TLR4 mRNA Levels Research Articles

Toll-like receptors and nuclear factor kappa B signaling pathway involvement in hepatorenal oxidative damage induced by some food preservatives in rats

Chemical food preservatives are extensively found in various processed food products in the human environment. Hence, this study aimed to investigate the effect of long-term exposure to five food preservatives (potassium sorbate (PS), butylated hydroxyanisole (BHA), sodium benzoate (SB), calcium propionate (CP), and boric acid (BA)) on the liver and kidney in rats and the probable underlying mechanisms. For 90 days, sixty male albino rats were orally given either water (control), 0.09 mg/kg b.wt BHA, 4.5 mg/kg b.wt PS, 0.9 mg/kg b.wt SB, 0.16 mg/kg b.wt BA, or 0.18 mg/kg b.wt CP. Liver and kidney function tests were assessed. Hepatic and renal oxidative stress biomarkers were estimated. Histologic examination analysis of liver and kidney tissues was achieved. Toll-like receptors 2 and 4 (TLR-2 and TLR-4), tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) mRNA expression levels were measured. The results revealed that long-term oral dosing of the five food preservatives resulted in significant increases in alkaline phosphatase, alanine transaminase, aspartate transaminase, urea, uric acid, and creatinine levels. There were significant reductions in hepatic and renal antioxidant enzymes, an increase in MDA concentrations, and pathological alterations in renal and hepatic tissues. The mRNA levels of TLR-4, TLR-2, NF-κB, and TNF-α were elevated in the food preservatives-exposed groups. Conclusively, the current findings revealed that long-term exposure to PS, BHA, SB, CP, and BA has a negative impact on liver and kidney function. Furthermore, these negative effects could be mediated via oxidative stress induction, inflammatory reactions, and cytokine production.

Dichotomine B Attenuates Neuroinflammatory Responses by Regulating TLR4/MyD88-mTOR Signaling Pathway in BV2 Cells.

Dichotomine B is a [Formula: see text]-Carboline alkaloid isolated from Stellariae Radix. Stellariae Radix, also known as Yin Chai Hu, is a common Chinese medicine in clinical practice. This herb has been demonstrated to have anti-inflammatory activity. This study aimed to investigate the effects and mechanisms of Dichotomine B on neuroinflammation by BV2 microglia induced by lipopolysaccharide (LPS) and adenosine triphosphate (ATP). The experiment was divided into a control group, a model group (10 µg/mL LPS + 5 mM ATP), a model+ TLR4 inhibitor (TAK-242, 10 µmol/L) group, model+ Dichotomine B (20, 40 and 80 µmol/L) groups and Dichotomine B (80 µmol/L) group. The BV2 cell viability was detected by MTT assay, the morphology of BV2 cells was observed by inverted microscope, and the levels of IL-6, IL-1[Formula: see text] and TNF-[Formula: see text] in BV2 cells were determined by ELISA. The expression levels of TLR4, MyD88, p-mTOR/mTOR, p62, p-RPS6/RPS6, LC3II/LC3I and Beclin-1 proteins were detected by western blot assay. The expression levels of TLR4, MyD88, mTOR, p62, RPS6, LC3B and Beclin-1 mRNA were detected by PCR assay. Finally, molecular docking was performed to predict the affinity of Dichotomine B with TLR4, MyD88 and mTOR by LibDock of Discovery Studio and MOE. The results showed that compared with the model group, the survival rates of damaged cells were significantly increased by TAK-242 and Dichotomine B, and the morphology of these BV2 cells improved. The levels of IL-6, IL-1[Formula: see text] and TNF-[Formula: see text] were significantly decreased by TAK-242 and Dichotomine B in LPS/ATP-induced BV2 cells. 80 µmol/L Dichotomine B has no effect on normal BV2 cells. Further mechanism investigation showed that TAK-242 and Dichotomine B significantly inhibited the protein and mRNA expression levels of TLR4, MyD88, p-mTOR/mTOR (mTOR), p62, p-RPS6/RPS6 (RPS6) and increased the protein and mRNA expression levels of LC3II/LC3I (LC3B), Beclin-1. Docking study showed the LibDock scores of Dichotomine B with TLR4, MyD88 and mTOR were all higher than those of positive drugs (Diazepam). These findings indicated that Dichotomine B attenuated neuroinflammatory responses in LPS/ATP-induced BV2 microglia, and its mechanism may be related to TLR4/MyD88-mTOR signaling pathway and autophagy.

