after the treatment with total triterpenoids from C. speciosa, including the down-regulation of the mRNA and protein expression levels of TLR4, MyD88, Bax and Bad, the protein expression levels of p-IKKβ, p-IκBα, NLRP3, ASC, pro-caspase-1, caspase-1, TXNIP, pro-IL-1β, pro-IL-18, cytochrome C, Apaf-1, cleaved-caspase-9, cleaved-caspase-3, cleaved-PARP-1 and nuclear NF-κB p65, reduction of p-IKKβ/IKKβ and p-IκBα/IκBα ratios and up-regulation of the mRNA and protein expression levels of Bcl-2 and Bcl-xl, up-regulation of pro-caspase-9, pro-caspace-3, cytosol NF-κB p65 protein expression levels and Bcl-2/Bax and Bcl-xl/Bad ratios in gastric tissue. These aforementioned results suggest that the total triterpenoids from C. speciosa have significant protective effects against CAG induced by Hp, and its mechanism may be related to enhancing the function of endogenous antioxidant system, suppressing the oxidative stress and inflammatory reaction induced by Hp, correcting lysosomal dysfunction and inflammatory activation of TLR4/NF-κB/NLRP3 inflammasome signaling pathway and thus inhibiting mitochondria-mediated apoptosis.

The Expression of Toll-Like Receptor 4 mRNA in PBMCs Is Upregulated in Smokers and Decreases Upon Smoking Cessation.

BackgroundStudies have shown in vitro that cigarette smoke condensate stimulates monocytes to express toll-like receptor 4 (TLR4), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule 1 (ICAM-1), and enhances their adhesion to the endothelium. However, the same effects of cigarette smoking have not been explored in vivo. This study is to investigate the effect of cigarette smoking and smoking cessation on their mRNA expression in human peripheral blood mononuclear cells (PBMCs).MethodsA group of 97 smokers and 62 nonsmokers were enrolled. The RNA from PBMCs was assessed with real-time polymerase chain reaction (PCR) to determine the levels of ICAM-1, TNF-α, and TLR4. The same markers in PBMCs of 87 quitters were examined before and at one week, one month, and two months after smoking cessation.ResultsOf the 97 smokers, 85 (87.6%) were males, and 30 (48.4%) of the nonsmokers were males (p < 0.0001). The mean (SD) age of the smokers was 43.24 (10.89) years, which was younger than 43.45 (11.41) years of nonsmokers (p < 0.0001). The incidence of cardiovascular diseases was 13.4% in smokers, which was higher than 1.6% in nonsmokers (p < 0.05). Both ICAM-1 and TNF-α mRNA levels in PBMCs were higher among the smokers (p < 0.0001). In addition, TLR4 mRNA levels in PBMCs were statistically elevated in the smokers (p < 0.0001) comparing with those in the nonsmokers. The mRNA levels of TLR4 and TNF-α in PBMCs decreased in those who had quit smoking for 2 months (p < 0.0001).ConclusionsICAM-1, TNF-α, and TLR4 mRNA expression levels in PBMCs increased in smokers and decreased after being on a smoking cessation program for 2 months. This finding suggested that TLR4 expression may mediate the atherogenic inflammatory process induced by smoking.

The miR-1906 mimic attenuates bone loss in osteoporosis by down-regulating the TLR4/MyD88/NF-κB pathway.

