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Background Contact urticaria (CU) is a representative chronic inflammatory skin disease, and the symptoms progressing in stages can cause life-threatening conditions such as anaphylaxis. Mesenchymal stem cells (MSCs) are attracting attention as therapeutic agents for immune diseases. However, studies on the efficacy and mechanism of stem cell therapy for the treatment of intractable urticaria are lacking. Methods The regulatory role of administered embryonic-stem-cell-derived multipotent MSCs (M-MSCs) was evaluated on mice with CU. The functions of the M-MSCs on splenic T cells and mast cells were measured by flow cytometry analysis, histological analysis, RT-PCR, and other molecular biological approaches. The mechanism of action of the M-MSCs was examined using TGF-β neutralization in vitro and in vivo. Results The therapeutic effects of administering M-MSCs were evaluated in the developed TMA-induced urticaria model, and it was confirmed to inhibit urticarial reactions in various conditions, such as edema, itchiness, and wheal formation. In addition, M-MSC administration enabled control of the effector T cell activities in major lymphoid and peripheral tissues as well as inhibited mast-cell degranulation in the peripheral tissues. Further, the M-MSC-mediated inhibitory effects were confirmed to be dependent on TGF-β. Conclusions Our findings prove that M-MSCs promote alleviation of urticaria by controlling the activation of the inflammatory effector T cells and mast cells. We also confirm that the TGF-β mechanism is an important contributor to M-MSC-mediated inhibition of urticaria.

IL-10+ regulatory B (Breg) cells play a vital role in regulating the immune responses in experimental autoimmune encephalomyelitis, colitis, and contact hypersensitivity (CHS). Several stimulants such as lipopolysaccharide (LPS), CD40 ligand, and IL-21 spur the activation and maturation of IL-10+ Breg cells, while the epigenetic mechanism for the IL-10 expression remains largely unknown. It is well accepted that the histone acetylation/ deacetylation is an important mechanism that regulates the expression of IL-10. We found that entinostat, an HDAC inhibitor, stimulated the induction of IL-10+ Breg cells by LPS in vitro and the formation of IL-10+ Breg cells to suppress CHS in vivo. We further demonstrated that entinostat inhibited HDAC1 from binding to the proximal region of the IL-10 expression promoter in splenic B cells, followed by an increase in the binding of NF-κB p65, eventually enhancing the expression of IL-10 in Breg cells. [BMB Reports 2021; 54(10): 534-539].

Mast cells (MCs) are systemically distributed and secrete several allergic mediators such as histamine and leukotrienes to cause type I hypersensitivity. Dasatinib is a type of anti-cancer agent and it has also been reported to inhibit human basophils. However, dasatinib has not been reported for its inhibitory effects on MCs or type I hypersensitivity in mice. In this study, we examined the inhibitory effect of dasatinib on MCs and MC-mediated allergic response in vitro and in vivo. In vitro, dasatinib inhibited the degranulation of MCs by antigen stimulation in a dose-dependent manner (IC50, ~34 nM for RBL-2H3 cells; ~52 nM for BMMCs) without any cytotoxicity. It also suppressed the secretion of inflammatory cytokines IL-4 and TNF-α by antigen stimulation. Furthermore, dasatinib inhibited MC-mediated passive cutaneous anaphylaxis (PCA) in mice (ED50, ~29 mg/kg). Notably, dasatinib significantly suppressed the degranulation of MCs in the ear tissue. As the mechanism of its effect, dasatinib inhibited the activation of Syk and Syk-mediated downstream signaling proteins, LAT, PLCγ1, and three typical MAP kinases (Erk1/2, JNK, and p38), which are essential for the activation of MCs. Interestingly, in vitro tyrosine kinase assay, dasatinib directly inhibited the activities of Lyn and Fyn, the upstream tyrosine kinases of Syk in MCs. Taken together, dasatinib suppresses MCs and PCA in vitro and in vivo through the inhibition of Lyn and Fyn Src-family kinases. Therefore, we suggest the possibility of repositioning the anti-cancer drug dasatinib as a treatment for various MC-mediated type I hypersensitive diseases.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) performed by O-GlcNAc transferase (OGT) is a nutrient-responsive post-translational modification (PTM) via the hexosamine biosynthetic pathway (HBP). Various transcription factors (TFs) are O-GlcNAcylated, affecting their activities and significantly contributing to cellular processes ranging from survival to cellular differentiation. Given the pleiotropic functions of O-GlcNAc modification, it has been studied in various fields, however, the role of O-GlcNAcylation during osteoclast differentiation remains to be explored. Kinetic transcriptome analysis during receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that the nexus of major nutrient metabolism, HBP was critical for this process. We observed that the critical genes related to HBP activation, including Nagk, Gfpt1, and Ogt, were upregulated, while the global O-GlcNAcylation was increased concomitantly during osteoclast differentiation. The O-GlcNAcylation inhibition by the small-molecule inhibitor OSMI-1 reduced osteoclast differentiation in vitro and in vivo by disrupting the translocation of NF-κB p65 and nuclear factor of activated T cells c1 (NFATc1) into the nucleus by controlling their PTM O-GlcNAcylation. Furthermore, OSMI-1 had a synergistic effect with bone target therapy on osteoclastogenesis. Lastly, knocking down Ogt with shRNA (shOgt) mimicked OSMI-1’s effect on osteoclastogenesis. Targeting O-GlcNAcylation during osteoclast differentiation may be a valuable therapeutic approach for osteoclast-activated bone diseases.

