Kappa Light Polypeptide Gene Enhancer Research Articles

Cryptosporidium parvum upregulates miR-942-5p expression in HCT-8 cells via TLR2/TLR4-NF-\u03baB signaling

BackgroundMicro (mi)RNAs are small noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. This study investigated host miRNA activity in the innate immune response to Cryptosporidium parvum infection.MethodsIn vitro infection model adopts HCT-8 human ileocecal adenocarcinoma cells infected with C. parvum. The expression of miR-942-5p was estimated using quantitative real-time polymerase chain reaction (qPCR). The TLRs-NF-κB signaling was confirmed by qPCR, western blotting, TLR4- and TLR2-specific short-interfering (si)RNA, and NF-κB inhibition.ResultsHCT-8 cells express all known toll-like receptors (TLRs). Cryptosporidium parvum infection of cultured HCT-8 cells upregulated TLR2 and TLR4, and downstream TLR effectors, including NF-κB and suppressed IκBα (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha). The expression of miR-942-5p was significantly upregulated at 4, 8, 12 and 24 h post-infection, and especially at 8 hpi. The results of TLR4- and TLR2-specific siRNA and NF-κB inhibition showed that upregulation of miR-942-5p was promoted by p65 subunit-dependent TLR2/TLR4-NF-κB pathway signaling.ConclusionsmiR-942-5p of HCT-8 cells was significantly upregulated after C. parvum infection, especially at 8 hpi, in response to a p65-dependent TLR2/TLR4-NF-κB signaling. TLR4 appeared to play a dominant role.

Effect of electroacupuncture on intestinal Toll-like receptor 4 and nuclear factor-kappa B in obese rats

To investigate the effect of electroacupuncture (EA) on intestinal Toll-like receptor 4 (TLR4) and nuclear factor-kappa B (NF-κB) in obese rats, so as to explore the mechanism of action of acupuncture in losing weight. A total of 50 male Wistar rats were randomly divided into control and model groups. High-fat feed was used to establish a rat model of obesity, and after modeling, the 24 rats were randomly divided into model group, TLR4 inhibitor group, and EA group, with 8 rats in each group. The rats in the EA group were given EA at "Guanyuan" (CV4), "Zhongwan "(CV12), "Zusanli" (ST36), and" Fenglong" (ST40), 10 minutes each time, 3 times a week, and those in the TLR4 inhibitor group were given intraperitoneal injection of TAK-242 three times a week; the course of treatment was 8 weeks for both groups. Body weight and blood glucose were measured every two weeks. Co-immunoprecipitation was used to observe the interaction between TLR4 and NF-κB p65 in the intestinal tissue; electrophoretic mobility shift assay was used to measure the activity of NF-κB p65; Western blot was used to measure the protein expression of TLR4, phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (p-IκBα), and NF-κB p65; quantitative real-time PCR was used to measure the mRNA expression of TLR4, NF-κB p65, and IκBα. Compared with the control group, the model group had significant increases in body weight, blood glucose, and protein and mRNA expression of TLR4 and NF-κB p65 (P<0.01, P<0.05), as well as significant enhancement in the interaction between TLR4 and NF-κB p65 and activity of NF-κB p65 (P<0.05，P<0.01). Compared with the model group, the EA group had a significant reduction in body weight (P<0.05), both of the EA group and the TLR4 inhibitor group had significant reductions in blood glucose, and protein and mRNA expression of TLR4, p-IκBα， and NF-κB p65 (P<0.05，P<0.01), as well as significant reductions in the activity of NF-κB p65 (P<0.01). EA can effectively regulate intestinal TLR4, inhibit the interaction between TLR4 and NF-κB p65, and reduce the activity of NF-κB p65, which may be a potential mechanism of EA in reducing body weight and blood glucose in obese rats.

Orobol, A Derivative of Genistein, Inhibits Heat-Killed Propionibacterium acnes-Induced Inflammation in HaCaT Keratinocytes.

