Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection.

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

Immunomodulation Effects of Schizonepeta tenuifolia Briq. on the IgE-Induced Allergic Model of RBL-2H3 Cells.

Schizonepeta tenuifolia (ST) Briq. is a traditional herbal medicine commonly used to treat allergic skin diseases, where the inflammation process is closely related to symptom severity. This study aimed to explore the immunomodulatory effect of ST by using immunoglobulin E- (IgE-) stimulated RBL-2H3 cell cultures, a common cell line for studying mast cell degranulation and inflammatory cytokine release in vitro. After stimulating the RBL-2H3 cells with IgE, ST at concentrations of 10, 50, or 100 µg/mL was added to the cell cultures. Cell viability, inflammatory cytokines (IL-6, IL-13, IL-4, TNF-α, and IFN-γ), anti-inflammatory cytokine IL-10, and degranulation ability were examined 48 and 72 hours after administration of ST. The markers of inflammation and allergic reaction, IFN-γ, TNF-α, IL-4, and IL-6, were suppressed, especially after treatment with 100 µg/mL ST. However, the anti-inflammation marker IL-10 was also suppressed by ST. Trend analysis showed that a higher ST concentration was associated with lower IFN-γ and TNF-α levels. Moreover, degranulation of RBL-2H3 cells was assessed by measuring the release of ß-hexosaminidase, which was suppressed by ST at 10 µg/mL. This study showed an immunomodulatory effect of ST at the cellular level and suggests the role of ST in treating allergic diseases.

Anti-enterovirus 71 activities of Melissa officinalis extract and its biologically active constituent rosmarinic acid.

Enterovirus 71 (EV71) infection is endemic in the Asia-Pacific region. No specific antiviral drug has been available to treat EV71 infection. Melissa officinalis (MO) is a medicinal plant with long history of usage in the European and Middle East. We investigated whether an aqueous solution of concentrated methanolic extract (MOM) possesses antiviral activity. MOM inhibited plaque formation, cytopathic effect, and viral protein synthesis in EV71-infected cells. Using spectral techniques, we identified rosmarinic acid (RA) as a biologically active constituent of MOM. RA reduced viral attachment and entry; cleavage of eukaryotic translation initiation factor 4 G (eIF4G); reactive oxygen species (ROS) generation; and translocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from nucleus to cytoplasm. It alleviated EV71-induced hyperphosphorylation of p38 kinase and EPS15. RA is likely to suppress ROS-mediated p38 kinase activation, and such downstream molecular events as hnRNP A1 translocation and EPS15-regulated membrane trafficking in EV71-infected cells. These findings suggest that MO and its constituent RA possess anti-EV71 activities, and may serve as a candidate drug for therapeutic and prophylactic uses against EV71 infection.

Antiviral activities of Schizonepeta tenuifolia Briq. against enterovirus 71 in vitro and in vivo.

No effective drug is currently available for treatment of enterovirus 71 (EV71) infection. Schizonepeta tenuifolia Briq. (ST) has been used as a herbal constituent of traditional Chinese medicine. We studied whether the aqueous extract of Schizonepeta tenuifolia Briq (STE) has antiviral activity. STE inhibited replication of EV71, as evident by its ability to diminish plaque formation and cytopathic effect induced by EV71, and to inhibit the synthesis of viral RNA and protein. Moreover, daily single-dose STE treatment significantly improved the survival of EV71-infected mice, and ameliorated the symptoms. Mechanistically, STE exerts multiple effects on enteroviral infection. Treatment with STE reduced viral attachment and entry; the cleavage of eukaryotic translation initiation factor 4 G (eIF4G) by EV71 protease, 2Apro; virus-induced reactive oxygen species (ROS) formation; and relocation of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) from the nucleus to the cytoplasm. It was accompanied by a decline in EV71-associated hyperphosphorylation of p38 kinase and EPS15. It is plausible that STE may inhibit ROS-induced p38 kinase activation, and subsequent hnRNP A1 relocation and EPS15-mediated membrane trafficking in infected cells. These findings suggest that STE possesses anti-EV71 activities, and may serve as health food or candidate antiviral drug for protection against EV71.

