Toll-like Receptor 4 mRNA Expression Research Articles

Rumen-protected l-carnitine supplementation during mating period altered metabolic status and reproductive performance of ewes.

Current study hypothesized that dietary l-carnitine (LC) inclusion during the mating period ameliorates both metabolic status and reproductive performance of ewes. Seventy Baluchi ewes (52 ± 4.2 kg of bodyweight and 18 ± 6 months old of age) were enrolled in this study. Animals were randomly allocated into two dietary treatments, control (only basal diet) or basal diet plus supplementation with a rumen-protected LC (Carneon 20 Rumin-pro; 20% LC; Kaesler Nutrition GmbH) at the rate of 10 g/head/day from 21 days before until 35 days after introducing rams to the ewes (MP). Feed intake was monitored by subtracting the ort from feed offered. Blood sample collection was conducted on Days -10, +10 and +20 relative to MP. Pregnancy was confirmed on Day 30 post-MP. Feed intake of the ewes in the LC group was higher than the control (p < 0.05). LC supplementation increased the cholesterol concentration in the ewes (p < 0.05). Blood urea concentration of animals in the LC group was significantly lower than the control (p < 0.05). The mRNA expression of toll-like receptor 4 was evidently lower in animals supplemented with LC than the control (p < 0.05). Both lambing and fecundity rates in the LC group tended to be higher compared with the control. LC supplementation showed potential to alter certain metabolites in the ewes. A tendency for higher lambing rate may partly be driven by dams efficient energy partitioning to support foetal growth and maintaining pregnancy.

Macrophage specific restoration of the Nrf2 gene in whole-body knockout mice ameliorates steatohepatitis induced by lipopolysaccharide from Porphyromonas gingivalis through enhanced hepatic clearance.

Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (P.g.), which causes periodontal disease, contributes to the development of non-alcoholic steatohepatitis (NASH). We investigated the role of Nrf2, an antioxidative stress sensor, in macrophages in the development of NASH induced by LPS from P.g. We generated macrophage-specific Nrf2 gene rescue mice (Nrf2-mRes), which express Nrf2 only in macrophages, using the cre/loxp system. Wild-type (WT) mice, whole body Nrf2-knockout (Nrf2-KO) mice, and Nrf2-mRes mice were fed a high-fat diet for 18 weeks, and LPS from P.g. was administered intraperitoneally for the last 6 weeks. Nrf2-KO mice developed severe steatohepatitis with liver inflammation and fibrosis compared with WT mice, and steatohepatitis was ameliorated in Nrf2-mRes mice. The mRNA expressions of Toll-like receptor (Tlr)-2, which activates inflammatory signaling pathways after LPS binding, and α-smooth muscle actin (αSma), which promotes hepatic fibrosis, were reduced in Nrf2-mRes mice compared with Nrf2-KO mice. The protein levels of LPS-binding protein in livers were increased in Nrf2-KO mice compared with WT mice; however, the levels were reduced in Nrf2-mRes mice despite similar numbers of F4/80 positive cells, which reflect macrophage/Kupffer cell infiltration into the livers. Nrf2 in macrophages ameliorates NASH through the increased hepatic clearance of LPS.

Fatigue, Toll-Like Receptor 4, and Pro-Inflammatory Cytokines in Adults With Subarachnoid Hemorrhage: A 6-Month Longitudinal Study.

Fatigue is prevalent in subarachnoid hemorrhage (SAH) survivors. Biological mechanisms underlying fatigue post-SAH are not clear. Inflammation may contribute to the development of fatigue. This study aimed to examine the associations between inflammatory markers and fatigue during the first 6months post-SAH. Specific biomarkers examined included both early and concurrent expression of Toll-Like Receptor 4 (TLR4) messenger RNA (mRNA) and plasma concentrations of pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNF-α), Interleukin (IL)1β, and IL6. We conducted a 6-month longitudinal study with a convenience sample of 43 SAH survivors. We collected blood samples on days 2, 3, and 7 and 2, 3, and 6months post-SAH to assess biomarkers. Fatigue was assessed by the PROMIS Fatigue Scale at 2, 3, and 6months. Linear mixed models were used to test the associations between early (days 2, 3, and 7) and concurrent (2, 3, and 6months) TLR4 mRNA expression (TagMan gene expression assays) and TNF-α, IL1β, and IL6 plasma concentrations (multiplex assays) and concurrent fatigue. 28% of SAH survivors experienced fatigue during the first 6months post-SAH. Fatigue levels in SAH survivors were higher than those of the U.S. population and consistent during the 6months. Experience of fatigue during the 6months post-SAH was associated with higher IL1β plasma concentrations on day 7 and IL1β, IL6, and TNF-α plasma concentrations during the 6months post-SAH. Inflammation appears to underlie the development of fatigue in SAH survivors.