Acne is a chronic skin disease that typically occurs in the teens and twenties, and its symptoms vary according to age, sex, diet, and lifestyle. The condition is characterized by hyperproliferation of keratinocytes in the epidermis, sebum overproduction, excessive growth of Propionibacterium acnes, and P. acnes-induced skin inflammation. Interleukin (IL)-1α and IL-6 are predominant in the inflammatory lesions of acne vulgaris. These cytokines induce an inflammatory reaction in the skin in the presence of pathogens or stresses. Moreover, IL-1α accelerates the production of keratin 16, which is typically expressed in wounded or aberrant skin, leading to abnormalities in architecture and hyperkeratinization. Orobol (3',4',5,7-tetrahydroxyisoflavone) is a metabolite of genistein that inhibited the P. acnes-induced increases in IL-6 and IL-1α levels in human keratinocytes (HaCaTs) more effectively compared with salicylic acid. In addition, orobol decreased the IL-1α and IL-6 mRNA levels and inhibited the phosphorylation of inhibitor of kappa-B kinase, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha, and mitogen-activated protein kinase induced by P. acnes. Finally, the expression of Ki67 was decreased by orobol. Thus, orobol ameliorated the inflammation and hyperkeratinization induced by heat-killed P. acnes and thus has potential for use in functional foods and cosmetics.

Anti-Inflammatory and Chondroprotective Effects of Vanillic Acid and Epimedin C in Human Osteoarthritic Chondrocytes.

In osteoarthritis (OA), inhibition of excessively expressed pro-inflammatory cytokines in the OA joint and increasing the anabolism for cartilage regeneration are necessary. In this ex-vivo study, we used an inflammatory model of human OA chondrocytes microtissues, consisting of treatment with cytokines (interleukin 1β (IL-1β)/tumor necrosis factor α (TNF-α)) with or without supplementation of six herbal compounds with previously identified chondroprotective effect. The compounds were assessed for their capacity to modulate the key catabolic and anabolic factors using several molecular analyses. We selectively investigated the mechanism of action of the two most potent compounds Vanillic acid (VA) and Epimedin C (Epi C). After identification of the anti-inflammatory and anabolic properties of VA and Epi C, the Ingenuity Pathway Analysis showed that in both treatment groups, osteoarthritic signaling pathways were inhibited. In the treatment group with VA, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was inhibited by attenuation of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) phosphorylation. Epi C showed a significant anabolic effect by increasing the expression of collagenous and non-collagenous matrix proteins. In conclusion, VA, through inhibition of phosphorylation in NF-κB signaling pathway and Epi C, by increasing the expression of extracellular matrix components, showed significant anti-inflammatory and anabolic properties and might be potentially used in combination to treat or prevent joint OA.

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.

Anti-Inflammatory Effects of Lasia spinosa Leaf Extract in Lipopolysaccharide-Induced RAW 264.7 Macrophages.

Lasia spinosa (L.) Thwaites was used as a traditional medicine to treat many inflammatory diseases for centuries. However, its effects on the inflammatory response are not yet characterized. In this study, we investigated the anti-inflammatory activities of L. spinosa leaf extract in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. We found that ethanol extracts of L. spinosa leaves showed anti-oxidant activity due to the presence of high levels of polyphenolic compounds. Treatment with the leaf extract significantly repressed the production of inflammatory mediators such as nitric oxide and reactive oxygen species and the expression of pro-inflammatory cytokines in the LPS-stimulated RAW 264.7 cells. Moreover, L. spinosa leaf extract treatment prevented activation of the nuclear factor-kappa B pathway by inhibiting nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) degradation. Furthermore, the mitogen-activated kinase and phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathways were suppressed upon treatment with the leaf extract. In addition to suppressing inflammatory factors, the extract also activated the nuclear factor erythroid 2-related factor 2/heme-oxygenase-1 pathway. We propose that L. spinosa leaf extract has the potential as an effective therapeutic agent for alleviating oxidative stress and excessive inflammation.

Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations

An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes. To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations. In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional invitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling. We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents. We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.

