AP-1 DNA Binding Research Articles

Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation.

Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton) with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived) microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO), TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1) and perilipin 2 (PLIN2). Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia.

Effect of Combined Treatment with Ursolic Acid and Resveratrol on Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate.

In this study, the effects of combining ursolic acid + resveratrol, for possible combined inhibitory effects on skin tumor promotion, were evaluated. Ursolic acid, resveratrol, and the combination of ursolic acid + resveratrol were applied topically prior to 12-O-tetracanoylphorbol-13-acetate (TPA) treatment on mouse skin to examine their effect on TPA-induced signaling pathways, epidermal hyperproliferation, skin inflammation, inflammatory gene expression, and skin tumor promotion. The combination of ursolic acid + resveratrol produced a greater inhibition of TPA-induced epidermal hyperproliferation. The combination of ursolic acid + resveratrol inhibited TPA-induced signaling pathways, including EGFR, STAT3, Src, Akt, Cox-2, Fas, NF-κB, p38 MAPK, c-Jun, and JNK1/2 while increasing levels of tumor suppressors, such as p21 and PDCD4, to a greater extent compared with the groups treated with the individual compounds. Ursolic acid + resveratrol also induced a dramatic increase of p-AMPK-α(Thr172). Combined treatment with ursolic acid + resveratrol resulted in a greater inhibition of expression of proinflammatory cytokines, including Il1a, Il1b, and Il22. Furthermore, NF-κB, Egr-1, and AP-1 DNA binding activities after TPA treatment were dramatically decreased by the combination of ursolic acid + resveratrol. Treatment with ursolic acid + resveratrol during skin tumor promotion with TPA produced greater inhibition of tumor multiplicity and tumor size than with either agent alone. Collectively, the greater ability of the combination of ursolic acid + resveratrol to inhibit skin tumor promotion was due to the greater inhibitory effects on growth factor and inflammatory signaling, skin inflammation, and epidermal hyperproliferation induced by TPA treatment.

Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.

A novel CBF that regulates abiotic stress response and the ripening process in oil palm (Elaeis guineensis) fruits

A new member of the C-repeat binding factor (CBF) encoding an AP2-domain protein was isolated from ripening oil palm (Elaeis guineensis var. Dura × Pisifera) fruit. Bioinformatics analysis revealed that EgCBF3 has an AP2 DNA binding domain containing 57 amino acids. According to the signature sequences found upstream and downstream of the AP2 domain, it was predicted that the deduced amino acid sequence belongs to the A-1 group of the CBF/DREB1 subfamily. EgCBF3 is differentially expressed in the fruit mesocarp tissue with the highest transcript accumulation in ripening fruit at 17 weeks after anthesis (w.a.a.). In the vegetative tissues, it showed higher expression levels in the roots compared to the leaves. In the mesocarp tissue, expression of EgCBF3 was upregulated after 2 h and peaked after 24 h of cold, ethylene and ABA treatments, while for drought and salt stresses, it exhibited the highest expression after 4 and 8 h, respectively. GFP fusion protein of EgCBF3 was localized to the nucleus of the onion’s epidermal cells. The in vitro and in vivo DNA-protein transactivation assay using electrophoretic mobility shift assay (EMSA) and yeast one-hybrid analysis revealed that EgCBF3 was able to bind to the DRE/CRT cis-element. Tomato was chosen as a model system for the climacteric oil palm fruits. Expression profile of the ethylene and abscisic acid biosynthesis-related genes and ripening-related genes were affected by transient overexpression of EgCBF3 in the tomato fruits at different developmental stages. These results suggest that EgCBF3 can mediate abiotic stress response in ripening fruits and may function as a regulator of ripening process-related genes.

The CO donor CORM‐2 inhibits LPS‐induced vascular cell adhesion molecule‐1 expression and leukocyte adhesion in human rheumatoid synovial fibroblasts

