Biomedical subjects

Junjun Huang

Publications and source records attributed to Junjun Huang.

6 recordsLinked to original sources

PubMed2006

Ascorbic acid protects SH-SY5Y neuroblastoma cells from apoptosis and death induced by beta-amyloid.

beta-Amyloid causes apoptosis and death in cultured neurons that may be mediated by generation of reactive oxygen species. Since ascorbic acid concentrations are relatively high in brain, we tested whether and how this antioxidant might protect cultured SH-SY5Y neuroblastoma cells from apoptotic cell death. SH-SY5Y cells did not contain ascorbate in culture but readily took it up to achieve intracellular concentrations several-fold those of GSH. Treatment of cells with 2-10 microM beta-amyloid(25-35) decreased both intracellular ascorbate and GSH without affecting rates of ascorbate transport, which suggests that the peptide induces an oxidant stress in the cells. Overnight culture of cells with 10-20 microM beta-amyloid(25-35) induced apoptosis in SH-SY5Y cells when measured as externalization of phosphatidylserine by annexin V binding, as DNA fragmentation in the TUNEL assay, and as caspase-3 activity in cell lysates. Pre-loading cells with ascorbate substantially prevented apoptosis measured by these assays as well as cell death. In addition to preventing apoptosis, ascorbate loading of SH-SY5Y cells also decreased basal rates of generation of endogenous beta-amyloid. Together, these results support the notion that beta-amyloid induces apoptosis and death in neurons due to oxidant stress and suggest that intracellular ascorbate effectively prevents this toxicity.

Amyloid beta-Peptides↗

PubMed2005

Macrophage uptake and recycling of ascorbic acid: response to activation by lipopolysaccharide.

To test whether ascorbic acid might be involved in the antioxidant defenses of inflammatory cells, we studied ascorbate uptake and recycling by quiescent and lipopolysaccharide-activated RAW264.7 murine macrophages. These cells concentrated ascorbate 100-fold in overnight culture, achieving steady-state concentrations of more than 10 mM at extracellular concentrations of 20-100 muM. This steep gradient was generated by high-affinity sodium-dependent ascorbate transport. The latter likely reflects function of the SVCT2 (SLC23A2), since this protein was detected on immunoblots. Dehydroascorbate, the two-electron oxidized form of ascorbate, was also taken up and reduced to ascorbate by the cells. Dehydroascorbate reduction required rapid recycling of GSH from GSSG by glutathione reductase. Activation of ascorbate-containing macrophages with lipopolysaccharide transiently depleted intracellular ascorbate without affecting GSH. Recovery of intracellular ascorbate required function of the SVCT2 transporter, the activity of which was modestly enhanced by lipopolysaccharide. Lipopolysaccharide treatment nearly doubled intracellular GSH concentrations over 2 h. Despite lipopolysaccharide-induced oxidant stress, this GSH increase was associated with a comparable increase in reduction of dehydroascorbate to ascorbate. These results show that macrophages maintain millimolar concentrations of ascorbate through function of the SVCT2 and that activated cells have an enhanced ability to transport and recycle ascorbate, possibly reflecting its role as an intracellular antioxidant.

Animals↗

PubMed2005

Ascorbate uptake and antioxidant function in peritoneal macrophages.

Since activated macrophages generate potentially deleterious reactive oxygen species, we studied whether ascorbic acid might function as an antioxidant in these cells. Thioglycollate-elicited murine peritoneal macrophages contained about 3 mM ascorbate that was halved by culture in ascorbate-free medium. However, the cells took up added ascorbate to concentrations of 6-8 mM by a high-affinity sodium-dependent transport mechanism. This likely reflected the activity of the SVCT2 ascorbate transporter, since its message and protein were present in the cells. Activation of the cells by phagocytosis of latex particles depleted intracellular ascorbate, although not below the basal levels present in the cells in culture. Glutathione (GSH) was unaffected by phagocytosis, suggesting that ascorbate was more sensitive to the oxidant stress of phagocytosis than GSH. Phagocytosis induced a modest increase in reactive oxygen species as well as a progressive loss of alpha-tocopherol, both of which were prevented in cells loaded with ascorbate. These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis.

