Promotion of neurite outgrowth by 3,5,7,3',4'-pentamethoxyflavone is mediated through ERK signaling pathway in Neuro2a cells.

In this study, the effects of 3,5,7,3',4'-pentamethoxyflavone (KP1), a major bioactive ingredient isolated from the Kaempferia parviflora rhizomes, on a neurite outgrowth in Neuro2a cells and its mechanism have been investigated. KP1 increased concentration-dependently the percentage of neurite-bearing cells. KP1 showed a remarkable capability to elicit neurite outgrowth in Neuro2a cells, as evidenced by morphological alterations and immunostaining using anti-class III ß-tubulin and anti-NeuN antibodies. KP1 also displayed a higher neurogenic activity than retinoic acid (RA), a promoter of neurite outgrowth in Neuro2a cells. KP1 treatment caused significant elevation in phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38 MAPK) and glycogen synthase kinase-3ß (GSK-3ß). However, KP1-triggered neurite outgrowth was markedly inhibited by treatment with the ERK inhibitor U0126, whereas p38 MAPK inhibitor SB203580 and GSK-3ß inhibitor SB216763 did not influence KP1-induced neurite outgrowth. These results demonstrate that KP1 elicits neurite outgrowth and triggers cell differentiation of Neuro2a cells through ERK signal pathway.

Human ST3Gal II and ST6GalNAc IV genes increase human serum-mediated cytotoxicity to xenogeneic cells.

Carbohydrate-antigens widely existed on glycoproteins and glycosphingolipids of all mammalian cells play a crucial role in self-defense and immunity. Xeno-reactive antibodies included in natural human sera play a protecting role in an acute phase-rejection of xenotransplantation. In this study, we investigated the effect of an alteration of glycosylation-pattern, caused by human sialyltransferases such as hST3Gal II or hST6GalNAc IV, on human serum mediated cytotoxicity in pig kidney PK15 cells. From LDH cytotoxicity assay, cytotoxicity to human serum was significantly increased in hST3Gal II and hST6GalNAc IV-transfected PK15 cells, as compared to the control. In the hST6Gal I-carrying cells, the cytotoxicity to human serum was rather decreased. Moreover, flow cytometry analysis revealed that an alteration of pig glycosylation-pattern by hST3Gal II or hST6GalNAc IV influences on a binding of human IgM or IgG, respectively, in pig kidney cells, regardless of Gal antigen alteration. Finally, we found that hST6GalNAc IV contributed to increase of terminal disialylated tetrasaccharide structure, disialyl T antigen, as evidenced by increase of the MAL II lectin binding capacity in the hST6GalNAc IV-transfected PK15 cells, compared with control. Therefore, our results suggest that carbohydrate antigens, such as disialyl T antigen, newly synthesized by the ST3Gal II- and ST6GalNAc IV are potentially believed to be new xeno-reactive elements.

T7 phage display reveals NOLC1 as a GM3 binding partner in human breast cancer MCF-7 cells.

Ganglioside GM3 is a simple monosialoganglioside (NeuAc-Gal-Glc-ceramide) that modulates cell adhesion, proliferation, and differentiation. Previously, we reported isolation of GM3-binding vascular endothelial growth factor receptor and transforming growth factor-ß receptor by the T7 phage display method (Chung et al., 2009; Kim et al., 2013). To further identify novel proteins interacting with GM3, we extended the T7 phage display method in this study. After T7 phage display biopanning combined with immobilized biotin-labeled 3'-sialyllactose prepared on a streptavidin-coated microplate, we isolated 100 candidate sequences from the human lung cDNA library. The most frequently detected clones from the blast analysis were the human nucleolar and coiled-body phosphoprotein 1 (NOLC1) sequences. We initially identified NOLC1 as a molecule that possibly binds to GM3 and confirmed this binding ability using the glutathione S-transferase fusion protein. Herein, we report another GM3-interacting protein, NOLC1, that can be isolated by the T7 phage display method. These results are expected to be helpful for elucidating the functional roles of ganglioside GM3 with NOLC1. When human breast cancer MCF-7 cells were examined for subcellular localization of NOLC1, immunofluorescence of NOLC1 was observed in the intracellular region. In addition, NOLC1 expression was increased in the nucleolus after treatment with the anticancer drug doxorubicin. GM3 and NOLC1 levels in the doxorubicin-treated MCF-7 cells were correlated, indicating possible associations between GM3 and NOLC1. Therefore, direct interactions between carbohydrates and cellular proteins can pave the path for new signaling phenomena in biology.

