Inhibitory Effects of Roseoside and Icariside E4 Isolated from a Natural Product Mixture (No-ap) on the Expression of Angiotensin II Receptor 1 and Oxidative Stress in Angiotensin II-Stimulated H9C2 Cells.

Hypertension is a major risk factor for the development of cardiovascular diseases. This study aimed to elucidate whether the natural product mixture No-ap (NA) containing Pine densiflora, Annona muricate, and Monordica charantia, or its single components have inhibitory effects on hypertension-related molecules in Angiotensin II (Ang II)-stimulated H9C2 cells. Individual functional components were isolated and purified from NA using various columns and solvents, and then their structures were analyzed using ESI⁻MS, ¹H-NMR, and 13H-NMR spectra. H9C2 cells were stimulated with 300 nM Ang II for 7 h. NA, telmisartan, ginsenoside, roseoside (Roseo), icariside E4 (IE4), or a combination of two components (Roseo and IE4) were administered to the cells 1 h before Ang II stimulation. The expression and activity of hypertension-related molecules or oxidative molecules were determined using RT-PCR, western blot, and ELISA. Ang II stimulation increased the expression of Ang II receptor 1 (AT1), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), tumor growth factor-ß (TGF-ß) mRNA, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and the levels of hydrogen peroxide (H2O2) and superoxide anion (•O2-) and reduced anti-oxidant enzyme activity. NA significantly improved the expression or activities of all hypertension-related molecules altered in Ang II-stimulated cells. Roseo or IE4 pretreatment either decreased or increased the expression or activities of all hypertension-related molecules similar to NA, but to a lesser extent. The pretreatment with a combination of Roseo and IE4 (1:1) either decreased or increased the expression of all hypertension-related molecules, compared to each single component, revealing a synergistic action of the two compounds. Thus, the combination of single components could exert promising anti-hypertensive effects similar to NA, which should be examined in future animal and clinical studies.

Inflammatory mediators resulting from transglutaminase 2 expressed in mast cells contribute to the development of Parkinson's disease in a mouse model.

This study aimed to investigate the role of transglutaminase 2 (TG2) expressed in mast cells in substantia nigra (SN) in Parkinson's disease (PD) model or human PD patients. C57BL/6 mice received 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by ip injection to induce PD. Bone marrow-derived mast cells (BMMCs) were adoptively transferred to TG2 knockout (KO or TG2-/-) mice by iv injection 1 day before MPTP injection or stimulated by 1 methyl-4-phenylpyridinium (MMP+). KO-MPTP mice showed reduced expression of tyrosine hydroxylase (TH) and dopamine (DA) transporter (DAT) and loss of TH+ DA neurons, and expression of markers (c-kit, tryptase, FcεRI), mediators' release (histamine, leukotrienes, cytokines), and TG2 related to mast cells, and co-localization of DA neuronal cells and mast cells in SN tissues or release of mediators and TG2 activity in SN tissues and sera versus those in WT (wild type)-MPTP or BM + KO-MPTP mice. KO-MPTP mice reversed the alterations of behavior. KO-BMMCs-transferred KO-MPTP (BM + KO-MPTP) mice had restoration of all the responses versus the KO-MPTP mice. MPP+-stimulated BMMCs had increased mediators' release, which were inhibited by TG2 inhibitor (R2 peptide). All the mediators and TG2 activity were also increased in the sera of human PD patients. The data suggest that TG2 expressed in mast cells recruited into SN tissues might contribute to neuroinflammation, which is known as one of the important features in pathogenesis of PD, via up-regulating the release of various mediators.

Inhibition of Osteoarthritis-Related Molecules by Isomucronulatol 7-O-ß-d-glucoside and Ecliptasaponin A in IL-1ß-Stimulated Chondrosarcoma Cell Model.