Genomic DNA Extracted from Lactiplantibacillus plantarum Attenuates Porphyromonas gingivalis Lipopolysaccharide (LPS)-Induced Inflammatory Responses via Suppression of Toll-Like Receptor (TLR)-Mediated Mitogen-Activated Protein Kinase (MAPK) and Nuclear Factor-κB (NF-κB) Signaling Pathways.

In the present study, we aimed to examine the inhibition of genomic DNA from Lactiplantibacillus plantarum (LpDNA) on Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammatory responses in RAW264.7 cells. Pretreatment with LpDNA for 15 h significantly inhibited PgLPS-induced mRNA expression and protein secretion of interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein-1. LpDNA pretreatment also reduced the mRNA expression of Toll-like receptor (TLR)2 and TLR4. Furthermore, LpDNA inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and the activation of nuclear factor-κB (NF-κB) induced by PgLPS. Taken together, these findings demonstrate that LpDNA attenuates PgLPS-induced inflammatory responses by regulating MAPKs and NF-κB signaling pathways through the suppression of TLR2 and TLR4 expression.

Dietary resveratrol supplementation on growth performance, immune function and intestinal barrier function in broilers challenged with lipopolysaccharide

This study discusses the effects of resveratrol (RES) on the productive performance, immune function and intestinal barrier function of broiler chickens challenged with lipopolysaccharide (LPS). Two hundred and forty 1-day-old male Arbor Acres broilers were randomly divided into 4 groups of 6 replicates each, with 10 broilers per replicate. This experiment used a 2×2 factorial design with dietary factors (basal diets or basal diets supplemented with 400 mg/kg RES were administered from d 1 to 21) and stress factors (intraperitoneal injection of 0.5 mg/kg BW of saline or LPS at 16, 18 and 20 d of age). The results showed that LPS challenge had a significant adverse effect on average daily gain (ADG) in broilers at 16 to 21 d of age (P < 0.05), whereas the addition of RES to the diet inhibited the LPS-induced decrease in ADG (P < 0.05). RES also alleviated LPS-induced immune function damage in broilers, which was manifested by the decrease of spleen index (P < 0.05) and the recovery of serum immunoglobulin M and ileal secretory immunoglobulin A content (P < 0.05). The LPS challenge also disrupts intestinal barrier function and inflammation, and RES mitigates these adverse effects in different ways. RES attenuated LPS-induced reduction of villus height in the jejunum and ileum of broilers (P < 0.05). LPS also caused an abnormal increase in plasma D-lactic acid levels in broilers (P < 0.05), which was effectively mitigated by RES (P < 0.05). LPS challenge resulted in a significant decrease in mRNA expression of occludin in the intestinal mucosa (P < 0.05), which was mitigated by the addition of RES (P < 0.05). RES significantly decreased the mRNA expression of toll-like receptor 4, nuclear factor kappa-B and tumor necrosis factor alpha in the ileum tissue stimulated by LPS (P < 0.05). Taken together, this study shows that RES exerts its beneficial effect on broilers challenged with LPS by alleviating immune function damage, relieving intestinal inflammation and barrier damage, and thus improving growth performance.

Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-β1(TGF-β1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-β1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Muscle fiber characteristics and expression level of Troponin T3, Toll-like receptor 2, and Toll-like receptor 4 genes in chicken meat with white striping.

