Tumor Necrosis Factor-α mRNA Research Articles

Upregulation of polymeric immunoglobulin receptor expression in flounder (Paralichthys olivaceus) gill cells by cytokine tumor necrosis factor-α via activating PI3K and NF-κB signaling pathways

The polymeric immunoglobulin receptor (pIgR) transports secretory immunoglobulins across mucosal epithelial cells into external secretions, playing critical roles in mucosal surface defenses, but the regulation mechanism of pIgR expression is not clarified in teleost fish. In this study, the dynamic changes of flounder (Paralichthys olivaceus) pIgR (fpIgR) and pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) mRNA expression in mucosal tissues were first analyzed post inactivated Vibrio anguillarum immunization, and increased production of TNF-α was found to correlate with increased expression of fpIgR. To determine that cytokine TNF-α influenced fpIgR expression, following confirming that natural fpIgR expressed on flounder gill (FG) cells, FG cells were incubated with various concentrations of recombinant TNF-α for different time, the results showed that the expressions of fpIgR were significantly upregulated at gene and protein levels in a dose-dependent and time-dependent manner, and similar change trend was observed for free secretory component (SC) secreted by fpIgR into the culture supernatant. After FG cells were treated with TNF-α, specific phosphoinositide 3-kinase (PI3K) inhibitor wortmannin, nuclear factor kappa-B (NF-κB) inhibitor Bay11−7082, and the mixtures of TNF-α and wortmannin / Bay11−7082 respectively, the fpIgR protein and mRNA levels, together with SC secretion, obviously decreased in wortmannin- and Bay11−7082-treated cells compared with the untreated control, and cotreatment with wortmannin / Bay11−7082 plus TNF-α resulted in lower expression compared with that upon treatment with TNF-α alone, indicating that the inhibition of PI3K and NF-κB both blocked the ability of TNF-α to increase cellular fpIgR and SC levels. Furthermore, the gene expressions of PI3K and NF-κB were upregulated and present a tendency to increase first and then decrease after TNF-α treatment of FG cells; However, the expression of PI3K mRNA was inhibited significantly by wortmannin but not by Bay11−7082, and the expression of NF-κB mRNA was suppressed obviously by Bay11−7082 but not by wortmannin, suggesting that inhibition of PI3K or NF-κB had no influence on each other. All these results collectively revealed that TNF-α could transcriptionally upregulate fpIgR expression and SC production, and this TNF-α-induced pIgR expression was regulated by complex mechanisms that involved PI3K and NF-κB signaling pathways, which provided evidences for pro-inflammatory cytokine TNF-α acting as a regulator in pIgR expression and better understanding of regulation mechanism of pIgR expression in teleost fish.

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines.

PurposeIsorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis.MethodsIn vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR.ResultsIn HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1β, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro.ConclusionsIsorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.

Effects and mechanisms of Xuezhikang on preventing contrast-induced acute kidney injury in diabetic rats

Objective: To explore the effects and mechanisms of Xuezhikang on preventing contrast-induced nephropathy (CIN) in diabetic rats. Methods: Streptozotocin (65 mg/kg) was injected intraperitoneally to establish a diabetes model in 7-week-old male Sprague-Dawley (SD) rats. After 4 weeks of modeling, 24 diabetic rats were randomly divided into 4 groups: sham group, CIN group, CIN+vehicle (Veh) group and Xuezhikang group. All animals were sacrificed at 24 hours after administration of contrast. Blood and kidney tissues were collected to detect biochemical, inflammation-related, oxidative stress-related and pathological indicators. Results: After administration of contrast agent, the renal function-related indicators were decreased in Xuezhikang group compared with CIN+Veh group [serum creatinine (SCr): (59.3±3.3) μmol/L vs (73.2±4.1) μmol/L; blood urea nitrogen (BUN): (13.8±0.5) mmol/L vs (16.3±0.6) mmol/L; serum neutrophil gelatinase-associated lipocalin (sNGAL): (41.4±2.0) ng/ml vs (54.9±4.4) ng/ml; urinary kidney injury moleculer-1 (uKIM-1): (11.1±0.5) ng/ml vs (16.6±0.5) ng/ml] (all P<0.05). Histological analysis showed that the severity of renal tubule dilatation, brush border loss and renal tubular cell necrosis in Xuezhikang group was better than that of CIN+Veh group. Additionally, the oxidative stress-related indicators of Xuezhikang group improved compared with those of CIN+Veh group [malondialdehyde (MDA): (12.1±0.7) nmol/mg vs (15.5±0.8) nmol/mg, superoxide dismutase (SOD): (35.0±2.2) U/mg vs (23.7±3.4) U/mg, renal nitrite: (1.7±0.1) nmol/mg vs (1.2±0.1) nmol/mg, all P<0.05]. Meanwhile, Xuezhikang pretreatment downregulated the mRNA and protein expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (both P<0.05). Conclusion: The current study suggests that Xuezhikang protects against CIN in diabetic rats by inhibiting oxidative stress and inflammation.

