Lipoprotein lipase liberates free fatty acids to inhibit HCV infection and prevent hepatic lipid accumulation.

Lipoprotein lipase (LPL) has been identified as an anti-hepatitis C virus (HCV) host factor, but the cellular mechanism remains elusive. Here, we investigated the cellular mechanism of LPL involving in anti-HCV. The functional activation of peroxisome proliferator-activated receptor (PPAR) α signal by LPL transducing into hepatocytes was investigated in HCV-infected cells, primary human hepatocytes, and in HCV-core transgenic mice. The result showed that the levels of transcriptional transactivity and nuclear translocation of PPARα in Huh7 cells and primary human hepatocytes were elevated by physiologically ranged LPL treatment of either very-low density lipoprotein or HCV particles. The LPL-induced hepatic PPARα activation was weakened by blocking the LPL enzymatic activity, and by preventing the cellular uptake of free unsaturated fatty acids with either albumin chelator or silencing of CD36 translocase. The knockdowns of PPARα and CD36 reversed the LPL-mediated suppression of HCV infection. Furthermore, treatment with LPL, like the direct activation of PPARα, not only reduced the levels of apolipoproteins B, E, and J, which are involved in assembly and release of HCV virions, but also alleviated hepatic lipid accumulation induced by core protein. HCV-core transgenic mice exhibited more hepatic miR-27b, which negatively regulates PPARα expression, than did the wild-type controls. The induction of LPL activity by fasting in the core transgenic mice activated PPARα downstream target genes that are involved in fatty acid ß-oxidation. Taken together, our study reveals dual beneficial outcomes of LPL in anti-HCV and anti-steatosis and shed light on the control of chronic hepatitis C in relation to LPL modulators.

Apolipoprotein J, a glucose-upregulated molecular chaperone, stabilizes core and NS5A to promote infectious hepatitis C virus virion production.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to glucose abnormality. HCV depends on lipid droplets (LDs) and very-low density lipoproteins for assembly/releasing; however, the components and locations for this process remain unidentified. Apolipoprotein J (ApoJ), upregulated by glucose, functions as Golgi chaperone of secreted proteins and resides abundantly in very-low density lipoproteins. This study investigates the interplay between glucose, ApoJ and HCV virion production. METHODS: The effects of high glucose on ApoJ expression and HCV production were evaluated with cultivated HuH7.5, primary human hepatocytes, and in treatment naive chronic hepatitis C patients. How ApoJ affects HCV lifecycle was assessed using siRNA knockdown strategy in JFH1 infected and subgenomic replicon cells. The interactions and locations of ApoJ with viral and host components were examined by immunoprecipitation, immunofluorescence and subcellular fractionation experiments. RESULTS: HCV infection increased ApoJ expression, which in parallel with HCV infectivity was additionally elevated with high glucose treatment. Serum ApoJ correlated positively with fasting blood glucose concentration and HCV-RNA titre in patients. ApoJ silencing reduced intracellular and extracellular HCV infectivity and extracellular HCV-RNA, but accumulated intracellular HCV-RNA in HCV-infected cells. ApoJ interacted with HCV core and NS5A and stabilized the dual protein complex. HCV infection dispersed cytoplasmic ApoJ from the compact zones of the Golgi to encircle LDs, where co-localization of the core, NS5A, HCV-RNA, subcellular markers for LDs, endoplasmic reticulum (ER), Golgi, and membrane contact sites occurred. CONCLUSIONS: ApoJ facilitates infectious HCV particle production via stabilization of core/NS5A, which might surround LDs at the ER-Golgi membrane contact site.

Blockade of reactive oxygen species and Akt activation is critical for anti-inflammation and growth inhibition of metformin in phosphatase and tensin homolog-deficient RAW264.7 cells.

