OBJECTIVE: To explore the mechanism of effects of total saponin fraction from Dioscorea Nipponica Makino (TSDN) on M1/M2 polarization of monocytes/macrophages and arachidonic acid (AA) pathway in rats with gouty arthritis (GA). METHODS: Seventy-two Sprague Dawley rats were randomly divided into 4 groups (n=18 in each): normal, model, TSDN at 160 mg/kg, and celecoxib at 43.3 mg/kg. Monosodium urate crystal (MSU) was injected into the rats' ankle joints to induce an experimental GA model. Blood and tissue samples were collected on the 3rd, 5th, and 8th days of drug administration. Histopathological changes in the synovium of joints were observed via hematoxylin and eosin (HE) staining. The expression levels of arachidonic acid (AA) signaling pathway were assessed via real-time polymerase chain reaction (qPCR) and Western blot. Flow cytometry was used to determine the proportion of M1 and M2 macrophages in the peripheral blood. An enzyme-linked immunosorbent assay (ELISA) was used to detect interleukine (IL)-1 β, tumor necrosis factor-alpha (TNF-α), IL-4, IL-10, prostaglandin E₂ (PGE₂), and leukotriene B4 (LTB4). RESULTS: HE staining showed that TSDN improved the synovial tissue. qPCR and Western blot showed that on the 3rd, 5th and 8th days of drug administration, TSDN reduced the mRNA and protein expressions of cyclooxygenase (COX)2, microsomal prostaglandin E synthase-1 derived eicosanoids (mPGES-1), 5-lipoxygenase (5-LOX), recombinant human mothers against decapentaplegic homolog 3 (Smad3), nucleotide-binding oligomerization domain-like receptor protein 3 (NALP3), and inducible nitric oxide synthase (iNOS) in rats' ankle synovial tissues (P<0.01). TSDN decreased COX1 mRNA and protein expression on 3rd and 5th day of drug administration and raised it on the 8th day (both P<0.01). It lowered CD68 protein expression on days 3 (P<0.01), as well as mRNA and protein expression on days 5 and 8 (P<0.01). On the 3rd, 5th, and 8th days of drug administration, TSDN elevated the mRNA and protein expression of Arg1 and CD163 (P<0.01). Flow cytometry results showed that TSDN decreased the percentage of M1 macrophages while increasing the percentage of M2 in peripheral blood (P<0.05 or P<0.01). ELISA results showed that on the 3rd, 5th, and 8th days of drug administration, TSDN decreased serum levels of IL-1 β, TNF-α, and LTB4 (P<0.01), as well as PGE₂ levels on days 3rd and 8th days (P<0.05 or P<0.01); on day 8 of administration, TSDN increased IL-4 serum levels and enhanced IL-10 contents on days 5 and 8 (P<0.05 or P<0.01). CONCLUSION The anti-inflammatory effect of TSDN on rats with GA may be achieved by influencing M1/M2 polarization through AA signaling pathway.
Rats ; Humans ; Animals ; Arthritis, Gouty/drug therapy* ; Monocytes/pathology* ; Interleukin-10/metabolism* ; Arachidonic Acid/pharmacology* ; Dioscorea/chemistry* ; Rats, Wistar ; Tumor Necrosis Factor-alpha/metabolism* ; Saponins/therapeutic use* ; Interleukin-4/metabolism* ; Leukotriene B4/pharmacology* ; Rats, Sprague-Dawley ; Macrophages ; Signal Transduction ; RNA, Messenger/metabolism*

