Zinc in Cardiovascular Functions and Diseases: Epidemiology and Molecular Mechanisms for Therapeutic Development

Zinc is an essential trace element that plays an important physiological role in numerous cellular processes. Zinc deficiency can result in diverse symptoms, such as impairment of the immune response, skin disorders, and impairments in cardiovascular functions. Recent reports have demonstrated that zinc acts as a signaling molecule, and its signaling pathways, referred to as zinc signals, are related to the molecular mechanisms of cardiovascular functions. Therefore, comprehensive understanding of the significance of zinc-mediated signaling pathways is vital as a function of zinc as a nutritional component and of its molecular mechanisms and targets. Several basic and clinical studies have reported the relationship between zinc level and the onset and pathology of cardiovascular diseases, which has attracted much attention in recent years. In this review, we summarize the recent findings regarding the effects of zinc on cardiovascular function. We also discuss the importance of maintaining zinc homeostasis in the cardiovascular system and its therapeutic potential as a novel drug target

The anti-inflammatory effects of flavanol-rich lychee fruit extract in rat hepatocytes.

Flavanol (flavan-3-ol)-rich lychee fruit extract (FRLFE) is a mixture of oligomerized polyphenols primarily derived from lychee fruit and is rich in flavanol monomers, dimers, and trimers. Supplementation with this functional food has been shown to suppress inflammation and tissue damage caused by high-intensity exercise training. However, it is unclear whether FRLFE has in vitro anti-inflammatory effects, such as suppressing the production of the proinflammatory cytokine tumor necrosis factor α (TNF-α) and the proinflammatory mediator nitric oxide (NO), which is synthesized by inducible nitric oxide synthase (iNOS). Here, we analyzed the effects of FRLFE and its constituents on the expression of inflammatory genes in interleukin 1β (IL-1β)-treated rat hepatocytes. FRLFE decreased the mRNA and protein expression of the iNOS gene, leading to the suppression of IL-1β-induced NO production. FRLFE also decreased the levels of the iNOS antisense transcript, which stabilizes iNOS mRNA. By contrast, unprocessed lychee fruit extract, which is rich in flavanol polymers, and flavanol monomers had little effect on NO production. When a construct harboring the iNOS promoter fused to the firefly luciferase gene was used, FRLFE decreased the luciferase activity in the presence of IL-1β, suggesting that FRLFE suppresses the promoter activity of the iNOS gene at the transcriptional level. Electrophoretic mobility shift assays indicated that FRLFE reduced the nuclear transport of a key regulator, nuclear factor κB (NF-κB). Furthermore, FRLFE inhibited the phosphorylation of NF-κB inhibitor α (IκB-α). FRLFE also reduced the mRNA levels of NF-κB target genes encoding cytokines and chemokines, such as TNF-α. Therefore, FRLFE inhibited NF-κB activation and nuclear translocation to suppress the expression of these inflammatory genes. Our results suggest that flavanols may be responsible for the anti-inflammatory and hepatoprotective effects of FRLFE and may be used to treat inflammatory diseases

FRLFE suppresses iNOS induction by the IL-1β signaling pathway.

<p>A pathway to activate the <i>iNOS</i> gene and the action of FRLFE are schematically depicted. The bold arrows indicate the decreases caused by FRLFE in this study. The proinflammatory cytokine IL-1β binds to its receptor (type I IL-1 receptor, IL1R1) to activate NF-κB through the IκB kinase (IKK) signaling pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Takimoto1" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Lawrence1" target="_blank">[25]</a>. Activated IKK phosphorylates IκB-α, resulting in the degradation of IκB-α. A circled P denotes protein phosphorylation. Active NF-κB enters into the nucleus, binds to the <i>iNOS</i> gene promoter (κB sites), and activates transcription. FRLFE inhibits the phosphorylation of IκB-α and the nuclear translocation of NF-κB, resulting in a decrease in the nuclear levels of NF-κB. Because NF-κB also regulates the transcription of the iNOS asRNA, FRLFE reduces the level of iNOS asRNA, which interacts with and stabilizes the iNOS mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Matsui1" target="_blank">[17]</a>. Furthermore, FRLFE may interfere with the iNOS mRNA–asRNA interaction at a posttranscriptional level. Therefore, FRLFE significantly decreases the level of iNOS mRNA.</p

Constituents of FRLFE, unprocessed lychee fruit extract, and green tea extract.

<p>*Ratio of weight is expressed as mean ± standard deviation in percentage (<i>n</i> = 10) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Kitadate1" target="_blank">[10]</a>; Amino Up Chemical Co., Ltd., unpublished data).</p><p>**Ratio of weight is expressed as mean ± standard deviation in percentage (<i>n</i> = 14) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#pone.0093818-Kitadate1" target="_blank">[10]</a>; Amino Up Chemical Co., Ltd., unpublished data).</p><p>***Described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093818#s2" target="_blank"><i>Materials and Methods</i></a>.</p>#<p>Total polyphenols – (monomers + dimers + trimer).</p

The effects of FRLFE on the NF-κB-dependent transcription of the <i>iNOS</i> gene.