Rian/miR-210-3p/Nfkb1 Feedback Loop Promotes Hypoxia-Induced Cell Apoptosis in Myocardial Infarction Through Deactivating the PI3K/Akt Signaling Pathway.

Myocardial infarction (MI) is a severe disease that could lead to reversible or irreversible ischemic heart damage. A previous study has revealed that microRNA mmu-miR-210-3p expression is downregulated in fat-1 transgenic mice post-MI. Nevertheless, the specific mechanism of miR-210-3p in MI remains obscure. In this study, we observed that miR-210-3p expression was downregulated in the mice's left ventricle post-MI, and miR-210-3p expression was suppressed while cell apoptosis was promoted in H9c2 cells under hypoxia condition. Besides, miR-210-3p overexpression could enhance cell proliferation and inhibit cell apoptosis in hypoxia-treated H9c2 cells. Then, molecular mechanism assays revealed that miR-210-3p overexpression could activate the PI3K/Akt pathway, and nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (Nfkb1) was the target of miR-210-3p. In addition, RNA imprinted and accumulated in nucleus (Rian), a long noncoding RNA, could sponge miR-210-3p to upregulate Nfkb1 expression. Besides, Nfkb1 was verified to facilitate the transcription of Rian by binding with a Rian promoter. Furthermore, rescue assays revealed that both Nfkb1 and PI3K/Akt pathway are engaged in the Rian-mediated cell proliferation and apoptosis in hypoxia-treated H9c2 cells. In conclusion, a Rian/miR-210-3p/Nfkb1 feedback loop enhances hypoxia-induced cell apoptosis in MI through deactivating the PI3K/Akt pathway.

Potential mechanisms and serum biomarkers involved in sex differences in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a disease associated with high mortality, but notable sex differences have been observed between males and females. For this reason, further research on the mechanisms underlying sex differences in PAH is required to better understand and treat the disease. This study mainly focused on gene expression levels to investigate potential differences in the pathogenesis and progression of PAH between the male and female sexes.Sex-specific differentially expressed genes (DEGs) were analyzed using the Gene Expression Omnibus datasets GSE117261 and GSE38267. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted, and a protein-protein interaction (PPI) network was established based on the identified DEGs to predict potential mechanisms involved in the observed sex differences in the pathogenesis of PAH.We identified 26 female- and 53 male-specific DEGs from lung tissue and 498 female-specific DEGs in blood samples. No male-specific DEGs were identified from blood samples. GO and KEGG pathway analyses revealed that female-specific DEGs in lung tissue were enriched in inflammatory response and cytokine-cytokine receptor interaction, whereas male-specific DEGs were mainly enriched in cellular chemotaxis and the nuclear factor of kappa light polypeptide gene enhancer in B-cell (NF-kappa B) signaling pathway. Lipocalin 2 (LCN2) was the only gene that was differentially expressed in both the lung tissue and the blood of female patients.In conclusion, inflammation and immunity may play key roles in the pathogenesis of female PAH, and LCN2 may act as a serum biomarker of female PAH, whereas the pathogenesis in males is more complicated.

