Neurotrophic Effect of Citrus 5-Hydroxy-3,6,7,8,3′,4′-Hexamethoxyflavone: Promotion of Neurite Outgrowth via cAMP/PKA/CREB Pathway in PC12 Cells

5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5-OH-HxMF), a hydroxylated polymethoxyflavone, is found exclusively in the Citrus genus, particularly in the peels of sweet orange. In this research, we report the first investigation of the neurotrophic effects and mechanism of 5-OH-HxMF in PC12 pheochromocytoma cells. We found that 5-OH-HxMF can effectively induce PC12 neurite outgrowth accompanied with the expression of neuronal differentiation marker protein growth-associated protein-43(GAP-43). 5-OH-HxMF caused the enhancement of cyclic AMP response element binding protein (CREB) phosphorylation, c-fos gene expression and CRE-mediated transcription, which was inhibited by 2-naphthol AS-E phosphate (KG-501), a specific antagonist for the CREB-CBP complex formation. Moreover, 5-OH-HxMF-induced both CRE transcription activity and neurite outgrowth were inhibited by adenylate cyclase and protein kinase A (PKA) inhibitor, but not MEK1/2, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) or calcium/calmodulin-dependent protein kinase (CaMK) inhibitor. Consistently, 5-OH-HxMF treatment increased the intracellular cAMP level and downstream component, PKA activity. We also found that addition of K252a, a TrKA antagonist, significantly inhibited NGF- but not 5-OH-HxMF-induced neurite outgrowth. These results reveal for the first time that 5-OH-HxMF is an effective neurotrophic agent and its effect is mainly through a cAMP/PKA-dependent, but TrKA-independent, signaling pathway coupling with CRE-mediated gene transcription. A PKC-dependent and CREB-independent pathway was also involved in its neurotrophic action

A comprehensive characterization of aggravated aging-related changes in T lymphocytes and monocytes in end-stage renal disease: The iESRD study

Background: Patients with end-stage renal disease (ESRD) exhibit a premature aging phenotype of the immune system. Nevertheless, the etiology and impact of these changes in ESRD patients remain unknown. Results: Compared to healthy individuals, ESRD patients exhibit accelerated immunosenescence in both T cell and monocyte compartments, characterized by a dramatic reduction in naïve CD4+ and CD8+ T cell numbers but increase in CD8+ TEMRA cell and proinflammatory monocyte numbers. Notably, within ESRD patients, aging-related immune changes positively correlated not only with increasing age but also with longer dialysis vintage. In multivariable-adjusted logistic regression models, the combination of high terminally differentiated CD8+ T cell level and high intermediate monocyte level, as a composite predictive immunophenotype, was independently associated with prevalent coronary artery disease as well as cardiovascular disease, after adjustment for age, sex, systemic inflammation and presence of diabetes. Levels of terminally differentiated CD8+ T cells also positively correlated with the level of uremic toxin p-cresyl sulfate. Conclusions: Aging-associated adaptive and innate immune changes are aggravated in ESRD and are associated with cardiovascular diseases. For the first time, our study demonstrates the potential link between immunosenescence in ESRD and duration of exposure to the uremic milieu

Association between Circulation Indole-3-Acetic Acid Levels and Stem Cell Factor in Maintenance Hemodialysis Patients : A Cross-Sectional Study

Protein-bound uremic toxin is a cardiovascular (CV) risk factor for patients with end-stage renal disease. Indole-3-acetic acid (IAA) was found to be associated with CV disease but the detailed pathophysiology remains unknown. Moreover, mitogen-activated protein kinase (MAPK) signaling cascades play an important role in the pathogenesis of CV disease. Thus, we explored the association between circulating IAA levels and forty MAPK cascade associated proteins in patients undergoing hemodialysis (HD). Circulating total form IAA was quantified by mass spectrometry and forty MAPK cascade associated proteins by a proximity extension assay in 331 prevalent HD patients. Accounting for multiple testing, and in multivariable-adjusted linear regression models, circulating total form IAA levels were positively associated with stem cell factor (beta coefficient 0.13, 95% confidence interval 0.04 to 0.21, p = 0.004). A bioinformatics approach using the search tool for interactions of chemicals (STITCH) tool provided information that IAA may be involved in the regulation of cell proliferation, hematopoietic cells, and the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. The knowledge gained here can be generalized, thereby impacting the non-traditional CV risk factors in patients with kidney disease. Further in vitro work is necessary to validate the translation of the mechanistic pathways

Luteolin Induces microRNA-132 Expression and Modulates Neurite Outgrowth in PC12 Cells

<div><p>Luteolin (3′,4′,5,7-tetrahydroxyflavone), a food-derived flavonoid, has been reported to exert neurotrophic properties that are associated with its capacity to promote neuronal survival and neurite outgrowth. In this study, we report for the first time that luteolin induces the persistent expression of microRNA-132 (miR-132) in PC12 cells. The correlation between miR-132 knockdown and a decrease in luteolin-mediated neurite outgrowth may indicate a mechanistic link by which miR-132 functions as a mediator for neuritogenesis. Furthermore, we find that luteolin led to the phosphorylation and activation of cAMP response element binding protein (CREB), which is associated with the up-regulation of miR-132 and neurite outgrowth. Moreover, luteolin-induced CREB activation, miR-132 expression and neurite outgrowth were inhibited by adenylate cyclase, protein kinase A (PKA) and MAPK/ERK kinase 1/2 (MEK1/2) inhibitors but not by protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase II (CaMK II) inhibitors. Consistently, we find that luteolin treatment increases ERK phosphorylation and PKA activity in PC12 cells. These results show that luteolin induces the up-regulation of miR-132, which serves as an important regulator for neurotrophic actions, mainly acting through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells.</p> </div

Hypothetical mechanism of luteolin mediation of neurite outgrowth in PC12 cells.