Effects of Empagliflozin on a Model of Chronic Depression and Brain Toll-Like Receptors Gene Expression in Male Rats

Background: Empagliflozin is an antidiabetic medication having anti-inflammatory and antioxidant properties. Objectives: To develop a chronic unpredictable stress (CUS) model in male rats, evaluate empagliflozin’s antidepressant effects, and describe the link between stress, an antioxidant enzyme, and Toll-like receptor (TLR)-4 gene expression in male rats. Materials and Methods: In this experiment, 50 rats were divided into five groups: G1–G5. The forced swimming test (FST) was used to examine the antidepressant effects of fluoxetine and empagliflozin, and real-time polymerase chain reaction was used to measure TLR-4 gene expression. FST was performed on each rat on days 0, 10, and 25. CUS was performed on each rat for 24 days. Results: By the end of day 10, all animals subjected to the CUS program had a substantial (P < 0.05) increase in immobility duration compared with day 0. The immobility duration in the CUS group was statistically significantly greater than the baseline. Stressed rats demonstrated a statistically significant reduction in immobility duration compared with CUS group G2. In comparison to the CUS group, fluoxetine significantly reduced immobility duration (P < 0.05). In terms of gene expression, the mean of fold changes in TLR-4 mRNA level in group 2 was considerably greater than in group 1. When compared with group 2, the means of the fold changes in TLR-4 mRNA level were considerably lower in groups 3, 4, and 5. Conclusions: Empagliflozin has antidepressant-like effects and can counteract the impact of stress-induced TLR-4 overexpression in the hippocampus and elevate the activity of antioxidant enzymes in the brains of depressed rats.

What do we know about Toll-Like Receptors Involvement in Gout Arthritis?

Toll-like receptors (TLRs) are a well-characterized family of cell-bound pattern recognition receptors able to identify and respond to conserved structures of external microorganisms or Pathogen Molecular-Associated Pattern (PAMPs). They can also interact with Damage-Associated Molecular Patterns (DAMPs) involved with any infectious and sterile cell stress of tissue injury. Accumulated knowledge about TLRs has revealed that these receptors and intracellular signaling pathways triggered through TLR activation contribute to the physiopathology of different inflammatory diseases, including arthritic conditions. Mostly, the literature focuses on exploring TLRs in rheumatoid and osteoarthritis. However, TLRs also seem to be an essential mediator for monosodium urate (MSU) crystals-induced gouty arthritis, both in animal models and humans. Accordingly, naked MSU crystals have a highly negatively charged surface recognized by TLRs; intracellular adapter protein MyD88 are significant mediators of MSU crystals-induced IL1β production in mice, and gouty patients demonstrate a robust positive correlation between TLR4 mRNA level and serum IL1β. Here, we revised the literature evidence regarding the involvement of TLRs in gout arthritis pathogenesis, with particular reference to TLR2 and TLR4, by analyzing the actual literature data.

Effect of dietary Saccharomyces-derived prebiotic refined functional carbohydrates as antibiotic alternative on growth performance and intestinal health of broiler chickens reared in a commercial farm

The search for effective in-feed antibiotic alternative is growing due to the global trend to reduce or ban the utilization of antibiotics as growth promotors in poultry diets. This study was processed to assess the effect of dietary refined functional carbohydrates (RFCs) replacing antibiotic growth promoters (AGP) on growth performance, intestinal morphologic structure and microbiota, as well as intestinal immune function and barrier function of broilers reared on a commercial broilers farm. Trials contained 3 treatments with 4 replicate broiler houses, with about 25,000 birds each room. The treatments were control group (CON), RFCs group (CON+100 mg/kg RFCs), and AGP group (CON+50 mg/kg bacitracin methylene disalicylate (BMD), respectively. Results showed that RFCs and AGP group significantly increased (P < 0.05) average daily gain (ADG) during d 22 to 45 in contrast to control. Compared with the control and AGP-treated groups, feeding RFCs increased (P < 0.05) jejunal villus height to crypt depth ratio. AGP addition reduced (P < 0.05) the jejunal villi surface area compared to broilers fed control and RFC supplemented diets. Supplementation of RFCs promoted (P < 0.05) the growth of Lactobacillus but inhibited Escherichia coli and Salmonella proliferation compared with the control group. Inclusion of RFCs and BMD enhanced (P < 0.05) antibody titers against avian influenza virus H9 compared with control. RFCs and AGP both down-regulated (P < 0.05) intestinal TLR4 mRNA levels, whereas RFCs tended to up-regulate (P=0.05) IFN-γ gene expression compared to control. Expression of intestinal tight junction genes was not affected by either AGP or RFCs supplementation. Based on above observation, we suggested that RFCs could replace in-feed antibiotic BMD in broiler diets for reducing intestinal pathogenic bacteria and modulating immunity of broilers.