Osteoarthritis (OA) is the common form of arthritis and is characterized by disability and cartilage degradation. Although natural product extracts have been reported to have anti-osteoarthritic effects, the potential bioactivity of Ryupunghwan (RPH), a traditional Korean medicinal botanical formula that contains Astragalus membranaceus, Turnera diffusa, Achyranthes bidentata, Angelica gigas, Eclipta prostrata, Eucommia ulmoides, and Ilex paraguariensis, is not known well. Therefore, the inhibitory effects of single compounds isolated from RPH on the OA-related molecules were investigated using IL-1ß-stimulated chondrosarcoma SW1353 (SW1353) cell model. Two bioactive compounds, isomucronulatol 7-O-ß-d-glucoside (IMG) and ecliptasaponin A (ES) were isolated and purified from RPH using column chromatography, and then the structures were analyzed using ESI-MS, ¹H-NMR, and 13C-NMR spectrum. The expression or amount of matrix metalloproteinase 13 (MMP13), COX1/2, TNF-α, IL-1ß or p65 was determined by RT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA). RPH pretreatment reduced the expression and amounts of MMP13, and the expression of collagen II, COX1/2, TNF-α, IL-1ß or p65, which were increased in IL-1ß-stimulated SW1353 cells. IMG reduced the expression of all OA-related molecules, but the observed inhibitory effect was less than that of RPH extract. The other single compound ES showed the reduced expression of all OA-related molecules, and the effect was stronger than that in IMG (approximately 100 fold). Combination pretreatment of both single components remarkably reduced the expression of MMP13, compared to each single component. These synergic effects may provide potential molecular modes of action for the anti-osteoarthritic effects of RPH observed in clinical and animal studies.

Association of TG2 from mast cells and chronic spontaneous urticaria pathogenesis.

BACKGROUND: Mast cells and their mediators play important roles in chronic spontaneous urticaria (CSU) pathogenesis. Transglutaminase 2 (TG2) is expressed in activated mast cells and contributes to airway inflammation in allergic asthma. OBJECTIVE: To investigate the role of TG2 in CSU. METHODS: Patients with CSU (n = 72) and healthy controls (n = 51) were evaluated. Skin biopsy specimens were obtained from 5 patients with CSU and 2 healthy controls. Cord blood-derived human mast cells and peripheral blood-derived human mast cells were activated with IgE. TG2 activity and inflammatory mediators, such as histamine, leukotriene C4, and cytokines, were measured in serum or supernatant from cultured mast cells by enzyme-linked immunosorbent assay. Colocalization of mast cells and TG2 was determined in skin tissues by immunofluorescence. RESULTS: TG2 activity was significantly higher in serum samples from patients with CSU than in serum samples from healthy controls (P < .001). Colocalization of mast cell surface marker c-kit and TG2 was significantly increased in the lesional skin of patients with CSU compared with that in healthy controls. The levels of histamine, leukotriene C4, tumor necrosis factor α, transforming growth factor ß, and interleukins 4, 5, and 6 were significantly higher in patients with CSU than in healthy controls (P < .001). Serum TG2 levels had positive correlations with each inflammatory mediator (P < .001). TG2 activity was increased in cord blood-derived human mast cells (CBMCs) and peripheral blood-derived human mast cells activated with IgE compared with those without activation (P < .05). CONCLUSION: Our findings suggest that TG2 expressed in and released from mast cells plays an important role in CSU pathogenesis.

Transglutaminase 2 expressed in mast cells recruited into skin or bone marrow induces the development of pediatric mastocytosis.

BACKGROUND: Mastocytosis is characterized by a pathological increase in mast cells in organs such as skin and bone marrow. Transglutaminase 2 (TG2) expressed in mast cells contributes to allergic diseases, but its role in mastocytosis has not been investigated. This study aimed to investigate whether TG2 contributes to pediatric mastocytosis. METHODS: Serum, various skin tissues or bone marrow (BM) biopsy and aspirates were obtained from pediatric normal control or patients with indolent systemic mastocytosis (SM), mastocytoma, and urticaria pigmentosa (UP). Tryptase, individual cytokines, leukotriene C4 (LTC4 ), and TG2 activity in the serum were determined by enzyme-linked immunosorbent assay, mast cell population by May-Grünwald-Giemsa, CD 117 by immunofluorescence, cell surface molecules by Western blot, and colocalization of c-kit and TG2 or IL-10-expressing cells, CD25, and FOXP3 by immunohistochemistry. RESULTS: Infiltration of CD25(+) CD117(+) CD2(-) mast cells into BM and scalp/trunk/ear dermis; expression of FcεRI, tryptase, c-kit, FOXP3, CCL2/CCR2, and vascular cell adhesion molecule-1; and colocalization of c-kit and TG2 were enhanced in patient's skin tissues or BM, particularly SM, but colocalization of c-kit and IL-10-expressing cells was decreased vs. normal tissues. Amounts of LTC4 and inflammatory cytokines, expression of tryptase or TG2 activity were increased in patient's serum, BM aspirates, or ear/scalp skin tissues, respectively, vs. normal persons, but IL-10 level was decreased. CONCLUSION: The data suggest that mast cells, recruited in the skin and BM by CCL2/CCR, may induce the development of pediatric mastocytosis through reducing IL-10 due to upregulating TG2 activity via transcription factor nuclear factor-κB. Thus, TG2 may be used in diagnosis of pediatric mastocytosis, particularly SM.