IL-10+ regulatory B (Breg) cells play a vital role in regulating the immune responses in experimental autoimmune encephalomyelitis, colitis, and contact hypersensitivity (CHS). Several sti-mulants such as lipopolysaccharide (LPS), CD40 ligand, and IL-21 spur the activation and maturation of IL-10+ Breg cells, while the epigenetic mechanism for the IL-10 expression remains largely unknown. It is well accepted that the histone acetylation/deacetylation is an important mechanism that regulates the expression of IL-10. We found that entinostat, an HDAC inhibitor, stimulated the induction of IL-10+ Breg cells by LPS in vitro and the formation of IL-10+ Breg cells to suppress CHS in vivo. We further demonstrated that entinostat inhibited HDAC1 from binding to the proximal region of the IL-10 expression promoter in splenic B cells, followed by an increase in the binding of NF-κB p65, eventually enhancing the expression of IL-10 in Breg cells.

Discovery and development of new potentially selective anticancer agents are necessary to prevent a global cancer health crisis. Currently, alternative medicinal agents derived from plants have been extensively investigated to develop anticancer drugs with fewer adverse effects. Among them, steroidal alkaloids are conventional secondary metabolites that comprise an important class of natural products found in plants, marine organisms and invertebrates, and constitute a judicious choice as potential anti-cancer leads. Traditional medicine and modern science have shown that representatives from this compound group possess potential antimicrobial, analgesic, anticancer and anti-inflammatory effects. Therefore, systematic and recapitulated information about the bioactivity of these compounds, with special emphasis on the molecular or cellular mechanisms, is of high interest. In this review, we methodically discuss the in vitro and in vivo potential of the anticancer activity of natural steroidal alkaloids and their synthetic and semi-synthetic derivatives. This review focuses on cumulative and comprehensive molecular mechanisms, which will help researchers understand the molecular pathways involving steroid alkaloids to generate a selective and safe new lead compound with improved therapeutic applications for cancer prevention and therapy. In vitro and in vivo studies provide evidence about the promising therapeutic potential of steroidal alkaloids in various cancer cell lines, but advanced pharmacokinetic and clinical experiments are required to develop more selective and safe drugs for cancer treatment.

Fetuin-B is a serum protein linked to the regulation of physiological or pathophysiological events such as fertility, energy metabolism, and liver disease. Recently, fetuin-B has been reported to be involved in the modulation of the rupture of atherosclerotic plaques associated with acute myocardial infarction. However, the exact mechanism involved in the modulation of atherosclerotic plaque rupture event by fetuin-B is not fully elucidated yet. In the present study, we investigated whether fetuin-B could influence atherosclerotic plaque rupture through vascular smooth muscle cells (VSMCs). Immunoprecipitation assay using membrane proteins from VSMCs revealed that fetuin-B tightly bound to transforming growth factor-β receptor (TGF-βR). Fetuin-B treatment elevated TGF-βR signals (e.g., phosphorylation of Smad2 and Smad3) in VSMCs. Fetuin-B also stimulated nuclear translocation of phosphorylated Smads. Phosphorylation of Smad and its nuclear translocation by treatment with fetuin-B were inhibited in VSMCs by treatment with SB431542, a selective inhibitor of TGF-βR. Fetuin-B enhanced expression levels of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-2 (MMP-2) in VSMCs through its epigenetic modification including recruitments of both histone deacetylase 1 and RNA polymerase II. These epigenetic alterations in VSMCs were also inhibited by treatment with SB431542. In vivo administration of fetuin-B protein increased expression levels of PAI-1 and MMP-2 in the vascular plaque. However, these increases in expression were inhibited by the administration of SB43154. These results indicate that fetuin-B may modulate vascular plaque rupture by promoting expression of PAI-1 and MMP-2 in VSMCs via TGF-βR-mediated Smad pathway.

Mast cells are critical effector cells in IgE-mediated allergic responses. The aim of this study was to investigate the anti-allergic effects of 3-[(aminocarbonyl)amino]-5-(3-fluorophenyl)-N-(3S)-3-piperidinyl-2-thiophenecarboxamide (AZD7762) in vitro and in vivo. AZD7762 inhibited the antigen-stimulated degranulation from RBL-2H3 (IC50, ∼27.9 nM) and BMMCs (IC50, ∼99.3 nM) in a dose-dependent manner. AZD7762 also inhibited the production of TNF-α and IL-4. As the mechanism of its action, AZD7762 inhibited the activation of Syk and its downstream signaling proteins, such as Linker of activated T cells (LAT), phospholipase (PL) Cγ1, Akt, and mitogen-activated protein (MAP) kinases (Erk1/2, p38, and JNK) in mast cells. In in vitro protein kinase assay, AZD7762 inhibited the activity of Lyn and Fyn kinases, which are important for the activation of Syk in mast cells. Furthermore, AZD7762 also suppressed the degranulation of LAD2 human mast cells (IC50, ∼49.9 nM) and activation of Syk in a dose-dependent manner. As observed in experiments with mast cells in vitro, AZD7762 inhibited antigen-mediated passive cutaneous anaphylaxis in mice (ED50, ∼35.8 mg/kg). Altogether, these results suggest that AZD7762 could be used as a new therapeutic agent for mast cell-mediated allergic diseases.