OBJECTIVE: To explore the expression patterns, prognostic implications, and biological role of leukotriene B4 receptor (LTB4R) in patients with acute myeloid leukemia (AML). METHODS: We collected the data of mRNA expression levels and clinical information of patients with AML from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database for mRNA expression analyses, survival analyses, Cox regression analyses and correlation analyses using R studio to assess the expression patterns and prognostic value of LTB4R. The correlation of LTB4R expression levels with clinical characteristics of the patients were analyzed using UALCAN. The co-expressed genes LTB4R were screened from Linkedomics and subjected to functional enrichment analysis. A protein-protein interaction network was constructed using STRING. GSEA analyses of the differentially expressed genes (DEGs) were performed based on datasets from TCGA-LAML stratified by LTB4R expression level. We also collected peripheral blood mononuclear cells (PBMCs) from AML patients and healthy donors for examination of the mRNA expression levels of LTB4R and immune checkpoint genes using qRT-PCR. We also examined serum LTB4R protein levels in the patients using ELISA. RESULTS: The mRNA expression level of LTB4R was significantly increased in AML patients (4.898±1.220 vs 2.252±0.215, P < 0.001), and an elevated LTB4R expression level was correlated with a poor overall survival (OS) of the patients (P=0.004, HR=1.74). LTB4R was identified as an independent prognostic factor for OS (P=0.019, HR=1.66) and was associated with FAB subtypes, cytogenetic risk, karyotype abnormalities and NPM1 mutations. The co- expressed genes of LTB4R were enriched in the functional pathways closely associated with AML leukemogenesis, including neutrophil inflammation, lymphocyte activation, signal transduction, and metabolism. The DEGs were enriched in differentiation, activation of immune cells, and cytokine signaling. Examination of the clinical serum samples also demonstrated significantly increased expressions of LTB4R mRNA (P=0.044) and protein (P=0.008) in AML patients, and LTB4R mRNA expression was positively correlated with the expression of the immune checkpoint HAVCR2 (r= 0.466, P=0.040). CONCLUSION LTB4R can serve as a novel biomarker and independent prognostic indicator of AML and its expression patterns provide insights into the crosstalk of leukemogenesis signaling pathways involving tumor immunity and metabolism.
Humans ; Leukemia, Myeloid, Acute/metabolism* ; Leukocytes, Mononuclear/metabolism* ; Prognosis ; RNA, Messenger/metabolism* ; Receptors, Leukotriene B4/genetics*

OBJECTIVE: To elucidate the pathogenic role of leukotriene B4 (LTB4) in pulmonary hyper-permeability and inflammation induced by lung-protective mechanical ventilation (LPMV) in rabbits. METHODS: Thirty-two healthy Japanese white rabbits were randomized into 4 groups for treatment with vehicle or bestatin (a leukotriene A4 hydrolase inhibitor that inhibits LTB4 production) administered intragastrically at the daily dose of 8 mg/kg for 5 days, followed by sham operation (group S and group BS, respectively, in which the rabbits were anesthetized only) or LPMV (group PM and group BPM, respectively, in which the rabbits received ventilation with 50% oxygen at a tidal volume of 8 mL/kg for 5 h). The concentrations of LTB4 and cyclic adenosine monophosphate (cAMP) in the lung tissues were analyzed by ELISA. cAMP content, protein kinase A (PKA) protein expression and the Rap1-GTP protein to total Rap1 protein ratio were determined to assess the activities of cAMP/PKA and Rap1 signaling pathways. The lung injury was evaluated by assessing lung permeability index, lung wet/dry weight ratio, polymorphonuclear leukocyte (PMN) count in bronchoalveolar lavage fluid (BALF), pulmonary myeloperoxidase (MPO) activity and lung histological scores. RESULTS: None of the examined parameters differed significantly between group S and group BS. All the parameters with the exception of lung histological score increased significantly in group PM and group BPM as compared to those in group S ( CONCLUSIONS LPMV can induce LTB4 overproduction to down-regulate cAMP/PKA and Rap1 signaling pathways in the lungs of rabbits, which results in lung hyper-permeability and inflammation. Bestatin can inhibit LTB4 production in the lungs to protect against LPMV-induced lung hyper-permeability and inflammation.
Animals ; Bronchoalveolar Lavage Fluid ; Leukotriene B4 ; Lung ; Lung Injury/prevention & control* ; Neutrophils ; Rabbits ; Respiration, Artificial/adverse effects*