Effect of electroacupuncture on Notch/NF-κB signaling pathway in colonic mucosa of mice with ulcerative colitis

To observe the protective effect of electroacupuncture (EA) on the intestinal mucosal barrier and its relationship with the Notch/NF-κB signaling pathway in mice with ulcerative colitis (UC), so as to explore its mechanism of treating UC. Male C57BL/6J mice were randomized into control, model and EA groups, with 6 mice in each group. The UC model was established by giving the mice with 2% Dextran Sulfate Sodium (DSS) for 7 days. EA (2 Hz/15 Hz, 0.2 mA) was applied at bilateral "Zusanli" (ST36) for 30 min, once a day for 7 days. The disease activity indexes ［DAI=(body weight index score+stool score+bleeding score)/3; 0-4 points］ of mice were calculated. The morphological changes of colonic tissues of mice in each group were observed by HE staining, and serum contents of TNF-α and IL-6 were detected by ELISA. Claudin-1 protein expression in colon tissue was detected by immunofluorescence, while the protein expression levels of Muc-2, Notch-1, MMP-9 in colon tissue were detected by immunohistochemistry. The real-time PCR method was used to detect the expression levels of Notch-1, Hes-1, NF-κB, TLR-4 and AKT mRNA in colon tissues. After modeling, the DAI, serum TNF-α and IL-6 contents, Notch-1 and MMP-9 protein expression, the relative expression levels of Notch-1, Hes-1, NF-κB, TLR-4 and AKT mRNA in the colonic tissue were significantly increased (P<0.001, P<0.01) in the model group relevant to the control group. At the same time, Claudin-1 and Muc-2 protein expression were significantly reduced (P<0.01). After the EA intervention, the increased DAI score, TNF-α and IL-6 contents, Notch-1 and MMP-9 protein expression, the relative expressions of Notch-1, Hes-1, NF-κB, TLR-4 and AKT mRNA, and the decreased Claudin-1 and Muc-2 protein expression were all reversed compared with the model group (P<0.05, P<0.01, P<0.001). H.E. staining of the colonic tissue showed damage and infiltration of inflammatory cells in the model group, and those were significantly improved in the EA group. EA can promote the recovery of intestinal mucosal barrier function and reduce inflammatory reaction in UC mice, which may be associated with its effects in inhibiting the excessive activation of the Notch/NF-κB signaling pathway.

Dietary Bacillus licheniformis shapes the foregut microbiota, improving nutrient digestibility and intestinal health in broiler chickens.

Bacillus licheniformis is considered a potential alternative to antibiotic growth promoters of animal growth and health. However, the effects of Bacillus licheniformis on the foregut and hindgut microbiota, and their relationships with nutrient digestion and health, in broiler chickens remain unclear. In this study, we aimed to identify the effects of Bacillus licheniformis BCG on intestinal digestion and absorption, tight junctions, inflammation, and the fore- and hind-gut microbiota. We randomly assigned 240 1-day-old male AA broilers into three treatment groups: CT (basal diet), BCG1 (basal diet + 1.0 × 108 CFU/kg B. licheniformis BCG), and BCG2 (basal diet + 1.0 × 109 CFU/kg B. licheniformis BCG). On day 42, the jejunal and ileal chyme and mucosa were subjected to analysis of digestive enzyme activity, nutrient transporters, tight junctions, and signaling molecules associated with inflammation. The ileal and cecal chyme were subjected to microbiota analysis. Compared with the CT group, the B. licheniformis BCG group showed significantly greater jejunal and ileal α-amylase, maltase, and sucrase activity; moreover, the α-amylase activity in the BCG2 group was higher than that in the BCG1 group (P < 0.05). The transcript abundance of FABP-1 and FATP-1 in the BCG2 group was significantly greater than that in the CT and BCG1 groups, and the GLUT-2 and LAT-1 relative mRNA levels were greater in the BCG2 group than the CT group (P < 0.05). Dietary B. licheniformis BCG resulted in significantly higher ileal occludin, and lower IL-8 and TLR-4 mRNA levels than observed in the CT group (P < 0.05). B. licheniformis BCG supplementation significantly decreased bacterial community richness and diversity in the ileum (P < 0.05). Dietary B. licheniformis BCG shaped the ileac microbiota by increasing the prevalence of f_Sphingomonadaceae, Sphingomonas, and Limosilactobacillus, and contributed to nutrient digestion and absorption; moreover, it enhanced the intestinal barrier by increasing the prevalence of f_Lactobacillaceae, Lactobacillus, and Limosilactobacillus. Dietary B. licheniformis BCG decreased microbial community diversity by diminishing Desulfovibrio, Alistipes, Campylobacter, Vibrio, Streptococcus, and Escherichia coli-Shigella levels, and down-regulating inflammatory associated molecule expression. Therefore, dietary B. licheniformis BCG contributed to digestion and absorption of nutrients, enhanced the intestinal physical barrier, and decreased intestinal inflammation in broilers by decreasing microbial diversity and optimizing the microbiota structure.