Bortezomib alleviates experimental pulmonary arterial hypertension.

Vascular remodeling and endothelial dysfunction are important pathogenic features of pulmonary arterial hypertension (PAH). There is a growing body of evidence that proteasome inhibitors may be beneficial in vascular diseases by inhibiting proliferation of vascular smooth muscle cells (VSMCs) and ameliorating endothelial dysfunction. Here, we evaluated whether bortezomib (BTZ) could alleviate hypoxia- and monocrotaline (MCT)-induced PAH. BTZ (at doses from 1 to 100 µg/kg, or a dose of 100 µg/kg) was administered to mice every other day for the last 2 weeks of a 5-week hypoxia (10% O(2)) period, or to rats once daily from Day 22 to Day 34 after MCT challenge, respectively. BTZ treatment substantially suppressed elevation of right ventricular (RV) systolic pressure, RV hypertrophy, and pulmonary vascular remodeling in hypoxia-exposed mice. Similarly, BTZ treatment inhibited RV hypertrophy and vascular remodeling in MCT-injected rats. Strikingly, BTZ rescued 70% of MCT-injected rats up to Day 60, along with a considerable reduction in RV systolic pressure and suppression of vascular remodeling, whereas, among MCT-injected rats not administered BTZ, there were no survivors by Day 41. BTZ significantly suppressed proliferation of pulmonary VSMCs in vivo and in vitro. Furthermore, BTZ increased not only endothelial nitric oxide (NO) synthase (eNOS), phosphorylated eNOS, and NO production in vitro, but also eNOS and p-eNOS in hypoxia-exposed mice and MCT-injected rats, respectively. In contrast to the beneficial effects, BTZ increased active caspase-3 in cardiac ventricles of MCT-injected rats. Taken together, with caution for cardiotoxicity, BTZ could be a potential therapeutic strategy in PAH, possibly acting by inhibition of VSMC proliferation and amelioration of endothelial dysfunction.

Cigarette smoke induces Akt protein degradation by the ubiquitin-proteasome system.

Emphysema is one of the characteristic features of chronic obstructive pulmonary disease, which is caused mainly by cigarette smoking. Recent data have suggested that apoptosis and cell cycle arrest may contribute to the development of emphysema. In this study, we addressed the question of whether and how cigarette smoke affected Akt, which plays a critical role in cell survival and proliferation. In normal human lung fibroblasts, cigarette smoke extract (CSE) caused cell death, accompanying degradation of total and phosphorylated Akt (p-Akt), which was inhibited by MG132. CSE exposure resulted in preferential ubiquitination of the active Akt (myristoylated), rather than the inactive (T308A/S473A double mutant) Akt. Consistent with cytotoxicity, CSE induced a progressive decrease of phosphorylated human homolog of mouse double minute homolog 2 (p-HDM2) and phosphorylated apoptosis signal regulating kinase 1 (p-ASK1) with concomitant elevation of p53, p21, and phosphorylated p38 MAPK. Forced expression of the active Akt reduced both CSE-induced cytotoxicity and alteration in HDM2/p53/p21 and ASK1/p38 MAPK, compared with the inactive Akt. Of note, CSE induced expression of the tetratrico-peptide repeat domain 3 (TTC3), known as a ubiquitin ligase for active Akt. TTC3 siRNAs suppressed not only CSE-induced Akt degradation but also CSE-induced cytotoxicity. Accordingly, rat lungs exposed to cigarette smoke for 3 months showed elevated TTC3 expression and reduced Akt and p-Akt. Taken together, these data suggest that cigarette smoke induces cytotoxicity, partly through Akt degradation via the ubiquitin-proteasome system, in which TTC3 acts as a ubiquitin ligase for active Akt.

Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage.

Cigarette smoking causes apoptotic death, senescence, and impairment of repair functions in lung fibroblasts, which maintain the integrity of alveolar structure by producing extracellular matrix (ECM) proteins. Therefore, recovery of lung fibroblasts from cigarette smoke-induced damage may be crucial in regeneration of emphysematous lung resulting from degradation of ECM proteins and subsequent loss of alveolar cells. Recently, we reported that bone marrow-derived mesenchymal stem cell-conditioned media (MSC-CM) led to angiogenesis and regeneration of lung damaged by cigarette smoke. In this study, to further investigate reparative mechanisms for MSC-CM-mediated lung repair, we attempted to determine whether MSC-CM can recover lung fibroblasts from cigarette smoke-induced damage. In lung fibroblasts exposed to cigarette smoke extract (CSE), MSC-CM, not only inhibited apoptotic death, but also induced cell proliferation and reversed CSE-induced changes in the levels of caspase-3, p53, p21, p27, Akt, and p-Akt. MSC-CM also restored expression of ECM proteins and collagen gel contraction while suppressing CSE-induced expression of cyclooxygenase-2 and microsomal PGE(2) synthase-2. The CSE-opposing effects of MSC-CM on cell fate, expression of ECM proteins, and collagen gel contraction were partially inhibited by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. In rats, MSC-CM administration also resulted in elevation of p-Akt and restored proliferation of lung fibroblasts, which was suppressed by exposure to cigarette smoke. Taken together, these data suggest that MSC-CM may recover lung fibroblasts from cigarette smoke-induced damage, possibly through inhibition of apoptosis, induction of proliferation, and restoration of lung fibroblast repair function, which are mediated in part by the PI3K/Akt pathway.

Signaling pathways in the activation of mast cells cocultured with astrocytes and colocalization of both cells in experimental allergic encephalomyelitis.

Mast cells in the CNS participate in the pathophysiology of chronic neurodegenerative inflammatory diseases. This study aimed to investigate the signaling pathway of mast cells activated in an environment cocultured with astrocytes and to explore the role of their colocalization in brain of experimental allergic encephalomyelitis. Human mast cell line-1 cells and human U87 glioblastoma cell lines (U87) or mouse bone marrow-derived mast cells and mouse cerebral cortices-derived astrocytes were cocultured. Intracellular Ca(2+) was measured by confocal microscopy; histamine by fluorometric analyzer; leukotrienes by ELISA; small GTPases, protein kinase Cs, MAPK, c-kit, CD40, and CD40L by Western blot; NF-kappaB and AP-1 by EMSA; cytokines by RT-PCR; and colocalization of mast cells and astrocytes in brain by immunohistochemistry. Mast cells cocultured with astrocytes showed time-dependent increases in intracellular Ca(2+) levels, release of histamine and leukotrienes, and cytokine production. Mast cells or astrocytes showed enhanced surface expression of CD40L and CD40, respectively, during coculture. Mast cells cocultured with astrocytes induced small GTPases (Rac1/2, cdc42), protein kinase Cs, MAPK, NF-kappaB, and AP-1 activities. These changes were blocked by anti-CD40 Ab pretreatment or CD40 small interfering RNA. Mast cells increased in the thalamus of experimental allergic encephalomyelitis model, particularly colocalized with astrocytes in the thalamic border region of the habenula. In conclusion, the data suggest that activation of mast cells cocultured with astrocytes induces release of mediators by small GTPases/Ca(2+) influx through CD40-CD40L interactions to participate in the pathophysiology of chronic neurodegenerative inflammatory diseases, such as multiple sclerosis.

Signal pathways in astrocytes activated by cross-talk between of astrocytes and mast cells through CD40-CD40L.