In this study, the ability of microRNA-1906 (miR-1906) to attenuate bone loss in osteoporosis was evaluated by measuring the effects of a miR-1906 mimic and inhibitor on the cellular toxicity and cell viability of MC3T3-E1 cells. Bone marrow-derived macrophage (BMM) cells were isolated from female mice, and tartrate-resistant acid phosphatase signalling was performed in miR-1906 mimic-treated, receptor-activated nuclear factor kappa-B (NF-κB) ligand (RANKL)-induced osteoclasts. In-vivo, osteoporosis was induced by ovariectomy (OVX). Rats were treated with 500 nmol/kg of the miR-1906 mimic via intrathecal administration for 10 consecutive days following surgery. The effect of the miR-1906 mimic on bone mineral density (BMD) in OVX rats was observed in the whole body, lumbar vertebrae and femur. Levels of biochemical parameters and cytokines in the serum of miR-1906 mimic-treated OVX rats were analysed. The mRNA expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), p-38 and NF-κB in tibias of osteoporotic rats (induced by ovariectomy) was observed using quantitative reverse-transcription polymerase chain reaction. Treatment with the miR-1906 mimic reduced cellular toxicity and enhanced the cell viability of MC3T3-E1 cells. Furthermore, osteoclastogenesis in miR-1906 mimic-treated, RANKL-induced osteoclast cells was reduced, whereas the BMD in the miR-1906 mimic-treated group was higher than in the OVX group of rats. Treatment with the miR-1906 mimic also increased levels of biochemical parameters and cytokines in the serum of ovariectomised rats. Finally, mRNA expression levels of TLR4, MyD88, p-38 and NF-κB were lower in the tibias of miR-1906 mimic-treated rats than in those of OVX rats. In conclusion, the miR-1906 mimic reduces bone loss in rats with ovariectomy-induced osteoporosis by regulating the TLR4/MyD88/NF-κB pathway.

Emodin protects against intestinal and lung injury induced by acute intestinal injury by modulating SP-A and TLR4/NF-κB pathway.

Objective: Our aim was to investigate the effect of emodin on intestinal and lung injury induced by acute intestinal injury in rats and explore potential molecular mechanisms.Methods: Healthy male Sprague–Dawley (SD) rats were randomly divided into five groups (n=10, each group): normal group; saline group; acute intestinal injury model group; model + emodin group; model+NF-κB inhibitor pynolidine dithiocarbamate (PDTC) group. Histopathological changes in intestine/lung tissues were observed by Hematoxylin and Eosin (H&E) and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling (TUNEL) staining. Serum IKBα, p-IKBα, surfactant protein-A (SP-A) and toll-like receptor 4 (TLR4) levels were examined using enzyme-linked immunosorbent assay (ELISA). RT-qPCR was performed to detect the mRNA expression levels of IKBα, SP-A and TLR4 in intestine/lung tissues. Furthermore, the protein expression levels of IKBα, p-IKBα, SP-A and TLR4 were detected by Western blot.Results: The pathological injury of intestinal/lung tissues was remarkedly ameliorated in models treated with emodin and PDTC. Furthermore, the intestinal/lung injury scores were significantly decreased after emodin or PDTC treatment. TUNEL results showed that both emodin and PDTC treatment distinctly attenuated the apoptosis of intestine/lung tissues induced by acute intestinal injury. At the mRNA level, emodin significantly increased the expression levels of SP-A and decreased the expression levels of IKBα and TLR4 in intestine/lung tissues. According to ELISA and Western blot, emodin remarkedly inhibited the expression of p-IKBα protein and elevated the expression of SP-A and TLR4 in serum and intestine/lung tissues induced by acute intestinal injury.Conclusion: Our findings suggested that emodin could protect against intestinal and lung injury induced by acute intestinal injury by modulating SP-A and TLR4/NF-κB pathway.

Toll-like receptor 4 deficiency ameliorates β2-microglobulin induced age-related cognition decline due to neuroinflammation in mice