Animals↗

PubMed2004

Antioxidant effects of dihydrocaffeic acid in human EA.hy926 endothelial cells.

Dihydrocaffeic acid (DHCA) is a metabolite of caffeic acid with potent antioxidant properties. Since DHCA has been detected in human plasma following coffee ingestion, we tested the hypothesis that DHCA protects the endothelium from oxidative stress in a model in human-derived EA.hy926 endothelial cells. During culture for 16-24 hours, the cells accumulated DHCA against a concentration gradient to low millimolar concentrations. In alpha-tocopherol-loaded cells, DHCA spared alpha-tocopherol during overnight culture in a dose-dependent manner. In response to oxidant stress induced by a water-soluble free radical initiator, both alpha-tocopherol and DHCA diminished oxidation of cis-parinaric acid that had been incorporated into the cells, although their antioxidant activities were not additive. DHCA also decreased intracellular oxidation of dihydrofluorescein due to redox cycling by menadione. This suggests that the protective effects of DHCA were caused by scavenging of intracellular reactive oxygen species. DHCA also increased nitric oxide synthase activity in a dose-dependent manner in cultured cells, which was associated with a comparable increase in endothelial nitric oxide synthase protein. Although the DHCA concentrations required for these effects are higher than those likely to be present in plasma or the interstitium, these results indicate that DHCA can function as an intracellular antioxidant.

Antioxidants↗

PubMed2003

Ascorbic acid spares alpha-tocopherol and decreases lipid peroxidation in neuronal cells.

Ascorbic acid is considered an antioxidant in the central nervous system, but direct evidence that ascorbate protects neuronal cells from oxidant stress is lacking. Differentiated SH-SY5Y cells in culture took up ascorbic acid on the sodium-dependent vitamin C transporter Type 2 and retained it much more effectively than dehydroascorbic acid. Intracellular ascorbate spared alpha-tocopherol, both in cells loaded with alpha-tocopherol in culture and in cells under oxidant stress due to extracellular ferricyanide. Sparing of alpha-tocopherol in response to ferricyanide was associated with protection against lipid peroxidation in cell membranes. These results show that neuronal cells concentrate ascorbate, and that intracellular ascorbate, either directly or through sparing of alpha-tocopherol, protects them against oxidant stress.

Ascorbic Acid↗

PubMed2003

Ascorbic acid spares alpha-tocopherol and prevents lipid peroxidation in cultured H4IIE liver cells.

Ascorbic acid, or vitamin C, can recycle alpha-tocopherol in lipid bilayers, but even sparing of alpha-tocopherol has not been a consistent finding in intact cells. Therefore, we tested the ability of ascorbate loading to spare alpha-tocopherol and to prevent lipid peroxidation of cultured H4IIE rat liver cells. Although alpha-tocopherol was undetectable in H4IIE cells, its cell content was increased by overnight incubation with alpha-tocopherol in culture. Cells incubated with ascorbate 2-phosphate accumulated ascorbate to concentrations as high as 0.6 mM after overnight loading, but also released ascorbate into the medium. Ascorbate loading of alpha-tocopherol-treated cells spared alpha-tocopherol in a concentration-dependent manner during overnight incubation. Lipid peroxidative damage, measured as a decrease in fluorescence of cell-bound cis-parinaric acid, was decreased in cells loaded with either alpha-tocopherol or ascorbate 2-phosphate, and showed an additive effect. These results suggest that ascorbate loading of H4IIE cells spares cellular alpha-tocopherol and either directly or through recycling of alpha-tocopherol prevents lipid peroxidative damage due to oxidant stress in culture.

Animals↗