Dimethyl fumarate alleviates the nitroglycerin (NTG)-induced migraine in mice

BackgroundOxidative stress and inflammatory pathways are involved in migraine and endogenous antioxidant defense system has a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of the most pharmacologically effective molecules among the fumaric acid esters (FAEs), dimethyl fumarate, nuclear factor E2-related factor 2/antioxidant response element (Nrf-2/ARE) pathway-mediated, in regulating the hypersensitivity in a mouse model of nitroglycerine (NTG)-induced migraine.MethodsMice were orally administered with DMF at the doses of 10, 30, and 100 mg/kg, 5 min after NTG intraperitoneal injections. We performed histological and molecular analysis on the whole brain and behavioral tests after 4 h by NTG-migraine induction. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-кB) subunit p65, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), inducible nitrite oxide synthase (iNOS), cyclooxygenase 2 (COX-2), Nrf-2, manganese superoxide dismutase (Mn-SOD), and heme-oxygenase-1 (HO-1) were detected by Western blot. Tail flick, hot plate, orofacial formalin, and photophobia tests were used to evaluate migraine-like pain and migraine-related light sensitivity.Moreover, we evaluate Nrf-2-dependent mechanism by the in vitro stimulation of cells extracted by trigeminal ganglia with diethylenetriamine/nitric oxide (DETA/NO), a nitric oxide (NO) donor. The cells were pre-treated with DMF and an antagonist of Nrf-2, trigonelline (TR) 2 h before DETA/NO stimulation.ResultsDMF treatment notably reduced histological damage as showed by cresyl violet staining; also, regulating both NF-κB and Nrf-2 pathway, DMF treatment decreased the severity of inflammation and increased the protective antioxidant action. Moreover, the headache was significantly reduced. The protective effect of DMF treatment, via Nrf-2, was confirmed in in vitro studies, through inhibition of Nrf-2 by trigonelline. Cytotoxicity, iNOS, and MnSOD expression were evaluated.ConclusionThese results provided the evidence that DMF, by Nrf-2 modulation, has a protective effect on central sensitization induced by NTG, suggesting a new insight into the potential application of DMF as novel candidates in drug development for migraine.

NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells.

The dimeric transcription factor NF-κB regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-κB is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα). Activation of NF-κB requires the degradation of IκBα, which then exposes a nuclear localization signal on NF-κB and promotes its trafficking to the nucleus. Once in the nucleus, NF-κB binds to the promotor region of NF-κB target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression. The activation of NF-κB occurs independently of transcription or translation. Therefore, the activation state of NF-κB must be measured either by quantifying NF-κB specifically in the nucleus, or by quantifying expression of NF-κB target genes. In this protocol, cells stably transfected with an NF-κB::luciferase reporter construct are assayed for NF-κB activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-κB, which traffics to the nucleus and binds to κB sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-κB activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

Apigenin Attenuates Allergic Responses of Ovalbumin-Induced Allergic Rhinitis Through Modulation of Th1/Th2 Responses in Experimental Mice

Background:Allergic rhinitis (AR) is an immunoglobulin E (IgE)-mediated immune-inflammatory response mainly affecting nasal mucosa. Apigenin, a flavonoid, has been documented to possess promising anti-allergic potential.Aim:To determine the potential mechanism of action of apigenin against ovalbumin (OVA)-induced AR by assessing various behavioral, biochemical, molecular, and ultrastructural modifications.Materials and Methods:Allergic rhinitis was induced in BALB/c mice (18-22 grams) by sensitizing it with OVA (5%, 500 μL, intraperitoneal [IP] on each consecutive day, for 13 days) followed by intranasal challenge with OVA (5%, 5 μL per nostril on day 21). Animals were treated with either vehicle (distilled water, 10 mg/kg, IP) or apigenin (5, 10, and 20 mg/kg, IP).Results:Intranasal challenge of OVA resulted in significant induction (P < .05) of AR reflected by an increase in nasal symptoms (sneezing, rubbing, and discharge), which were ameliorated significantly (P < .05) by apigenin (10 and 20 mg/kg) treatment. It also significantly inhibited (P < .05) OVA-induced elevated serum histamine, OVA-specific IgE, total IgE, and IgG1 and β-hexosaminidase levels. Ovalbumin-induced increased levels of interleukin (IL)-4, IL-5, IL-13, and interferon (IFN)-γ in nasal lavage fluid were significantly decreased (P < .05) by apigenin. Ovalbumin-induced alterations in splenic GATA binding protein 3 (ie, erythroid transcription factor) (GATA3), T-box protein expressed in T cells (T-bet), signal transducer and activator of transcription-6 (STAT6), suppressor of cytokine signaling 1 (SOCS1), nuclear factor-kappa B (NF-κB), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha messenger RNA, as well as protein expressions were significantly inhibited (P < .05) by apigenin. It also significantly ameliorated (P < .05) nasal and spleen histopathologic and ultrastructure aberration induced by OVA.Conclusion:Apigenin regulates Th1/Th2 balance via suppression in expressions of Th2 response (IgE, histamine, ILs, GATA3, STAT6, SOCS1, and NF-κB) and activation of Th1 response (IFN-γ and T-bet) to exert its anti-allergic potential in a murine model of OVA-induced AR.