Regulatory mechanism for the human glioblastoma cell-specific expression of the human GD1c/GT1a/GQ1b synthase (hST8Sia V) gene.

In this study we observed that human GD1c/GT1a/GQ1b synthase (hST8Sia V) is particularly expressed in human glioblastoma cells. To address the mechanism regulating human glioblastoma-specific gene expression of the hST8Sia V, after the transcription start site (TSS) was identified by the 5'-rapid amplification of cDNA end with total RNA from human glioblastoma U87MG cells, the 5'-flanking region (2.5 kb) of the hST8Sia V gene was isolated and its promoter activity was examined. By luciferase reporter assay, this 5'-flanking region revealed strong promoter activity in only U-87MG cells, but not in other tissue-derived cancer cells. 5'-deletion mutant analysis showed that the region from -1140 to -494 is crucial for transcription of the hST8Sia V gene in U87MG cells. This region contains the activator protein-1 (AP-1) binding site, the main target of the c-Jun N-terminal kinase (JNK) downstream. The AP-1 binding site at -1043/-1037 was proved to be indispensable for the hST8Sia V gene-specific expression in U87MG cells by site-directed mutagenesis. Moreover, the transcriptional activation of hST8Sia V gene in U87MG cells was strongly inhibited by a specific JNK inhibitor, SP600125. These results suggest that the hST8Sia V gene-specific expression in U87MG cells is controlled by JNK/AP-1 signaling pathway.

Upregulation of human GD3 synthase (hST8Sia I) gene expression during serum starvation-induced osteoblastic differentiation of MG-63 cells.

In this study, we have firstly elucidated that serum starvation augmented the levels of human GD3 synthase (hST8Sia I) gene and ganglioside GD3 expression as well as bone morphogenic protein-2 and osteocalcin expression during MG-63 cell differentiation using RT-PCR, qPCR, Western blot and immunofluorescence microscopy. To evaluate upregulation of hST8Sia I gene during MG-63 cell differentiation by serum starvation, promoter area of the hST8Sia I gene was functionally analyzed. Promoter analysis using luciferase reporter assay system harboring various constructs of the hST8Sia I gene proved that the cis-acting region at -1146/-646, which includes binding sites of the known transcription factors AP-1, CREB, c-Ets-1 and NF-κB, displays the highest level of promoter activity in response to serum starvation in MG-63 cells. The -731/-722 region, which contains the NF-κB binding site, was proved to be essential for expression of the hST8Sia I gene by serum starvation in MG-63 cells by site-directed mutagenesis, NF-κB inhibition, and chromatin immunoprecipitation (ChIP) assay. Knockdown of hST8Sia I using shRNA suggested that expressions of hST8Sia I and GD3 have no apparent effect on differentiation of MG-63 cells. Moreover, the transcriptional activation of hST8Sia I gene by serum starvation was strongly hindered by SB203580, a p38MAPK inhibitor in MG-63 cells. From these results, it has been suggested that transcription activity of hST8Sia I gene by serum starvation in human osteosarcoma MG-63 cells is regulated by p38MAPK/NF-κB signaling pathway.