<p>Luteolin induces the up-regulation of miR-132, which may serve as a mediator for neurite outgrowth through the activation of cAMP/PKA- and ERK-dependent CREB signaling pathways in PC12 cells. In addition, ERK- or PKC-dependent but CREB/miR-132-independent pathways may also partially contribute to the mediation of neurite outgrowth by luteolin in PC12 cells.</p

Luteolin induces miR-132 expression in PC12 cells.

<p>PC12 cells were seeded on poly-L-lysine-coated 6-well plates in low-serum medium (1% horse serum and 0.5% FBS) for 24 h prior to exposure to forskolin (10 µM) for 2 h, vehicle (0.1% DMSO) or luteolin (20 µM) for an additional 2–8 h. Cellular RNA was then prepared, and the levels of immature miR-132 (pri- and pre-miR-132) (<b>A</b>) and mature miR-132 (<b>B</b>) were detected by reverse transcription quantitative PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. Data represent the mean ± SD from three independent experiments. **<i>p</i><0.01 represents significant differences compared to vehicle-treated cells.</p

Effects of the CREB protein knockdown on the luteolin-mediated miR-132 induction and neurite outgrowth in PC12 cells.

<p>PC12 cells were transfected transiently with siRNA negative control (si-NC) or with CREB-specific siRNA (si-CREB) before vehicle and luteolin (20 µM) treatment. (A) CREB and Phospho-CREB-Ser¹³³ (p-CREB) proteins were determined by Western blotting analysis after luteolin treatment for 60 min. β-actin protein is as an internal control. The immunoblot experiments were replicated at least three times, and a representative blot is shown. The normalized intensity of CREB or p-CREB versus β-actin is presented as the mean ± SD of three independent experiments. ##<i>p</i><0.01 represents significant differences compared to the siRNA negative control-transfected group. (B) Effect of CREB knockdown on miR-132 levels after luteolin treatment for 2 h. The levels of miR-132 were determined by RT-Q-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. (C) Effect of CREB knockdown on neurite outgrowth of PC12 cells after luteolin treatment for 72 h. Neurite-bearing cells were analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. Data represent the mean ± SD from three independent experiments. **<i>p</i><0.01 represents significant differences compared to the siRNA negative control-transfected group.</p

Involvement of ERK, PKC, CaMK and cAMP-dependent PKA signaling in luteoln-mediated CREB activation.

<p>(<b>A</b>) PC12 cells were seeded on poly-L-lysine-coated 24-well plates in DMEM containing 10% horse serum and 5% FBS for 24 h. Cells were then transfected with a CRE-mediated luciferase reporter construct and <i>Renilla</i> luciferase control plasmid as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. After transfection, the cells were pre-treated for 30 min with the following inhibitors: 10 µM U0126, 2.5 µM BIM, 10 µM H-89, 500 µM SQ22536, 10 µM KN-62 or vehicle (0.1% DMSO) followed by exposure to luteolin (20 µM) for 8 h. The intensities of the luciferase reactions measured in the lysates of the transfectants were normalized to their <i>Renilla</i> luciferase control activity. (<b>B</b>) PC12 cells were seeded on poly-L-lysine-coated 100 mm dishes in normal medium for 24 h and then shifted to low-serum medium (1% horse serum and 0.5% FBS) for an additional 24 h of culture. Cells were treated with the inhibitors U0126, H-89 or SQ22536 for 30 min prior to their exposure to vehicle (0.1% DMSO) or luteolin (20 µM) for 60 min. Phospho-CREB-Ser¹³³ (p-CREB) and CREB were analyzed by Western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. The immunoblot experiments were replicated at least three times, and a representative blot is shown. The normalized intensity of p-CREB versus CREB is presented as the mean ± SD of three independent experiments. **<i>p</i><0.01 represents significant differences compared to vehicle-treated cells. ##<i>p</i><0.01 represents significant differences compared to the respective inhibitor-non-treated group.</p

Luteolin increases the phosphorylation and activity of CREB in PC12 cells.

<p>(<b>A</b>) PC12 cells were seeded on poly-L-lysine-coated 100 mm dishes in normal medium for 24 h and then shifted to low-serum medium (1% HS and 0.5% FBS) for 24 h prior to their exposure to the indicated agents. Adherent PC12 cells were treated with luteolin (20 µM) for 0–240 min. Phospho-CREB-Ser¹³³ (p-CREB) and CREB proteins were analyzed by Western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. The immunoblot experiments were replicated at least three times, and a representative blot is shown. The normalized intensity of p-CREB versus CREB is presented as the mean ± SD of three independent experiments. *<i>p</i><0.05 and **<i>p</i><0.01 represents significant differences compared to the 0 min group. (<b>B</b>) PC12 cells were seeded on poly-L-lysine-coated 24-well plates in DMEM containing 10% horse serum and 5% FBS for 24 h. Cells were then transfected with a CRE-mediated luciferase reporter construct and <i>Renilla</i> luciferase control plasmid as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. After transfection, PC12 cells were treated with vehicle (0.1% DMSO) or luteolin (10 or 20 µM) for 8 h. Cells were harvested, and the luciferase activities were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043304#s4" target="_blank">Materials and Methods</a>. The intensities of the luciferase reactions measured in the lysates of the transfectants were normalized to their <i>Renilla</i> luciferase control activity. Data represent the mean ± SD from three independent experiments. *<i>p</i><0.05 and **<i>p</i><0.01 represent significant differences compared to vehicle-treated cells.</p