The poultry industry faces an emerging muscular defect in chicken meat called white striping (WS). The biological processes associated with WS myopathy are immune system activation, angiogenesis, hypoxia, cell death, and striated muscle contraction. We examined the Troponin T3 (TNNT3), Toll-like receptor 2 (TLR2), and Toll-like receptor 4 (TLR4) genes based on their functions related to muscle contraction and the innate immune system. This study aimed to determine the muscle fiber characteristics (MFCs) and expression level of TNNT3, TLR2, and TLR4 genes in white striping chicken meat (WSCM). A total of 428 breast samples were randomly collected from a commercial poultry processing plant. The samples were classified into four levels: 0 (normal), 1 (moderate WS), 2 (severe WS), and 3 (extreme WS). Five samples per group were selected to evaluate MFCs, including total number of muscle fibers, muscle fiber diameter, cross-sectional area, endomysium thickness, and perimysium thickness. Five samples per group were selected for ribonucleic acid (RNA) isolation to evaluate the messenger RNA (mRNA) expression levels of TNNT3, TLR2, and TLR4 genes related to WS. Statistical analysis revealed that the total number of fibers, endomysium thickness, and perimysium thickness significantly differed between groups (p < 0.05). Muscle fiber diameter and cross-sectional area did not significantly differ (p > 0.05). The expression of the TNNT3 gene did not significantly differ among groups (p > 0.05). Toll-like receptor 2 and TLR4 mRNA expression significantly differed among groups (p < 0.05). These detailed MFCs will provide baseline information to observe WS in chicken meat. Toll-like receptor 2 and TLR4 genes may play a role in the occurrence of WS in chicken meat through non-specific immune reactions.

Arjunolic acid modulate pancreatic dysfunction by ameliorating pattern recognition receptor and canonical Wnt pathway activation in type 2 diabetic rats

BackgroundArjunolic acid (AA) is a potent phytochemical with multiple therapeutics effects. In this study, AA is evaluated on type 2 diabetic (T2DM) rats to understand the mechanism of β-cell linkage with Toll-like receptor 4 (TLR-4) and canonical Wnt signaling. However, its role in modulating TLR-4 and canonical Wnt/β-catenin crosstalk on insulin signaling remains unclear during T2DM.AimThe current study is aimed to examine the potential role of AA on insulin signaling and TLR-4-Wnt crosstalk in the pancreas of type 2 diabetic rats. MethodMultiple methods were used to determine molecular cognizance of AA in T2DM rats, when treated with different dosage levels. Histopathological and histomorphometry analysis was conducted using masson trichrome and H&E stains. While, protein and mRNA expressions of TLR-4/Wnt and insulin signaling were assessed using automated Western blotting (jess), immunohistochemistry, and RT-PCR. ResultsHistopathological findings revealed that AA had reversed back the T2DM-induced apoptosis and necrosis caused to rats pancreas. Molecular findings exhibited prominent effects of AA in downregulating the elevated level of TLR-4, MyD88, NF-κB, p-JNK, and Wnt/β-catenin by blocking TLR-4/MyD88 and canonical Wnt signaling in diabetic pancreas, while IRS-1, PI3K, and pAkt were all upregulated by altering the NF-κB and β-catenin crosstalk during T2DM. ConclusionOverall results, indicate that AA has potential to develop as an effective therapeutic in the treatment of T2DM associated meta-inflammation. However, future preclinical research at multiple dose level in a long-term chronic T2DM disease model is warranted to understand its clinical relevance in cardiometabolic disease.

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.

Sperm activate TLR2/TLR1 heterodimerization to induce a weak proinflammatory response in the bovine uterus.

Toll-like receptor 2 (TLR2) signaling pathway is involved in the sperm-triggered uterine inflammatory response at insemination, but its precise mechanism at molecular-level remains unknown. According to the ligand specificity, TLR2 forms a heterodimer with TLR1 or TLR6 as an initial step to mediate intracellular signaling, leading to a specific type of immune response. Hence, the present study aimed to identify the active TLR2 heterodimer (TLR2/1 or TLR2/6) that is involved in sperm-uterine immune crosstalk in bovine using various models. First, in-vitro (bovine endometrial epithelial cells, BEECs) and ex-vivo (bovine uterine explant) models were employed to test different TLR2 dimerization pathways in endometrial epithelia after exposure to sperm or TLR2 agonists; PAM3 (TLR2/1 agonist), and PAM2 (TLR2/6 agonist). Additionally, in-silico approaches were performed to confirm the dimer stability using de novo protein structure prediction model for bovine TLRs. The in-vitro approach revealed that sperm triggered the mRNA and protein expression of TLR1 and TLR2 but not TLR6 in BEECs. Moreover, this model disclosed that activation of TLR2/6 heterodimer, triggers a much stronger inflammatory response than TLR2/1 and sperm in bovine uterine epithelia. In the ex-vivo model that mimics the intact uterine tissue at insemination, sperm also induced the protein expression of both TLR1 and TLR2, but not TLR6, in bovine endometrium, particularly in uterine glands. Importantly, PAM3 and sperm induced similar and low mRNA expression of pro-inflammatory cytokines and TNFA protein to a lesser extent than PAM2 in endometrial epithelia. This implied that sperm might trigger a weak inflammatory response via TLR2/TLR1 activation which is similar to that of PAM3. Additionally, the in-silico analyses showed that the existence of bridging ligands is essential for heterodimer stability in bovine TLR2 with either TLR1 or TLR6. Altogether, the present findings revealed that sperm utilize TLR2/1, but not TLR2/6, heterodimerization to trigger a weak physiological inflammatory response in the bovine uterus. This might be the way to remove excess dead sperm remaining in the uterine lumen without tissue damage for providing an ideal uterine environment for early embryo reception and implantation.