DJ-1 and sphingosine-1-phosphate (S1P) receptors (S1PRs) are implicated in the control of physiology and pathophysiology of cardiovascular systems such as blood pressure, atherosclerosis, and restenosis. Here, we investigated whether DJ-1 with antioxidant function participates in the regulation of S1PR1 and S1PR2 expression in vascular smooth muscle cells (VSMCs) and whether this response is related to vascular neointima formation. In vitro studies used cellular migration assay, western blot, reverse transcriptase and real-time PCR analysis, and immunocytochemistry. In vivo studies were performed using the carotid artery ligation model together with immunohistochemistry in DJ-1 knockout (DJKO) and corresponding wild-type (DJWT) mice. S1P stimulated migration of VSMCs from DJKO and DJWT mice. VSMC migration was suppressed by S1PR1 inhibitor but was elevated by S1PR2 inhibitor. Compared with DJWT mice, S1PR1 expression was higher in VSMCs and neointimal plaque from DJKO mice, but S1PR2 expression was lower. Overexpression of DJ-1 in DJKO VSMCs reduced S1PR1 expression and elevated S1PR2 expression. Compared with DJWT mice, histone deacetylase-1 recruitment and histone H3 acetylation at the S1PR1 promoter region were lower and higher, respectively, but this pattern was reversed at the S1PR2 promoter region in DJKO VSMCs. S1PR expressions and epigenetic changes at S1PR promoter regions in DJWT VSMCs treated with H2O2 showed similar patterns to those in DJKO VSMCs. Our findings suggest that DJ-1 may be involved in the regulation of S1PR1 and S1PR2 expression via H2O2-mediated histone modification in VSMCs. Consequently, this modification may affect S1P-induced VSMC migration and be related to vascular neointima formation.

The pathogenesis of atopic dermatitis (AD) involves T helper 2 (Th2) cells, and effective therapies remain elusive due to the paucity of animal models. We aimed to develop a mouse model of an immune system aberration caused by allergen. Experiments were conducted in two phases. In experiment 1, BALB/c mice were sensitized with one of four chemical allergens - toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), trimellitic anhydride (TMA), or 2,4-dinitrochlorobenzene (DNCB) - for 3 weeks. Based on results of experiment 1, immunological features were compared between TMA-sensitized BALB/c mice and NC/Nga mice, after exposure to mite extracts, harmful chemicals and detergents in experiment 2. Sensitization by allergen caused a large number of pathological changes in the skin, and an increase in mast cell number. TMA-sensitized BALB/c mice models showed higher sensitivity to an environmental allergen than NC/Nga mice did. Overall, the initial sensitization with TMA leads to disturbances in Th2-mediated immunity.

Regulatory B cells, also well-known as IL-10-producing B cells, play a role in the suppression of inflammatory responses. However, the epigenetic modulation of regulatory B cells is largely unknown. Recent studies showed that the bromodomain and extra-terminal domain (BET) protein inhibitor JQ1 controls the expression of various genes involving cell proliferation and cell cycle. However, the role of BET proteins on development of regulatory B cells is not reported. In this study, JQ1 potently suppressed IL-10 expression and secretion in murine splenic and peritoneal B cells. While bromodomaincontaining protein 4 (BRD4) was associated with NF-κB on IL-10 promoter region by LPS stimulation, JQ1 interfered the interaction of BRD4 with NF-κB on IL-10 promoter. In summary, BRD4 is essential for toll like receptor 4 (TLR4)-mediated IL-10 expression, suggesting JQ1 could be a potential candidate in regulating IL-10-producing regulatory B cells in cancer. [BMB Reports 2017; 50(12): 640-646].

Mast cells trigger IgE-mediated allergic reactions by releasing various allergic mediators. 8-Formyl-7-hydroxy-4-methylcoumarin, also called 4μ8C, was originally known as an inositol-requiring enzyme 1 (IRE1) suppressant, but no study has examined its relationship with mast cells and allergic diseases. Therefore, the purpose of this study was to determine whether 4μ8C is effective in suppressing allergic reactions in mast cells and in IgE-mediated allergic animal model. 4μ8C suppressed the degranulation of IgE-mediated mast cells (IC50=3.2μM) and the production of cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) in a dose-dependent manner. 4μ8C also suppressed passive cutaneous anaphylaxis (PCA) in mice (ED50=25.1mg/kg). In an experiment on mast cell signaling pathways stimulated by antigen, the phosphorylation and activation of Syk was decreased by 4μ8C, and phosphorylation of downstream signaling molecules, such as linker for activated T cells (LAT), Akt, and the three MAP kinases, ERK, p38, and JNK, were suppressed. Mechanistic studies showed that 4μ8C inhibited the activity of Lyn and Fyn in vitro. Based on the results of those experiments, the suppressor mechanism of allergic reaction by 4μ8C involved reduced activity of Lyn and Fyn, which is pivotal in an IgE-mediated signaling pathway. In summary, for the first time, this study shows that 4μ8C inhibits Lyn and Fyn, thus suppressing allergic reaction by reducing the degranulation and the production of inflammatory cytokines. This suggests that 4μ8C can be used as a new medicinal candidate to control allergic diseases such as seasonal allergies and atopic dermatitis.

Mast cells induce regulatory B cells to maintain peripheral tolerance in contact hypersensitivity., The function of regulatory immune cells in peripheral tissues is crucial to the onset and severity of various diseases. Interleukin-10 (IL-10)–producing regulatory B (IL-10+ Breg) cells are known to suppress various inflammatory diseases. However, evidence for the mechanism by which IL-10+ Breg cells are generated and maintained is still very limited. Here, we found that IL-10+ Breg cells suppress the activation of IL-13–producing type 2 innate lymphoid cells (IL-13+ ILC2s) in an IL-10–dependent manner in mice with oxazolone-induced severe contact hypersensitivity (CHS). Mast cell (MC) IL-5 was important for maintaining the population of IL-10+ Breg cells in peripheral lymphoid tissues. Overall, these results uncover a previously unknown mechanism of MCs as a type of immunoregulatory cell and elucidate the cross-talk among MCs, IL-10+ Breg cells, and IL-13+ ILC2s in CHS.