BACKGROUND: Astrocytes, which play an active role in chronic inflammatory diseases like multiple sclerosis, exist close to mast cells with which they share perivascular localization. We previously demonstrated the possibility that astrocytes and mast cells interact in vitro and in vivo. This study aimed to investigate the signaling pathways and the role for astrocytes in the interaction of astrocytes and mast cells. METHODS: We co-cultured human U87 glioblastoma (U87) and human mast cell-1 (HMC-1) cell lines, and mouse cerebral cortices-derived astrocytes and mouse bone marrow-derived mast cells (BMMCs). Intracellular Ca2+ ([Ca2+]i) was measured by confocal microscopy; CD40 siRNA by Silencer Express Kit; small GTPases by GTP-pull down assay; PKCs, MAPKs, CD40, CD40L, Jak1/2, STAT1, TNF receptor 1 (TNFR1) by Western blot; NF-κB and AP-1 by EMSA; cytokines by RT-PCR. An experimental allergic encephalomyelitis (EAE) model was induced using myelin oligodendrocyte glycoprotein (MOG) peptide and pertussis toxin in mice. Co-localization of TNFR1 and astrocytes in EAE brain tissues was determined by immunohistochemistry. RESULTS: Each astrocyte co-culture had increases in [Ca2+]i levels, release of cytokines and chemokines; activities of Rho-family GTPases, NF-κB/AP-1/STAT1727, and Jack1/2, STAT1701. These effects were inhibited by anti-CD40 antibody or CD40 siRNA, and signaling pathways for Jak1/2 were inhibited by anti-TNFR1 antibody. EAE score, expression of TNFR1, and co-localization of TNFR1 and astrocytes were enhanced in brain of the EAE model. Anti-CD40 antibody or 8-oxo-dG pretreatment reduced these effects in EAE model. CONCLUSIONS: These data suggest that astrocytes activated by the CD40-CD40L interaction in co-culture induce inflammatory cytokine production via small GTPases, and the secreted cytokines re-activate astrocytes via Jak/STAT1701 pathways, and then release more cytokines that contribute to exacerbating the development of EAE. These findings imply that the pro-inflammatory mediators produced by cell-to-cell cross-talk via interaction of CD40-CD40L may be as a promising therapeutic target for neurodegenerative diseases like MS.

Cigarette smoke exacerbates mouse allergic asthma through Smad proteins expressed in mast cells.

BACKGROUND: Many studies have found that smoking reduces lung function, but the relationship between cigarette smoke and allergic asthma has not been clearly elucidated, particularly the role of mast cells. This study aimed to investigate the effects of smoke exposure on allergic asthma and its association with mast cells. METHODS: BALB/c mice were sensitized and challenged by OVA to induce asthma, and bone marrow-derived mast cells (BMMCs) were stimulated with antigen/antibody reaction. Mice or BMMCs were exposed to cigarette smoke or CSE solution for 1 mo or 6 h, respectively. The recruitment of inflammatory cells into BAL fluid or lung tissues was determined by Diff-Quik or H&E staining, collagen deposition by Sircol assay, penh values by a whole-body plethysmography, co-localization of tryptase and Smad3 by immunohistochemistry, IgE and TGF-ß level by ELISA, expressions of Smads proteins, activities of signaling molecules, or TGF-ß mRNA by immunoblotting and RT-PCR. RESULTS: Cigarette smoke enhanced OVA-specific IgE levels, penh values, recruitment of inflammatory cells including mast cells, expressions of smad family, TGF-ß mRNA and proteins, and cytokines, phosphorylations of Smad2 and 3, and MAP kinases, co-localization of tryptase and Smad3, and collagen deposition more than those of BAL cells and lung tissues of OVA-induced allergic mice. CSE solution pretreatment enhanced expressions of TGF-ß, Smad3, activities of MAP kinases, NF-κB/AP-1 or PAI-1 more than those of activated-BMMCs. CONCLUSIONS: The data suggest that smoke exposure enhances antigen-induced mast cell activation via TGF-ß/Smad signaling pathways in mouse allergic asthma, and that it exacerbates airway inflammation and remodeling.

Antiallergic herbal composition from Scutellaria baicalensis and Phyllostachys edulis.