Lemon Balm and Dandelion Leaf Extracts Synergistically Protect against Carbon Tetrachloride-Induced Acute Liver Injury in Mice

Lemon balm and dandelion are commonly used medicinal herbs exhibiting numerous pharmacological activities that are beneficial for human health. In this study, we explored the protective effects of a 2:1 (w/w) mixture of lemon balm and dandelion extracts (MLD) on carbon tetrachloride (CCl4)-induced acute liver injury in mice. CCl4 (0.5 mL/kg; i.p.) injection inhibited body weight gain and increased relative liver weight. Pre-administration of MLD (50–200 mg/kg) for 7 days prevented these CCl4-mediated changes. In addition, histopathological analysis revealed that MLD synergistically alleviated CCl4-mediated hepatocyte degeneration and infiltration of inflammatory cells. MLD decreased serum aspartate aminotransferase and alanine transferase activities and reduced the number of liver cells that stained positive for cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase, suggesting that MLD protects against CCl4-induced hepatic damage via the inhibition of apoptosis. Moreover, MLD attenuated CCl4-mediated lipid peroxidation and protein nitrosylation by restoring impaired hepatic nuclear factor erythroid 2-related factor 2 mRNA levels and its dependent antioxidant activities. Furthermore, MLD synergistically decreased mRNA and protein levels of tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the liver. Together, these results suggest that MLD has potential for preventing acute liver injury by inhibiting apoptosis, oxidative stress, and inflammation.

Dietary nucleotide supplementation as an alternative to in-feed antibiotics in weaned piglets

Nucleotides are important to cell growth and division and are crucial to the rapid proliferation of such cells as the intestinal mucosa and immune cells. Accordingly, the nucleotide requirements of animals are high during periods of rapid growth and periods of stress like post-weaning period. Thus, nucleotide supplementation may be a possible alternative to in-feed antibiotics as growth promoter in this phase. The study aimed to evaluate dietary nucleotide supplementation as an alternative to in-feed antibiotics on performance and gut health of weaned piglets. Ninety-six 21-day-old piglets, weighing 7.44 ± 0.65 kg, were allocated into 1 of 3 treatments (8 pens per treatment; 4 pigs per pen) in a 14-day trial. Dietary treatments consisted of control: corn-soybean meal-based diet; nucleotides: control +2 g/kg of a nutritional additive with purified nucleotides; and antibiotic: control +0.8 g/kg of antibiotic growth promoter based on colistin and tylosin. Performance variables and fecal score were not affected (P > 0.05) by supplementing nucleotide or antibiotic. Nucleotides treatment had similar effect to antibiotic and superior to control (P < 0.05) on enhancing duodenum villus height, jejunum crypt depth, and reduction of Paneth cellular area. Duodenum and ileum of animals supplemented with nucleotides or antibiotics had higher (P < 0.05) number of proliferating cells than did those of control animals, whereas the jejunum of animals that received antibiotic diets presented more (P < 0.05) proliferating cells than either the nucleotides or control animals. Jejunum of nucleotide-treated piglets showed a greater number of apoptotic cells than those fed antibiotic or control diets (P < 0.05). Nucleotides and antibiotic treatments decreased the B lymphocyte counts in duodenum and ileum (P < 0.05) but increased in the jejunum (P < 0.05), when compared to the control treatment. Relative abundance of mitogen-activated protein kinases-6, haptoglobin, and tumor necrosis factor-α mRNA was not influenced (P > 0.05) by treatments. In the ileal, antibiotic supplementation reduced total bacteria quantification compared to nucleotide supplementation or the control (P < 0.05), whereas nucleotides supplementation increased enterobacteria proliferation compared to the antibiotic or control diets (P < 0.05). However, nucleotides and antibiotic reduced (P < 0.05) colon total bacteria quantification when compared to control. These results suggest that the nucleotides source used to weaned piglets improved gut health by modulating the local immune response and modulating intestinal mucosa development, and, therefore, nucleotides may be an alternative to antibiotics as growth promoters.

Temporal Effects of Different Vehicles on Wound Healing Potentials of Quercetin: Biochemical, Molecular, and Histopathological Approaches.