Toll-like receptor 4 (TLR4) is a crucial receptor in neuroinflammation and apoptotic neuronal death, and increasing evidences indicated that β2-microglobulin (B2M) is thought to be a major contributor to age-related cognitive decline. In present study, we designed to investigate the effects of TLR4 on B2M-induced age-related cognitive decline. Wild-type (WT) C57BL/6, TLR4 knockout (TLR4 -KO) mice and hippocampal neurons from the two type mice were respectively divided into two groups: (1) Veh group; (2) B2M-treated group. The behavioral responses of mice were measured using Morris Water Maze. Hippocampal neurogenesis and neuronal damage, inflammatory response, apoptosis, synaptic proteins and neurotrophic factors, and TLR4/MyD88/NF-κB signaling pathway proteins were examined using molecular biological or histopathological methods. The results showed that WT mice received B2M in the DG exhibited age-related cognitive declines, increased TLR4 mRNA expression and high levels of interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α) and apoptotic neuronal death in the hippocampus, which were partially attenuated in TLR4-KO mice. Moreover, in absence of TLR4, B2M treatment improved hippocampus neurogenesis and increased synaptic related proteins. Our cell experiments further demonstrated that deletion of TLR4 could significantly increase synaptic related protein, decrease neuroinflammatory fators, inhibited apoptotic neuronal death, and regulated MyD88/NF-κB signal pathway after B2M treatment. In summary, our results support the TLR4 contributes to B2M-induced age-related cognitive decline due to neuroinflammation and apoptosis through TLR4/MyD88/NF-κB signaling pathway via a modulation of hippocampal neurogenesis and synaptic function. This may provide an important neuroprotective mechanism for improving age-related cognitive decline.

MiR-885-3p is down-regulated in peripheral blood mononuclear cells from T1D patients and regulates the inflammatory response via targeting TLR4/NF-κB signaling.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the progressive destruction of insulin-production pancreatic β cells. Recently, microRNAs (miRNAs) have emerged as important regulators in T1D. The present study aimed to determine miR-885-3p expression in T1D patients and to examine the effects of miR-885-3p on the inflammatory response in human monocytes. Relevant gene expression levels were determined by a quantitative polymerase chain reaction; western blotting and enzyme-linked immunosorbent assays determined the respective protein levels; and the interaction between miRNA and the downstream targets was evaluated using a luciferase reporter assay. MiR-885-3p is down-regulated and the levels of pro-inflammatory cytokines are increased in peripheral blood mononuclear cells (PBMCs) from T1D patients compared to healthy controls. MiR-885-3p overexpression suppressed mRNA expression and secreted protein levels of pro-inflammatory cytokines in THP-1. A luciferase reporter assay showed that miR-885-3p directly targeted the 3'-untranslated region of Toll-like receptor 4 (TLR4) and miR-885-3p overexpression down-regulated TLR4 expression in THP-1 cells. The TLR4 mRNA expression level was increased in PBMCs isolated from T1D patients compared to heathy controls. TLR4 overexpression increased the secretion of pro-inflammatory cytokines and enhanced the activity of NF-κB signaling, and also attenuated the inhibitory effects of miR-885-3p overexpression on pro-inflammatory cytokine secretion and the activity of NF-κB signaling in THP-1 cells. The present study identified the down-regulation of miR-885-3p and up-regulation of TLR4 in PBMCs isolated from T1D patients. Further mechanistic data demonstrated that miR-885-3p overexpression represses the production of pro-inflammatory cytokines via targeting TLR4/NF-κB signaling in THP-1 cells.

TLR4 promotes liver inflammation by activating the JNK pathway.