<p>(<b>A</b>) FRLFE decreases the iNOS promoter activity. Hepatocytes were transfected with an iNOS promoter–luciferase construct (pRiNOS-Luc-3′UTR; top) and pCMV-LacZ (internal control) and were subsequently treated with IL-1β and/or FRLFE. κB, NF-κB-binding site; TATA, TATA box. The luciferase activity in the transfected cells was normalized to the β-galactosidase activity, and the fold activation was calculated by dividing the normalized luciferase activity by the luciferase activity in the presence of IL-1β alone. The data represent the mean ± SD (<i>n</i> = 3). *<i>P</i><0.05, **<i>P</i><0.01 versus IL-1β alone. (<b>B</b>) Nuclear extracts from FRLFE-treated hepatocytes decrease the DNA-binding activity to an NF-κB-binding site. Nuclear extracts were prepared from the cells and were analyzed using an EMSA to detect the NF-κB that was bound to a radiolabeled DNA probe harboring an NF-κB-binding site (κB). Competitor (Comp), cold DNA probe that was added to the reaction mixture at 100-fold molar excess to the radiolabeled probe. (<b>C</b>) FRLFE does not directly inhibit the DNA-binding activity of nuclear NF-κB. To induce NF-κB, hepatocytes were treated with IL-1β alone for 0.5 h, and a nuclear extract (NE) was prepared from these cells. The nuclear extract was directly mixed with FRLFE and analyzed using an EMSA to detect the NF-κB that was bound to the DNA probe harboring an NF-κB-binding site, similarly to (B). (<b>D</b>) FRLFE decreases the phosphorylation of IκB-α. Hepatocytes were treated with IL-1β and/or FRLFE for the indicated times. Hepatocyte extracts were immunoblotted with an anti-IκB-α, anti-phosphorylated IκB-α (p-IκB-α), or anti-β-tubulin antibody (internal control).</p

FRLFE suppresses the induction of the <i>iNOS</i> gene in the hepatocytes.

<p>(<b>A</b>) FRLFE suppresses NO induction. Hepatocytes were treated with or without IL-1β (0.1 nM) and/or FRLFE (100 μg/ml) for the indicated times. The NO levels in the medium were measured in duplicate. (<b>B</b>) FRLFE decreases the expression levels of both iNOS mRNA and its asRNA. Hepatocytes were treated with IL-1β and/or FRLFE, and total RNA from the cells was analyzed using strand-specific RT-PCR. The iNOS mRNA and its asRNA, as well as GAPDH mRNA (internal control), were detected by agarose gel electrophoresis of the PCR products. RT(−) indicates the negative PCR control without RT, which was used to monitor contamination with genomic DNA.</p

FRLFE suppresses NO production in IL-1β-treated hepatocytes.

<p>(<b>A</b>) Structures of the flavanol monomers in green tea catechins (left and center) and a flavanol polymer in FRLFE (right). Due to the two asymmetric carbons (C-2 and C-3), a flavanol monomer has four diastereoisomers, such as (+)-catechin [2<i>R</i>,3<i>S</i>] (left) and (−)-epicatechin [2<i>R</i>,3<i>R</i>] (center). (−)-Epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) are galloyl esters of (−)-epicatechin and (−)-epigallocatechin (EGC), respectively. G =  galloyl group. A flavanol oligomer from FRLFE (right) was synthesized by creating a covalent bond between (+)-catechin and the lychee fruit procyanidin. (<b>B</b>) FRLFE suppresses the induction of NO production and iNOS protein expression. Rat hepatocytes were treated with or without FRLFE for 8 h. Simultaneously, 0.1 nM IL-1β was added to the cells. The NO levels in the medium were measured in triplicate (mean ± SD), and the cell extracts were immunoblotted with an anti-iNOS or anti-β-tubulin antibody (internal control). *<i>P</i><0.05, **<i>P</i><0.01 versus IL-1β alone. (<b>C</b>) FRLFE suppresses the induction of NO production. FRLFE, unprocessed lychee fruit extract, or green tea extract were added to the medium in the presence of 0.1 nM IL-1β. The NO levels in the medium were measured in duplicate (mean). Cytotoxicity was not observed at these concentrations (data not shown).</p

Inhibition of nitric oxide production by the flavanol monomers.

<p>When IL-1β increased nitric oxide (NO) in the medium, the NO level in the presence of IL-1β was set to 100%, whereas the NO level in the absence of IL-1β was set to 0%. Gallic acid was used as a positive control to monitor the suppression of IL-1β-indcuced NO production.</p><p>IC₅₀, half-maximal (50%) inhibitory concentration of NO production in IL-1β-treated hepatocytes.</p><p>NA, not applied because 50% suppression of NO production was not observed.</p><p>ND, not determined because NO levels increased.</p

Transcripts reduced by FRLFE in rat hepatocytes.

<p>*The fold-change in signal ratios observed by microarray analysis of the mRNA levels at 2.5 h is indicated.</p><p>**mRNA decrease represents the ratio of the mRNA levels determined by real-time RT-PCR (FRLFE + IL-1β versus IL-1β) at 4 h. The mRNA level of IL-1β was set to 100%.</p><p>(+), asRNA was experimentally detected.</p><p>iNOS, inducible nitric oxide synthase; IL-23A, interleukin 23, α subunit p19; CX3CL1, chemokine (C-X₃-C motif) ligand 1; CCL, chemokine (C-C motif) ligand; TNF-α, tumor necrosis factor α; CXCL1, chemokine (C-X-C motif) ligand 1; PSMB10, proteasome subunit, β type 10; NF-κB, nuclear factor κB; IκB-α, nuclear factor κB inhibitor α.</p