Development of novel drugs or formulations to accelerate the wound healing process is the need of current era. Quercetin (Q), a bioflavonoid, at 0.3% concentration has showed some wound healing potential in our preliminary studies. The present study was aimed to explore the wound healing potential of 0.3% quercetin formulated in 3 different vehicles, that is, dimethyl sulfoxide (DMSO; 10%), ointment base, and corn oil. Ninety experimentally wounded rats were grouped in 6 groups. The 0.3% quercetin mixed with DMSO, ointment base, and corn oil was topically applied once daily for 21 days on the wounds of groups 2, 4, and 6, respectively. DMSO, ointment base, and corn oil alone was applied similarly in groups 1, 3, and 5, respectively. Gross evaluation and wound contraction results revealed accelerated wound closure in all quercetin-treated groups. The mRNA expressions of vascular endothelial growth factor, transforming growth factor-β1, and interluekin-10 were markedly upregulated in healing tissues of quercetin-treated groups. Tumor necrosis factor-α mRNA expression and protein levels were lowered by quercetin treatment. Quercetin-treated groups also showed increased activities of SOD (superoxide dismutase) and catalase, and levels of total thiols in wound tissues on day 7. Levels of superoxide anion radicals and malondialdehyde were markedly lower in quercetin-treated groups. Histologically, wound sections of quercetin-treated groups showed early dominance of fibroblasts, increased blood vessels, marked collagen deposition, and regenerated epithelial layer. The significant effects were more pronounced in ointment + Q group among all the quercetin-treated groups. In conclusion, 0.3% quercetin mixed in ointment base produced the fastest and better wound healing in rats.

Paneth cells mediated the response of intestinal stem cells at the early stage of intestinal inflammation in the chicken

The rapid renewal and repair of the intestinal mucosa are based on intestinal stem cells (ISC), which are located at the crypt bottom. Paneth cells are an essential component in the crypt, which served as the niche for ISC development. However, in the chicken, how the function of Paneth cells changes during intestinal inflammation is unclear and is the key to understand the mechanism of mucosal repair. In the present study, 36 HyLine White chickens (7 d of age, n = 6) were randomly divided into 1 control and 5 lipopolysaccharide (LPS) injection groups. The chickens were injected (i.p.) with PBS in the control group, however, were injected (i.p.) with LPS (10 mg/kg BW) in the LPS injection groups, which would be sampled at 5 time points (1 h postinjection [hpi], 2 hpi, 4 hpi, 6 hpi, and 8 hpi). Results showed that tumor necrosis factor-α mRNA transcription in duodenal tissue increased gradually since 1 hpi, peaked at 4 hpi, and then reduced remarkably, indicating that 4 hpi of LPS was the early stage of intestinal inflammation. Meanwhile, the MUC2 expression in duodenal tissue was dramatically reduced since 1 hpi of LPS. The ISC marker, Lgr5 and Bmi1, in the duodenal crypt were reduced from 1 hpi to 4 hpi and elevated later. Accordingly, the hydroethidine staining showed that the reactive oxygen species level, which drives the differentiation of ISC, in the duodenal crypt reduced obviously at 1 hpi and recovered gradually since 4 hpi. The analysis of Paneth cells showed that many swollen mitochondria appeared in Paneth cells at 4 hpi of LPS. Meanwhile, the Lysozyme transcription in the duodenal crypt was substantially decreased since 1 hpi of LPS. However, the Wnt3a and Dll1 in duodenal crypt decreased at 1 hpi of LPS, then increased gradually. In conclusion, Paneth cells were impaired at the early stage of intestinal inflammation, then recovered rapidly. Thus, the ISC activity was reduced at first and recovery soon.

Intramammary inoculation with lactic acid bacteria at dry-off triggers an immunomodulatory response in dairy cows.

The use of antibiotics to prevent bovine mastitis is responsible for the emergence and selection of resistant strains. Lactic acid bacteria (LAB) could be introduced into animal feed as an alternative prevention method that would bypass the risk of resistance development. In previous research, we demonstrated that two probiotic LAB strains isolated from bovine milk were capable of stimulating the production of antibodies and the host's immune cellular response in the udder. The present study aimed to elucidate whether the antibodies of animals inoculated with these strains were able to increase phagocytosis by neutrophils and inhibit the growth of different mastitis-causing pathogens. Moreover, the effect of LAB on the expression of pro-inflammatory cytokines was assessed. Ten animals were inoculated intramammarily with 106 cells of the two strains at dry-off. The blood serum was tested for its ability to opsonize bovine mastitis pathogens, the in vitro bactericidal activity of bovine blood and milk against these pathogens was determined, and cytokine mRNA expression was quantified in milk somatic cells. The inoculated animals did not show abnormal signs of sensitivity to the LAB. Their blood serum significantly enhanced the phagocytosis of Staphylococcus spp. and the LAB. Escherichia coli and Streptococcus uberis were inhibited by the milk serum but not the blood serum, whereas Staphylococcus aureus and Staphylococcus haemolyticus were inhibited by both. In regard to cytokine expression, interleukin (IL)-1β increased markedly for up to 4 h post-inoculation, and an increase in IL-8 was observed 4, 12 and 24 h after inoculation. Tumour necrosis factor-α mRNA increased 1 and 2 h after inoculation and a significant difference was registered at 6 h for interferon-γ. This rapid immunomodulatory response shows that inoculating animals with LAB at dry-off, when they are especially susceptible, could be a useful strategy for the prevention of bovine mastitis.