Infection with Gram-negative bacteria has been recognized as an initiator of rheumatoid arthritis, which is characterized by chronic inflammation and infiltration of immune cells. Carbon monoxide (CO) exhibits anti-inflammatory properties. Here we have investigated the detailed mechanisms of vascular cell adhesion molecule-1 (VCAM-1) expression induced by LPS and if CO inhibited LPS-induced leukocyte adhesion to synovial fibroblasts by suppressing VCAM-1 expression. Human rheumatoid arthritis synovial fibroblasts (RASFs) were incubated with LPS and/or the CO-releasing compound CORM-2. Effects of LPS on VCAM-1 levels were determined by analysing mRNA expression, promoter activity, protein expression, and immunohistochemical staining. The molecular mechanisms were investigated by determining the expression, activation, and binding activity of transcriptional factors using target signal antagonists. CORM-2 significantly inhibited inflammatory responses in LPS-treated RASFs by down-regulating the expression of adhesion molecule VCAM-1 and leukocyte infiltration. The down-regulation of LPS-induced VCAM-1 expression involved inhibition of the expression of phosphorylated-NF-κB p65 and AP-1 (p-c-Jun, c-Jun and c-Fos mRNA levels). These results were confirmed by chromatin immunoprecipitation assay to detect NF-κB and AP-1 DNA binding activity. LPS-mediated formation of the TLR4/MyD88/TRAF6/c-Src complex regulated NF-κB and MAPKs/AP-1 activation leading to VCAM-1 expression and leukocyte adhesion. CORM-2, which liberates CO to elicit direct biological activities, attenuated LPS-induced VCAM-1 expression by interfering with NF-κB and AP-1 activation, and significantly reduced LPS-induced immune cell infiltration of the synovium.

Pirfenidone Attenuates the IL-1β–Induced Hyaluronic Acid Increase in Orbital Fibroblasts From Patients With Thyroid-Associated Ophthalmopathy

This study aimed to investigate the effect of pirfenidone on the IL-1β-induced hyaluronic acid (HA) increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). Primary cultured orbital fibroblasts were obtained from patients with TAO, and the excreted levels of HA from IL-1β-treated cells with or without pirfenidone were measured. The effect of pirfenidone on IL-1β-induced hyaluronic acid synthase (HAS) expression was evaluated. The relevance of the mitogen-activated protein kinase (MAPK)-mediated signaling pathway in IL-1β-induced HAS expression was assessed using specific inhibitors to p38, extracellular signal-regulated kinase (ERK), or c-Jun N-terminal kinase (JNK). The phosphorylation level of each MAPK in IL-1β-treated cells with or without pirfenidone and the level of AP-1 DNA binding were measured. The inhibitory potency of pirfenidone on HA production was evaluated using dexamethasone as a reference agent. Pirfenidone strongly attenuated the IL-1β-induced HA release in a dose-dependent manner. The IL-1β-induced HAS expression was decreased significantly following cotreatment with pirfenidone at the mRNA and protein levels. The production of mRNAs was halted by cotreatment with inhibitors of ERK and p38, but not by inhibitors of JNK. The IL-1β-induced ERK and p38 phosphorylation, and AP-1 DNA binding were attenuated in the presence of pirfenidone. Pirfenidone showed greater potency than dexamethasone in inhibiting increases in IL-1β-induced HA. Pirfenidone attenuates the IL-1β-induced HA production in orbital fibroblasts from patients with TAO, at least in part, through suppression of the MAPK-mediated HAS expression. These results support the potential use of pirfenidone for treatment of patients with TAO.

PRDX6 promotes lung tumor progression via its GPx and iPLA2 activities

PRDX6 is a bifunctional protein with both glutathione peroxidase (GPx) and calcium-independent phospholipase A2 (iPLA2) activities, which are concomitantly increased with the expression of PRDX6. PRDX6 promoted lung tumor growth in an in vivo allograft model. Herein, we further studied the vital roles in tumor progression of PRDX6 in lung cancer using nude mice bearing PRDX6-overexpressing lung cancer cells. Nude mice xenografted with PRDX6 showed increases in tumor size and weight compared to control mice. Histopathological and Western blotting examination demonstrated that expression of proliferating cell nuclear antigen, vascular endothelial growth factor, metalloproteinases 2 and 9, and cyclin-dependent kinases accompanied by increased iPLA2 and GPx activities were increased in the tumor tissues of PRDX6-overexpressing nude mice. In tumor tissues of PRDX6-overexpressing mice, the activation of mitogen-activated protein kinases and AP-1 DNA binding were also increased. The growth of lung cancer cell lines (A549 and NCI-H460) was enhanced by the increase in iPLA2 and GPx activities of PRDX6. In addition, mutant PRDX6 (C47S) attenuated PRDX6-mediated p38, ERK1/2, and AP-1 activities as well as its enzyme activities in the A549 and NCI-H460 lines. Furthermore, tumor growth and p38, ERK1/2, and AP-1 activities were also inhibited in nude mice bearing mutant PRDX6 (C47S) compared to PRDX6. Therefore, our findings indicate that PRDX6 promotes lung tumor growth via increased glutathione peroxidase and iPLA2 activities.