Trifolirhizin mitigates ovalbumin-induced lung inflammation and tissue damage in neonatal rats via inhibition of the NF-κB signaling pathway

Purpose: To investigate the effect of trifolirhizin on neonatal rat model of asthma, and the mechanism of action involved. Methods: Neonatal rats (n = 50) were randomly assigned to 5 groups (10 pups/group): sham, asthma and three treatment groups. With the exception of sham group, the rat pups were sensitized intraperitoneally with ovalbumin (OVA) at a dose of 20 µg/kg on days 7 and 21 postpartum. Rats in the treatment groups received trifolirhizin intragastrically at doses of 2, 4 and 5 mg/kg on day 7 postpartum. Eosinophils in bronchoalveolar lavage fluid (BALF) were counted using hematological analyzer. Serum immunoglobulin (Ig)E and interleukin (IL)-4, IL-5 and IL-13 levels in BALF were determined using their respective enzyme-linked immunosorbent assay (ELISA) kits. Messenger RNA (mRNA) expressions of mucin 5AC (Muc5AC), mucin 5B (Muc5B), tumor necrosis factor α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) were determined using immunohistochemical staining, while the protein expression of inhibitor of nuclear factor of kappa light polypeptide gene enhancer in B-cells alpha (IκBα) was assayed by Western blotting. Results: Serum IgE level was significantly higher in asthma group than in sham group, but was significantly and dose-dependently reduced after treatment with trifolirhizin (p < 0.05). Lung tissue damage was also significantly mitigated in the treatment groups, relative to asthma group (p < 0.05). Trifolirhizin treatment significantly and dose-dependently downregulated the mRNA expressions of Muc5AC, Muc5B, TNF-α and ICAM-1, but upregulated IκBα protein expression significantly and dosedependently (p < 0.05). Bronchoalveolar lavage fluid (BALF) levels of IL-4, IL-5 and IL-13 were significantly higher in asthma group, but were significantly and dose-dependently reduced after treatment with trifolirhizin (p < 0.05). Conclusion: These results indicate that trifolirhizin mitigates OVA-induced lung inflammation and tissue damage in neonatal rats via inhibition of NF-κB signaling pathway, thus affording a potential therapeutic strategy for the management of asthma

Activation of CREB Protein With Tabersonine Attenuates STAT3 During Atherosclerosis in Apolipoprotein E-Deficient Mice

Objective:Atherosclerosis is a pathological condition of fat deposition in the arteries, which causes cardiovascular disorders. Management of atherosclerosis remains a challenge and conventional drugs used for its management have several limitations. This study evaluated the protective effect of tabersonine against atherosclerosis and assessed its molecular mechanism of action.Methods:Atherosclerosis was induced by feeding apolipoprotein E (ApoE)-deficient mice a high-fat diet. Mice were treated with 20 or 40 mg/kg of tabersonine intraperitoneally for the 12-week duration of the study. Atherosclerosis markers and nitric oxide were measured in the sera of ApoE-deficient mice. Mediators of inflammation and markers of oxidative stress were assessed using enzyme-linked immunosorbent assays. Western blotting, quantitative reverse transcriptase polymerase chain reaction, and immunohistochemistry analyses were conducted to determine the protein expression in aortic tissue.Results:The tabersonine-treatment groups had an improved lipid profile and enhanced liver function, compared to the ApoE treatment group. Tabersonine treatment resulted in reduced levels of nitric oxide, cytokines, and oxidative stress, compared to the ApoE group. The altered expression levels of protein inhibitor activated STAT-3 (PIAS3), signal transducer and activator of transcription-3 (STAT-3), and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα) in ApoE-deficient mice were ameliorated by tabersonine treatment. Moreover, cAMP-response-element-binding (CREB) expression was elevated in aortic tissue of tabersonine treatment groups, compared to the ApoE group.Conclusion:These results suggested that tabersonine ameliorates the expression of STAT-3 by activating CREB protein in atherosclerotic ApoE-deficient mice.