Contributions of Lactobacillus plantarum PC170 administration on the recovery of gut microbiota after short-term ceftriaxone exposure in mice.

This study aimed to determine the impact of Lactobacillus plantarum PC170 concurrent with antibiotic treatment and/or during the recovery phase after antibiotic treatment on the body weight, faecal bacterial composition, short-chain fatty acids (SCFAs) concentration, and splenic cytokine mRNA expression of mice. Orally administrated ceftriaxone quantitatively and significantly decreased body weight, faecal total bacteria, Akkermansia muciniphila, and Lactobacillus plantarum, and faecal SCFAs concentration. Ceftriaxone treatment also dramatically altered the faecal microbiota with an increased Chao1 index, decreased species diversities and Bacteroidetes, and more Firmicutes and Proteobacteria. After ceftriaxone intervention, these changes all gradually started to recover. However, faecal microbiota diversities were still totally different from control by significantly increased α- and β-diversities. Bacteroidetes all flourished and became dominant during the recovery process. However, mice treated with PC170 both in parallel with and after ceftriaxone treatment encouraged more Bacteroidetes, Verrucomicrobia, and Actinobacteria, and the diversity by which to make faecal microbiota was very much closer to control. Furthermore, the expression of splenic pro-inflammatory cytokine tumour necrosis factor-α mRNA in mice supplemented with PC170 during the recovery phase was significantly lower than natural recovery. These results indicated that antibiotics, such as ceftriaxone, even with short-term intervention, could dramatically damage the structure of gut microbiota and their abilities to produce SCFAs with loss of body weight. Although such damages could be partly recovered with the cessation of antibiotics, the implication of antibiotics to gut microbiota might remain even after antibiotic treatment. The selected strain PC170 might be a potential probiotic because of its contributions in helping the host animal to remodel or stabilise its gut microbiome and enhancing the anti-inflammatory response as protection from the side effects of antibiotic therapy when it was administered in parallel with and after antibiotic treatment.

Soybean protein-derived peptides inhibit inflammation in LPS-induced RAW264.7 macrophages via the suppression of TLR4-mediated MAPK-JNK and NF-kappa B activation.

This study aimed to determine the effect of soybean protein-derived peptides (SBP) on the inhibition of lipopolysaccharide (LPS)-induced RAW264.7 cell inflammation. The mRNA of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), Lymphocyte Antigen 96 (LY96), and nuclear factor-κB (NF-κB) were detected with RT-qPCR. The concentrations of cytokines (TNF-α, IL-6, and IL-1β) secreted were detected by ELISA Kit. The results indicated that SBP inhibited the inflammatory stress induced by LPS in RAW264.7 cells. Western blot analysis was used to examine this anti-inflammatory molecular mechanism. The findings showed that SBP impeded the increase of toll-like receptor 4 activity by restricting LY96, while also inhibiting the mitogen-activated protein kinase-c-Jun N-terminal kinase pathway in cells, as well as LPS-induced NF-κB activation caused by the degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα). Consequently, the release of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) was inhibited, preventing LPS-induced inflammation of RAW 264.7 cells. Therefore, this research highlighted the potential application of SBP in the development of anti-inflammatory foods that prevented inflammatory-immune diseases. PRACTICAL APPLICATIONS: Inflammation is the root cause of almost all pathology and is related to many human diseases, including arthritis, obesity, cancer and atherosclerosis. Therefore, the development of products that can regulate and intervene inflammation has a broad application prospect. Soybean protein and soybean peptide have many functional properties, including immunoregulation, anti-inflammatory, anti-oxidation and so on. However, there are still some shortcomings in the development of soy protein supplements, such as solubility and absorption. Compared with soybean protein, derived peptide is easy to digest, and has high solubility. As a good nutritional supplement, the nutritional support of soybean protein-derived peptides may help to reduce inflammation and improve the level of cytokines combined with drugs.

Carboxyamidotriazole exerts anti-inflammatory activity in lipopolysaccharide-induced RAW264.7 macrophages by inhibiting NF-κB and MAPKs pathways.