AdipoRon Effect on Expression of Lipid Metabolism Genes in Cultured Human Primary Macrophages

Atherosclerosis is characterized by excessive uptake of cholesterol-rich low-density lipoprotein (LDL) by vascular wall macrophages. The macrophages are transformed into foam cells, lipids accumulate in the intima of arteries, atherosclerotic plaques arise, and cardiovascular diseases develop. Adiponectin is an adipose tissue adipokine and possess anti-atherogenic and anti-inflammatory activities, which are mediated by adiponectin binding to its receptors AdipoR1 and AdipoR2. To exert its anti-atherogenic effect, adiponectin may regulate the reverse cholesterol transport and prevent foam cells formation. The small-molecule adiponectin receptor agonist AdipoRon was assumed to modulate expression of reverse cholesterol transport and inflammation genes in human macrophages. Several AdipoRon concentrations (0, 5, 10, and 20 μM) were tested for effect on expression of the lipid metabolism genes ABCA1, ABCG1, APOA1, NR1H3 (LXRα), NR1H2 (LXRβ), PPARG, and ACAT1 and the inflammation genes IL6, TNFA, and TLR4 in cultured human primary macrophages and the THP-1 macrophage cell line. Cell viability was measured using the MTS assay. ABCA1, ABCG1, APOA1, NR1H3, NR1H2, PPARG, ACAT1, IL6, TNFA, and TLR4 mRNA levels in human primary macrophages were assessed by real-time PCR. The PPARG and ABCA1 relative mRNA levels were found to increase in human primary macrophages treated with 5 or 10 μM AdipoRon for 24 h. A higher AdipoRon concentration (20 μM) was cytotoxic to macrophages, especially THP-1 cells. The effect of AdipoRon on human macrophages and potential adiponectin receptor agonists are of interest to study in view of the need to develop new approaches to atherosclerosis prevention and treatment.

Effects of Reticuloendotheliosis virus on TLR-3/IFN-Β pathway in specific pathogen-free chickens

Birds infected by Reticuloendotheliosis virus (REV) are vulnerable to other microorganisms. This immunosuppression is related to the immune organs (thymus, bursa of Fabricius, and spleen) damaged by REV. The regulation of IFN-β greatly depends on pattern recognition receptor TLR-3 and nuclear factors IRF-7, NF-κB. To address if and how the TLR-3/IFN-β pathway is disturbed by REV, 60 one-day-old specific-pathogen-free chickens were intraperitoneally injected with RE virus dilution (n = 30) or stroke-physiological saline solution (n = 30). At 1, 3, 7, 21, and 28 days post-infection, after collecting thymuses, bursas, and spleens, we monitor the kinetics of TLR-3, IFN-β, NF-κB p65, and IRF-7 at transcriptional and translational levels using qPCR, Western blotting, and ELISA separately. As a result, compared with control chickens, the mRNA levels of TLR-3, IRF-7, and NF-κB p65 showed increasingly differences in the early period of REV infection. Synchronal changes occurred at translation levels. In the latter infection period, a decrease of NF-κB p65 was contemporaneous with a fall in IFN-β at both transcriptional and translational levels in the thymuses and bursas. These data suggest that the changes of IFN-β content are closely related to NF-κB p65 when REV invades chicken central immune organs. That reveals new insights into the immunosuppression mechanism of REV in avian.

Exposure to polystyrene microplastics triggers lung injury via targeting toll-like receptor 2 and activation of the NF-κB signal in mice

Microplastics are ubiquitous pollutants with a wide range of plastic applications. More recently, microplastics are in the air and can be inhaled into the lungs, causing respiratory diseases. Knowledge of the underlying mechanisms by which microplastics may induce respiratory disease is still limited. This study used intranasal instillation to develop a model of lung injury. The histopathology result showed that the mouse lung had severe inflammatory responses, apoptosis and collagen deposition with chronic exposure to different sizes (Small: 1–5 μm and Large: 10–20 μm) of polystyrene microplastics (PS-MPS), and the damage of smaller sizes was obvious. The expression levels of the Toll-like receptors (TLRs) family, evolutionarily conserved pattern recognition receptors, were detected, and the levels of TLR2 mRNA was significantly increased. In transfection experiments, PS-MPS increased the inflammatory response in HEK293 cells with TLR2 expression. Furthermore, exposure to small polystyrene microplastics promoted oxidative stress and apoptosis, and accelerated the process of fibrosis. Interestingly, inhibition of the NF-κB signal relieves inflammation and oxidative stress, reduces apoptosis, and thus controls the fibrosis process. These results suggested that PS-MPS targeted binding to TLR2 and further exacerbated fibrosis by facilitating inflammation, oxidative stress, and apoptosis with the activation of NF-κB signal.

