Toll-like Receptor 4 mRNA Levels Research Articles

Effects of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination on inflammatory responses in atherosclerotic mice

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P<0.05), thickened intima(P<0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P<0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P<0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P<0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P<0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P<0.01), alleviated the intimal hyperplasia(P<0.01), and reduced the plasma TNF-α and IL-6 levels(P<0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P<0.05), inhibited the expression of VCAM-1 and iNOS(P<0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P<0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.

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 Improvement of Recombinant Plasmids of the Interleukin-7 Gene in Chitosan-Derived Nanoparticles and Their Elevation of Mice Immunity.

To investigate a safe and effective approach for enhancing the in vivo expression of recombinant genes and improving the systemic immunity of animals against infectious diseases, we employed the interleukin-7 (IL-7) gene from Tibetan pigs to construct a recombinant eukaryotic plasmid (VRTPIL-7). We first examined VRTPIL-7's bioactivity on porcine lymphocytes in vitro and then encapsulated it with polyethylenimine (PEI), chitosan copolymer (CS), PEG-modified galactosylated chitosan (CS-PEG-GAL) and methoxy poly (ethylene glycol) (PEG) and PEI-modified CS (CS-PEG-PEI) nanoparticles using the ionotropic gelation technique. Next, we intramuscularly or intraperitoneally injected mice with various nanoparticles containing VRTPIL-7 to evaluate their immunoregulatory effects in vivo. We observed a significant increase in neutralizing antibodies and specific IgG levels in response to the rabies vaccine in the treated mice compared to the controls. Treated mice also exhibited increased leukocytes, CD8+ and CD4+ T lymphocytes, and elevated mRNA levels of toll-like receptors (TLR1/4/6/9), IL-1, IL-2, IL-4, IL-6, IL-7, IL-23, and transforming growth factor-beta (TGF-β). Notably, the recombinant IL-7 gene encapsulated in CS-PEG-PEI induced the highest levels of immunoglobulins, CD4+ and CD8+ T cells, TLRs, and cytokines in the mice's blood, suggesting that chitosan-PEG-PEI may be a promising carrier for in vivo IL-7 gene expression and enhanced innate and adaptive immunity for the prevention of animal diseases.

RAGE-TLR4 Crosstalk Is the Key Mechanism by Which High Glucose Enhances the Lipopolysaccharide-Induced Inflammatory Response in Primary Bovine Alveolar Macrophages.

The receptor of advanced glycation end products (RAGE) and Toll-like receptor 4 (TLR4) are important receptors for inflammatory responses induced by high glucose (HG) and lipopolysaccharide (LPS) and show crosstalk phenomena in inflammatory responses. However, it is unknown whether RAGE and TLR4 can influence each other's expression through a crosstalk mechanism and whether the RAGE-TLR4 crosstalk related to the molecular mechanism of HG enhances the LPS-induced inflammatory response. In this study, the implications of LPS with multiple concentrations (0, 1, 5, and 10 μg/mL) at various treatment times (0, 3, 6, 12, and 24 h) in primary bovine alveolar macrophages (BAMs) were explored. The results showed that a 5 μg/mL LPS treatment at 12 h had the most significant increment on the pro-inflammatory cytokine interleukin 1β (IL-1β), IL-6, and tumor necrosis factor (TNF)-α levels in BAMs (p < 0.05) and that the levels of TLR4, RAGE, MyD88, and NF-κB p65 mRNA and protein expression were upregulated (p < 0.05). Then, the effect of LPS (5 μg/mL) and HG (25.5 mM) co-treatment in BAMs was explored. The results further showed that HG significantly enhanced the release of IL-1β, IL-6, and TNF-α caused by LPS in the supernatant (p < 0.01) and significantly increased the levels of RAGE, TLR4, MyD88, and NF-κB p65 mRNA and protein expression (p < 0.01). Pretreatment with FPS-ZM1 and TAK-242, the inhibitors of RAGE and TLR4, significantly alleviated the HG + LPS-induced increment of RAGE, TLR4, MyD88, and NF-κB p65 mRNA and protein expression in the presence of HG and LPS (p < 0.01). This study showed that RAGE and TLR4 affect each other's expression through crosstalk during the combined usage of HG and LPS and synergistically activate the MyD88/NF-κB signaling pathway to promote the release of pro-inflammatory cytokines in BAMs.