Carboxyamidotriazole (CAI), originally developed as a non-cytotoxic anti-cancer drug, was shown to have anti-inflammatory activity according to recent studies in a number of animal models of inflammation. However, its mechanism of action has not been characterized. Therefore, the present study was performed to identify the anti-inflammatory action of CAI in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and to identify the signal transduction pathways involved. The in vitro results revealed that CAI had no direct effect on the activity of cyclooxygenase (COX), suggesting a different anti-inflammatory mechanism compared with that of COX-inhibiting non-steroidal anti-inflammatory drugs. Further investigation in RAW264.7 macrophages revealed that CAI decreased the production of nitric oxide via decreasing the LPS-stimulated expression of inducible nitric oxide synthase, and downregulated both mRNA and protein expression levels of the cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. CAI also significantly reduced the increased DNA-binding activity of nuclear factor (NF)-κB induced by LPS stimulation. With respect to the mechanisms involved on NF-κB activity, CAI exhibited suppression of the phosphorylation and degradation of the inhibitor of nuclear factor-κBα (IκB), and decreased the phosphorylation levels of the p65 subunit and its subsequent nuclear translocation. In addition, CAI significantly decreased the phosphorylated forms of p38, JNK and ERK, which were increased following LPS stimulation, while the total expression levels of p38, JNK and ERK remained unaltered. The results in the present study indicate that CAI alleviates the inflammatory responses of RAW 264.7 macrophages in response to LPS stimulation via attenuating the activation of NF-κB and MAPK signaling pathways and decreasing the levels of pro-inflammatory mediators. This offers a novel perspective for understanding the anti-inflammatory mechanism of CAI and suggests its potential use as a therapeutic treatment in inflammatory diseases with excessive macrophage activation.

Cotinine inhibits TLR4/NF-κB signaling pathway and improves deep vein thrombosis in rats.

Background: The present study was designed to explore the regulatory mechanisms and influences of cotinine on deep vein thrombosis (DVT) in rats via the toll-like receptor 4/nuclear factor κ binding (TLR-4/NF-κB) pathway. Methods: In this experimental study, 30 SD rats were randomly assigned to control group, sham operation group, model group, cotinine (10 μg/kg) group, and model + cotinine (10 μg/kg) group. The thromboxane B2 (TXB2), 6-keto-PGF1α, plasminogen activator inhibitor (PAI), tissue plasminogen activator (t-PA), TLR4, NF-κB, and p65 mRNA and protein expression and tissue changes were analyzed by ELISA, Hematoxylin–Eosin (HE) staining, RT-PCR, and Western blot. Results: There was no significant difference between the control and sham operation groups (P>0.05). The model and cotinine groups showed significantly higher mRNA and protein levels of TXB2, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), PAI, TLR-4, and NF-κB, and significantly lower levels of 6-keto-PGF1α and t-PA than the control and sham operation groups (P<0.05), and the model + cotinine group showed significantly higher mRNA and protein levels of TXB2, IL-6 and TNF-α, PAI, TLR-4, and NF-κB and significantly lower levels of 6-keto-PGF1α and t-PA than the model group (P<0.05). Conclusion: Cotinine can aggravate thrombus and inflammation in rats with DVT, and the mechanism may be associated with the activation of the TLR-4/NF-κB inflammatory signaling pathway.

Lemon balm and dandelion leaf extract synergistically alleviate ethanol-induced hepatotoxicity by enhancing antioxidant and anti-inflammatory activity.

We investigated the effect of a 2:1 (w/w) mixture of lemon balm and dandelion extracts (LD) on ethanol (EtOH)-mediated liver injury and explored the underlying mechanisms. Administration of LD synergistically reduced relative liver weight and decreased the levels of serum biomarkers of hepatic injury. Histopathological and biochemical analyses indicated that LD synergistically attenuated hepatic accumulation of triacylglycerides (TGs) and restored the levels of mRNAs related to fatty acid metabolism. In addition, LD significantly reduced EtOH-induced hepatic oxidative stress by attenuating the reduction in levels of nuclear factor E2-related factor 2 (Nrf2) mRNA and enhancing antioxidant activity. Moreover, LD decreased the EtOH-mediated increase in levels of hepatic tumor necrosis factor-α (TNF-α) mRNA. In vitro, LD significantly scavenged free radicals, increased cell viability against tert-butylhydroperoxide (tBHP), and transactivated Nrf2 target genes in HepG2 cells. Furthermore, LD decreased levels of pro-inflammatory mediators in lipopolysaccharide-stimulated Raw264.7 cells. Therefore, LD shows promise for preventing EtOH-mediated liver injury. PRACTICAL APPLICATIONS: There were no approved therapeutic agents for preventing and/or treating alcoholic liver diseases. In this study, a 2:1 (w/w) mixture of lemon balm and dandelion leaf extract (DL) synergistically ameliorated EtOH-induced hepatic injury by inhibiting lipid accumulation, oxidative stress, and inflammation. Our findings will enable the development of a novel food supplement for the prevention or treatment of alcohol-mediated liver injury.