Berberine attenuates high glucose-induced proliferation and extracellular matrix accumulation in mesangial cells: Involvement of suppression of cell cycle progression and NF-κB/AP-1 pathways

Berberine has been shown to have renoprotective effects on diabetes through attenuating TGF-β1 and fibronectin (FN) expression. However, how berberine regulates TGF-β1 and FN is not fully clear. Here we investigated whether berberine inhibited TGF-β1 and FN expression in high glucose-cultured mesangial cells. Berberine significantly inhibited mesangial cell proliferation and hypertrophy by increasing the cell population in G1-phase and reducing that in S-phase. In addition, berberine reversed high glucose-induced down-regulation of cyclin-dependent kinase inhibitor p21Waf1/Cip1 and p27Kip1. Berberine inhibited p65 translocation to the nucleus and c-jun phosphorylation induced by high glucose. Furthermore, berberine attenuated high glucose-induced expression of TGF-β1 and FN. Using a luciferase reporter assay, we found that high glucose-induced transcription activity of NF-κB and AP-1 was blocked by berberine. Electrophoretic mobility shift assay showed that high glucose increased that NF-κB and AP-1 DNA binding activity. These data indicate that berberine inhibited mesangial cell proliferation and hypertrophy by modulating cell cycle progress. In addition, berberine suppressed high glucose-induced TGF-β1 and FN expression by blocking NF-κB/AP-1 pathways.

Studies on Molecular Characterization of DREB Gene in Indica Rice (Oryza sativa L.)

Number of potential candidate genes (CGs) has been identified by transcriptome and transgenic approaches involved in the adaptive responses to drought in cereals. One of these genes conferring tolerance to drought is DREB gene family. The exploration of genetic/allelic variation within the DREB gene family is an important prerequisite towards a better understanding of stress tolerance. The present investigation aims to study allelic variation in nucleotide sequence within DREB gene of different rice cultivars need for crop improvement programme. Ten upland as well as lowland rice cultivars was used for molecular characterization and allele mining. The results showed that the DREB gene was found in all the tested germplasm and submitted to NCBI GenBank (accession numbers KF 545561 to KF 545569). Bioinformatics analysis showed that 100 percent identity with AP2 DNA binding domain of 59 amino acids which showed conserved three sheets with 14th valine and 19th glutamic acid conserved residues. AP2 domain of putative DREB protein were found rich in alanine (17.6%) and arginine (15.7%) amino acids with a predicted molecular mass of 5.89 kDa and iso-elecric point (pI) 10.38. It is observed that DREB gene nucleotide sequences were verified with DNA polymorphism analysis, out of which 196 invariable (monomorphic) and 8 variable (polymorphic) i.e. segregating sites were identified including 9 number of mutation and 5 haplotypes. The haplotypes (gene) diversity was 0.756; the variance and standard deviation diversity were 0.01678 and 0.130 respectively. The result showed that the variety ‘Khandagiri’ showed 97.5% similarity with all accessions DREB gene of rice and 0.036% evolutionary divergence with AK121956 accession present in the database. This might have enhanced the capability of DREB gene as a transcription factor and that lead to provide better adoption and tolerance capability of the cultivar during drought.

Chlorophyll-related compounds inhibit cell adhesion and inflammation in human aortic cells.