We aimed to find antiallergic agents from natural sources using mast cells activated during allergic reaction. We screened over 2000 plants for blockade of histamine release and identified two of them, S. baicalenesis and P. edulis. Bioassay-guided fractionation led to two main constituent flavonoids, baicalin from S. baicalenesis roots and isoorientin from P. edulis leaves. Based on these two compounds, two standardized extracts (SSBE and SPEE) and a combined standardized herb composition (SHC) were developed. SSBE, SPEE, and SHC remarkably inhibited histamine and leukotriene release from mast cells activated by anti-OVA/OVA binding, and SHC showed a stronger inhibition than either extract alone. SHC also showed greater inhibition potency than either aspirin or cromolyn, which are known antiallergic agents. Our results suggest that SHC reduce degranulation during mast cell activation and could be a promising candidate for the treatment of immune/allergic diseases related to mast cells.

Cytokine production through PKC/p38 signaling pathways, not through JAK/STAT1 pathway, in mast cells stimulated with IFNgamma.

IFNgamma is strongly related to mast cell-associated diseases. There are many reports that IFNgamma inhibits mast cell degranulation. However, inflammatory cytokine production in mast cells stimulated with IFNgamma has not yet been clearly investigated. Therefore, we aimed to investigate the signaling pathways of cytokine production in mast cells stimulated with IFNgamma. Human mast cell line (HMC)-1 or mouse bone marrow-derived mast cells (BMMCs) were stimulated with IFNgamma (100 units) for time periods indicated. Expressions of proteins and mRNAs of cytokines were determined by ELISA and RT-PCR, respectively, activities of MAP kinases, PKC, JAK1/2, and STAT1 on tyrosine 701 and serine 727 by immunoblotting, the DNA-binding activity of the transcription factors by electrophoretic mobility shift assay. IFNgamma-stimulated mast cells showed increase in expressions of proteins and mRNAs of inflammatory cytokines, phosphorylations of MAP kinases, PKCalpha and betaI, JAK1/2, and STAT1 on tyrosine 701 and serine 727. JAK inhibitor or PKC inhibitors inhibited the phosphorylations of p38 kinase, STAT1 on serine 727, and activities of NF-kappaB and AP-1 compared to IFNgamma stimulation alone. These data suggest that IFNgamma-stimulated mast cells induce productions of inflammatory cytokines through PKC/p38/NF-kappaB and AP-1 pathways, not through classical JAK/STAT1 pathway, in both mast cells.

Simvastatin inhibits induction of matrix metalloproteinase-9 in rat alveolar macrophages exposed to cigarette smoke extract.

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.

Hyaluronic acid targets CD44 and inhibits FcepsilonRI signaling involving PKCdelta, Rac1, ROS, and MAPK to exert anti-allergic effect.

Effects of hyaluronic acid (HA) on allergic inflammation were investigated. HA exerted negative effects on beta-hexoaminidase secretion and histamine release in antigen-stimulated rat basophilic leukemia (RBL2H3) cells. HA inhibited interaction between IgE and FcepsilonRI and between FcepsilonRI and PKCdelta. HA inhibited CD44 interaction with PKCalpha, indicating that HA targets CD44. PKCalpha and -delta were responsible for increased Rac1 activity and expression of p47(phox), p67(phox). HA inhibited phosphorylation of PKCalpha and -delta. Rac1 was responsible for increased ROS, and NADPH oxidase was the main source for ROS. The inhibition of PKC prevented antigen from increasing phosphorylation of ERK and p38 MAPK. ERK, p38 MAPK, and ROS, were responsible for secretion of beta-hexosaminidase, histamine release, and induction of chemokines. HA suppressed induction of chemokines, such as MIP-2 and Sprr-2a. CD44 mediated effect of antigen on phosphorylation of ERK, p38MAPK, ROS production, secretion of beta-hexosaminidase, and histamine release. GPCR did not mediate allergic function of antigen or affect anti-allergic function of HA. In vivo anti-allergic effect of HA was investigated using Nc/Nga mice model of DNFB-induced atopic dermatitis. HA reduced skin lesions in Nc/Nga mice treated with DNFB, decreased expression levels of MIP-2, Sprr-2a, and serum IgE level. In conclusion, hyaluronic acid exerts negative effect on allergic inflammation by targeting CD44 and inhibiting FcepsilonRI signaling.