Chimonanthus nitens Oliv. leaves flavonoids alleviate hyperuricemia by regulating uric acid metabolism and intestinal homeostasis in mice

This study aimed to evaluate the alleviating effect of Chimonanthus nitens Oliv. leaves flavonoids (CLF) on hyperuricemia induced by potassium oxonate in mice. The results showed that CLF lowered the serum levels of uric acid (UA), creatinine and blood urea nitrogen, downregulated hepatic mRNA expressions of xanthine oxidase (XO), phosphate ribose pyrophosphate synthetase (PRPS) and adenosine deaminase (ADA) in hyperuricemia mice. In addition, CLF repaired renal injury by significantly down-regulating mRNA and protein expressions of renal UA reabsorption-related proteins and up-regulating the mRNA and protein expressions of UA secretory-related proteins. Finally, CLF inhibited UA synthesis and promoted UA excretion to alleviate hyperuricemia. Besides, CLF supplementation repaired the intestinal barrier function as demonstrated by significant increased mRNA levels of intestinal zonula occludens-1 (ZO-1), Occludin, mucin 2 (MUC2) and mucin 4 (MUC4), as well as decreased mRNA levels of toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in mice. Further research showed that CLF treatment restored intestinal homeostasis mediated by improving the composition of gut microbiota and elevating the abundance of beneficial bacteria like Lactobacillus, Alistipes, Prevotellaceae_UCG-001 and Parasutterella. Overall, our findings revealed a novel function of CLF as a promising therapeutic candidate for the treatment of hyperuricemia.

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.

Increases in Pro-inflammatory Cytokines in the Ventral Striatum Following Frequent Morphine Exposure Impose New Setpoints in the Expression of Biologically-Relevant Genes to Addiction and Neuroinflammation in Rats

Background: The ventral striatum is an integrated drug reward and addiction center. Objectives: We aimed to examine changes in pro-inflammatory cytokines and subsequent changes in the expression of biologically-relevant genes to addiction and neuroinflammation in the ventral striatum in response to frequent morphine exposure. Methods: Sixteen male Wistar rats were divided into two experimental groups, a control, and a morphine-treated group, receiving eight days of twice-daily injections of either saline or morphine sulfate (10 mg/kg). Results: The results revealed that frequent morphine treatment increased both gene and protein levels of pro-inflammatory cytokines, including tumor necrosis factor α, interleukin 1-β, and interleukin 6 in the ventral striatum. Frequent morphine treatment also induced significant upregulations in the mRNA levels of mu-opioid receptor, dopamine D1 receptor (Drd1), Fos, nuclear factor- kappa B, and pre-miRNAs expression including, mir-124, mir-133b, mir-339, mir-365, and Let-7c1 in the ventral striatum. On the contrary, frequent morphine injection significantly downregulated mRNA levels of toll-like receptor 4, cannabinoid CB1 and CB2 receptors, Drd2, Il1r, Il6r, tnfr, protein kinase Cγ, calcium/calmodulin-dependent protein kinase IIα, nitric oxide synthase, cAMP-response element-binding protein as well as p38 and Jnk3 MAP kinases in the ventral striatum. However, no group differences were detected in the expression of Erk1 and mir-219 in the ventral striatum. Conclusions: It can be concluded that dysregulations in pro-inflammatory cytokines and, subsequently, in the downstream signaling pathways impair physiological functions of the ventral striatum following chronic morphine exposure, affecting reward pathways and the expression of morphine tolerance and dependence.

Bushen Wenyang Huayu Decoction Targets TLR4/NF-κB Mediated Autophagy to Treat Endometriosis Effectively.