CONTEXT: Metformin is widely used for treatment of type 2 diabetes and has a potential application on the treatment of inflammation and cancer. Phosphatase and tensin homolog (PTEN) plays a critical role in cancer cell growth and inflammation; however, precise mechanisms remain unclear. OBJECTIVE: We aimed to investigate the possible mechanisms of how PTEN regulates metformin against cell growth and inflammation. MATERIALS AND METHODS: We established PTEN knockdown in RAW264.7 murine macrophages (shPTEN cells) to detect inflammatory mediators using commercial kits, production of reactive oxygen species (ROS) by flow cytometry, cell growth by MTT assay and phosphorylated levels of signal molecules by western blot. RESULTS: The shPTEN cells had a significant large amount of inflammatory mediators, such as inducible nitric oxide synthase (iNOS)/nitric oxide (NO) and cyclooxygenase-2 (COX-2)/prostaglandin E(2) (PGE(2)); and also elevated the production of ROS and increased cell proliferation. These effects were accompanied with the activation of Akt and p38 mitogen-activated protein kinase (MAPK), and the inactivation of an AMP-activated protein kinase (AMPK) activator and extracellular signal-regulated kinase 1/2. Pretreatment with metformin not only blocked these inflammatory mediators, but also caused growth inhibition induced by significant apoptosis. Furthermore, inactivation of Akt, blockade of ROS generation and independence of activations of AMPK and MAPK by metformin were also observed. CONCLUSION: Macrophages with PTEN deficiency developed a continuous inflammatory microenvironment, which further aggravated tumor cell growth. Moreover, metformin affected PTEN-deficient cells dependent of inhibition of ROS production and Akt activation against enlarged inflammatory mediators and/or cell growth in shPTEN cells.

Glycogen synthase kinase-3ß is critical for interferon-α-induced serotonin uptake in human Jurkat T cells.

Dysregulation of glycogen synthase kinase (GSK)-3ß contributes to the pathophysiology of mood disorders. However, how its regulation is responsible for the functioning of serotonin (5-HT) requires further investigation. Although enhancement of T-cell function may present an alternative strategy to treat depression, the precise mechanisms have yet to be established. Our previous studies have found that interferon-alpha (IFN-α) up-regulates serotonin transporter (5-HTT) expression and induces 5-HT uptake in T cells. The present study is to examine GSK-3ß regulation on IFN-α-induced 5-HTT functions. GSK-3ß short hairpin RNAs (shRNAs) or GSK-3ß inhibitors decreased IFN-α-induced 5-HT uptake and 5-HTT expression. Src activation and calcium/calcium-activated calmodulin kinase II (CaMKII) were involved in IFN-α-induced phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) (Tyr402) and GSK-3ß (Tyr216), which regulated 5-HT uptake. GSK-3ß knockdown blocked the IFN-α-induced phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 (Thr202/Tyr204) and signal transducer and transactivator (STAT) 1. In addition to inhibiting ERK, a selective 5-HTT inhibitor fluoxetine blocked IFN-α-induced activations of Src, CaMKII-regulated Pyk2/GSK-3ß cascade, as well as STAT1 activation and translocation. These results indicated that calcium/CaMKII- and Src-regulated Pyk2 participated in IFN-α-induced GSK-3ß activation and GSK-3ß-regulated 5-HT uptake. GSK-3ß signaling facilitated IFN-α-activated STAT1 by regulating ERK1/2, which controlled 5-HT uptake. Fluoxetine interfered with the Pyk2/GSK-3ß cascade, thereby inhibiting IFN-α-induced 5-HT uptake.

Glucosylceramide synthase inhibitor PDMP sensitizes chronic myeloid leukemia T315I mutant to Bcr-Abl inhibitor and cooperatively induces glycogen synthase kinase-3-regulated apoptosis.

Inactivation of glycogen synthase kinase (GSK)-3 has been implicated in cancer progression. Previously, we showed an abundance of inactive GSK-3 in the human chronic myeloid leukemia (CML) cell line. CML is a hematopoietic malignancy caused by an oncogenic Bcr-Abl tyrosine kinase. In Bcr-Abl signaling, the role of GSK-3 is not well defined. Here, we report that enforced expression of constitutively active GSK-3 reduced proliferation and increased Bcr-Abl inhibition-induced apoptosis by nearly 1-fold. Bcr-Abl inhibition activated GSK-3 and GSK-3-dependent apoptosis. Inactivation of GSK-3 by Bcr-Abl activity is, therefore, confirmed. To reactivate GSK-3, we used glucosylceramide synthase (GCS) inhibitor PDMP to accumulate endogenous ceramide, a tumor-suppressor sphingolipid and a potent GSK-3 activator. We found that either PDMP or silence of GCS increased Bcr-Abl inhibition-induced GSK-3 activation and apoptosis. Furthermore, PDMP sensitized the most clinical problematic drug-resistant CML T315I mutant to Bcr-Abl inhibitor GNF-2-, imatinib-, or nilotinib-induced apoptosis by >5-fold. Combining PDMP and GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis. The synergistic efficacy was Bcr-Abl restricted and correlated to increased intracellular ceramide levels and acted through GSK-3-mediated apoptosis. This study suggests a feasible novel anti-CML strategy by accumulating endogenous ceramide to reactivate GSK-3 and abrogate drug resistance.