Differential innate immune response of endometrial cells to porcine reproductive and respiratory syndrome virus type 1 versus type 2.

Modification of cellular and immunological events due to porcine reproductive and respiratory syndrome virus (PRRSV) infection is associated with pathogenesis in lungs. PRRSV also causes female reproductive dysfunction and persistent infection which can spread to fetus, stillbirth, and offspring. In this study, changes in cellular and innate immune responses to PRRSV type 1 or type 2 infection, including expression of PRRSV mediators, mRNA expression of Toll-like receptors (TLRs) and cytokine, and cytokine secretion, were examined in primary porcine glandular endometrial cells (PGE). Cell infectivity as observed by cytopathic effect (CPE), PRRSV nucleocapsid proteins, and viral nucleic acids was detected as early as two days post-infection (2 dpi) and persisted until 6 dpi. A higher percentage of CPE and PRRSV-positive cells were observed in type 2 infections. PRRSV mediator proteins, CD151, CD163, sialoadhesin (Sn), integrin and vimentin, were upregulated following type 1 and type 2 infection. CD151, CD163 and Sn were upregulated by type 2. In both PRRSV types, mRNA expression of TLR1 and TLR6 was upregulated. However, TLR3 was upregulated by type 1, but TLR4 and TLR8 mRNA and protein were downregulated by type 2 only. Interleukin (IL)-1β, IL-6 and tumor necrotic factor (TNF)-α were upregulated by type 2, but IL-8 was upregulated by type 1. Both PRRSV type 1 and 2 stimulated IL-6 but suppressed TNF-α secretion. In addition, IL-1β secretion was suppressed only by type 2. These findings reveal an important mechanism underlying the strategy of PRRSV infection in the endometrium and associated with the viral persistence.

Lambda-cyhalothrin enhances inflammation in nigrostriatal region in rats: Regulatory role of NF-κβ and JAK-STAT signaling

The risk to develop neurobehavioural abnormalities in humans on exposure to lambda-cyhalothrin (LCT) - a type II synthetic pyrethroid has enhanced significantly due to its extensive uses in agriculture, homes, veterinary practices and public health programs. Earlier, we found that the brain dopaminergic system is vulnerable to LCT and affects motor functions in rats. In continuation to this, the present study is focused to unravel the role of neuroinflammation in LCT-induced neurotoxicity in substantia nigra and corpus striatum in rats. Increase in the mRNA expression of proinflammatory cytokines (TNF- α, IL-1β, IL-6) and iNOS whereas decrease in anti-inflammatory cytokine (IL-10) was distinct both in substantia nigra and corpus striatum of rats treated with LCT (0.5, 1.0, 3.0 mg/kg body weight, p.o, for 45 days) as compared to control rats. Further, LCT-treated rats exhibited increased levels of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba-1), the glial marker proteins both in substantia nigra and corpus striatum as compared to controls. Exposure of rats to LCT also caused alterations in the levels of heat shock protein 60 (HSP60) and mRNA expression of toll-like receptors (TLR2 and TLR4) in the substantia nigra and corpus striatum. An increase in the phosphorylation of key proteins involved in NF-kβ (P65, Iκβ, IKKα, IKKβ) and JAK/STAT (STAT1, STAT3) signaling and alteration in the protein levels of JAK1 and JAK2 was prominent in LCT-treated rats. Histological studies revealed damage of dopaminergic neurons and reactive gliosis as evidenced by the presence of darkly stained pyknotic neurons and decrease in Nissl substance and an increase in infiltration of immune cells both in substantia nigra and corpus striatum of LCT-treated rats. Presence of reactive microglia and astrocytes in LCT-treated rats was also distinct in ultrastructural studies. The results exhibit that LCT may damage dopaminergic neurons in the substantia nigra and corpus striatum by inducing inflammation as a result of stimulation of neuroglial cells involving activation of NF-κβ and JAK/STAT signaling.