Endometriosis has been found to be closely related to autophagy. This study aimed to elucidate the possible mechanism of Bushen Wenyang Huayu Decoction (BWHD) in treating endometriosis (EMs) by targeting TLR4/NF-κB-mediated autophagy. Autologous grafting was used to generate the EMs model in rats. Once the model was developed, BWHD high-dose and low-dose groups received intragastric administration of BWHD, and the gestrinone group served as a positive control. Immunofluorescence labeling and Western blotting were used for the protein expression of toll-like receptor 4 (TLR4), nuclear transcription factor-κB (NF-κB), Beclin-1, and selective autophagy connector protein P62 (P62). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze mRNA levels of TLR4, NF-κB, Beclin-1, and P62. We found that BWHD significantly reduced the size of ectopic lesions in rats with EMs, regulated reproductive hormone levels, and alleviated the cell autophagy level. It suggested that BWHD could be an effective treatment of EMs by targeting TLR4/NF-κB signaling pathway.

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

Sini Decoction inhibits TLR4/NF-κB signaling pathway to improve airway remodeling of allergic asthmatic mice

This study aims to explore the effect of Sini Decoction on Toll-like receptor 4(TLR4)/nuclear factor-κB(NF-κB) signaling pathway in the mice with allergic asthma(AA). Forty-eight SPF-grade BALB/c mice were randomly assigned into a blank control group, a model group, a dexamethasone group, and high-, medium-, and low-dose Sini Decoction groups, with 8 mice in each group. The sensitization solution made of ovalbumin and aluminum hydroxide powder was injected intraperitoneally in other groups except the blank control group which was injected with an equal volume of normal saline. The solution(or normal saline) was injected three times in total with an interval of 7 days. At the same time of sensitization, external cold stimulation and ice water were administered in a 4 ℃ climate box for 20 min every day. After modeling, the mice in each group were administrated with corresponding drugs by gavage for 3 weeks. At the end of administration, pentobarbital sodium(30 mg·kg~(-1)) was used for anesthesia, and then the samples were collected for the determination of various indexes. The phenol red test was conducted to evaluate tracheal excretion function. The histopathological changes of lung tissue were observed via hematoxylin-eosin(HE) staining. Masson staining was employed to reveal the deposition of blue collagen fibers around bronchi in lung tissue and the area occupied by blue collagen fibers was calculated. Immunofluorescence method was used to measure the expression of bronchial type Ⅰ collagen(Col-Ⅰ) and α-smooth muscle actin(α-SMA). The protein and mRNA levels of TLR4, NF-κB, cysteinyl aspartate specific proteinase-1(caspase-1), and interleukin-13(IL-13) were determined by Western blot and real-time fluorescence quantitative polymerase chain reaction(real-time PCR), respectively. Compared with the model group, Sini Decoction significantly increased the phenol red excretion from trachea, lowered the lung inflammation score, reduced subepithelial collagen deposition, and decreased Col-Ⅰ and α-SMA levels. Furthermore, the decoction down-regulated the protein and mRNA levels of TLR4, NF-κB, caspase-1, and IL-13 in mouse lung tissue. In conclusion, Sini Decoction can improve air remodeling by inhibiting the TLR4/NF-κB signaling pathway.