Dermatophagoides pteronyssinus 2 regulates nerve growth factor release to induce airway inflammation via a reactive oxygen species-dependent pathway.

Group 2 allergen of Dermatophagoides pteronyssinus 2 (Der p2) induces airway inflammation without protease activity, and elevated nerve growth factor (NGF) levels are also found in this inflammation. How the allergen Der p2 regulates NGF release via reactive oxygen species (ROS) to induce inflammation remains unclear. In the present study, intratracheal administration of Der p2 to mice led to inflammatory cell infiltration, mucus gland hyperplasia, and NGF upregulation in the bronchial epithelium, as well as elevated ROS and NGF production in bronchoalveolar lavage fluids. In addition, Der p2 caused fibrocyte accumulation and mild fibrosis. p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) inhibitors inhibited Der p2-induced NGF release in LA4 lung epithelial cells and MLg lung fibroblasts. Pretreatment with an antioxidant, tiron, reduced the Der p2-induced ROS production, NGF expression and release, p38 MAPK or JNK phosphorylation, and airway inflammation. These results suggest that Der p2 allergen-induced airway inflammation and elevated NGF release were through increasing ROS production and a MAPK-dependent pathway. The use of an antioxidant, tiron, may provide a new therapeutic modality for the treatment of allergic asthma.

Glycogen synthase kinase-3beta facilitates IFN-gamma-induced STAT1 activation by regulating Src homology-2 domain-containing phosphatase 2.

Glycogen synthase kinase-3beta (GSK-3beta)-modulated IFN-gamma-induced inflammation has been reported; however, the mechanism that activates GSK-3beta and the effects of activation remain unclear. Inhibiting GSK-3beta decreased IFN-gamma-induced inflammation. IFN-gamma treatment rapidly activated GSK-3beta via neutral sphingomyelinase- and okadaic acid-sensitive phosphatase-regulated dephosphorylation at Ser(9), and proline-rich tyrosine kinase 2 (Pyk2)-regulated phosphorylation at Tyr(216). Pyk2 was activated through phosphatidylcholine-specific phospholipase C (PC-PLC)-, protein kinase C (PKC)-, and Src-regulated pathways. The activation of PC-PLC, Pyk2, and GSK-3beta was potentially regulated by IFN-gamma receptor 2-associated Jak2, but it was independent of IFN-gamma receptor 1. Furthermore, Jak2/PC-PLC/PKC/cytosolic phospholipase A(2) positively regulated neutral sphingomyelinase. Inhibiting GSK-3beta activated Src homology-2 domain-containing phosphatase 2 (SHP2), thereby preventing STAT1 activation in the late stage of IFN-gamma stimulation. All these results showed that activated GSK-3beta synergistically affected IFN-gamma-induced STAT1 activation by inhibiting SHP2.

Insulin resistance without obesity induced by cotton pellet granuloma in mice.

Obesity leads to insulin resistance because the larger adipocytes in obese persons secrete proinflammatory cytokines that cause chronic inflammation in adipose tissue. This, in turn, leads to the alteration of adipokine secretion, which can induce insulin resistance. However, the development of insulin resistance without obesity is still obscure. We aimed to use an animal inflammation model with cotton pellet granuloma (CPG) in adipose tissue to characterize insulin resistance formation. We found that CPG in epididymal white adipose tissue (WAT), rather than in interscapular brown adipose tissue, impaired insulin sensitivity, and glucose utilization, and that it decreased levels of phosphoinsulin receptor and phospho-Akt in both muscle and liver tissue, but that it did not modify the body weight or food intake in mice. Macrophage infiltration in adipose tissue, leukocyte counts, monocyte chemoattractant protein-1, and interleukin-6 were elevated in CPG-treated mice. However, we found a marked decrease of plasma adiponectin only in the WAT group, which might have been because of the lower level of peroxisome proliferator-activated receptor-gamma in WAT. These results show that granuloma formation in WAT by implantation of a cotton pellet may induce insulin resistance under nonobese condition through circulating inflammatory mediators, especially the low level of adiponectin.

Activation of muscarinic M-3 receptor may decrease glucose uptake and lipolysis in adipose tissue of rats.