Biochemical disorders associated with antiproliferative effect of dehydroepiandrosterone in hepatoma cells as revealed by LC-based metabolomics.

DHEA is known to have chemopreventive and antiproliferative activities, and was initially thought to be mediated by inhibition of G6PD. Our previous study has shown that DHEA may act through interference with energy metabolism. To study the effect of pharmacological dose of DHEA on cellular metabolism, and to further delineate the mechanism underlying its antiproliferative effect, we applied a metabolomic approach to globally profile the changes in metabolites in SK-Hep1 cells underexpressing G6PD (Sk-Gi) and control cells (Sk-Sc) after DHEA treatment. RRLC-TOF-MS was used to identify metabolites, and tandem mass spectrometry was used to confirm their identity. DHEA induced changes in glutathione metabolism, lipid metabolism, s-adenosylmethionine (SAM) metabolism, as well as lysine metabolism. Elevation in level of glutathione disulfide, together with a concomitant decrease in level of reduced glutathione, was indicative of increased oxidative stress. Depletion of carnitine and its acyl derivatives reflected decline in fatty acid catabolism. These changes were associated with mitochondrial malfunction and reduction in cellular ATP content. Cardiolipin (CL) and phosphatidylcholine (PC) levels decreased significantly, suggesting that alterations in lipid composition are causally related to decline in mitochondrial function after DHEA treatment. The decline in cellular SAM content was accompanied by decreased expression of methionine adenosyltransferase genes MAT2A and MAT2B. SAM supplementation partially rescued cells from DHEA-induced growth stagnation. Our findings suggest that DHEA causes perturbation of multiple pathways in cellular metabolism. Decreased SAM production, and cardiolipin depletion and the resulting mitochondrial dysfunction underlie the antiproliferative effect of DHEA.

Dehydroepiandrosterone induces growth arrest of hepatoma cells via alteration of mitochondrial gene expression and function.

DHEA is known to have anti-proliferative effect. The mechanism is not completely understood. We investigated the mechanism underlying DHEA-induced growth arrest of hepatoma cells. Growth inhibition was associated with increased G6PD activity, and insensitive to reversal by mevalonate. Thus, DHEA does not act via inhibition of G6PD and HMGR. Instead, growth stagnation was accompanied by reduced expression of nucleus-encoded mitochondrial genes; morphological and functional alterations of mitochondria; and depletion of intracellular ATP. Conversely, pyruvate supplementation alleviated DHEA-induced growth inhibition. It is likely that DHEA suppresses cell growth by altering mitochondrial gene expression, morphology and functions.

Green tea polyphenol epigallocatechin-3-gallate protects cells against peroxynitrite-induced cytotoxicity: modulatory effect of cellular G6PD status.

Glucose-6-phosphate dehydrogenase (G6PD) plays important roles in the maintenance of cellular redox balance. It was not until recently that the importance of G6PD in regulation of cellular growth and apoptosis emerged. In the present study, we found that G6PD-deficient fibroblasts were more susceptible to peroxynitrite-induced cytotoxicity. Treatment with peroxynitrite generator 3-morpholinosydnonimine (SIN-1) hydrochloride caused apoptosis in human fibroblast in a dose-dependent manner. This was preceded by a decrease in the intracellular level of glutathione (GSH) as well as accumulation of p53. The extent of apoptosis and glutathione depletion were greater in G6PD-deficient fibroblasts than in the normal counterpart. Pretreatment with green tea polyphenol epigallocatechin-3-gallate (EGCG) effectively blocked peroxynitrite-induced glutathione depletion, p53 accumulation, and apoptosis in both normal and G6PD-deficient cells. EGCG, administered to cells alone or as pretreatment, caused activation of Akt. The protective effect was abolished by phosphatidylinositol 3-kinase (PI3K) inhibitors, wortmannin, and LY294002. Our findings suggest that G6PD deficiency enhances the toxicity of peroxynitrite and that EGCG initiates cell survival signaling via the PI3K/akt pathway.