The objectives of this study were to investigate the effects of chlorophyll-related compounds (CRCs) and chlorophyll (Chl) a+b on inflammation in human aortic endothelial cells. Adhesion molecule expression and interleukin (IL)-8, nuclear factor (NF)-κB p65 protein, and NF-κB and activator protein (AP)-1 DNA binding were assessed. The effects of CRCs on inflammatory signaling pathways of signal transducers and activators of transcription 3 (STAT3) and mothers against decapentaplegic homolog 4, respectively induced by IL-6 and transforming growth factor (TGF)-β, in human aortic smooth muscle cells cultured in vitro were also investigated. HAECs were pretreated with 10 μM of CRCs, Chl a+b, and aspirin (Asp) for 18 h followed by tumor necrosis factor (TNF)-α (2 ng/mL) for 6 h, and U937 cell adhesion was determined. TNF-α-induced monocyte-endothelial cell adhesion was significantly inhibited by CRCs. Moreover, CRCs and Chl a+b significantly attenuated vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and IL-8 expressions. Treatments also significantly decreased in NF-κB expression, DNA binding, and AP-1 DNA binding by CRCs and Asp. Thus, CRCs exert anti-inflammatory effects through modulation of NF-κB and AP-1 signaling. Ten micromoles of CRCs and Asp upregulated the expression of mothers against decapentaplegic homolog 4 (Drosophila) (SMAD4) in the TGF-β receptor signaling pathway, and SMAD3/4 transcription activity was also increased. Ten micromoles of CRCs were able to potently inhibit STAT3-binding activity by repressing IL-6-induced STAT3 expression. Our results provide a potential mechanism that explains the anti-inflammatory activities of these CRCs.

Glial cell line-derived neurotrophic factor induces cell migration in human oral squamous cell carcinoma

Perineural invasion is a prominent clinical feature of various cancers, which causes difficulty in curative resection. Glial cell-derived neurotrophic factor (GDNF), a potent neurotrophic factor, plays an important role in the invasive and metastatic behavior of various cancers. The aim of this study was to examine the role of GDNF on oral squamous cell carcinoma. GDNF expression in tissue samples was analyzed by immunohistochemistry. Transwell assay, zymography, Western blot, reverse transcription-PCR, and electrophoretic mobility shift assay (EMSA) were carried out to assess the effects of GDNF on oral cancer cells. Human oral cancer tissues showed higher GDNF expression than that in normal tissues. We also found that application of human GDNF enhanced the cell migration ability of human oral cancers. Moreover, treatment with GDNF increased matrix metalloproteinase (MMP)-9 and MMP-13 expression in oral cancer. Inhibition of MMP-9 and MMP-13 in oral cancer cells by pharmacological inhibitors or neutralizing antibodies reduced GDNF-enhanced cell migration. Moreover, transfection with siRNA against MMP-13 inhibited GDNF-enhanced cell migration. Treatment with GDNF also increased ERK, p38 and JNK phosphorylation, and AP-1 DNA binding activity in human oral cancer cells. Inhibition of MAP kinase or AP-1 also reduced GDNF-induced oral cancer cell migration. In migration-prone sublines, oral cancer cells showed a higher migration ability than that of the original oral cancer cells. Surprisingly, the enhancement of cell migratory activity in migration-prone sublines was reduced by a GDNF-neutralizing antibody. Importantly, migration-prone sublines of oral cancer revealed higher GDNF expression. These results indicate a regulatory effect on cell migration by GDNF in oral squamous cancer.

Epigallocatechin-3-gallate prevents heat shock-induced MMP-1 expression by inhibiting AP-1 activity in human dermal fibroblasts

The anti-skin aging effects of epigallocatechin-3-gallate (EGCG) have been studied extensively in vitro and in vivo models. Accumulating data suggest that EGCG possesses important antioxidant and photoprotective properties. Our previous study demonstrated that heat exposure induces cutaneous angiogenesis and inflammatory cellular infiltration, disrupts the dermal extracellular matrix by inducing matrix metalloproteinases, and alters dermal structural proteins, thereby causing premature skin aging. In the present study, we examined whether EGCG may inhibit expression of MMP-1 in heat-stimulated human dermal fibroblasts. Furthermore, we investigated the inhibitory mechanism of EGCG on heat-induced MMP-1 expression. Western blot analysis and MMP-1 promoter assay revealed that EGCG markedly inhibited heat shock-induced MMP-1 expression in human dermal fibroblasts. In addition, we found that heat shock increased AP-1 DNA binding activity, and c-Jun was found to be increased mostly by heat stimulation in a supershift assay, which were suppressed by EGCG treatment. Also, in Western blotting, EGCG significantly inhibited the heat-induced expression of AP-1 constituent proteins, c-Jun, JunB and c-Fos. These results demonstrated that EGCG has abilities to inhibit heat-induced collagenolytic MMP-1 production via interfering with AP-1 pathways. Therefore, we propose that EGCG may be a potential agent for the prevention and treatment for heat shock-induced skin aging (thermal skin aging).