Up-regulation of inflammatory reactions by MPT32, a secreted protein of Mycobacterium tuberculosis in RAW264.7 macrophages.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is still one of the global health burdens. The occurrence of various cases and multidrug resistance confirm that TB has not been completely conquered. For these reasons, the present research has been conducted to explore TB vaccine and drug candidate possibility using Mtb-secreted proteins. Among these proteins, MPT32 is known to have antigenicity and immunogenicity. There has not been a report on the host immune responses and regulation in macrophage cells. The present study was conducted with MPT32 in RAW 264.7 murine macrophage cells that control immune responses by sensing pathogen invasion and environmental change. We have found that MPT32 could activate lipopolysaccharide (LPS)-induced gene expression of metalloproteinase-9 (MMP-9) and inflammation in RAW 264.7 cells. After treating cells with MPT32, the increase in pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß (IL-1ß) and IL-6, was observed. In addition, activated macrophages expressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to generate various inflammatory mediator molecules, such as nitric oxide (NO). The increase in iNOS and COX-2 levels, which are up-regulators of MMP-9 expression, was also confirmed. The biochemical events are involved in the downstream of activated MAPK signaling and translocation of NF-κ B transcription factor. The present results prove the immunomodulatory effect of MPT32 in the RAW 264.7 murine macrophage cells. it claims the possibility of a TB vaccination and drug candidate using MPT32, contributing to the prevention of TB.

Transcription of human ß-galactoside α2,6-sialyltransferase (hST6Gal I) is downregulated by curcumin through AMPK signaling in human colon carcinoma HCT116 cells.

BACKGROUND: In this study, we observed that in human colon carcinoma HCT116 cells mRNA level of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was decreased by curcumin. FACS analysis using the α2,6-sialyl-specific lectin (SNA) also showed a noticeable decrease in binding to SNA by curcumin. OBJECTIVE: To investigate the mechanism for curcumin-triggered downregulation of hST6Gal I transcription. METHODS: The mRNA levels of nine kinds of hST genes were assessed by RT-PCR after curcumin was treated in HCT116 cells. The level of hST6Gal I product on cell surface was examined by flow cytometry analysis. Luciferase reporter plasmids with 5'-deleted constructs and mutants of the hST6Gal I promoter were transiently transfected into HCT116 cells, and the luciferase activity was measured after treatment with curcumin. RESULTS: Curcumin led to significant transcriptional repression of the hST6Gal I promoter. Promoter analysis using deletion mutants proved that the - 303 to - 189 region of the hST6Gal I promoter is required for transcriptional repression in response to curcumin. Among putative binding sites for transcription factors IK2, GATA1, TCF12, TAL1/E2A, SPT, and SL1 in this region, by site-directed mutagenesis analysis the TAL/E2A binding site (nucleotides - 266/- 246) was proved to be crucial for curcumin-triggered downregulation of hST6Gal I transcription in HCT116 cells. The transcription activity of hST6Gal I gene in HCT116 cells was markedly suppressed by compound C, an AMP-activated protein kinase (AMPK) inhibitor. CONCLUSION: These indicate that gene expression of hST6Gal I in HCT116 cells is controlled through AMPK/TAL/E2A signal pathway.

Involvement of the p38 MAPK-NLRC4-Caspase-1 Pathway in Ionizing Radiation-Enhanced Macrophage IL-1ß Production.

Macrophages are abundant immune cells in the tumor microenvironment and are crucial in regulating tumor malignancy. We previously reported that ionizing radiation (IR) increases the production of interleukin (IL)-1ß in lipopolysaccharide (LPS)-treated macrophages, contributing to the malignancy of colorectal cancer cells; however, the mechanism remained unclear. Here, we show that IR increases the activity of cysteine-aspartate-specific protease 1 (caspase-1), which is regulated by the inflammasome, and cleaves premature IL-1ß to mature IL-1ß in RAW264.7 macrophages. Irradiated RAW264.7 cells showed increased expression of NLRC4 inflammasome, which controls the activity of caspase-1 and IL-1ß production. Silencing of NLRC4 using RNA interference inhibited the IR-induced increase in IL-1ß production. Activation of the inflammasome can be regulated by mitogen-activated protein kinase (MAPK)s in macrophages. In RAW264.7 cells, IR increased the phosphorylation of p38 MAPK but not extracellular signal-regulated kinase and c-Jun N-terminal kinase. Moreover, a selective inhibitor of p38 MAPK inhibited LPS-induced IL-1ß production and NLRC4 inflammasome expression in irradiated RAW264.7 macrophages. Our results indicate that IR-induced activation of the p38 MAPK-NLRC4-caspase-1 activation pathway in macrophages increases IL-1ß production in response to LPS.