Osteoclasts (OCs) have been well-known involved in the exacerbation of bone-related diseases. However, the role of metabolites on osteoclastogenesis has not been well characterized. Herein, we found osteoclastogenesis was negatively regulated by α-ketoglutarate (αKG) in vitro and in vivo (C57BL/6 mouse). Kinetic transcriptome analysis revealed the upregulation of solute carrier family 7 member 11 (Slc7a11), a subunit of the cysteine/glutamate antiporter, as well as the downregulation of typical OC maker genes through αKG treatment. Given that Slc7a11 could control ROS level through glutathione import, we measured intracellular ROS, then RANKL-induced ROS production was inhibited by αKG. Notably, we highlight that αKG plays an epigenetic co-factor at the Slc7a11 promoter by demethylating repressive histone H3K9 methylation and simultaneously increasing the nuclear factor erythroid 2-related factor (Nrf2) binding, a critical transcription factor through chromatin immunoprecipitation (ChIP) analysis. Together, we suggested that αKG could be a therapeutic strategy for OC activated diseases.

Background Afrocyclamin A, an oleanane-type triterpene saponin, was isolated from Androsace umbellata which used as a traditional herbal medicine. Purpose This study aimed to explore the anticancer activity of afrocyclamin A on human prostate cancer cells in vitro as well as in vivo. Methods Cytotoxicity, cell cycle distribution, apoptosis, and autophagic cell death were measured following exposure to afrocyclamin A. In vivo antitumor activity of afrocyclamin A was assessed in a xenograft model. The protein levels of p-Akt, p-mTOR, Bax, Bcl-2, caspase-3, and caspase-9 were quantified using western blot analysis. Results In DU145 cells, afrocyclamin A increased cytotoxicity, caused changes in cell morphology, and induced sub-G0/G1 phase indicating increased apoptosis. Afrocyclamin A robustly induced autophagic cell death as demonstrated by the conversion of LC3B-I to LC3B-II, and the formation of autophagic vacuoles as revealed by western blot analysis and fluorescence staining, respectively. Afrocyclamin A also inhibited the phosphorylation of PI3K, Akt, and mTOR, suggesting their role in afrocyclamin A induced cell death. In addition, afrocyclamin A inhibited cell migration and invasion in concentration and time-dependent manners. In an in vivo xenograft model, afrocyclamin A inhibited the growth of DU145 cells. Conclusion Afrocyclamin A has anticancer activity via the PI3K/Akt/mTOR pathway, which leads to cell death.

Mast cells are the most prominent effector cells of Type 1 hypersensitivity immune responses. CYC116 [4-(2-amino-4-methyl-1,3-thiazol-5-yl)-N-[4-(morpholin-4-yl)phenyl] pyrimidin-2-amine] is under development to be used as an anti-cancer drug, but the inhibitory effects of CYC116 on the activation of mast cells and related allergy diseases have not reported as of yet. In this study, we demonstrated, for the first time, that CYC116 inhibited the degranulation of mast cells by antigen stimulation (IC50, ~1.42 μM). CYC116 also inhibited the secretion of pro-inflammatory cytokines including TNF-α (IC50, ~1.10 μM), and IL-6 (IC50, ~1.24 μM). CYC116 inhibited the mast cell-mediated allergic responses, passive cutaneous anaphylaxis (ED50, ~22.5 mg/kg), and passive systemic anaphylaxis in a dose-dependent manner in laboratory experiments performed on mice. Specifically, CYC116 inhibited the activity of Fyn in mast cells and inhibited the activation of Syk and Syk-dependent signaling proteins including LAT, PLCγ, Akt, and MAP kinases. Our results suggest that CYC116 could be used as an alternative therapeutic medication for mast cell-mediated allergic disorders, such as atopic dermatitis and allergic rhinitis.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthaquinone) has displayed antitumor activity in vitro and in animal models; however, the underlying molecular mechanisms have not been fully explored. The aim of this study was to investigate the anticancer effects of plumbagin isolated from Nepenthes alata against MCF-7 breast cancer cells. We examined the cytotoxicity, cell cycle regulation, apoptotic cell death, and generation of intracellular reactive oxygen species (ROS) in MCF-7 cells. Plumbagin exhibited potent cytotoxicity in MCF-7 cells (wild-type p53) compared to that in SK-OV-3 (null-type) human epithelial ovarian cancer cells. Specifically, plumbagin upregulated the expression of p21CIP1/WAF1 in MCF-7 cells, causing cell cycle arrest in the G2/M phase through inhibition of cyclin B1 levels. Plumbagin also significantly increased the ratio of Bax/Bcl-2 and release of cytochrome c, resulting in apoptotic cell death in MCF-7 cells. Furthermore, plumbagin dramatically increased the intracellular ROS level, whereas pretreatment with the ROS scavenger N-acetyl cysteine protected against plumbagin-induced cytotoxicity, suggesting that ROS formation plays a pivotal role in antitumor activity in MCF-7 cells. In mice bearing MCF-7 cell xenografts, plumbagin significantly reduced tumor growth and weight without apparent side effects. We therefore concluded that plumbagin exerts anticancer activity against MCF-7 cells through the generation of intracellular ROS, resulting in the induction of apoptosis via a p53-dependent pathway. This study thus identifies a new anticancer mechanism of plumbagin against p53-dependent breast cancer cells and suggests a novel strategy for overcoming of breast cancer therapy.