Ferulic acid alleviates sciatica by inhibiting neuroinflammation and promoting nerve repair via the TLR4/NF-κB pathway.

Sciatica causes intense pain. No satisfactory therapeutic drugs exist to treat sciatica. This study aimed to probe the potential mechanism of ferulic acid in sciatica treatment. Thirty-two SD rats were randomly divided into 4 groups: sham operation, chronic constriction injury (CCI), mecobalamin, and ferulic acid. We conducted RNA sequencing, behavioral tests, ELISA, PCR, western blotting, and immunofluorescence analysis. TAK-242 and JSH23 were administered to RSC96 and GMI-R1 cells to explore whether ferulic acid can inhibit apoptosis and alleviate inflammation. RNA sequencing showed that TLR4/NF-κB pathway is involved in the mechanism of sciatica. CCI induced cold and mechanical hyperalgesia; destroyed the sciatic nerve structure; increased IL-1β, IL-6, TNF-α, IL-8, and TGF-β protein levels and IL-1β, IL-6, TNF-α, TGF-β, TLR4, and IBA-1 mRNA levels; and decreased IL-10 and INF-γ protein levels and IL-4 mRNA levels. Immunohistochemistry showed that IBA-1, CD32, IL-1β, iNOS, nNOS, COX2, and TLR4 expression was increased while S100β and Arg-1 decreased. CCI increased TLR4, IBA-1, IL-1β, iNOS, Myd88, p-NF-κB, and p-p38MAPK protein levels. Treatment with mecobalamin and ferulic acid reversed these trends. Lipopolysaccharide (LPS) induced RSC96 cell apoptosis by reducing Bcl-2 and Bcl-xl protein and mRNA levels and increasing Bax and Bad mRNA and IL-1β, TLR4, Myd88, p-NF-κB, and p-p38MAPK protein levels, while ferulic acid inhibited cell apoptosis by decreasing IL-1β, TLR4, Myd88, p-NF-κB, and p-p38MAPK levels and increasing Bcl-2 and Bcl-xl levels. In GMI-R1 cells, Ferulic acid attenuated LPS-induced M1 polarization by decreasing the M1 polarization markers IL-1β, IL-6, iNOS, and CD32 and increasing the M2 polarization markers CD206, IL-4, IL-10 and Arg-1. After LPS treatment, IL-1β, iNOS, TLR4, Myd88, p-p38MAPK, and p-NF-κB levels were obviously increased, and Arg-1 expression was reduced, while ferulic acid reversed these changes. Ferulic acid can promote injured sciatic nerve repair by reducing neuronal cell apoptosis and inflammatory infiltration though the TLR4/NF-κB pathway.

Toll-Like Receptor 7 a Novel Non-Invasive Inflammatory Genetic Sensor for Ulcerative Colitis Remission Monitoring.

Ulcerative colitis (UC) and Crohn's disease (CD) are two major types of inflammatory bowel diseases (IBDs). Toll-like receptors (TLRs) are expressed in the innate immune system compartments, in charge of identifying a wide range of microorganisms. The aim of the present study was to evaluate the expression of TLR-2, -7, and -8 in peripheral blood mononuclear cells (PBMC) of UC patients as a novel non-invasive primary inflammation sensor for monitoring the clinical course of UC candidates. In this cross-sectional study, total RNA was extracted from the PBMC of 42 UC patients along with 20 healthy donors. The mRNA levels of TLR-2, -7, and -8 were assessed using the quantitative real-time polymerase chain (qRT-PCR) reaction. The present research study demonstrated no significant changes in TLR-2 mRNA expression in UC patients in comparison with the control group (P = 0.1264), whereas significant elevation (P = 0.0008) was distinguished in the TLR-7 expression of UC participants specifically during the remission course compared with healthy donors and flareup patients (P = 0.0004 and P = 0.0063, respectively). The last selected TLR, TLR-8 was not shown remarkable changes either between UC patients and the control group or between clinical courses of the disease. Here, among three nominated TLRs for predicting UC patients, TLR-7 was potentially selected according to the significant difference in mRNA expression in flareup UC patients and control donors. TLR-7 could be used as a novel non-invasive biomarker for monitoring UC patients in the active course of the disease.