IL-1Ra Protects Hepatocytes from CCl4-Induced Hepatocellular Apoptosis via Activating the ERK1/2 Pathway

AbstractInterleukin-1 receptor antagonist is an important acute-phase protein and an immune mediator, and its expression is associated with the development of hepatitis or acute liver failure. The aim of this study was to investigate whether recombinant human interleukin-1 receptor antagonist directly targets and improves cell survival in a carbon tetrachloride-induced hepatocyte injury model in vitro. A human hepatoma cell line and a mouse hepatocyte cell line were used to establish carbon tetrachloride-induced cell injury models in vitro, and cell viability, apoptosis, and reactive oxygen species level were determined to assess the degree of hepatocellular damage. Quantitative real-time polymerase chain reaction was used to analyze the level of interleukin-1β, interleukin-6, and tumor necrosis factor-α mRNA in cells; extracellular regulated protein kinases 1/2 phosphorylation in hepatocytes was analyzed using western blotting. Recombinant human interleukin-1 receptor antagonist could directly target hepatocytes, improve cell survival, and decrease carbon tetrachloride-induced cell apoptosis in vitro. In hepatocytes, recombinant human interleukin-1 receptor antagonist remarkably downregulated expression of interleukin-1β, interleukin-6, and tumor necrosis factor-α in hepatocytes exposed to carbon tetrachloride. It also decreased accumulation of reactive oxygen species and abrogated the suppression of extracellular regulated protein kinases 1/2 phosphorylation induced by carbon tetrachloride. However, stimulation of cells with an extracellular regulated protein kinases 1/2 inhibitor blocked the recombinant human interleukin-1 receptor antagonist-induced upregulation of extracellular regulated protein kinase1/2 activation and abrogated the improvement in hepatocyte survival following carbon tetrachloride treatment. Collectively, these findings provide new insights into the hepatocyte-protective mechanism of recombinant human interleukin-1 receptor antagonist.

Avicularin Reduces the Expression of Mediators of Inflammation and Oxidative Stress in Bradykinin-Treated MG-63 Human Osteoblastic Osteosarcoma Cells.

BackgroundAvicularin is a plant-derived flavonoid used in traditional Chinese medicine to treat conditions that include ankle fracture. Bradykinin stimulated MG-63 human osteoblastic osteosarcoma cells has previously been studied in an in vitro model. This study aimed to investigate the effects of avicularin on bradykinin-treated MG-63 human osteoblastic osteosarcoma cells in vitro.Material/MethodsMG-63 cells were treated with increasing concentrations of avicularin for 48 hours, followed by 1 μM of bradykinin for 24 h. The MTT assay was used to measure cell viability. Quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to measure the expression of inflammatory mediators, interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) mRNA and protein, respectively. The expression of oxidative stress factors, malondialdehyde (MDA), superoxide dismutase (SOD), and catalase were measured. Western blot and qRT-PCR were performed to determine p38, p65, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) protein levels and mRNA expression, respectively.ResultsAvicularin had no cytotoxic effect on MG-63 cells. Avicularin significantly upregulated the expression levels IL-1β, IL-6, and TNF-α in the bradykinin treated group in a dose-dependent manner. Avicularin reduced the level of MDA and the activity of SOD and catalase in the bradykinin treated MG-63 cells, reduced p-p38, p-p65, iNOS, and COX-2 expression, and decreased the p-p38/p38 ratio and the p-p65/p65 ratio in bradykinin treated MG-63 cells in a dose-dependent manner.ConclusionsAvicularin reduced the expression of inflammatory cytokines and the levels of markers of oxidative stress in MG-63 human osteoblastic osteosarcoma cells in vitro.

Neosaxitoxin Inhibits the Expression of Inflammation Markers of the M1 Phenotype in Macrophages.

(1) Background: Neosaxitoxin (NeoSTX) has been used as a local anesthetic, but its anti-inflammatory effects have not been well defined. In the present study, we investigate the effects of NeoSTX on lipopolysaccharide (LPS)-activated macrophages. (2) Methods: Raw 264.7 and equine PBMC cells were incubated with or without 100 ng/mL LPS in the presence or absence of NeoSTX (1µM). The expression of inflammatory mediators was assessed: nitric oxide (NO) content using the Griess assay, TNF-α content using the ELISA assay, and mRNA of inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) using a real-time polymerase chain reaction. (3) Results: NeoSTX (1 μM) significantly inhibited the release of NO, TNF-α, and expression of iNOS, IL-1β, and TNF-α in LPS-activated macrophages of both species studied. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by NeoSTX. Additionally, NeoSTX deactivated polarized macrophages to M1 by LPS without compromising its polarization towards M2. (4) Conclusions: NeoSTX inhibits LPS-induced release of inflammatory mediators from macrophages, and these effects may be mediated by the blockade of voltage-gated sodium channels (VGSC).

Exacerbated Imiquimod-Induced Psoriasis-Like Skin Inflammation in IRF5-Deficient Mice.