Anti-inflammatory activity of Zanthoxylum rhetsa bark fractions via suppression of nuclear factor-kappa B in lipopolysaccharide-stimulated macrophages

Background: Zanthoxylum rhetsa is a plant used in traditional medicine and is known to possess health benefits such as antibacterial, antidiabetic, and anti-diarrheal activities. Objectives: The objective of this study was to explore and demonstrate the anti-inflammatory activity of various solvent fractions of Z. rhetsa bark. Materials and Methods: The effect of crude methanolic extract and its fractions (hexane, chloroform, ethyl acetate, and butanol) on the levels of pro-inflammatory cytokines (interleukin-1β, tumor necrosis factor-alpha, and interleukin-6) and inflammatory factors was tested via targeting nuclear factor-kappa B (NF-κB) signaling pathways in lipopolysaccharide (LPS)-stimulated mouse RAW 264.7 macrophages. Results: Treatment with all the solvent fractions at various concentrations (50, 100, and 200 μg/ml) suppressed pro-inflammatory cytokine levels in a dose-dependent manner. Among the fractions, the chloroform fractions exhibited a significant inhibition of LPS-induced inflammation. Moreover, these fractions effectively suppressed the expression of various NF-κB signaling targets, including NF-κB, nitric oxide synthase, and cyclooxygenase-2 as well as inhibited the degradation of nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (inhibitor of kappa B alpha [IκBα]). This anti-inflammatory effect was mediated by the prevention of IκBα degradation, as this protein prevents the translocation of NF-κB from the cytoplasm to the nucleus, initiating the transcription of pro-inflammatory genes. Conclusion: Thus, the Z. rhetsa chloroform fraction may be an effective natural anti-inflammatory agent against inflammation-associated diseases.

Synthesis, Characterization and in vitro, in vivo and in silico Anti-Inflammatory Studies of the Novel Hybrid Based on Ibuprofen and 3-Hydroxy-Copalic Acid Isolated from Copaiba Oil (Copaifera multijuga)

A novel anti-inflammatory hybrid 3-ibuprofenyl-copalic acid (3-IbuCA) was synthesized from 3-hydroxy-copalic acid isolated from Amazonian copaiba oil (Copaifera multijuga Hayne), and the anti-inflammatory ibuprofen. After full characterization, several assays to verify its anti-inflammatory effects were performed in vitro, in vivo and in silico (molecular docking). Induced fit docking was performed to observe the interactions with the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). In vitro tests of cytotoxicity and tumor necrosis factor (TNF)-α inhibition, and in vivo tests of pleurisy, protein expression and gastrocytotoxicity were performed. Molecular docking studies with COX-1 and 2 showed binding free energies (ΔG) of -2.2 and -7.8 kcal mol-1, respectively, while for mofezolac and indomethacin, the binding free energies ΔG presented values of -8.5 and -10.1 kcal mol-1, which makes 3-IbuCA selective for COX-2 inhibition. This hybrid showed no toxicity against human macrophage at concentrations up to 2 µM, and inhibited TNF-α production in lipopolysaccharide (LPS)-stimulated macrophages. In the pleurisy assays, 3-IbuCA reduced the total leukocytes and mononuclear cells, which was followed by reduction of p-IKBα (phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) protein expression. Compared with ibuprofen alone, the hybrid caused less gastric damage. Thus, the docking, together with in vitro and in vivo studies suggest that this novel hybrid has potential as a new anti-inflammatory agent.