Granule formation in NGF-cultured mast cells is associated with expressions of pyruvate kinase type M2 and annexin I proteins.

BACKGROUND: Nerve growth factor (NGF) is a potent mediator, which regulates characteristics of mast cells, but its biological function is not well characterized. This study aimed to screen proteins associated with the maturation of human mast cells-1 (HMC-1) or mouse bone marrow-derived mast cells (BMMCs) cultured with NGF, and to examine the functions of proteins involved. METHODS: NGF (10 ng/ml) was added to cell culture medium every other day for 10 days for HMC-1 or twice a week for 5 weeks for BMMCs. Granule formation was determined by electron microscopy or May-Grunwald-Giemsa staining, TNF-alpha by ELISA, expressions of various proteins by two-dimensional gel electrophoresis (2-DE), siRNA transfection by Lipofectamine 2000, and the expressions of pyruvate kinase and annexin I by immunoblotting. RESULTS: After NGF treatment, granule formation and total amounts of granular mediator, TNF-alpha increased in both mast cells. This TNF-alpha was released by calcium ionophore or by antigen/antibody reaction. Expressions of pyruvate kinase and annexin I obtained by 2-DE were confirmed by immunoblotting and siRNA-transfected HMC-1 cells. Expressions of proteins, granule formation and TNF-alpha content were blocked by both the TrkA inhibitor, K252a, and the ERK inhibitor, PD98059, but not by the PI3 kinase inhibitors, LY294002 and wortmannin. CONCLUSION: These data suggest that pyruvate kinase and annexin I expressed by NGF contribute to granule formation containing TNF-alpha as well as other mediators in mast cells, which play a major role in allergic diseases via a TrkA/ERK pathway.

Inhibitory mechanism of anti-allergic peptides in RBL2H3 cells.

Here we report the identification and functional characterization of several anti-allergic peptides identified through biopanning of pooled sera of patients with various allergies. Several peptides, including LSYLLWRSRLP (LSY), LVAHVGAGGVL (LVA), RVSSCRGRNHIV (RVS), ETIGARWVRIE (ETI), TDGVTYTNDCL (TDG), RVVRYDADFWI (RVV), GFWCRRSGLVGV (GFW), were further characterized. These peptides inhibited the release of histamine from antigen-stimulated mast cells isolated from lung tissues of guinea pigs. Furthermore, the peptides inhibited calcium influx in ovalbumin-stimulated mast cells isolated from lung tissues of guinea pigs. Likewise, the peptides inhibited the release of beta-hexosaminidase from antigen-stimulated rat basophilic leukemia (RBL2H3) cells and decreased calcium influx and intracellular reactive oxygen species production as well. We found that the peptides significantly decreased phosphorylation of extracellular regulated kinase (ERK) and that this was responsible for the decreased calcium influx and beta-hexosaminidase in antigen-stimulated RBL2H3 cells, suggesting that ERK plays an important role in allergic reactions. The peptides identified in this study also affected upstream signaling of allergic inflammation. In other words, these peptides decreased phosphorylation of Lyn, PKCalpha, and -delta. Lyn and PKC are known to be responsible for the phosphorylation of FcepsilonRI, in response to receptor aggregation. The peptides inhibited interaction between IgE and FcepsilonRI, suggesting that these peptides exert anti-allergic effects by inhibiting receptor cross-linking. These peptides also inhibited interaction between FcepsilonRI and PKCdelta. Taken together, these data suggest that peptides exert anti-allergic effect through the inhibition of upstream signaling, involving receptor cross-linking, and downstream multiple signaling.

Anti-inflammatory mechanism of simvastatin in mouse allergic asthma model.