Curcumin-mediated transcriptional regulation of human N-acetylgalactosamine-α2,6-sialyltransferase which synthesizes sialyl-Tn antigen in HCT116 human colon cancer cells.

Human N-acetylgalactosamine-α2,6-sialyltransferase (hST6GalNAc I) is the major enzyme involved in the biosynthesis of sialyl-Tn antigen (sTn), which is known to be expressed in more than 80% of human carcinomas and correlated with poor prognosis in cancer patients. Athough high expression of hST6GalNAc I is associated with augmented proliferation, migration and invasion in various cancer cells, transcriptional mechanism regulating hST6GalNAc I gene expression remains largely unknown. In this study, we found that hST6GalNAc I gene expression was markedly augmented by curcumin in HCT116 human colon carcinoma cells. To understand the molecular mechanism for the upregulation of hST6GalNAc I gene expression by curcumin in HCT116 cells, we first determined the transcriptional start site of hST6GalNAc I gene by 5'-RACE and cloned the proximal hST6GalNAc I 5'-flanking region spanning about 2 kb by PCR. Functional analysis of the hST6GalNAc I 5' flanking region of hST6GalNAc I by sequential 5'-deletion, transient transfection of reporter gene constructs and luciferase reporter assays showed that -378/-136 region is essential for maximal activation of transcription in response to curcumin in HCT 116 cells. This region includes putative binding sites for transcription factors c-Ets-1, NF-1, GATA-1, ER-α, YY1, and GR-α. ChIP analysis and site-directed mutagenesis demonstrated that estrogen receptor α (ER-α) binding site (nucleotides -248/-238) in this region is crucial for hST6GalNAc I gene transcription in response to curcumin stimulation in HCT116 cells. The transcription activity of hST6GalNAc I gene induced by curcumin in HCT116 cells was strongly inhibited by PKC inhibitor (Gö6983) and ERK inhibitor (U0126). These results suggest that curcumin-induced hST6GalNAc I gene expression in HCT116 cells is modulated through PKC/ERKs signal pathway.

Avenanthramide C Suppresses Matrix Metalloproteinase-9 Expression and Migration Through the MAPK/NF- κB Signaling Pathway in TNF-α-Activated HASMC Cells.

In oat ingredients, flavonoids and phenolic acids are known to be the most important phenolic compounds. In phenolic compounds, wide-ranging biological responses, including antioxidative, anti-inflammatory, anti-allergic, and anti-cancer properties, were reported. Avenanthramide C (Avn C), a component of the phenolic compound of oats, has been reported to be highly antioxidant and anti-inflammatory, but its role in an anti-atherosclerosis response is unknown. The aim of this research was to assess the effect of Avn C on expression of MMP-9 on TNF-α-activated human arterial smooth-muscle cells (HASMC) and signaling involved in its anti-atherosclerosis activity. HASMC cells are known to produce inflammatory cytokines involving IL-6, IL-1ß, and TNF-α during arteriosclerosis activity. Avn C specifically reduced IL-6 secretion in HASMC cells. Furthermore, we investigated whether Avn C could inhibit NF-κB nuclear protein translocation. Avn C suppressed nuclear protein translocation of NF-κB in TNF-α-stimulated HASMCs. The MMP-9 enzyme activity and expression are controlled through the MAPKs signaling path during the Avn C treatment. We confirmed that the levels of wound healing (p-value = 0.013, *p < 0.05) and migration (p-value = 0.007, **p < 0.01) are inhibited by 100 ng/ml TNF-α and 100 µM Avn C co-treated. Accordingly, Avn C inhibited the expression of MMP-9 and cell migration through the MAPK/NF-κB signaling pathway in TNF-α-activated HASMC. Therefore, Avn C can be identified and serve as disease prevention material and remedy for atherosclerosis.

4-O-methylascochlorin attenuates inflammatory responses induced by lipopolysaccharide in RAW 264.7 macrophages.