Therapeutic Effect of Artesunate on Influenza A Viral Pneumonia

To investigate the therapeutic effect of artesunate (ART) on influenza A viral pneumonia. A total of 36 mice were evenly and randomly assigned to six groups, a normal control group (C group), a solvent control group (M group, 10% DMSO), a positive drug group (P group, oseltamivir, 1.25 mg/kg/day), ART high-dose group (ART-G group, 120 mg/kg/day), ART medium-dose group (ART-Z group, 60 mg/kg/day), and ART low-dose group (ART-D group, 30 mg/kg/day). Except for group C, which did not receive any influenza A virus intervention or intraperitoneal injection, mice in the five other groups were infected with influenza A virus through intranasal drip. Then, after 12 hours, mice in the five other groups received intraperitoneal injection of the assigned drugs and dosage once a day. The signs, body weight, and survival of the mice were observed over the course of treatment. After 7 days of treatment, the lung tissue of the mice was collected and weighed, and the lung index was calculated accordingly. HE staining was performed to observe the pathological changes in the lung tissue. The mRNA and protein expression levels of Toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB [p65]), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and IL-1β were examined with RT-qPCR and Western blot, respectively. Compared with those in C group, mice in the M group had worse physical signs and lower body mass and survival, increased lung index, severe pathological changes in lung tissue, and increased levels of TLR4, NF-κB (p65), TNF-α, IL-6 and IL-1β mRNA and protein expression in their lung tissue ( P<0.05). Compared with those in M group, the mice in the ART groups had better physical signs, higher body mass and survival rate, decreased lung index, improvement of pathological changes in the lung tissue, and decreased levels of level of TLR4, NF-κB (p65), TNF-α, IL-6 and IL-1β mRNA and protein expression in the lung tissue ( P<0.05). Furthermore, the most prominent changes in these indexes were observed in the ART-G group. ART has therapeutic effects on influenza A viral pneumonia, and the mechanisms are related to the inhibition of TLR4/p65 signaling pathway activation and anti-inflammation.

Effect of Berberine on Activation of TLR4-NFκB Signaling Pathway and NLRP3 Inflammasome in Patients with Gout.

To determine the effects of berberine (BBR) on the activation of toll-like receptor 4 (TLR4), nuclear factor (NF)κB (NF-κB) signaling and NLRP3 inflammasome in patients with gout. Peripheral blood mononuclear cells (PBMCs) were obtained from 24 acute (AP) and 41 non-acute (NAP) phases of primary gout patients, respectively, as well as 30 healthy controls (HC). TLR4, NF-κB (p65), NLRP3, apoptosis-associated specklike protein containing a CARD (PYCARD), cysteinyl aspartate specific proteinase-1 (CASP1), and interleukin-1β (IL-1β) mRNA expression levels in PBMCs were measured by quantitative reverse transcriptase polymerase chain reaction. The protein levels of TLR4, myeloid differentiation factor 88 (MyD88), NF-κB (p50/65), inhibitor of kappa B kinase α/β (IKKα/β), NF-κB inhibitor α (IKBα), phospho-IKKα/β (p-IKKα/β), NLRP3, PYCARD, and CASP1 were monitored by Western blotting. Serum IL-1β protein level was measured using enzyme-linked immunosorbent assay (ELISA). In addition, PBMCs from HC and macrophages derived from a spontaneously immortalized monocyte-like cell line (THP-1) were stimulated using monosodium urate (MSU, 100 µg/mL), 0.1% dimethyl sulfoxide, 25 µmol/L BBR, and 10, 25, and 50 µmol/L BBR+100 µg/mL MSU for different time periods. The protein levels of IL-1β and IL-18 in cell culture supernatants was measured by ELISA, and the protein expressions of TLR4, MyD88, NF-κB (p50/p65), IKKα/β, I κBβ, p-IKKα/β, NLRP3, PYCARD, and CASP1 in macrophages were analyzed by Western blotting. (1) TLR4, NF-κB (p65), PYCARD, CASP1, and IL-1β mRNA levels in PBMCs were significantly higher in the AP group than in the HC group (P<0.05). The NLRP3 mRNA expression levels in PBMCs were found to be significantly lower in the AP and NAP groups than in the HC group (P<0.05, P<0.01). (2) The protein levels of TLR4, IKKβ, MyD88, NF-κB, p-IKKα/β, PYCARD, and CASP1 in PBMCs were significantly higher, and those of IκBα, IKKα, and NLRP3 were found to be significantly lower in the AP group than in the HC group (P<0.05 or P<0.01). (3) The serum IL-1β protein levels were significantly higher in the AP and NAP groups than in the HC group (P<0.01). (4) The IL-1β protein level was significantly lower in the culture supernatants of the PBMCs stimulated with MSU for 3 and 6 h in the 25 and 50 µmoL/L BBR groups compared with that in the MSU group (P<0.01). (5) The protein levels of IL-1β and IL-18 were also significantly lower in the culture supernatants of macrophages stimulated with MSU for 3 and 6 h in BBR groups compared with those in the MSU group (P<0.01). (6) The protein levels of TLR4, MyD88, NF-κB (p50, p65, p105), IKKα/β, p-IκBα, p-IKKα/β, PYCARD, and CASP1 were significantly differed between the macrophages stimulated with MSU for 0.5 and 6 h in BBR groups compared with those in the MSU group (P<0.05 or P<0.01). Activation of TLR4-NFκB signaling and NLRP3 inflammasome by MSU crystals drives the progression of gout inflammation. BBR ameliorates gouty inflammation, which is mechanistically associated with its regulation of TLR4-NF-κB signaling and NLRP3 inflammasome expression.