BACKGROUND: Brennan’s rodent paw incision model has been extensively used for understanding mechanisms underlying postoperative pain in humans. However, alterations of physiological parameters like blood pressure and heart rate, or even feeding and drinking patterns after the incision have not been documented as yet. Moreover, though eicosanoids like prostaglandins and leukotrienes contribute to inflammation, tissue levels of these inflammatory mediators have never been studied. This work further investigates the antinociceptive effect of protein C after intra-wound administration. METHODS: Separate groups of Sprague–Dawley rats were used for quantitation of cyclooxygenase (COX) activity and leukotriene B4 level by enzyme-linked immunosorbent assay, as well as estimation of cardiovascular parameters and feeding and drinking behavior after paw incision. In the next part, rats were subjected to incision and 10 μg of protein C was locally administered by a micropipette. Both evoked and non-evoked pain parameters were then estimated. RESULTS: COX, particularly COX-2 activity and leukotriene B4 levels increased after incision. Hemodynamic parameters were normal. Feeding and drinking were affected on days 1 and 3, and on day 1, respectively. Protein C attenuated non-evoked pain behavior alone up to day 2. CONCLUSIONS: Based upon current observations, Brennan’s rodent paw incision model appears to exhibit a prolonged period of nociception similar to that after surgery, with minimal interference of physiological parameters. Protein C, which is likely converted to activated protein C in the wound, attenuated the guarding score, which probably represents pain at rest after surgery in humans.
Animals ; Blood Pressure ; Drinking ; Drinking Behavior ; Eicosanoids ; Enzyme-Linked Immunosorbent Assay ; Heart Rate ; Hemodynamics ; Humans ; Inflammation ; Leukotriene B4 ; Leukotrienes ; Nociception ; Pain, Postoperative ; Prostaglandin-Endoperoxide Synthases ; Prostaglandins ; Protein C ; Rats ; Rodentia ; Wounds and Injuries

PURPOSE: Platycodon grandiflorum (a domestic diploid variety, DV-PG) has been used as a food and component of various traditional oriental medicines. Although DV-PG is known to have an anti-allergic effect, little is known about the beneficial health effects of the tetraploid ‘Etteum’ variety in the Platycodon grandiflorum (TV-PG), which is a recently developed variety. In this study, we investigated the effect of TV-PG on the rat basophilic leukemia mast cell (RBL-2H3)-mediated allergic response. METHODS: To examine the effects of TV-PG on the allergic response, RBL-2H3 cells were sensitized with dinitropheny (DNP)-immunoglobin E, treated with various concentrations of TV-PG, and challenged with DNP-human serum albumin. We estimated cell granulation by measuring the release of β-hexosaminidase and production of inflammatory mediators by ELISA. RESULTS: TV-PG had no effect on the proliferation or cytotoxicity of RBL-2H3 cells within the concentration range of 0 to 200 µg/mL. TV-PG inhibited degranulation of RBL-2H3 cells by antigen stimulation in a dose-dependent manner. TV-PG also suppressed the production of inflammatory cytokines and mediators such as interleukin-4, tumor necrosis factor-α, prostagladin E2, and leukotriene B4 in RBL-2H3 cells by antigen stimulation. CONCLUSION: These results indicate that TV-PG exhibits anti-allergic activity via inhibition of degranulation as well as suppression of inflammatory mediators and cytokine release. These findings suggest that TV-PG may have potential as a preventive and therapeutic agent for the treatment of various allergic diseases.
Animals ; Basophils ; Cytokines ; Diploidy ; Enzyme-Linked Immunosorbent Assay ; Functional Food ; Hypersensitivity ; Inflammation Mediators ; Interleukin-4 ; Leukemia ; Leukotriene B4 ; Mast Cells ; Medicine, East Asian Traditional ; Necrosis ; Platycodon* ; Rats ; Serum Albumin ; Tetraploidy*

Recently, single-nucleotide polymorphisms (SNPs) in G-protein-coupled receptors (GPCRs) have been suggested to contribute to physiopathology and therapeutic effects. Leukotriene B4 receptor 2 (BLT2), a member of the GPCR family, plays a critical role in the pathogenesis of several inflammatory diseases, including cancer and asthma. However, no studies on BLT2 SNP effects have been reported to date. In this study, we demonstrate that the BLT2 SNP (rs1950504, Asp196Gly), a Gly-196 variant of BLT2 (BLT2 D196G), causes enhanced cell motility under low-dose stimulation of its ligands. In addition, we demonstrated that Akt activation and subsequent production of reactive oxygen species (ROS), both of which act downstream of BLT2, are also increased by BLT2 D196G in response to low-dose ligand stimulation. Furthermore, we observed that the ligand binding affinity of BLT2 D196G was enhanced compared with that of BLT2. Through homology modeling analysis, it was predicted that BLT2 D196G loses ionic interaction with R197, potentially resulting in increased agonist-receptor interaction. To the best of our knowledge, this report is the first to describe a SNP study on BLT2 and shows that BLT2 D196G enhances ligand sensitivity, thereby increasing cell motility in response to low-dose ligand stimulation.
Asthma ; Cell Movement* ; Humans ; Ligands ; Reactive Oxygen Species ; Receptors, G-Protein-Coupled ; Receptors, Leukotriene B4 ; Therapeutic Uses