Dietary Scenedesmus ovalternus improves disease resistance of overwintering gibel carp (Carassius gibelio) by alleviating toll-like receptor signaling activation

This study was conducted to investigate the effect of dietary Scenedesmus ovalternus on the growth and disease resistance of gibel carp (Carassius gibelio) during overwintering. Gibel carp (initial body weight: 90.39 ± 0.33 g) were fed with diets containing 0% or 4% Scenedesmus ovalternus (DS0 and DS4) for 4 weeks during the early overwintering period, and then all fish were left unfed during the late overwintering period. A bacterial challenge test using Aeromonas hydrophila was subsequently conducted. The 4% Scenedesmus ovalternus diet had no effect on the growth of gibel carp (P > 0.05), but did improve the survival rate after the challenge (P ≤ 0.05). In the DS0 group, the bacterial challenge decreased the contents of complement 3 (C3), immunoglobulin M (IgM), interleukin 2 (IL2) and tumor necrosis factor α (TNFα) in fish (P < 0.05); in the DS4 group, the challenge increased total antioxidant capacity (T-AOC) and myeloperoxidase (MPO) activity but decreased IL2 and TNFα contents (P < 0.05). The activities of MPO and contents of C3, IgM and TNFα were higher in the DS4 group than that fed the DS0 diet after bacterial challenge (P < 0.05). Compared to pre challenge, the expression levels of toll like receptor 2 (TLR2), toll like receptor 3 (TLR3), toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), TIR-domain-containing adapter-inducing interferon β (TRIF), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα), transforming growth factor β (TGFβ), interleukin 1β (IL1β), tumor necrosis factor α1 (TNFα1) and interleukin 10 (IL10) in the head kidney of gibel carp were induced after challenge (P < 0.05). Gibel carp fed the DS4 diet showed lower expression of TGFβ in head kidney before the challenge and lower expression of TLR2, TLR3, TLR4, TIRAP, TRIF, IκBα, TNFα1, IL10 and TGFβ after the challenge than that fed the DS0 diet (P < 0.05). Overall, Scenedesmus ovalternus supplement enhanced the resistances of gibel carp against A. hydrophila after overwintering via the TLR signaling pathway.

Piperine, a functional food alkaloid, exhibits inhibitory potential against TNBS-induced colitis via the inhibition of IκB-α/NF-κB and induces tight junction protein (claudin-1, occludin, and ZO-1) signaling pathway in experimental mice

Inflammatory bowel disease is a chronic immunoinflammatory disease of the gastrointestinal tract. Piperine, an alkaloid, has been reported to possess antioxidant, anti-inflammatory, antiapoptotic, and antiulcer potential. To elucidate the plausible mechanisms of action of piperine on experimental trinitrobenzenesufonic acid (TNBS)-induced colitis by assessing various biochemical, molecular, histological, and ultrastructural modifications. Colitis was induced in male Sprague-Dawley rats via intrarectal instillation of TNBS. Then, the rats were treated with piperine (10, 20, and 40 mg/kg, p.o.) for 14 days. TNBS induced significant (p < 0.05) colonic damage, which was assessed by disease activity index, macroscopic score, and stool consistency. The administration of piperine (20 and 40 mg/kg) significantly inhibited (p < 0.05) these damages. Treatments with piperine (20 and 40 mg/kg) notably inhibited (p < 0.05) the TNBS-induced elevation of oxido-nitrosative stress (superoxide dismutase, glutathione, malondialdehyde, and nitric oxide), 5-hydroxytryptamine, and hydroxyproline content in the colon. Furthermore, colonic inducible nitric oxide synthase (iNOs), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, interferon-gamma, and cyclooxygenase-2 (COX-2) messenger RNA (mRNA) expressions were upregulated after TNBS instillation and piperine (20 and 40 mg/kg) significantly attenuated (p < 0.05) these elevated mRNA expressions. TNBS decreased the expressions of tight junction (TJ) protein (claudin-1, occludin, and zonula occludens-1 (ZO-1)) and increased the expressions of proapoptotic (caspase-1) protein. These expressions were markedly inhibited (p < 0.05) by piperine treatment. Histological and ultrastructural studies of transmission electron microscopy suggested that piperine significantly ameliorated (p < 0.05) TNBS-induced colonic aberrations. Piperine ameliorated the progression of TNBS-induced colitis by modulating the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-alpha/nuclear factor-kappa B signaling pathway, thus inhibiting the overexpression of proinflammatory cytokines (TNF-α and IL's), COX-2, iNOs, oxido-nitrosative stress, and proapoptotic proteins (caspase-1) that may improve the expression of TJ protein (claudin-1, occludin, and ZO-1).