Resveratrol Attenuates Heat-Stress-Impaired Immune and Inflammatory Responses of Broilers by Modulating Toll-Like Receptor-4 Signaling Pathway

This study investigated the effect of resveratrol on the immune and inflammatory responses and the mRNA levels of splenic toll-like receptor (TLR)-4 signaling pathway-related genes of broilers under heat stress (HS). One hundred and sixty-two birds were allocated to three groups, each with 6 replicates, for 21 continuous days. The three treatments were as follows: the control group (22 ± 1 °C), the HS (33 ± 1 °C for 10 h d-1 and 22 ± 1 °C for the remaining time) group and the HS + resveratrol (400 mg kg-1) group. At the end of the trial, one bird per replicate close to the average body weight (BW) was selected, exsanguinated, and slaughtered. Compared with the control group, the HS treatment decreased (p<0.05) final BW, average daily gain (ADG), average daily feed intake (ADFI), relative weight of bursa of Fabricius and spleen, serum immunoglobulin (Ig) Y, IgA and interleukin (IL)-10 contents, and splenic IL-10 mRNA level, while it increased (p<0.05) feed/gain, mRNA levels of splenic tumor necrosis factor-α (TNF-α), TLR-4, nuclear factor-kappa-B (NF-κB), IL-1β, and IL-6. Compared to the HS group, the HS+resveratrol group exhibited increased (p<0.05) final BW, ADG, relative weight of bursa of Fabricius and spleen, serum IgY, IgA and IL-10 contents, and splenic IL-10 mRNA level, while it exhibited lower (p<0.05) TNF-α, IL-1β and IL-6 contents in serum, and splenic TLR4, TNF-α, IL-1β, and NF-κB mRNA levels. In conclusion, resveratrol prevented a HS-impairment of the immune function of broilers by blocking the abnormal activation of the TLR4 signaling pathway.

A blend of formic acid, benzoic acid, and tributyrin alleviates ETEC K88-induced intestinal barrier dysfunction by regulating intestinal inflammation and gut microbiota in a murine model

This study aimed to investigate the effects of an organic acid (OA) blend on intestinal barrier function, intestinal inflammation, and gut microbiota in mice challenged with enterotoxigenic Escherichia coli K88 (ETEC K88). Ninety female Kunming mice (7weeks old) were randomly allotted to five treatments with six replicates per treatment and three mice per replicate. The five treatments were composed of the non-ETEC K88 challenge group and ETEC K88 challenge+OA blend groups (0, 0.6%, 1.2%, and 2.4% OA blend). The OA blend consisted of 47.5% formic acid, 47.5% benzoic acid, and 5% tributyrin. The feeding trial lasted for 15days, and mice were intraperitoneally injected with PBS or ETEC K88 solution on day 15. At 24h post-challenge, one mouse per replicate was selected for sample collection. The results showed that a dosage of 0.6% OA blend alleviated the ETEC K88-induced intestinal barrier dysfunction, as indicated by the elevated villus height and the ratio of villus height to crypt depth of jejunum, and the reduced serum diamine oxidase (DAO) and D-lactate levels, as well as the up-regulated mRNA levels of ZO-1, Claudin-1, and Occludin in jejunum mucosa of mice. Furthermore, dietary addition with 0.6% OA blend decreased ETEC K88-induced inflammation response, as suggested by the decreased TNF-α and IL-6 levels, and the increased IgA level in the serum, as well as the down-regulated mRNA level of TNF-α, IL-6, IL-1β, TLR-4, MyD88, and MCP-1 in jejunum mucosa of mice. Regarding gut microbiota, the beta-diversity analysis revealed a remarkable clustering between the 0.6% OA blend group and the ETEC K88 challenge group. Supplementation of 0.6% OA blend decreased the relative abundance of Firmicutes, and increased the relative abundance of Bacteroidota, Desulfobacterota, and Verrucomicrobiota of colonic digesta in mice. Also, the butyric acid content in the colonic digesta of mice was increased by dietary 0.6% OA blend supplementation. Collectively, a dosage of 0.6% OA blend could alleviate the ETEC K88-induced intestinal barrier dysfunction by regulating intestinal inflammation and gut microbiota of mice.

Prolonged alcohol consumption influences microRNA expression in the nucleus accumbens of the rat brain.

The microRNA (miR) species analyzed in this study are involved in molecular mechanisms of TLR4 and TLR7 signaling, mediating the development of neuroinflammation and neurodegeneration. We have investigated the expression levels of miR-let7b, miR-96, miR-182, miR-155, and the mRNA content of HMGB1, TLR3, TLR4 in the nucleus accumbens (NAc) of the brain of rats exposed to long-term alcoholization. The long-term alcoholization caused a decrease in miR-let7b, miR-96, miR-182, and TLR7 mRNA levels; this was accompanied by an increase in miR-155, TLR4, and Hmgb1 mRNA levels in the NAc of rat brain. TLR7 is functionally linked to miR-let7b. The data of a simultaneous decrease in miR-let7b and TLR7 mRNA are of interest for further studies; they may indicate on the lack of functionally significant links between Hmgb1 and the miR-let7b-TLR7 system in NAc. The existing evidence of a functional relationship between TLR4 with miR-155 and miR-182 and our observations on their expression changes during chronic alcoholization are very interesting and require further investigation. The suggestion about the development of neuroinflammatory process in NAc under prolonged alcohol exposure are relevant for studying the level of TLR gene expression in NAc, as well as the expression of miR species, which may have a functional relationship with the TLR system.