Chrysanthemum zawadskiivar.latilobum(CZ) has been used for beverage or tea and also as folk medicine for the remedy of diverse inflammatory diseases. Nevertheless, the therapeutic effect of CZ on arthritis remains to be unknown. In this paper we aim to investigate the CZ’s antiarthritic effect and mechanism of action both in vitro and in vivo. To assess CZ’s antiarthritic effect, mouse models of type II collagen-induced arthritis (CIA) were used. Mice were used to gauge clinical arthritis index and histopathological changes. Reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, electrophoretic mobility shift assay (EMSA), and other biological methods were adopted to measure CZ’s effect on arthritis and to understand the veiled mechanism of action. CZ greatly suppressed CIA, histopathological score, bone erosion, and osteoclast differentiation. Mechanistically, CZ inhibited the production of various inflammatory and arthritic mediators like inflammatory cytokines, matrix metalloproteinases (MMPs), and chemokines. Of note, CZ significantly suppressed the activation of the NF-κB pathway in vivo. CZ exerted an antiarthritic effect in CIA mice by curbing the production of crucial inflammatory and arthritis mediators. This study warrants further investigation of CZ for the use in human rheumatoid arthritis (RA).

The discovery of new biomarkers for early detection of drug-induced acute kidney injury (AKI) is clinically important. In this study, sensitive metabolomic biomarkers identified in the urine of rats were used to detect cisplatin-induced AKI. Cisplatin (10 mg kg(-1), i.p.) was administered to Sprague-Dawley rats, which were subsequently euthanized after 1, 3 or 5 days. In cisplatin-treated rats, mild histopathological alterations were noted at day 1, and these changes were severe at days 3 and 5. Blood urea nitrogen (BUN) and serum creatinine (SCr) levels were significantly increased at days 3 and 5. The levels of new urinary protein-based biomarkers, including kidney injury molecule-1 (KIM-1), glutathione S-transferase-α (GST-α), tissue inhibitor of metalloproteinase-1 (TIMP-1), vascular endothelial growth factor (VEGF), calbindin, clusterin, neutrophil, neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin, were significantly elevated at days 3 and 5. Among urinary metabolites, trigonelline and 3-indoxylsulfate (3-IS) levels were significantly decreased in urine collected from cisplatin-treated rats prior to histological kidney damage. However, carbon tetrachloride (CCl4), a hepatotoxicant, did not affect these urinary biomarkers. Trigonelline is closely associated with GSH depletion and results in insufficient antioxidant capacity against cisplatin-induced AKI. The predominant cisplatin-induced AKI marker appeared to be reduced in urinary 3-IS levels. Because 3-IS is predominantly excreted via active secretion in proximal tubules, a decrease is indicative of tubular damage. Further, urinary excretion of 3-IS levels was markedly reduced in patients with AKI compared to normal subjects. The area under the curve receiver operating characteristics (AUC-ROC) for 3-IS was higher than for SCr, BUN, lactate dehydrogenase (LDH), total protein, and glucose. Therefore, low urinary or high serum 3-IS levels may be more useful for early detection of AKI than conventional biomarkers.

Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p

Background Psammaplin A (PsA) is a natural product isolated from marine sponges, which has been demonstrated to have anticancer activity against several human cancer cell lines via the induction of cell cycle arrest and apoptosis. New drugs that are less toxic and more effective against multidrug-resistant cancers are urgently needed. Methods We tested cell proliferation, cell cycle progression and autophagic cell death pathway in doxorubicin-resistant MCF-7 (MCF-7/adr) human breast cancer cells. The potency of PsA was further determined using an in vivo xenograft model. Results and conclusion PsA significantly inhibited MCF-7/adr cells proliferation in a concentration-dependent manner, with accumulation of cells in G2/M phase of the cell cycle. PsA significantly decreased SIRT1 enzyme activity and reduced expression of SIRT1 protein in the cultured cells with greater potency than sirtinol or salermide. Acetylation of p53, a putative target of SIRT1, increased significantly following PsA treatment. In addition, PsA markedly increased the expression levels of autophagy-related proteins. In support of this, it was found that PsA significantly increased the expression of damage-regulated autophagy modulator (DRAM), a p53-induced protein. General significance The results of this study suggest that PsA is sufficient to overcome multidrug-resistant cancer via SIRT1-mediated autophagy in MCF-7/adr breast cancer cells, indicating that PsA has therapeutic potential for clinical use.

Synaptosomal-associated protein 23 (SNAP23) is involved in microvesicle trafficking and exocytosis in various cell types, but its functional role in blood pressure (BP) regulation has not yet been defined. Here, we found that lipid raft SNAP23 expression was much lower in vascular smooth-muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) than in those from normotensive Wistar-Kyoto (WKY) rats. This led us to investigate the hypothesis that this lower expression may be linked to the spontaneous hypertension found in SHR. The expression level of lipid raft SNAP23 and the fluidity in the plasma membrane of VSMCs were lower in SHR than in WKY rats. Cholesterol content in the VSMC membrane was higher, but the secreted cholesterols found in VSMC-conditioned medium and in the blood serum were lower in SHR than in WKY rats. SNAP23 knockdown in WKY rat VSMCs reduced the membrane fluidity and increased the membrane cholesterol level. Systemic overexpression of SNAP23 in SHR resulted in an increase of cholesterol content in their serum, a decrease in cholesterol in their aorta and the reduction of their BP. Our findings suggest that the low expression of the lipid raft SNAP23 in VSMCs might be a potential cause for the characteristic hypertension of SHR.