Maresin-1 reduces the pro-inflammatory response of bronchial epithelial cells to organic dust

BackgroundExposure to organic dust causes detrimental airway inflammation. Current preventative and therapeutic measures do not adequately treat resulting disease, necessitating novel therapeutic interventions. Recently identified mediators derived from polyunsaturated fatty acids exhibit anti-inflammatory and pro-resolving actions. We tested the potential of one of these mediators, maresin-1 (MaR1), in reducing organic dust-associated airway inflammation.MethodsAs bronchial epithelial cells (BECs) are pivotal in initiating organic dust-induced inflammation, we investigated the in vitro effects of MaR1 on a human BEC cell line (BEAS-2B). Cells were pretreated for 1 hour with 0–200 nM MaR1, followed by 1–24 hour treatment with 5% hog confinement facility-derived organic dust extract (HDE). Alternatively, a mouse lung slice model was utilized in supportive cytokine studies. Supernatants were harvested and cytokine levels determined via enzyme-linked immunosorbent assays. Epithelial cell protein kinase C (PKC) isoforms α and ϵ, and PKA activities were assessed via radioactivity assays, and NFκB and MAPK-related signaling mechanisms were investigated using luciferase vector reporters.ResultsMaR1 dose-dependently reduced IL-6 and IL-8 production following HDE treatment of BECs. MaR1 also reduced HDE-stimulated cytokine release including TNF-α in a mouse lung slice model when given before or following HDE treatment. Previous studies have established that HDE sequentially activates epithelial PKCα and PKCϵ at 1 and 6 hours, respectively that regulated TNF-α, IL-6, and IL-8 release. MaR1 pretreatment abrogated these HDE-induced PKC activities. Furthermore, HDE treatment over a 24-hour period revealed temporal increases in NFκB, AP-1, SP-1, and SRE DNA binding activities, using luciferase reporter assays. MaR1 pretreatment did not alter the activation of NFκB, AP-1, or SP-1, but did reduce the activation of DNA binding at SRE.ConclusionsThese observations indicate a role for MaR1 in attenuating the pro-inflammatory responses of BECs to organic dust extract, through a mechanism that does not appear to rely on reduced NFκB, AP-1, or SP-1-related signaling, but may be mediated partly through SRE-related signaling. These data offer insights for a novel mechanistic action of MaR1 in bronchial epithelial cells, and support future in vivo studies to test MaR1’s utility in reducing the deleterious inflammatory effects of environmental dust exposures.

Sulforaphane controls TPA-induced MMP-9 expression through the NF-κB signaling pathway, but not AP-1, in MCF-7 breast cancer cells

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells. [BMB Reports 2013; 46(4): 201-206]

Tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats

Tetramethylpyrazine (TMP) has been used to treat ischemic stroke. However, scientific evidence related to its effectiveness or precise modes of neuroprotective action is largely unclear. This study provides evidence of an alternative target for TMP and sheds light on the mechanism of its physiological benefits. We report a global inhibitory effect of TMP on intracerebral cellular inflammatory response in a rat model of permanent cerebral ischemia. TMP exhibited a neuroprotective effect against ischemic deficits by reduction of behavioral disturbance, brain infarction, and edema. The results of immunohistochemistry, enzymatic assay, Western blot, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and flow cytometric analysis revealed that TMP reduced the percentages of activated macrophages/microglia and infiltrative lymphocytes, neutrophils, and macrophages and pro-inflammatory cytokine expression after cerebral ischemia. In parallel with these immunosuppressive phenomena, TMP also attenuated the activities of ischemia-induced inflammation-associated signaling molecules and transcription factors. Another finding in this study was that the anti-inflammatory and neuroprotective effects of TMP were accompanied by a further elevated expression of NF-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in ipsilateral neurons and macrophages/microglia after cerebral ischemia. Taken together, our results suggest that both the promotion of endogenous defense capacity and the attenuation of the extent and composition percentage of the major cellular inflammatory responses via targeting of macrophages/microglia by elevating Nrf2/HO-1 expression might actively contribute to TMP-mediated neuroprotection against cerebral ischemia.