Inflammation is implicated in various diseases, such as inflammatory bowel disease and cancer. Ascochlorin (ASC) and its derivatives have been shown to modulate inflammatory responses in many previous studies. However, the effects of 4-O-methylascochlorin (MAC), one of the ASC derivatives, on inflammatory responses have yet to be reported. In addition, the consequences of chemical modification of ASC on protein signaling and immunity have yet to be fully understood. The fourth carbon in MAC is methylated, which may result in modulation of immune response differently compared with ASC. Hence, we have investigated the role of MAC in inflammatory response induced by lipopolysaccharide in murine macrophage cells. Here, we found that MAC treatment decreased the inflammatory response by murine macrophages. When murine macrophages were treated with MAC, the transcription and translation of various pro-inflammatory indicators such as iNOS and COX-2 decreased. In addition, the ELISA results showed that the expression of TNF-α, IL-6, and IL-1ß, which are pro-inflammatory cytokines, was successfully decreased by MAC. Such effects of MAC appear to be mediated via downregulation of MAPK signaling and the transactivational activity of NF-κB. Lipopolysaccharide upregulates MAPK protein phosphorylation and NF-κB translocation, which in turn enhances the transactivation of genes related to NF-κB. Such results of lipopolysaccharide were attenuated by MAC. Collectively, our results indicate that MAC alleviated the inflammatory responses induced by lipopolysaccharide in murine macrophages successfully by modulating MAPK signaling pathway and NF-κB-related genes. This study shows that MAC, similar to other ASC derivatives, can potentially be used therapeutically to reduce the harmful damage induced by prolonged inflammation. In addition, the structural differences between ASC and its derivatives as well as their effect on intracellular signaling will also be discussed.

REDD1 Is Involved in Amyloid ß-Induced Synaptic Dysfunction and Memory Impairment.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neurological dysfunction, including memory impairment, attributed to the accumulation of amyloid ß (Aß) in the brain. Although several studies reported possible mechanisms involved in Aß pathology, much remains unknown. Previous findings suggested that a protein regulated in development and DNA damage response 1 (REDD1), a stress-coping regulator, is an Aß-responsive gene involved in Aß cytotoxicity. However, we still do not know how Aß increases the level of REDD1 and whether REDD1 mediates Aß-induced synaptic dysfunction. To elucidate this, we examined the effect of Aß on REDD1-expression using acute hippocampal slices from mice, and the effect of REDD1 short hairpin RNA (shRNA) on Aß-induced synaptic dysfunction. Lastly, we observed the effect of REDD1 shRNA on memory deficit in an AD-like mouse model. Through the experiments, we found that Aß-incubated acute hippocampal slices showed increased REDD1 levels. Moreover, Aß injection into the lateral ventricle increased REDD1 levels in the hippocampus. Anisomycin, but not actinomycin D, blocked Aß-induced increase in REDD1 levels in the acute hippocampal slices, suggesting that Aß may increase REDD1 translation rather than transcription. Aß activated Fyn/ERK/S6 cascade, and inhibitors for Fyn/ERK/S6 or mGluR5 blocked Aß-induced REDD1 upregulation. REDD1 inducer, a transcriptional activator, and Aß blocked synaptic plasticity in the acute hippocampal slices. REDD1 inducer inhibited mTOR/Akt signaling. REDD1 shRNA blocked Aß-induced synaptic deficits. REDD1 shRNA also blocked Aß-induced memory deficits in passive-avoidance and object-recognition tests. Collectively, these results demonstrate that REDD1 participates in Aß pathology and could be a target for AD therapy.

Regulation of human ß-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189 bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

Rubrofusarin Attenuates Chronic Restraint Stress-Induced Depressive Symptoms.