Rhoifolin protects cecal ligation and puncture induced sepsis mice model by regulating inflammatory pathway

Sepsis is a systemic infection affects several organs, which needs novel therapy for the management of it, thus protective effect of Rhoifolin was estimated against sepsis. Cecal ligation and puncture (CLP) method was used to induce sepsis and thereafter mice were treated with rhoifolin (20 and 40 mg/kg, i.p.) for one week. Food intake and survival rate was determined sepsis mice, moreover liver function test and cytokines was estimated in the serum of sepsis mice. In the lung tissue homogenate, oxidative stress parameters were determined, histopathological analysis was performed in liver and lung tissue of sepsis mice. Food intake and percentage of survival was improved in rhoifolin treated group than sham group. Level of liver function enzyme and cytokine was reduced significantly in the serum of rhoifolin treated sepsis mice. Treatment with rhoifolin ameliorates the altered oxidative stress parameters, and mRNA expression of Toll-like receptor 4 (TLR-4) in lung tissue of sepsis mice. Histopathological changes were also reverse in rhoifolin treated group than sham group of mice. In conclusion, result of report indicates Rhoifolin treatment reduces oxidative stress and inflammation in CLP induced sepsis mice, as it regulates TLR4/MyD88/NF-κB pathway.

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.

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.

Atorvastatin reduces contrast media-induced pyroptosis of renal tubular epithelial cells by inhibiting the TLR4/MyD88/NF-κB signaling pathway

BackgroundContrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure. However, there is no effective treatment of CI-AKI, and its mechanism is unknown. Interestingly, atorvastatin has been reported to be effective in renal injury. Therefore, the aim of this study was to explore the effect and possible molecular mechanism of atorvastatin in CI-AKI.MethodsOn the CI-AKI in vitro model, rat tubular epithelial cells (NRK-52E) were treated with 18 mg I/ml meglumine diatrizoate (MEG) and then pretreated with atorvastatin. pcDNA3.1-TLR4 treatment was performed to overexpress toll-like receptor 4 (TLR4) in NRK-52E cells. Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) kits were used to detect NRK-52E cell viability as well as LDH release in each group, respectively; qRT-PCR to determine mRNA expression of TLR4 in cells; western blot to detect protein expression levels of pyroptosis-related proteins (NLRP3, caspase-1, ASC, and GSDMD) and TLR4/MyD88/NF-κB signaling pathway-related proteins (TLR4, MyD88, NF-κBp65, and p-NF-κB p65) in cells.ResultsMEG treatment significantly inhibited the viability of NRK-52E cells, increased pro-inflammatory factor levels and promoted pyroptosis, representing successful establishment of a rat tubular epithelial cell (NRK-52E) CI-AKI in vitro model. Notably, atorvastatin increased the activity of MEG-treated NRK-52E cells and alleviated cell injury in a concentration-dependent manner. In addition, atorvastatin significantly down-regulated the expression of TLR4 in MEG-treated NRK-52E cells. However, overexpression of TLR4 inhibited the effects of atorvastatin on increasing cell viability, alleviating cell injury, reducing pro-inflammatory factors (IL-1β, IL-6, and TNF-α) levels, and inhibiting apoptosis (by down-regulating the expression of NLRP3, caspase-1, ASC, and GSDMD). Furthermore, atorvastatin also inhibited the expression of TLR4/MyD88/NF-κB pathway-related proteins (TLR4, MyD88, and p-NF-κB p65).ConclusionAtorvastatin can attenuate CI-AKI through increasing the activity of MEG-treated renal tubular epithelial cells, relieving cell injury, as well as inhibiting pyroptosis and inflammation. More importantly, the mechanism was achieved by inhibiting the TLR4//MyD88/NF-κB signaling pathway.