Bioinformatics analysis of ferroptosis in spinal cord injury.

Ferroptosis plays a key role in aggravating the progression of spinal cord injury (SCI), but the specific mechanism remains unknown. In this study, we constructed a rat model of T10 SCI using a modified Allen method. We identified 48, 44, and 27 ferroptosis genes that were differentially expressed at 1, 3, and 7 days after SCI induction. Compared with the sham group and other SCI subgroups, the subgroup at 1 day after SCI showed increased expression of the ferroptosis marker acyl-CoA synthetase long-chain family member 4 and the oxidative stress marker malondialdehyde in the injured spinal cord while glutathione in the injured spinal cord was lower. These findings with our bioinformatics results suggested that 1 day after SCI was the important period of ferroptosis progression. Bioinformatics analysis identified the following top ten hub ferroptosis genes in the subgroup at 1 day after SCI: STAT3, JUN, TLR4, ATF3, HMOX1, MAPK1, MAPK9, PTGS2, VEGFA, and RELA. Real-time polymerase chain reaction on rat spinal cord tissue confirmed that STAT3, JUN, TLR4, ATF3, HMOX1, PTGS2, and RELA mRNA levels were up-regulated and VEGFA, MAPK1 and MAPK9 mRNA levels were down-regulated. Ten potential compounds were predicted using the DSigDB database as potential drugs or molecules targeting ferroptosis to repair SCI. We also constructed a ferroptosis-related mRNA-miRNA-lncRNA network in SCI that included 66 lncRNAs, 10 miRNAs, and 12 genes. Our results help further the understanding of the mechanism underlying ferroptosis in SCI.

H3K9 acetylation modification and TLR9 immune regulation mechanism in patients after anti-HBV treatment

To improve the curative effect of anti-hepatitis B virus (HBV) drugs, methods such as thymosin and entecavir combination have become a focus of clinical investigation. The aim of this retrospective experimental study was to explore the potential mechanism of action of thymosin a1 (Ta1) combined with entecavir in the treatment of HBV infection. A total of 28 patients with chronic hepatitis B, 29 patients treated with thymosin a1 and entecavir combination, and 15 healthy individuals were enrolled in this study. RT-qPCR was conducted to evaluate the mRNA levels of TLR9 in peripheral blood mononuclear cells (PBMCs). The serum level of TLR9 protein was analyzed by ELISA. The binding of TLR9 gene to the protein H3K9Ac in PBMCs was assessed by chromatin immunoprecipitation, and serum inflammatory factors were detected by Luminex technology. The expression levels of TLR9 mRNA and serum TLR9 protein in patients with HBV infection were significantly lower than those in subjects in the control group before treatment but increased after treatment with the Ta1 and entecavir combination. Moreover, the acetylation protein H3K9Ac was significantly bound to the promoter region of the TLR9 gene in patients with HBV infection treated with the Ta1 and entecavir combination compared to that in patients with HBV infection without treatment. Furthermore, the expression levels of interleukin 6 (IL-6), interleukin 12 (IL-12), interferon gamma, and necrosis factor alpha in patients with HBV infection after the combination treatment were slightly decreased compared to those in patients with HBV infection without treatment. In conclusion, the histone acetylation modification of TLR9 was significantly improved in patients with HBV infection after treatment with the Ta1 and entecavir combination, which elevated the expression of TLR9 at the mRNA and protein levels and further regulated the expression of IL-6, IL-12, and other cytokines.

Association between the expression of toll-like receptors, cytokines, and homeostatic chemokines in SARS-CoV-2 infection and COVID-19 severity