The aim of this study was to examine whether rubrofusarin, an active ingredient of the Cassia species, has an antidepressive effect in chronic restraint stress (CRS) mouse model. Although acute treatment using rubrofusarin failed, chronic treatment using rubrofusarin ameliorated CRS-induced depressive symptoms. Rubrofusarin treatment significantly reduced the number of Fluoro-Jade B-positive cells and caspase-3 activation within the hippocampus of CRS-treated mice. Moreover, rubrofusarin treatment significantly increased the number of newborn neurons in the hippocampus of CRS-treated mice. CRS induced activation of glycogen synthase kinase-3ß and regulated development and DNA damage responses, and reductions in the extracellular-signal-regulated kinase pathway activity were also reversed by rubrofusarin treatment. Microglial activation and inflammasome markers, including nod-like receptor family pyrin domain containing 3 and adaptor protein apoptosis-associated speck-like protein containing CARD, which were induced by CRS, were ameliorated by rubrofusarin. Synaptic plasticity dysfunction within the hippocampus was also rescued by rubrofusarin treatment. Within in vitro experiments, rubrofusarin blocked corticosterone-induced long-term potentiation impairments. These were blocked by LY294002, which is an Akt inhibitor. Finally, we found that the antidepressant effects of rubrofusarin were blocked by an intracerebroventricular injection of LY294002. These results suggest that rubrofusarin ameliorated CRS-induced depressive symptoms through PI3K/Akt signaling.

3'-sialyllactose targets cell surface protein, SIGLEC-3, and induces megakaryocyte differentiation and apoptosis by lipid raft-dependent endocytosis.

3'-sialyllactose is one of the abundant components in human milk oligosaccharides (HMOs) that protect infants from various viral infections in early stages of immune system development. 3SL is a combination of lactose and sialic acid. Most sialic acids are widely expressed in animal cells and they bind to siglec proteins. In this study, we demonstrate that 3SL specifically binds to CD33. It induces megakaryocyte differentiation and subsequent apoptosis by targeting cell surface protein siglec-3 (CD33) in human chronic myeloid leukemia K562 cells. The 3SL-bound CD33 was internalized to the cytosol via caveolae-dependent endocytosis. At the molecular level, 3SL-bound CD33 recruits the suppressor of cytokine signaling 3 (SOCS3) and SH2 domain-containing protein tyrosine phosphatase 1 (SHP1). SOCS3 is degraded with CD33 by proteasome degradation, while SHP-1 activates extracellular signal-regulated kinase (ERK) to induce megakaryocytic differentiation and subsequent apoptosis. The present study, therefore, suggests that 3SL is a potential anti-leukemia agent affecting differentiation and apoptosis.

ß-Amyrin Ameliorates Alzheimer's Disease-Like Aberrant Synaptic Plasticity in the Mouse Hippocampus.

Alzheimer's disease (AD) is a progressive and most frequently diagnosed neurodegenerative disorder. However, there is still no drug preventing the progress of this disorder. ß-Amyrin, an ingredient of the surface wax of tomato fruit and dandelion coffee, is previously reported to ameliorate memory impairment induced by cholinergic dysfunction. Therefore, we tested whether ß-amyrin can prevent AD-like pathology. ß-Amyrin blocked amyloid ß (Aß)-induced long-term potentiation (LTP) impairment in the hippocampal slices. Moreover, ß-amyrin improved Aß-induced suppression of phosphatidylinositol-3-kinase (PI3K)/Akt signaling. LY294002, a PI3K inhibitor, blocked the effect of ß-amyrin on Aß-induced LTP impairment. In in vivo experiments, we observed that ß-amyrin ameliorated object recognition memory deficit in Aß-injected AD mice model. Moreover, neurogenesis impairments induced by Aß was improved by ß-amyrin treatment. Taken together, ß-amyrin might be a good candidate of treatment or supplement for AD patients.

Cryptotanshinone enhances neurite outgrowth and memory via extracellular signal-regulated kinase 1/2 signaling.

Neurite outgrowth is important process in synaptic formation and neuronal development. Many previous studies reported that natural compounds as well as neurotrophins induce neurite outgrowth through various signaling pathways. In this study, we tested the effect of cryptotanshinone (CPT), a constituent of Salvia miltiorrhiza Bunge, on neurite outgrowth using neuro2a cell line, a mouse neuroblastoma cell line. And then, we examined the effect of CPT on learning and memory. We first found that CPT facilitated neurite outgrowth in a concentration-dependent manner. Although CPT induced MTT reduction, CPT did not induce LDH release. Moreover, CPT suppressed cell proliferation. CPT increased ERK1/2 phosphorylation and ERK1/2 inhibitor blocked CPT-facilitated neurite outgrowth. CPT also enhanced learning and memory without affecting basal sensory conditions and increased ERK1/2 phosphorylation in the hippocampus in a dose-dependent manner. These results demonstrate that CPT facilitates neurite outgrowth and enhances learning and memory, which may be mediated by facilitating ERK1/2 signal.