Mast cells are major immune cells in allergy to secrete allergic mediators by a degranulation process and make and secrete inflammatory lipids and cytokines in response to antigen stimulation. An amino acid tryptophan regulates immune functions. Tryptophan ameliorates inflammatory colitis in which mast cells are engaged. However, its effects on mast cells remain to be solved. We investigated the effect of tryptophan on IgE-mediated allergic responses in the mast cells and mice. IgE-mediated passive cutaneous anaphylaxis (PCA) in mice were examined. Also IgE-mediated mast cell activation responses such as degranulation of stored granules and secretion of inflammatory lipid LTB₄ and cytokines (TNF-α and IL-4) were measured. Intraperitoneal administration of tryptophan suppressed PCA in mice. Also, in the cellular level tryptophan inhibited IgE-mediated mast cell activation such as IgE-mediated degranulation and the production of LTB₄. Also, it inhibited production of inflammatory cytokines TNF-α and IL-4. In summary, tryptophan suppressed IgE-mediated allergic activation in vivo and in vitro. Tryptophan supplementation is beneficial for IgE-mediated allergy.
Animals ; Colitis ; Cytokines ; Hypersensitivity ; Immunoglobulin E ; In Vitro Techniques ; Interleukin-4 ; Leukotriene B4 ; Mast Cells* ; Mice ; Passive Cutaneous Anaphylaxis ; Tryptophan*

Sjogren-Larsson syndrome (SLS) is a rare autosomal recessive neurocutaneous disorder with worldwide incidence of 0.4 per 100,000 people. It is characterized by the triad of congenital ichthyosis, spastic diplegia or quadriplegia, and mental retardation. Herein we report a 2-year-old male child with SLS, asthma, and recurrent pneumonia. SLS was confirmed by a molecular genetics study that revealed a deletion mutation in the ALDH3A2 gene. An ALDH3A2 gene mutation results in dysfunction of the microsomal enzyme fatty aldehyde dehydrogenase and impaired metabolism and accumulation of leukotriene B4, which is a key molecule and a pro-inflammatory mediator in developing allergic diseases, especially asthma. An increased level of leukotriene B4 has been reported in SLS patients. As far as we are aware, this is the first report of SLS associated with asthma and recurrent pneumonia. In conclusion, pediatricians should be aware of and evaluate patients with SLS for possible associated asthma and allergic disorders.
Aldehyde Dehydrogenase ; Asthma* ; Cerebral Palsy ; Child ; Child, Preschool ; Humans ; Ichthyosis ; Incidence ; Intellectual Disability ; Leukotriene B4 ; Male ; Metabolism ; Molecular Biology ; Neurocutaneous Syndromes ; Pneumonia* ; Quadriplegia ; Sequence Deletion ; Sjogren-Larsson Syndrome*

BACKGROUND/AIMS: This study aimed to examine the gastroprotective effects of PMK-S005, which is a synthetic S-allyl-L-cysteine (SAC; a sulfur-containing amino acid), against acute ethanol-induced gastric damage in rats. METHODS: Sprague-Dawley rats were divided into six groups, including a nonethanol group, groups treated with absolute ethanol 1 hour after pretreatment with various doses of PMK-S005 (1, 5, and 10 mg/kg) or rebamipide (50 mg/kg), and an absolute ethanol-only group. Ethanol-induced gross ulcer and mucus levels were measured. Myeloperoxidase, tumor necrosis factor α, interleukin 1β, PGE2, LTB4, cPLA2, COX-1, and COX-2 levels were estimated by enzyme-linked immunosorbent assay or Western blot analysis. Furthermore, the protein expression levels of antioxidant enzymes, including heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase 1 (NQO-1), GCLC, and GCLM, were assessed. RESULTS: PMK-S005 significantly attenuated the ethanol-induced gastric damage; it reduced mucosal inflammatory cytokine production and increased mucus levels. The expression levels of cPLA2, COX-1, and COX-2 were decreased by PMK-S005. PMK-S005 did not affect PGE2 synthesis, but LTB4 production was significantly suppressed. In addition, long-term administration of PMK-S005 significantly increased the expression of HO-1, NQO-1, GCLC, and GCLM. CONCLUSIONS: These results strongly suggest that PMK-S005 prevents gastric mucosal damage and that these gastroprotective activities are due to anti-inflammatory effects and enhancement of the gastric defense system, including antioxidant enzymes.
Animals ; Antioxidants ; Blotting, Western ; Dinoprostone ; Enzyme-Linked Immunosorbent Assay ; Ethanol ; Heme Oxygenase-1 ; Interleukins ; Leukotriene B4 ; Mucus ; Peroxidase ; Rats* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; Ulcer