Statins have anti-inflammatory property and immunomodulatory activity. In this study we aimed to investigate the inhibitory mechanism of simvastatin in allergic asthmatic symptoms in mice. BALB/c mice were sensitized and challenged by ovalbumin to induce asthma. Ovalbumin-specific serum IgE levels were measured by enzyme-linked immunosorbent assay (ELISA), and the recruitment of inflammatory cells into bronchoalveolar lavage fluid or lung tissues was measured by Diff-Quik staining and hematoxylin and eosin (H&E) staining, respectively, the expressions of CD40, CD40 ligand (CD40L), and vascular cell adhesion molecule-1 (VCAM-1) by immunohistochemistry, the mRNA and protein expressions of cytokines in lung tissues by reverse transcriptase-polymerase chain reaction (RT-PCR) or ELISA, epithelial hyperplasia by periodic acid-Schiff (PAS) staining, activities of matrix metalloproteinases (MMPs) by zymography, the activities of small G proteins, mitogen-activated protein (MAP) kinases and nuclear factor-kappa B (NF-kappaB) in bronchoalveolar lavage cells and lung tissues by western blot and EMSA, respectively. Simvastatin reduced ovalbumin-specific IgE level, the number of total inflammatory cells, macrophages, neutrophils, and eosinophils into bronchoalveolar lavage fluid, the expressions of CD40, CD40L or VCAM-1, the mRNA and protein levels of interleukin (IL)-4, IL-13 and tumor necrosis factor (TNF)-alpha, the numbers of goblet cells, activities of MMPs, and further small G proteins, MAP kinases and NF-kappaB activities in bronchoalveolar lavage cells and lung tissues increased in ovalbumin-induced allergic asthma in mice. Our data suggest that simvastatin may be used as a therapeutic agent in asthma, based on reductions of various allergic responses via regulating small G proteins/MAP kinases/NF-kappaB in mouse allergic asthma.

DA-9601, Artemisia asiatica herbal extract, ameliorates airway inflammation of allergic asthma in mice.

We previously reported that DA-9601, ethanol herbal extract of Artemisia asiatica, inhibited histamine and leukotriene releases in guinea pig lung mast cells activated with specific antigen/antibody reaction. This study aimed to evaluate the inhibitory effect of DA-9601 on the OVA-induced airway inflammation in allergic asthma mouse model. BALB/c mice were sensitized and challenged with OVA. DA-9601 was administered orally 1 h before every local OVA-challenge. OVA-specific serum IgE was measured by ELISA, recruitment of inflammatory cells in BAL fluids and lung tissues by Diff-Quik and H&E staining, respectively, the expressions of CD40, CD40L and VCAM-1 by immunohistochemistry, goblet cell hyperplasia by PAS staining, activities of MMPs by gelatin zymography, expressions of mRNA and proteins of cytokines by RT-PCR and ELISA, activities of MAP kinases by western blot, and activity of NF-KappaB by EMSA. DA-9601 reduced IgE level, recruitment of inflammatory cells into the BAL fluid and lung tissues, expressions of CD40, CD40L and VCAM-1 molecules, goblet cell hyperplasia, MMPs activity, expressions of mRNA and productions of various cytokines, activities of MAP kinases and NK-KappaB increased from OVA-challenged mice. These data suggest that DA-9601 may be developed as a clinical therapeutic agent in allergic diseases due to suppressing the airway allergic inflammation via regulation of various cellular molecules expressed by MAP kinases/NF-KappaB pathway.

Periostin and Interleukin-13 Are Independently Related to Chronic Spontaneous Urticaria.

Chronic spontaneous urticaria (CSU) is a complex idiopathic disease of the skin with various cellular infiltrations. Although mast cells are key effector cells in the pathogenesis of CSU, CD4+ T helper 2 cells also have particular roles in the development and maintenance of CSU. Periostin is known as a downstream molecule of interleukin (IL)-4 and IL-13, key cytokines of type 2 immune responses. In this study, we examined periostin and IL-13 levels in the sera of patients with CSU (n=84) and healthy normal controls (NCs, n=43). Periostin levels were significantly lower in the CSU group than in NCs (71.4±21.8 vs 85.1±22.4 ng/mL, P=0.04). Periostin levels were also lower in the severe CSU group than those in mild CSU (59.7±18.0 vs 73.4±22.0 ng/mL, P=0.04). However, IL-13 levels were significantly higher in patients with CSU than in NCs (508.5±51.2 vs 200.7±13.3 pg/mL, P=0.001). In conclusion, periostin and IL-13 may be independently related to the pathogenesis of CSU.