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40hiCD5+ B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40hi is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10−/−CD5+CD19+ B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40hiCD5+ Breg cells in mice. However, the population of CD40hiCD5+ B cells was minimal in IL-10−/− mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40hiCD5+ B cells and the autocrine effect of IL-10 is critical to the formation of CD40hiCD5+ Breg cells. [BMB Reports 2015; 48(1): 54-59]

An allergic reaction occurs when the immune system overreacts to harmless substance called allergen that gains access to the body. Food allergy is a hypersensitive immune reaction to food proteins and the number of patients with food allergy has recently increased. Aloe Vera is used for wellness and medicinal purposes. In particular, Aloe vera has been reported to enhance immunity. However, the effect of Aloe vera on food allergy is not yet known. In this study, we investigated the effects of processed Aloe vera gel (PAG) containing low molecular weight Aloe polysaccharide (AP) on ovalbumin (OVA)-induced food allergy in mice. Allergic symptoms, rectal temperature, and diarrhea were measured in OVA-induced food allergy mice. Other allergic parameters were also analyzed by RT-PCR, ELISA, flow cytometry, and other biochemical methods. As the results, PAG suppressed the decrease of body temperature, diarrhea, and allergic symptoms in OVA-induced food allergy mice. PAG also reduced serum concentrations of type 2 helper T cell (Th2) cytokines (Interleukin-(IL)-4, IL-5, and IL-13) as well as histamine, mast cell protease-1 (MCP-1), and immunoglobulin (Ig)E. PAG blocked the degranulation of mast cells and infiltration of eosinophils in intestine. Furthermore, PAG suppressed the population of Th2 cells in spleen and mesenteric lymph nodes. PAG also increased the production of IL-10 and population of type 1 regulatory T (Tr1) cells in mice with food allergy. Taken together, our findings suggest that PAG suppressed Th2 immune responses through, at least partially, stimulating the secretion of IL-10 in food allergy mice.

Mast cells are critical cells that prompt various allergic response-inducing factors, contributing to allergic diseases. While used as an antibiotic for livestock, there is no study on the effect of furaltadone on allergic response. This study investigated the effect of furaltadone on mast cells and passive cutaneous anaphylaxis (PCA). Furaltadone inhibited the degranulation of mast cells stimulated by antigen (IC50, ~ 3.9 μM), and also suppressed the production of tumor necrosis factor (TNF)-α and interleukin (IL)-4 in a concentration dependent manner. In addition, furaltadone inhibited allergic responses in an acute allergy animal model, PCA. Further investigation on the mechanism for these inhibitory effects of furaltadone found that the activities of Lyn/Syk and Syk-dependent downstream proteins such as mitogen-activated protein (MAP) kinases were inhibited by furaltadone in mast cells. Taken together, this study demonstrates that furaltadone inhibits the activation of mast cells by antigen via the suppression of the Lyn/Syk pathway and ameliorates allergic responses in vivo.

Angiotensin II (Ang II) is implicated in the development of cardiovascular disorders including hypertension and atherosclerosis. However, the role of Ang II in the interaction between apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and sphingosine-1-phosphate (S1P) signals in relation to vascular disorders remains to be clarified. This study aimed to determine whether APE/Ref-1 plays a role in epigenetic regulation of the S1P receptor (S1PR) in response to Ang II in vascular smooth muscle cell (VSMC) migration and vascular neointima formation. Ang II augmented the expression of S1PR1 in aortic smooth muscle cells of Sprague Dawley rats (RASMCs), which was attenuated by Ang II receptor (AT) 1 inhibitors, antioxidants, and APE/Ref-1 knockdown with small interference RNA. Ang II stimulation produced H2O2, and exogenous H2O2 elevated S1PR1 expression in RASMCs. Moreover, Ang II caused translocation of cytoplasmic APE/Ref-1 into the nucleus in RASMCs. H3 histone acetylation and APE/Ref-1 binding at the S1PR1 promoter were increased in RASMCs treated with Ang II. In addition, Ang II induced migration in RASMCs, which was suppressed by AT1 and S1PR1 inhibitors. The expression of S1PR1, and colocalization of APE/Ref-1 and acetylated histone H3 in vascular neointima, were greater in Ang II-infused rats compared with a control group. These findings demonstrate that Ang II stimulates the epigenetic regulation of S1PR1 expression via H2O2-mediated APE/Ref-1 translocation, which may consequently be involved in Ang II-induced VSMC migration and vascular neointima formation. Therefore, APE/Ref-1-mediated overexpression of S1PR1 may be implicated in the vascular dysfunction evoked by Ang II.

Background Inhaled protease allergens preferentially trigger T H 2-mediated inflammation in allergic asthma. The role of dendritic cells (DCs) on induction of T H 2 cell responses in allergic asthma has been well documented; however, the mechanism by which protease allergens induce T H 2-favorable DCs in the airway remains unclear. Objective We sought to determine a subset of DCs responsible for T H 2 cell responses in allergic asthma and the mechanism by which protease allergens induce the DC subset in the airway. Methods Mice were challenged intranasally with protease allergens or fibrinogen cleavage products (FCPs) to induce allergic airway inflammation. DCs isolated from mediastinal lymph nodes were analyzed for surface phenotype and T-cell stimulatory function. Anti-Thy1.2 and Mas-TRECK mice were used to deplete innate lymphoid cells and mast cells, respectively. Adoptive cell transfer, bone marrow DC culture, anti–IL-13, and Toll-like receptor (TLR) 4–deficient mice were used for further mechanistic studies. Results Protease allergens induced a remarkable accumulation of T H 2-favorable programmed cell death 1 ligand 2 (PD-L2) + DCs in mediastinal lymph nodes, which was significantly abolished in mice depleted of mast cells and, to a lesser extent, innate lymphoid cells. Mechanistically, FCPs generated by protease allergens triggered IL-13 production from wild-type mast cells but not from TLR4-deficient mast cells, which resulted in an increase in the number of PD-L2 + DCs. Intranasal administration of FCPs induced an increase in numbers of PD-L2 + DCs in the airway, which was significantly abolished in TLR4- and mast cell–deficient mice. Injection of IL-13 restored the PD-L2 + DC population in mice lacking mast cells. Conclusion Our findings unveil the "protease–FCP–TLR4–mast cell–IL-13" axis as a molecular mechanism for generation of T H 2-favorable PD-L2 + DCs in allergic asthma and suggest that targeting the PD-L2 + DC pathway might be effective in suppressing allergic T-cell responses in the airway.