OBJECTIVETo observe the effects of docosahexaenoic acid (DHA) on the expressions of TNF-α, IL-6, and leukotriene B4 (LTB4) in serum and expression of NF-κB in pulmonary tissue of rats with severe scald injury.

METHODSOne hundred and sixty SD rats were divided into sham injury (A), sham injury+DHA (B), scald (C), and scald+DHA (D) groups according to the random number table, with 40 rats in each group. Rats in groups A and B were sham injured, while rats in groups C and D were inflicted with 30% TBSA full-thickness scald on the back. Rats in groups B and D were injected with 0.5 mg/mL DHA solution with the dosage of 1 mL/kg via tail vein 5 minutes post injury, while rats in groups A and C with normal saline solution 1 mL/kg. At post injury hour (PIH) 3, 6, 12, 24, and 48, pulmonary tissue and abdominal aorta blood were collected from 8 rats in each group. The serum levels of TNF-α, IL-6, and LTB4 were determined with ELISA, and the protein expression of NF-κB p65 in pulmonary tissue was determined with Western blotting. Data were processed with analysis of variance of factorial design and LSD-t test.

RESULTS(1) The serum levels of TNF-α and IL-6 of rats in group A were similar to those of group B at each time point (with tTNF-α values from 0.223 to 0.947, tIL-6 values from 0.767 to 2.084, P values above 0.05). Compared with those of group A, the serum levels of TNF-α and IL-6 of rats in groups C and D were significantly higher at each time point (with tTNF-α values from 11.800 to 40.357, tIL-6 values from 10.334 to 39.321, P values below 0.01). The serum levels of TNF-α and IL-6 of rats in group D were significantly lower than those of group C at each time point (with tTNF-α values from -17.643 to -8.331, tIL-6 values from -21.596 to -6.332, P values below 0.01). The serum levels of TNF-α and IL-6 in groups C and D both showed a trend of increase earlier and decrease later, and they peaked at PIH 12, respectively (360.4 ± 13.2), (306.8 ± 7.2) pg/mL and (265.4 ± 12.3), (230.5 ± 2.2) pg/mL. (2) The serum level of LTB4 in group A was similar to that of group B at each time point (with t values from 0.787 to 1.096, P values above 0.05). The serum level of LTB4 was significantly higher in groups C and D than in group A at each time point (with t values from 7.501 to 38.962, P values below 0.01). The serum level of LTB4 in group D was obviously lower than that of group C at each time point (with t values from -19.244 to -2.532, P values below 0.01). The serum level of LTB4 in groups C and D both showed a trend of increase earlier and decrease later, and it peaked at PIH 12, (4.59 ± 0.29) and (2.85 ± 0.32) ng/mL respectively. (3) The protein expression of NF-κB p65 in pulmonary tissue in group A was similar to that of group B at each time point (with t values from 0.847 to 1.256, P values above 0.05). The protein expression of NF-κB p65 was significantly higher in groups C and D than in group A at each time point (with t values from 15.167 to 98.074, P values below 0.01). The protein expression of NF-κB p65 in group D was obviously lower than that of group C at each time point (with t values from -37.190 to -14.415, P values below 0.01). The protein expression of NF-κB p65 in groups C and D both showed a trend of increase earlier and decrease later, and it peaked at PIH 12, respectively 4.46 ± 0.12 and 2.94 ± 0.21.

CONCLUSIONSParenteral supply of DHA to rats with severe scald injury can reduce the levels of TNF-α, IL-6, and LTB4 in serum and decrease the expression of NF-κB in pulmonary tissue, thus alleviating the inflammation response.

Animals ; Blotting, Western ; Burns ; Cytokines ; Docosahexaenoic Acids ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; Interleukin-6 ; blood ; Leukotriene B4 ; blood ; Lung ; metabolism ; pathology ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum ; Soft Tissue Injuries ; Tumor Necrosis Factor-alpha ; blood ; genetics ; Up-Regulation ; physiology