Drug-induced liver injury has become a serious public health problem that cannot be ignored. Although the mechanism of acetaminophen (APAP)-induced liver injury has been investigated for several decades, there are still many deficiencies. However, only a deeper study of its mechanism can provide more effective measures of prevention and treatment for APAP-induced liver injury. The aim of this study was to investigate whether toll-like receptor 4 (TLR4) participates in and regulates APAP-induced liver injury, which may provide a new direction for the prevention and treatment of clinical drug-induced hepatitis. WT mice were treated with APAP (300 mg/kg) or equivalent PBS. The livers of mice were taken at 1 h, 3 h, 6 h and 12 h after treatment. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect TLR4 mRNA expression level in the liver. After TLR4 involving in APAP-induced liver injury was confirmed, we investigated the relationship between TLR4 expression and hepatic inflammation. WT and TLR4-/- mice received APAP (3000 mg/kg) intraperitoneal injection after 16 h of fasting; serum was collected after 8 h and 24 h, and serum alanine aminotransferase (ALT) and reduced glutathione (GSH) activity were measured. Rat liver tissue was observed for histological changes by hematoxylin and eosin (H&E) staining. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) assay were performed to analyze proinflammatory cytokines expression (such as TNF-α, IL-1β, MCP-1, IL-6). After isolating mononuclear cells (MNCs) in the liver of mice, flow cytometry was used to detect cell activation level and infiltration of macrophages and neutrophils. Western blotting was used to analyze the activation of phosphorylated JNK and p38 signaling pathways in livers of WT and TLR4-/- mice. In addition, after stimulated with APAP, the silence of TLR4 in RAW264.7 cells could activate phosphorylated JNK and p38 signaling pathways. After APAP stimulation, WT mice exhibited more severe liver injury than TLR4-/- mice, with higher ALT levels, lower GSH levels, and more necrotic or apoptotic cells. TLR4-/- mice have lower levels of inflammatory cytokines including MCP-1 and IL-6; at the same time, the number of infiltrating macrophages and neutrophils in liver tissue of TLR4-/- mice was significantly lower than that of WT mice. The activation of JNK signaling pathway was strikingly enhanced in WT mice treated with APAP, but no significant difference was observed in the activation of JNK phosphorylation in TLR4-/- mice after the same dose of APAP stimulation. Similarly, in RAW264.7 cells, the activation of phosphorylated JNK and p38 was remarkably inhibited by TLR4-siRNA, but was activated in the control group, which was consistent in vivo. APAP-treated TLR4-/- mice showed milder liver injury compared to WT mice. It was confirmed that TLR4 could activate the JNK signaling pathway to induce the secretion of inflammatory factors and the infiltration of macrophages to promote APAP-induced liver injury. This finding might provide a new prevention and treatment idea for clinical drug-induced hepatitis.

Dexmedetomidine protects against lung injury induced by limb ischemia-reperfusion via the TLR4/MyD88/NF-κB pathway.

Dexmedetomidine (DEX) can protect the lung from ischemia-reperfusion (I/R) injury, but the underlying mechanisms are not fully understood. The aims of this study were to determine whether DEX attenuates lung injury following lower extremity I/R and to investigate the related toll-like receptor 4 (TLR4) signaling pathway. Twenty-eight SD rats were divided into four groups (n = 7): Sham, I/R, I/R + DEX (25 μg/kg prior to ischemia), and I/R + DEX + Atip (250 μg/kg atipamezole before DEX treatment). Lower extremity I/R was induced by left femoral artery clamping for 3 hours and followed by 2 hours reperfusion. Quantitative alveolar damage and the wet/dry (W/D) ratio were calculated. Interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α in the bronchoalveolar lavage fluid (BALF) and serum and myeloperoxidase (MPO) in the lung were measured. The TLR4 and MyD88 mRNA expression levels were measured by RT-PCR, nuclear factor (NF)-κB, and phosphorylated NF-κB by western blot, respectively. Quantitative alveolar damage, W/D ratio, MPO, BALF and serum IL-1, IL-6, and TNF-α, and TLR4, MyD88, NF-κB, and p-NF-κB expression significantly increased in the I/R group relative to the Sham group. DEX preconditioning significantly reduced lung edema, and histological injury relative to the I/R group. Serum and BALF IL-1, IL-6, and TNF-α levels, MPO activity and TLR4, MyD88, NF-κB, and p-NF-κB expression were also significantly reduced in the I/R + DEX group compared with the I/R group. Atipamezole partially reversed all the aforementioned effects. DEX preconditioning protects the lungs against lower extremity I/R injury via α2-adrenoceptor-dependent and α2-adrenoceptor-independent mechanisms. It also suppresses the TLR4 pathway and reduces inflammation.