β-Agarase cleaves the β-1,4 linkages of agar to produce neoagarooligosaccharides (NAO), which are associated with various physiological functions. However, the immunological functions of NAO are still unclear. In this study, we demonstrated that β-agarase DagA-produced neoagarohexaose (DP6), an NAO product, promoted the maturation of dendritic cells (DCs) by Toll-like receptor 4 (TLR4). DP6 directly and indirectly enhanced the activation of natural killer (NK) cells in a TLR4-dependent manner in vitro and in vivo. Finally, the antitumor activity of DP6 against B16F1 melanoma cells was inhibited in NK cell-depletion systems by using NK-cell depleting antibodies in vivo. Collectively, the results indicated that DP6 augments antitumor immunity against B16F1 melanoma cells via the activation of DC-mediated NK cells in a TLR4-dependent manner. Thus, DP6 is a potential candidate adjuvant that acts as an immune cell modulator for the treatment of melanoma. [BMB Reports 2017; 50(5): 263-268].

Mast cells (MCs) play an important role as effector cells that cause allergic responses in allergic diseases. For these reasons, MC is considered an attractive therapeutic target for allergic disease treatment. In this study, we investigated the inhibitory effect of WZ3146, N-[3-[5-chloro-2-[4-(4-methylpiperazin-1-yl)anilino]pyrimidin-4-yl]oxyphenyl]prop-2-enamide, and the mechanisms of its actions on the MC activation and IgE-mediated allergic response by using three types of MCs such as rat basophilic leukemia (RBL)-2H3 cells, mouse bone marrow mast cells (BMMCs), and human Laboratory of Allergic Diseases 2 (LAD2) cells. WZ3146 inhibited antigen-stimulated degranulation in a dose-dependent manner (IC50, ~ 0.35 μM for RBL-2H3 cells; ~ 0.39 μM for BMMCs; ~ 0.41 for LAD2 cells). WZ3146 also suppressed the production of histamine, tumor necrosis factor (TNF)-α and interleukin (IL)-6, which mediate various allergic responses, in a dose-dependent manner. As the mechanism of WZ3146 to inhibit MCs, it inhibited the activation of spleen tyrosine kinase (Syk) and the downstream signaling proteins of Syk such as linker for activation of T cell (LAT) and phospholipase (PL) Cγ1 in the signaling pathway of FceRI. In addition, WZ3146 inhibited the activation of Akt, extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). However, WZ3146 did not inhibit degranulation of MCs by thapsigargin or ionomycin, which increase calcium concentration in cytosol. Notably, WZ3146 inhibited the activity of Lyn and Fyn, but not Syk. In an following animal experiment, WZ3146 inhibited IgE-dependent passive cutaneous anaphylaxis (PCA) in a dose-dependent manner (ED50, ~ 20 mg/kg). Taken together, in this study we show that the pyrimidine derivative, WZ3146, inhibits the IgE-mediated allergic response by inhibiting Lyn and Fyn Src-family kinases, which are initially activated by antigen stimulation in MCs. Therefore, we propose that WZ3146 could be used as a new therapeutic agent for the treatment of allergic diseases.

Histone deacetylase (HDAC) inhibitors are a new class of anticancer agents that act by inhibiting cancer cell proliferation and inducing apoptosis in various cancer cell lines. To investigate the anticancer effect of a novel histone deacetylase (HDAC) inhibitor MHY219, its efficacy was compared to that of suberoylanilide hydroxamic acid (SAHA) in human prostate cancer cells. The anticancer effects of MHY219 on cell viability, HDAC enzyme activity, cell cycle regulation, apoptosis and other biological assays were performed. MHY219 was shown to enhance the cytotoxicity on DU145 cells (IC 50 , 0.36 μM) when compared with LNCaP (IC 50 , 0.97 μM) and PC3 cells (IC 50 , 5.12 μM). MHY219 showed a potent inhibition of total HDAC activity when compared with SAHA. MHY219 increased histone H3 hyperacetylation and reduced the expression of class I HDACs (1, 2 and 3) in prostate cancer cells. MHY219 effectively increased the sub-G1 fraction of cells through p21 and p27 dependent pathways in DU145 cells. MHY219 significantly induced a G2/M phase arrest in DU145 and PC3 cells and arrested the cell cycle at G0/G1 phase in LNCaP cells. Furthermore, MHY219 effectively increased apoptosis in DU145 and LNCaP cells, but not PC3 cells, according to Annexin V/PI staining and Western blot analysis. These results indicate that MHY219 is a potent HDAC inhibitor that targets regulating multiple aspects of cancer cell death and might have preclinical value in human prostate cancer chemotherapy, warranting further investigation.