Interferon regulatory factors (IRFs) play diverse roles in the regulation of the innate and adaptive immune responses in various diseases. In psoriasis, IRF2 is known to be involved in pathogenesis, while studies on other IRFs are limited. In this study, we investigated the role of IRF5 in psoriasis using imiquimod-induced psoriasis-like dermatitis. Although IRF5 is known to play a critical role in the induction of proinflammatory cytokines by immune cells, such as dendritic cells (DCs), macrophages, and monocytes, IRF5 deficiency unexpectedly exacerbated psoriasiform skin inflammation. The interferon-α and tumor necrosis factor-α mRNA expression levels were decreased, while levels of Th17 cytokines including IL-17, IL-22, and IL-23 were increased in IRF5-deficient mice. Furthermore, IL-23 expression in DCs from IRF5-deficient mice was upregulated both in steady state and after toll-like receptor 7/8 agonist stimulation. Importantly, the expression of IRF4, which is also important for the IL-23 production in DCs, was augmented in DCs from IRF5-deficient mice. Taken together, our results suggest that IRF5 deficiency induces the upregulation of IRF4 in DCs followed by augmented IL-23 production, resulting in the amplification of Th17 responses and the exacerbation of imiquimod-induced psoriasis-like skin inflammation. The regulation of IRF4 or IRF5 expression may be a novel therapeutic approach to psoriasis.

Effects of an F18 enterotoxigenic Escherichia coli challenge on growth performance, immunological status, and gastrointestinal structure of weaned pigs and the potential protective effect of direct-fed microbial blends.

The objective of this experiment was to investigate the impact of an F18 enterotoxigenic Escherichia coli (ETEC) challenge on growth performance, aspects of intestinal function, and selected immune responses of piglets, as well as to evaluate potential protective effects of direct-fed microbial (DFM) blends. Seventy-two weaned piglets (6.4 ± 0.2 kg body weight [BW]; ~21 d of age) were assigned to one of four treatments: 1) NC: Nonchallenged (n = 10), 2) positive challenged control (PC): F18 ETEC-challenged (n = 10), 3) PC + DFM1 (n = 8; three strains of Bacillus amyloliquefaciens; 7.5 × 105 colony-forming units [cfu]/g), or 4) PC + DFM2 (n=8; 2 strains of B. amyloliquefaciens and one strain of Bacillus subtilis; 1.5 × 105 cfu/g). Feed intake and BW were recorded on day 0, 7, and 17. Pigs were sham-infected either with 6 mL phosphate-buffered saline or inoculated with 6 mL F18 ETEC (~1.9 × 109 cfu/mL) on day 7 (0 d postinoculation [dpi]). All ETEC-challenged pigs were confirmed to be genetically susceptible to F18. Pigs had ad libitum access to feed and water throughout the 17-d trial. Fecal scores were visually ranked and rectal temperatures were recorded daily. To evaluate ETEC shedding, fecal swabs were collected on dpi 0, 1, 2, 3, 5, 7, and 10. Blood samples were collected on dpi 0, 1, 2, 4, 7, and 10. Ileal tissues were collected at necropsy on dpi 10. All challenged treatments had lower final BW, decreased average daily gain (ADG), and average daily feed intake (ADFI) during the 10-d postchallenge period (P < 0.01). The DFM2 treatment increased E. coli shedding on dpi 2 and decreased iton dpi 7 (P < 0.05) compared with the PC. Rectal temperature decreased across all challenged treatments (P < 0.01). Ileal mRNA abundance of occludin (OCLN) and zonula occludens-1 (ZO-1) decreased in PC and DFM1 compared with NC (P < 0.05). Pigs fed DFM2 had intermediate ileal mRNA abundance of OCLN and increased ZO-1 mRNA compared with pigs in PC (P < 0.05). Interleukin 8 (IL-8) increased in the plasma of PC and DFM2 on dpi 2 compared with NC (P < 0.05). Mucosal IL-8 increased in PC compared with NC (P < 0.05). All challenged treatments tended to have elevated tumor necrosis factor-α (TNF-α) mRNA abundance compared with NC (P < 0.10). Challenged pigs had reduced secretory immunoglobulin A and villus height compared with NC pigs (P < 0.05). The impact of an ETEC challenge on intestinal function and the immune system has been revealed, information critical to developing improved treatment regimes.

Knockdown of Interleukin 17 Receptor A (IL‐17RA) in Paraventricular Nucleus of Hypothalamus Attenuates IL‐17‐induced Pressor Responses and Sympathetic Excitation