상백피에 의한 MC/9 비만세포의 활성 억제 조절 연구

ABSTRACT Objective : Morus alba Linne Root Bark (MRAL) is a medicinal herb in Korean Medicine, known for its anti-inflammatory and anti-allergic properties. However, its me chanisms of action and the cellular targets have not yet been found and the study was developed to investigate the allergic suppressive effect of MRAL. The purpose of this study is to investigate the allergic suppressive effects of MRAL on activation of MC/9 mast cells.Methods : Cytotoxic activity of MRAL (50, 100, 200, 400 µg/mL) on MC/9 mast cells measured using EZ-Cytox cell viability assay kit (WST reagent). The levels of interleuk in-5 (IL-5), IL-13 and IL-4, IL-5, IL-6, IL-13 mRNA expression were measured by enzyme-linked immunosorbent as say (ELISA) and real-time PCR respectively. The expression of transcription factors such as G ATA-1, GATA-2, NFAT, AP-1 and NF-κB p65 DNA binding activity were measured by western blot and electrop horesis mobility shift assay (EMSA).Results : Our results indicated that MRAL (50 µg/mL, 100 µg/mL) significantly inhibited PMA/Ionomycin-induced production of IL-5 and IL-13 and the expr ession of IL-4, IL-5, IL-6 and IL-13 mRNA in MC/9 mast cells. Moreover, MRAL (50 µg/mL, 100 µg/mL) inhibited PMA/Ionomycin-induced GATA-1, GATA-2, NFAT-1, NFAT-2, c-Fos protein expression and NF-κB p65 DNA binding activity in MC/9 mast cells.Conclusions : In conclusion, we suspect the anti-allergenic activities of MRAL, may be related to the regulation of transcription factors GATA-1, GATA-2, NFAT-1, NFAT-2, c-Fos and NF-κB p65 DNA binding assay causing inhibition of Th2 cytokines IL-5 and IL-13 in mast cells.Key words : Morus alba Linne Root Bark (MRAL), MC/9 mast cell line, Anti-allergy, Transcription factor, Th2 cytokines

Isolation and characterization of an AP2/ERF-type drought stress inducible transcription factor encoding gene from rice

A genomic DNA fragment of 964 bp corresponding to AP2/ERF family transcription factor was isolated from drought tolerant Oryza sativa L. cv. ‘N-22’ (AP2/ERF-‘N-22’) and Oryza sativa L. cv. ‘Teipei 309’ (AP2/ERF-jap). The cDNA sequence of AP2/ERF-‘N-22’ (732 bp) was amplified using gene specific primers. The gene contains two exons and a single intron. The deduced protein of the AP2/ERF-‘N-22’ contains a potential nuclear localization signal, a possible regulation domain and an AP2 DNA binding domain of 60 amino acids. Northern and RT-PCR analysis showed that transcript of AP2/ERF-‘N-22’ accumulates in response to WDS and the Southern analysis indicated the presence of single copy of AP2/ERF-‘N-22’ gene in Oryza sativa genome. Phylogenetic analysis of AP2/ERF family revealed that AP2/ERF-‘N-22’ belongs to group Va along with SHN clade of AP2/ERF protein which activates wax biosynthesis. A higher epicuticular wax content was observed at decreased RWC and positively correlated with AP2/ERF-‘N-22’ expression.

Abstract P1-10-01: Curcumin suppresses MMP-9 expression via inhibition of PKCα/MAPKs and NF-κB/AP-1 activation in MCF-7 cells

Abstract Background: Curcumin is a polyphenol derived from the plant turmeric (Curcuma longa), which is commonly used as a spice. It was recently reported for its anticancer effect on several types of cancer cells in vitro, however the molecular mechanisms of this anticancer effect are not fully understood. In the present study, we investigated the effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced matrix metalloproteinase-9(MMP-9) expression and cell invasion in MCF-7 breast cancer cells. Materials and Methods: The effect of curcumin on MCF-7 cell viability was determined using MTT assay. The cells invasion was demonstrated by the Matrigel-coated transwell assay. Western blot analysis was performed to detect the effect of curcumin on the expression of MMP-9. MMP-9 mRNA levels were analyzed by real-time PCR. NF-κB and AP-1 DNA binding was analyzed by EMSA. Results: Our results showed that curcumin inhibits TPA-induced MMP-9 expression and cell invasion through suppressing NF-κB and AP-1 activation. Curcumin strongly repressed the TPA-induced phosphorylation of p38 and JNK and also inhibited TPA-induced translocation of PKCa from the cytosol to the membrane, but did not affect the translocation of PKCδ. Conclusion: It is concluded that curcumin inhibits the TPA-induced MMP-9 expression and cell invasion through the suppression of the PKCα/MAPK/NF-kB/AP-1 pathway in MCF-7 breast cancer cells. Accordingly, curcumin may have the therapeutic potential in restricting breast cancer metastasis. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-10-01.