Spinosin Attenuates Alzheimer's Disease-Associated Synaptic Dysfunction via Regulation of Plasmin Activity.

Hippocampal synaptic dysfunction is a hallmark of Alzheimer's disease (AD). Many agents regulating hippocampal synaptic plasticity show an ameliorative effect on AD pathology, making them potential candidates for AD therapy. In the present study, we investigated spinosin as a regulating agent of synaptic plasticity in AD. Spinosin attenuated amyloid ß (Aß)-induced long-term potentiation (LTP) impairment, and improved plasmin activity and protein level in the hippocampi of 5XFAD mice, a transgenic AD mouse model. Moreover, the effect of spinosin on hippocampal LTP in 5XFAD mice was prevented by 6-aminocaproic acid, a plasmin inhibitor. These results suggest that spinosin improves synaptic function in the AD hippocampus by regulating plasmin activity.

Mycobacterium tuberculosis-Secreted Protein, ESAT-6, Inhibits Lipopolysaccharide-Induced MMP-9 Expression and Inflammation Through NF-κB and MAPK Signaling in RAW 264.7 Macrophage Cells.

-20pt?>Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes contagious tuberculosis (TB). Recently, Mtb-secreted proteins have been considered virulence factors and candidates for drugs and vaccines. Among these proteins, 6-kDa early secreted antigenic target (ESAT-6) is known to be able to induce component of matrix metalloproteinase-9 (MMP-9) in epithelial cells, leading to recruitment of macrophages. However, detailed function of ESAT-6 during macrophage recruitment to inflammatory sites remains unknown. Thus, the objective of the present study was to elucidate such function of EAST-6 and mechanism(s) involved. In the present study, we have found that recombinant ESAT-6 purified in the form of ESAT-6 double-connected structure (2E6D) could inhibit lipopolysaccharide (LPS)-induced potential of cell migration and inflammation in murine macrophage cells. Interestingly, 2E6D suppressed LPS-induced MMP-9 expression at both protein and mRNA levels as well as its enzyme activity. Levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzymes as known upregulators of MMP-9 were significantly decreased when 2E6D has been treated. In addition, nitric oxide (NO) as a second messenger was also significantly decreased by treatment with the purified 2E6D. Furthermore, 2E6D inhibited LPS-induced phosphorylation of IκB and translocation of NF-κB. Moreover, 2E6D suppressed phosphorylation of MAPK signaling proteins. Taken together, these results suggest that ESAT-6 can suppress LPS-induced MMP-9 and inflammation by downregulating COX-2, iNOS, and NO through NF-κB and MAPK signaling.

The effect of coniferaldehyde on neurite outgrowth in neuroblastoma Neuro2a cells.

Neurite outgrowth is the differentiation process by which neurons establish synapses. In the dentate gyrus of the hippocampus, new neurons are constantly produced and undergo neurite outgrowth to form synapses, and this process is involved in cognitive ability. Therefore, if an agent could modulate neurite outgrowth, it could potentially be developed as a compound for modulating cognitive ability. In this study, we examined whether coniferaldehyde, a natural compound, regulates neurite outgrowth in Neuro2a cells. We ascertained morphological changes and measured the percentage of neurite-bearing cells and neurite lengths. Coniferaldehyde significantly increased the percentage of neurite-bearing cells, and the length of neurites in a concentration-dependent manner, without inducing cell death. We then have identified that, coniferaldehyde activates the extracellular signals-regulated Kinase 1 and 2 (ERK1/2), and further noted that, U0126, an ERK1/2 inhibitor, blocks coniferaldehyde-facilitated neurite outgrowth. Moreover, Subchronic administration of CA enhanced learning and memory, and increased neurite length of newborn neurons in the hippocampus. These results suggest that coniferaldehyde induces neurite outgrowth by a process possibly mediated by ERK1/2 signaling and enhances learning and memory.