Oleanolic Acid Acetate Exerts Anti-Inflammatory Activity via IKKα/β Suppression in TLR3-Mediated NF-κB Activation.

Oleanolic acid acetate (OAA), a major triterpenoid compound of Vigna angularis (azuki bean, V. angularis), has been shown to downregulate inflammatory responses in macrophages. Here, we show the molecular basis for the effect of OAA on Toll-like receptor (TLR) downstream signaling. OAA treatment significantly inhibited the secretion of embryonic alkaline phosphatase (SEAP) induced by polyinosinic acid (poly(I), TLR3 ligand) in a dose-dependent manner and without cytotoxicity in THP1-XBlue cells. In addition, OAA downregulated the gene expression of poly(I) induced pro-inflammatory cytokines and chemokines genes such as MCP-1, IL-1β, IL-8, VCAM-1 and ICAM-1. Furthermore, we found that the inhibition activity of OAA was accompanied by decreased activation of not only nuclear factor-kappa B (NF-κB) signaling but also mitogen-activated protein kinase (MAPK) signaling upon stimulation with the TLR3 agonist. Interestingly, the interaction of OAA with IκB kinase α/β (IKKα/β) strongly attenuated the production of certain proteins and inflammatory cytokines in the TLR3 signaling pathway, such as nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IkBα), extracellular regulated kinases (ERK), and p38, in an in vitro model. The action of OAA was regulated by TLR3, demonstrating that TLR3 plays a critical role in mediating the physiologically-relevant anti-inflammatory action of OAA and that the interaction with IKKα/β is modulated through TLR3. These results reveal new insight into the understanding of the regulatory mechanisms of the downstream TLR3 signaling pathway and consequent inflammatory responses that are involved in the development and progression of inflammatory diseases.

IκBζ is a key player in the antipsoriatic effects of secukinumab

IκBζ plays a key role in psoriasis by mediating IL-17A-driven effects, but the molecular mechanism by which IL-17A regulates IκBζ expression is not clarified. We sought to explore the molecular transformation in patients with psoriasis during anti-IL-17A (secukinumab) treatment with a focus on IκBζ. The study was an open-label, single-arm, single-center secukinumab treatment study that included 14 patients with plaque psoriasis. Skin biopsy specimens and blood samples were collected on days 0, 4, 14, 42, and 84 and processed for microarray gene expression analysis. Furthermore, invitro experiments with human keratinocytes and synovial fibroblasts were conducted. Secukinumab improved clinical scores and histologic psoriasis features. Moreover, secukinumab altered the skin transcriptome. The major transcriptional shift appeared between day 14 and day 42 after treatment initiation, although 80 genes were differentially expressed already at day 4. Expression of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor (IκB) ζ (NFKBIZ, the gene encoding IκBζ) was reduced already after 4 days of treatment in the skin. NFKBIZ expression correlated to Psoriasis Area and Severity Index score, and NFKBIZ mRNA levels in the skin decreased during anti-IL-17A treatment. Moreover, specific NFKBIZ signature genes were significantly altered during anti-IL-17A treatment. Finally, we identified NF-κB activator 1 (Act1), p38 mitogen-activated protein kinase (MAPK), Jun NH2-terminal kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as key signaling pathways in NFKBIZ/IκBζ regulation. Our results define a crucial role for IκBζ in the antipsoriatic effect of secukinumab. Because IκBζ signature genes were regulated already after 4 days of treatment, this strongly indicates that IκBζ plays a crucial role in the antipsoriatic effects mediated by anti-IL-17A treatment.