Nasopharyngeal epithelial cells from patients with coronavirus disease 2019 express abnormal levels of Toll-like receptors

ABSTRACT Aberrant activation of the immune system has been attributed with etiology and pathogenesis of coronavirus disease 2019 (COVID-19). Here, the transcript levels of toll-like receptors (TLRs) were measured in the nasopharyngeal epithelial cells obtained from COVID-19 patients to assess the involvement of these molecules in the clinical outcome of COVID-19 patients. Nasopharyngeal swab samples were used to obtain epithelial cells from 120 COVID-19 patients and 100 healthy controls. COVID-19 cases were classified into those having clinical symptoms/needing for hospitalization, having clinical symptoms/not needing for hospitalization, and those without clinical symptoms‌. The mRNA expression levels of TLRs were measured in the nasopharyngeal epithelial cells. Overall, mRNA expression of TLR1, TLR2, TLR4, and TLR6 was significantly higher in COVID-19 cases compared to controls. The mRNA expression of TLRs were all higher significantly in the samples from COVID-19 patients having clinical symptoms and needing hospitalization as well as in those with clinical symptoms/not needing for hospitalization in comparison to controls. TLR expression was significantly higher in those with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization compared to COVID-19 cases without clinical symptoms. In cases with clinical symptoms/needing for hospitalization and those with clinical symptoms/not needing for hospitalization, there was a correlation between TLR expression and clinicopathological findings. In conclusion, aberrant expression of TLRs in the nasopharyngeal epithelial cells from COVID-19 cases may predict the severity of the diseases and necessity for supportive cares in the hospital.

Ginsenoside Rg3 treats acute radiation proctitis through the TLR4/MyD88/NF-κB pathway and regulation of intestinal flora.

This study aimed to investigate the protective effect of ginsenoside Rg3 (GRg3) against acute radiation proctitis (ARP) in rats. Wistar rats were randomly divided into control, model, dexamethasone-positive, GRg3 low-dose, GRg3 medium-dose, and GRg3 high-dose groups. The ARP rat model was established by a single 22-Gy irradiation of 6 MV) X-rays. The distribution and function of intestinal flora were detected using 16S rRNA high-throughput sequencing, rectal tissue was observed by hematoxylin and eosin (H&E) staining, the expression of interleukin 1β (IL-1β) and IL-10 inflammatory factors was detected by ELISA, and mRNA and protein expression of toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were detected by RT-qPCR and Western blotting, respectively. GRg3 improved the symptoms of ARP in rats in a dose-dependent manner. The species distribution of intestinal flora in GRg3 rats was significantly different from that in ARP rats. These differences were more significant in the high-dose group, where the numbers of Ruminococcus, Lactobacillus, and other beneficial bacteria were significantly increased, whereas those of Escherichia, Alloprevotella, and other harmful bacteria were decreased. In addition, GRg3 was closely related to amino acid metabolism. After GRg3 treatment, the mRNA and protein expression of TLR4, MyD88, and NF-κB in rectal tissue was significantly down-regulated, and the level of downstream inflammatory factor IL-1β decreased, whereas that of IL-10 increased. Our study indicated GRg3 as a new compound for the treatment of ARP by inhibiting the TLR4/MyD88/NF-κB pathway, down-regulating the expression of proinflammatory factors, thus effectively regulating intestinal flora and reducing inflammatory reactions.

Effects of the Co-Administration of Morphine and Lipopolysaccharide on Toll-Like Receptor-4/Nuclear Factor Kappa β Signaling Pathway of MDA-MB-231 Breast Cancer Cells.

The Toll-like receptor 4 (TLR4) gene promotes migration in adenocarcinoma cells. Morphine is an agonist for TLR4 that has a dual role in cancer development. The promoter or inhibitor role of morphine in cancer progression remains controversial. This study aims to evaluate the effects of morphine on the TLR4, myeloid differentiation primary response protein 88-dependent (MyD88), and nuclear factor-kappa B (NF-κB) expressions in the human MDA-MB-231 breast cancer cell line. The cells were examined after 24 hours of incubation with morphine using the Boyden chamber system. TLR4, MyD88, and NF-κB mRNA expressions were assessed using quantitative real-time polymerase chain reaction (RT-PCR). The concentration of interleukin-2 beta was also measured using the ELISA assay. According to the findings, three doses of morphine (0.25, 1.25, and 0.025 μM) increased the expression of the TLR4 and NF-κB genes, whereas no significant change was observed in the mRNA expression of MyD88. Furthermore, treatment with morphine and lipopolysaccharide (LPS) significantly decreased the expression of TLR4, MyD88, and NF-κB. However, no significant change was observed in interleukin 2 beta concentration. These findings confirmed the excitatory effects of morphine on TRL4 expression and the MYD88 signaling pathway in vitro.

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.