We isolated a sesquiterpene lactone from the methanol extract of the roots of Cosmos bipinnatus, namely, MDI (a mixture of dihydrocallitrisin and isohelenin). The anti-inflammatory activity of MDI was evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. MDI significantly inhibited the expression of inducible nitric oxide synthase and cyclooxygenase-2. Consistent with these results, the production of NO and prostaglandin E2 (PGE2) was suggested to be suppressed by MDI in a concentration-dependent manner (IC50 value was 0.94 and 2.88 µg mL(-1) for NO and PGE2, respectively). In addition, MDI significantly inhibited the expressions of pro-inflammatory cytokines such as IL-1β, IL-6, IFN-γ and TNF-α. Furthermore, MDI attenuated DNA-binding activity of NF-κB by inhibiting the phosphorylation of IκB. These results indicate that MDI isolated from the roots of C. bipinnatus shows anti-inflammatory activity in LPS-stimulated murine macrophages by modulating the NF-κB pathway.

The inward rectifier Kir2.1 current (IKir2.1) was reported to be facilitated by fluid flow. However, the mechanism underlying this facilitation remains uncertain. We hypothesized that during K+ influx or efflux, [K+] adjacent to the outer mouth of the Kir2.1 channel might decrease or increase, respectively, compared with the average [K+] of the bulk extracellular solution, and that fluid flow could restore the original [K+] and result in the apparent facilitation of IKir2.1. We recorded the IKir2.1 in RBL-2H3 cells and HEK293T cells that were ectopically over-expressed with Kir2.1 channels by using the whole-cell patch-clamp technique. Fluid-flow application immediately increased the IKir2.1, which was not prevented by either the pretreatment with inhibitors of various protein kinases or the modulation of the cytoskeleton and caveolae. The magnitudes of the increases of IKir2.1 by fluid flow were driving force-dependent. Simulations performed using the Nernst-Planck mass equation indicated that [K+] near the membrane surface fell markedly below the average [K+] of the bulk extracellular solution during K+ influx, and, notably, that fluid flow restored the decreased [K+] at the cell surface in a flow rate-dependent manner. These results support the “convection-regulation hypothesis” and define a novel interpretation of fluid flow-induced modulation of ion channels.

Chrysanthemum zawadskii var. latilobum (CZ) has been used for beverage or tea and also as folk medicine for the remedy of diverse inflammatory diseases. Nevertheless, the therapeutic effect of CZ on arthritis remains to be unknown. In this paper we aim to investigate the CZ's antiarthritic effect and mechanism of action both in vitro and in vivo. To assess CZ's antiarthritic effect, mouse models of type II collagen-induced arthritis (CIA) were used. Mice were used to gauge clinical arthritis index and histopathological changes. Reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting, electrophoretic mobility shift assay (EMSA), and other biological methods were adopted to measure CZ's effect on arthritis and to understand the veiled mechanism of action. CZ greatly suppressed CIA, histopathological score, bone erosion, and osteoclast differentiation. Mechanistically, CZ inhibited the production of various inflammatory and arthritic mediators like inflammatory cytokines, matrix metalloproteinases (MMPs), and chemokines. Of note, CZ significantly suppressed the activation of the NF-κB pathway in vivo. CZ exerted an antiarthritic effect in CIA mice by curbing the production of crucial inflammatory and arthritis mediators. This study warrants further investigation of CZ for the use in human rheumatoid arthritis (RA).

Trachelospermum jasminoides (Apocynaceae) has pharmacological effects that include anti-inflammatory, anti-bacterial and anti-viral activities, which have been observed from various studies. Of these pharmacological effects, the anti-inflammatory capacity of compounds from T. jasminoides is not yet known exactly. In this study, we investigated the compound that can be used for the suppression of lipopolysacchaide (LPS) stimulated inflammatory responses in macrophages among the five isolated compounds. β-sitosterol-β-D-glucoside (1) was found to reduce nitric oxide (NO) production from LPS-induced RAW 264.7 cells the most. In addition, compound 1 strongly inhibited the interleukin 6 (IL-6) activities of stimulated macrophages. Treatment of RAW 264.7 cells with compound 1 reduced secretion of inflammatory elements including tumour necrosis factor - alpha (TNF-α) and interleukin 1 beta (IL-1β). Thus, compound 1 may be a useful candidate for the development of new drugs to treat endotoxemia and inflammation accompanied by the overproduction of NO.

Osteoclasts (OCs) are the only bone-resorbing cells and are critically involved in various bone-associated diseases, including osteoporosis and rheumatoid arthritis. Differentiation of OCs from bone marrow macrophage cells (BMMs) is regulated by RANK and the adaptor protein (DAP12/FcRγ)-mediated costimulatory signals. However, it is unknown how RANKL/RANK signal stimulates phosphorylation of DAP12/FcRγ to initiate the costimulatory signals. As reported here, we found that OC differentiation and acquisition of bone resorption capacity were suppressed in RANKL-stimulated Fyn(-/-) or Fyn-siRNA-transfected BMMs, but could be restored by overexpression of Fyn kinase in Fyn(-/-) BMMs. However, the RANKL-stimulated proliferation of BMMs was unaffected by the absence of Fyn. In addition, RANKL-stimulated Fyn(-/-) BMMs no longer exhibited the optimal induction of typical OC markers such as NFATc1, c-Fos, c-Src, TRAF6, and cathepsin K or costimulatory signals such as the activating phosphorylations of Syk, PLCγ2, and Gab2. These were restored by overexpression of Fyn in Fyn(-/-) BMMs. Immunoprecipitation studies also indicated that the adaptor proteins DAP12/FcRγ and Syk interacted with RANK during RANKL stimulation in BMMs in a Fyn-dependent manner. Phosphorylation of the DAP12/FcRγ and the recruitment of Syk by DAP12/FcRγ were suppressed in Fyn(-/-) BMMs. This is the first demonstration that Fyn relays the initial RANK/RANKL signal to the ITAM-containing adaptors DAP12/FcRγ for OC differentiation.