Immunolocalization of antibacterial peptide S100A7 in mastitis goat mammary gland and lipopolysaccharide induces the expression and secretion of S100A7 in goat mammary gland epithelial cells via TLR4/NFκB signal pathway

The antimicrobial peptide S100A7, with antimicrobial activities for a broad spectrum of bacteria, has attracted more and more attention for the prevention and treatment of mastitis. However, there is little information about the expression and regulation mechanism of S100A7 in mastitis goats. This study revealed that S100A7 was mainly expressed in the stratified squamous epithelium of teat skin and streak canal, and S100A7 was present weakly in the healthy goat alveolus yet densely in the mastitis goat collapsed alveolus. Goat mammary epithelial cells (MECs) were isolated and treated with 2.5, 5, 10 and 20 µg/mL lipopolysaccharide (LPS) respectively for a different time, S100A7 mRNA expression and protein secretion were upregulated significantly with LPS treatment for 3 h, and the secretion level of S100A7 descended after 48 h treatment for all of these four groups. Moreover, after treatment with LPS, the mRNA levels of Toll-like receptor 4 (TLR4) and MyD88 were up-regulated, and the phosphorylation of p65 was up-regulated markedly. However, adding TLR4 inhibitor TAK-242 or/and NF-κB inhibitor QNZ significantly suppressed the phosphorylation of p65, and then inhibited the expression and secretion of S100A7 induced by LPS treatment. In conclusion, LPS induced the expression and secretion of S100A7 in goat MECs via TLR4/NF-κB signaling pathway.

Remnant Cholesterol and Dyslipidemia Are Risk Factors for Guillain-Barré Syndrome and Severe Guillain-Barré Syndrome by Promoting Monocyte Activation.

BackgroundGuillain–Barré syndrome (GBS) is the most common severe acute paralytic neuropathy, with a mortality rate of 5% and permanent sequelae rate of 10%. Currently, the cause of GBS remains unclear. Therefore, we sought to determine potential predictors for GBS and its severity.MethodsA case–control study was performed at Tiantan Hospital in Beijing from January 2017 to December 2021. Laboratory and clinical characteristics were assessed in recruited GBS patients and healthy control individuals (matched by sex and age). The potential risk factors for GBS and severe GBS were assessed using a logistic regression analysis. The mRNA levels of toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2) and nuclear factor κB (NF-κB) in GBS patients and control PBMCs were detected by fluorescence quantitative PCR. THP-1 cells were costimulated with LPS and free cholesterol to demonstrate the effect of free cholesterol on monocyte activation.ResultsA total of 147 GBS patients and 153 healthy individuals were included in the study. Logistic regression analyses showed that preceding infection, alcohol consumption, remnant cholesterol, homocysteine and the dyslipidemia index were correlated with a higher risk of GBS. In contrast, increased HDL cholesterol was correlated with a lower risk of GBS. Moreover, remnant cholesterol and the dyslipidemia index were significantly correlated with severe GBS. The mRNA levels of TLR4, TLR2 and NF-κB in the PBMCs of GBS patients were significantly higher than those of healthy individuals. LPS activated THP-1 cells, and free cholesterol treatment increased the expression of TLR4, TLR2, NF-κB and IL-1β mRNA in LPS-activated THP-1 cells.ConclusionDyslipidemia was correlated with the risk of GBS and severe GBS. Remnant cholesterol may promote the activation of monocytes in GBS patients. It may be valuable to control lipid levels in the prevention of GBS and severe GBS.