Inflammation contributes significantly to cardiovascular and autonomic dysfunction in hypertension and heart failure. Over the years, the pro‐inflammatory cytokine interleukin (IL)‐17 (also known as IL‐17A) has been recognized as a critical inflammatory regulator bridging immune responses and tissue inflammation. Our previous studies have demonstrated that peripherally or centrally administrated IL‐17A dramatically elevates blood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in rats, and that its receptors IL‐17RA are richly expressed in the brain. The present work sought to determine whether IL‐17RA in the hypothalamic paraventricular nucleus (PVN), a key cardiovascular/autonomic brain center, mediate hemodynamic and sympathetic responses to systemically administered IL‐17A. In urethane anesthetized male SD rats, Mean BP (MBP, mmHg), HR (beats/min) and RSNA (% change) were recorded before and for 6–8 hours after an intravenous (IV) injection of IL‐17A (500 ng). In age‐matched control rats, IV IL‐17A induced a large and long‐lasting increase (*p&lt;0.001 vs. baseline) in MBP (16.2 ± 2.3*), HR (49.8 ± 5.1*) and RSNA (165.6 ± 13.2 %*), beginning within 120 mins and peaking 6–8 hours after the injection. However, in rats pretreated with bilateral PVN microinjections of an IL‐17RA siRNA AAV virus and studied one week later, the IV IL‐17A‐elicited pressor responses in MBP (10.1 ± 2.1*), HR (22.1 ± 4.0*) and RSNA (68.5 ± 7.8 %*) were significantly (p&lt;0.05) attenuated. mRNA for tumor necrosis factor‐α (TNF‐α), IL‐1β, monocyte chemoattractant protein‐1 (MCP‐1) and IL‐17RA in the PVN was measured by real‐time qPCR. Knockdown efficiency of IL‐17RA siRNA was confirmed by a reduction (−55%) of IL‐17RA mRNA level in the PVN. Additionally, the mRNA levels of TNF‐α (1.12 ± 0.24 vs. 0.51 ± 0.11*), IL‐1β (1.05 ± 0.16 vs. 0.64 ± 0.09*), and MCP‐1 (1.01 ± 0.10 vs. 0.60 ± 0.10*) induced by IV IL‐17A were significantly reduced in the PVN in rats pretreated with IL‐17RA siRNA. These data indicate that systemically administered IL‐17A acts upon IL‐17RA in the PVN to increase the expression of inflammatory mediators that drive sympathetic excitation, suggesting that PVN plays an important role in mediating the effects of circulating IL‐17A on sympathetic drive and cardiovascular function in inflammatory conditions such as hypertension and heart failure. IL‐17RA in the PVN is a potential target for therapeutic intervention in these diseases.Support or Funding InformationThis research was supported by NIH/NHLBI RO1 HL139521.

The role of inflammation in a rat model of chronic thromboembolic pulmonary hypertension induced by carrageenan.

BackgroundChronic thromboembolic pulmonary hypertension (CTEPH) is a life-threatening condition arising from the thrombus and obstructive remodeling of the pulmonary arteries, which causes a significant morbidity and mortality. Although the modern treatment in CTEPH has been significant advanced both in surgical and medical treatment, none can claim to cure the disease, largely because of our limited understanding of the underlying pathogenesis of the disease and lack of a reliable CTEPH animal model to study for. Recently, inflammation has been accepted as a common pathway through which various risk factors trigger venous thrombo-embolism (VTE) formation, we describe a novel mouse model of CTEPH which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of CTEPH in humans, to open a new horizons of inflammation in CTEPH.MethodsBy administering a pulmonary embolism (PE) protocol (comprising 3 sequential left jugular vein injections of autologous blood clots) to 8-week-old male Sprague Dawley (SD) rats using tranexamic acid (200 mg/kg.d) to inhibit fibrinolysis and injecting additional carrageenan (20 mg/kg, once a week) to create perivascular inflammation, we successfully generated a CTEPH animal model. By monitoring the mean pulmonary artery pressure (mPAP) and the histopathological change to evaluate the CTEPH model. By detecting the RT-PCR, western blot, TUNEL, and immunohistochemistry in the sub-groups to find the potential mechanism of inflammation may work in the pulmonary vascular remolding.ResultsIn this study, rats with CTEPH exhibited pronounced pulmonary vascular remolding with higher vessel wall area/total area (WA/TA) ratio in comparison to the control rats (85.41%±7.37% vs. 76.41%±5.97%, P<0.05), the mPAP (25.51±1.13 vs. 15.92±1.13 mmHg, P<0.05). Significant differences in mean pulmonary artery pressure (mPAP) values were observed between rats injected solely with clots and those injected with both clots and carrageenan (25.51±1.13 vs. 29.82±1.26 mmHg, P<0.05, respectively). Furthermore, following the third embolization, thrombi and intimal hyperplasia occurred in the pulmonary artery. In addition, repeated embolization elevated mRNA and protein levels of tumor necrosis factor-α (TNF-α), NF-κB/p65, and B-cell lymphoma-2 (BCL-2), but decreased BAX expression in a time-dependent manner.ConclusionsTake advantage of the inflammation to trigger VTE formation, we successfully generated a CTEPH animal model. Inflammation may play a crucial role in the pathogenesis and progression of CTEPH by inhibiting endothelial cell apoptosis. Understanding the role of inflammation in CTEPH may not only help to determine the optimal treatment options but also may aid in the development of future preventative strategies, since current anticoagulation treatment regimens are not designed to inhibit inflammation.