Molecular mechanism of capillarisin-mediated inhibition of MyD88/TIRAP inflammatory signaling in in vitro and in vivo experimental models

Ethnopharmacological relevanceArtemisia capillaris Thunberg (Compositae) have been used as traditional medicine as a diuretic, liver protective agent, and for amelioration of inflammatory and analgesic disorders. The present study was carried out to establish the scientific rationale for treating inflammation and to find active principles from A. capillaris.The aim of the present study is to investigate the possible anti-inflammatory mechanism of the major component (capillarisin) isolated from A. capillaris via inhibition of MyD88/TIRAP inflammatory signaling both in vitro and in vivo models. Materials and methodsThe nitrite, PGE2, and TNF-α productions were evaluated by Griess reagent and ELISA kits. The protein and mRNA expression levels were investigated by Western blot and RT-PCR. The NF-κB and AP-1 DNA-binding was performed by electrophoretic mobility shift assay. The CFA- and carrageenan-induced paw edema was performed in ICR mice in which 20 and 80mg/kg body weight of capillarisin was administered intraperitoneally (i.p.). ResultsThe results demonstrated that pretreatment with capillarisin effectively inhibited the LPS-induced activation of NF-κB, Akt, and MAP kinase-activated inflammatory genes, which is mediated by MyD88 and TIRAP. Treatment with capillarisin reduced the mRNA and protein levels of iNOS and COX-2 in RAW 264.7 cells as assessed by RT-PCR and Western blot. Capillarisin suppressed LPS-induced inhibitory kappa kinase (IKK) phosphorylation and the degradation of inhibitory kappa B (IκBα) and prevented the nuclear translocation of p65 and p50. Capillarisin also exhibited a promising inhibitory effect on the LPS-induced NF-κB and AP-1 DNA binding activity based on an electrophoretic mobility shift assay. The LPS-induced activation of p-JNK, p-p38, p-ERK, and p-Akt was significantly inhibited. In addition, the TNF-α level in the media was effectively reduced by capillarisin. In vivo experimental analysis revealed that capillarisin (20 and 80mg/kg, i.p.) inhibited complete Freund's adjuvant (CFA)-and carrageenan-induced paw edema, nitrite production in plasma, and TNF-α, a pro-inflammatory cytokine production. ConclusionThe results presented here demonstrate that capillarisin has consistent anti-inflammatory properties and acts by inhibiting inflammatory mediators in in vitro and in vivo experimental models, and suggest its potential utility in the control of inflammatory disorders.

Emerging nonanticoagulant role of low molecular weight heparins on extravillous trophoblast functions and on heparin binding–epidermal growth factor and cystein-rich angiogenic inducer 61 expression

To examine the effects of low molecular weight heparins (LMWHs) on extravillous trophoblast (EVTC) invasiveness and on EVTC expression/secretion of heparin binding-EGF (HB-EGF) and cystein-rich angiogenic inducer 61 (Cyr61), both of which are involved in the process of EVTC invasion. Furthermore, to investigate the intracellular DNA binding activity of activator protein (AP)-1. Experimental study. Department of Obstetrics Gynecology, Università Cattolica del Sacro Cuore, Rome, Italy. Cultures of primary EVTC cells isolated from patients with first trimester unexplained recurrent miscarriage. The effects of LMWHs on EVTC invasiveness were examined by an in vitro matrigel invasion assay. Matrix metalloprotease-2 activity (MMP-2) was examined by gelatin zimography. HB-EGF and Cyr61 expression and secretion were studied by Western blot analysis and ELISA assay. AP-1 activity was measured through a multiwell colorimetric assay. The EVTC invasiveness, the expression/secretion of HB-EGF and Cyr61 proteins, and the AP-1 DNA binding activity in the presence of increasing concentrations of LMWHs were investigated. Both LMWHs, and primarily tinzaparin, increased EVTC invasiveness, by enhancing the MMP-2 proteolytic activity, and induced the expression/secretion of HB-EGF and Cyr61 in EVTC. This effect was mediated by an increased DNA binding activity of AP-1. Both LMWHs are able to promote EVTC development because they are able to stimulate the EVTC invasive properties. Our results may provide a possible biological rationale for the clinical use of LMWH for placental-mediated pregnancy complications unrelated to prothrombotic disorders.