Interferon-γ enhances the immunosuppressive ability of canine bone marrow-derived mesenchymal stem cells by activating the TLR3-dependent IDO/kynurenine pathway.

The immunomodulatory function of mesenchymal stem cells (MSCs) has been considered to be vital for MSC-based therapies. Many works have been devoted to excavate effective strategies for enhancing the immunomodulation effect of MSCs. Nonetheless, canine MSC-mediated immunomodulation is still poorly understood. The inflammatory microenvironment was simulated through the employment of interferon-γ (IFN-γ) in a culture system. Compared with unstimulated cBMSCs, IFN-γ stimulation increased the mRNA levels of Toll-like receptor 3 (TLR3) and indoleamine 2, 3-dioxygenase 1 (IDO-1), and simultaneously enhanced the secretion of immunosuppressive molecules, including interleukin (IL)-10, hepatocyte growth factor (HGF), and kynurenine in cBMSCs. IFN-γ stimulation significantly enhanced the ability of cBMSCs and their supernatant to suppress the proliferation of murine spleen lymphocytes. Lymphocyte subtyping evaluation revealed that cBMSCs and their supernatant diminished the percentage of CD3+CD4+ and CD3+CD8+ lymphocytes compared with the control group, with a decreasing CD4+/CD8+ ratio. Notably, exposure to IFN-γ decreased the CD4+/CD8+ ratio more effectively than unstimulated cells or supernatant. Additionally, IFN-γ-stimulation increased the mRNA levels of the Th1 cytokines TNF-α, and remarkably decreased the mRNA level of the Th2 cytokine IL-4 and IL-10. Our findings substantiate that IFN-γ stimulation can enhance the immunomodulatory properties of cBMSCs by promoting TLR3-dependent activation of the IDO/kynurenine pathway, increasing the secretion of immunoregulatory molecules and strengthening interactions with T lymphocytes, which may provide a meaningful strategy for the clinical application of cBMSCs in immune-related diseases.

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis

PurposeTo explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. ResultsCS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. ConclusionCS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

Ethyl Pyruvate Alleviating Inflammatory Response after Diabetic Cerebral Hemorrhage.

This study's purpose is to investigate the neuroprotective role of ethyl pyruvate (EP) in the pathogenesis of diabetic intracerebral hemorrhage. The present study used a mouse model of collagenase-induced intracerebral hemorrhage (ICH) and streptozotocin-induced diabetes. The C57BL/6 mice were randomly divided into 3 groups: sham operation, diabetic cerebral hemorrhage, and diabetic cerebral hemorrhage with EP. The EP (80 mg/kg) and EP (50 mg/kg) were injected intraperitoneally one day and one hour before modeling. The protein expression levels of high mobility group box 1 (HMGB1) and NOD-like receptors 3 (NLRP3) were detected with western blot. The mRNA levels of HMGB1 and toll-like receptor 4 (TLR4) were measured by quantitative real-time polymerase chain reaction (PCR). Immunofluorescence and ELISA were performed to confirm some inflammatory factors. Compared to the normal diabetic intracerebral hemorrhage group, the mRNA and protein expression levels of HMGB1 and TLR4 were downregulated in the EP-affected group with diabetic cerebral hemorrhage, together with the downregulation of the expression of inflammasomes, including NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), and caspase 1. EP can reduce the inflammatory response after diabetic intracerebral hemorrhage and may inhibit the activation of inflammasomes by the HMGB1/TLR4 pathway.