The recent identification of the involvement of the immune system response in the severity and mortality of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection highlights the importance of cytokines and chemokines as important factors in the clinical outcomes of COVID-19. However, the impact and roles of the BAFF/APRIL cytokine system, homeostatic chemokines (CXCL12, CXCL13, CCL19, and CCL21), as well as Toll-like receptor (TLR)-3/4 in COVID-19, have not been investigated. We sought to assess the expression levels and roles of TLR3/4, BAFF, APRIL, IFN-β, homeostatic chemokines (CXCL12, CXCL13, CCL19, and CCL21), SARS-CoV-2 IgG and IgM antibodies in patients with critical (ICU) and non-ICU (mild) COVID-19 and their association with mortality and disease severity. Significant high levels of TLR-4 mRNA, IFN-β, APRIL, CXCL13, and IgM and IgG antibodies were observed in ICU patients with severe COVID-19 compared to non-ICU COVID-19 patients and healthy controls. On the other hand, BAFF and CCL21 expression were significantly upregulated in non-ICU patients with COVID-19 compared with that in critical COVID-19 patients. The two groups did not differ in TLR-3, CXCL12, and CCL19 levels. Our findings show high expression levels of some inflammatory chemokines in ICU patients with COVID-19. These findings highlight the potential utility of chemokine antagonists as an immune-based treatment for the severe form of COVID-19. We also believe that selective targeting of TLR/spike protein interactions might lead to the development of a new COVID-19 therapy.

Hygiene of housing conditions and proinflammatory signals alter gene expressions in porcine adipose tissues and blood cells.

Adipose tissue is an organ with metabolic, endocrine and immune functions. In this tissue, the expressions of genes associated with several metabolic pathways, including lipid metabolism, have been shown to be affected by genetic selection for feed efficiency, an important trait to consider in livestock. We hypothesized that the stimulation of immune system caused by poor hygiene conditions of housing impacts the molecular and cellular features of adipose tissue and that the impact may differ between pigs that diverge in feed efficiency. At the age of 12 weeks, Large White pigs from two genetic lines divergent for residual feed intake (RFI) were housed in two contrasting hygiene conditions (good vs poor). After six weeks of exposure, pigs were slaughtered (n=36). Samples of blood, subcutaneous (SCAT) and perirenal (PRAT) adipose tissues were collected for cell response and gene expression investigations. The decrease in the relative weight of PRAT was associated with a decline in mRNA levels of FASN, ME, LCN2 and TLR4 (P<0.05) in pigs housed in poor conditions compared with pigs housed in good conditions for both RFI lines. In SCAT, the expressions of only two key genes (PPARG and TLR4) were significantly affected by the hygiene of housing conditions. Besides, the mRNA levels of both LCN2 and GPX3 were influenced by the RFI line (P<0.05). Because we suspected an effect of poor hygiene at the cellular levels, we investigated the differentiation of stromal vascular cells isolated from SCAT in vitro in the absence or presence of a pro-inflammatory cytokine, Tumor Necrosis Factor-α (TNF-α). The ability of these cells to differentiate in the absence or presence of TNF-α did not differ among the four groups of animals (P>0.05). We also investigated the expressions of genes involved in the immune response and lipid metabolism in whole blood cells cultured in the absence and presence of LPS. The hygiene conditions had no effect but, the relative expression of the GPX3 gene was higher (P<0.001) in high RFI than in low RFI pigs while the expressions of IL-10 (P=0.027), TGFβ1 (P=0.023) and ADIPOR2 (P=0.05) genes were lower in high RFI than in low RFI pigs. Overall, the current study indicates that the hygiene of housing had similar effects on both RFI lines on the expression of genes in adipose tissues and on the features of SCAT adipose cells and whole blood cells in response to TNF-α and LPS. It further demonstrates that the number of genes with expression impacted by housing conditions was higher in PRAT than in SCAT. It suggests a depot-specific response of adipose tissue to the current challenge.

Analysis of Macrophage Chemotactic Activity and TLR9 Signaling Pathway in the Mouse Model of Viral Acute Lung Injury.

This study focused on the chemotactic activity of macrophages and the role of the TLR9 signaling pathway in the pathogenesis of viral Acute Lung Injury (ALI). For this purpose, a total of 40 male SPF mice were used, aged 5-8 weeks. They were randomly divided into an experimental group and a control group. The experimental group was further divided into S1 and S2, and the control group was further divided into D1 and D2, with 10 in each. The different groups were detected for the expression of inflammatory cytokines and chemokines and the expression of alveolar macrophages. Results showed that as for the weight, survival status, arterial blood gas analysis, lung index and wet-to-dry value of lung tissue, and lung histopathological analysis results, the S2 group showed more obvious changes versus the D2 group, and the difference was statistically significant (P<0.05). S2 had higher levels of the inflammatory factors TNF-α, IL-1β, IL-6 and the chemokine CCL3 in the BALF supernatant versus the D2 Group, and the difference is statistically significant (P<0.05). S2 had higher expression levels of chemokines CCR5, TLR9, and JMJD1A mRNA versus the D2 group, and the difference was statistically significant (P<0.05). In conclusion, the establishment of a mouse ALI model induced by poly l:C was successful; AM has a certain chemotactic activity on CCL3; polyI:C can promote the expression activity and chemotactic activity of macrophages CCR5 through signal pathways, such as TLR9.