Role of muscarinic receptor in the regulation of glucose uptake or lipolysis in adipose tissue remained unclear. In epididymal white adipose tissue (WAT) isolated from Wistar rats, we observed that acetylcholine (ACh) attenuated the insulin-stimulated glucose uptake and the release of glycerol from WAT in a concentration-dependent manner. Using the blockade of specific antagonists, both actions of ACh were characterized mainly due to an activation of M3 receptors. In the presence of various inhibitors for PLC-PKC pathway, ACh-decreased glucose uptake was also reversed. Taken together, these results suggest that muscarinic M3 receptor is involved in the regulation of glucose uptake and/or lipolysis in adipose tissue.

Etifoxine, a TSPO Ligand, Worsens Hepatitis C-Related Insulin Resistance but Relieves Lipid Accumulation.

Etifoxine, an 18 kDa translocator protein (TSPO) agonist for the treatment of anxiety disorders in clinic, may be able to cause acute liver injury or cytolytic hepatitis. TSPO has been demonstrated to participate in inflammatory responses in infective diseases as well as to modulate glucose and lipid homeostasis. Hepatitis C virus (HCV) infection disrupts glucose and lipid homoeostasis, leading to insulin resistance (IR). Whether TSPO affects the HCV-induced IR remains unclear. Here, we found that the administration of etifoxine increased the TSPO protein expression and recovered the HCV-mediated lower mitochondrial membrane potential (MMP) without affecting HCV infection. Moreover, etifoxine reversed the HCV-induced lipid accumulation by modulating the expressions of sterol regulatory element-binding protein-1 and apolipoprotein J. On the other hand, in infected cells pretreated with etifoxine, the insulin-mediated insulin receptor substrate-1/Akt signals, forkhead box protein O1 translocation, and glucose uptake were blocked. Taken together, our results pointed out that etifoxine relieved the HCV-retarded MMP and reduced the lipid accumulation but deteriorated the HCV-induced IR by interfering with insulin signal molecules.

Role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the action of ginsenoside Rh2 against beta-amyloid-induced inhibition of rat brain astrocytes.

Ginsenoside, the active principles in Panax ginseng root, has been demonstrated to show neurotrophic and neuroprotective actions for prevention of neuron degeneration. Deposition of beta-amyloid peptide (Abeta) causes neurotoxicity through the formation of plaques in brains with Alzheimer's disease. Pituitary adenylate cyclase-activating polypeptide (PACAP) is introduced as a neurotrophic factor to promote cell survival. However, effect of Rh2, one of ginsenosides, on PACAP expression induced by Abeta remains unclear. In the present study, we found that Rh2 stimulates PACAP gene expression and cell proliferation in type I rat brain astrocytes (RBA1) cells and both effects were not modified by the estrogen antagonists (MPP or ICI 182780). Also, Rh2 ameliorates the RBA1 growth inhibition of Abeta. Moreover, blockade of PACAP receptor PAC1 using PACAP (6-38) inhibits all the actions of Rh2. These results suggest that Rh2 can induce an increase of PACAP to activate PAC1, but not estrogen receptor, and thereby leads to attenuate Abeta-induced toxicity. Thus, ginseng seems useful in the prevention of dementia.

Plasma Translocator Protein Levels and Outcomes of Acute Ischemic Stroke: A Pilot Study.

Translocator protein 18 kDa (TSPO) has been used as a biomarker of brain injury and inflammation in various neurological diseases. In this study, we measured the level of TSPO in acute ischemic stroke patients and determined its association with the degree of stroke severity and its ability to predict stroke functional outcomes. In total, 38 patients with moderate to severe acute ischemic stroke were enrolled. Demographic information, cerebral risk factors, and stroke severity were examined at the baseline. The National Institutes of Health Stroke Scale, modified Rankin Scale, and Barthal Index were assessed at discharge as measures of poor functional outcomes and severe disability. The baseline fasting plasma TSPO level was assessed within 24 h after the incident stroke and during hospitalization (on days 8-10). The proportion of patients with poor functional outcomes was significantly higher in the higher-TSPO group (compared to the lower group) in terms of clinical worsening (odds ratio (OR) = 11.69, 95% confidence interval (CI) = 2.08-65.6), poor functional outcomes (OR = 10.5, 95% CI = 1.14-96.57), and severe disability (OR = 4.8, 95% CI = 1.20-19.13). Plasma TSPO may be intimately linked with disease progression and worse functional outcomes in acute ischemic stroke patients.

Calcitriol Inhibits HCV Infection via Blockade of Activation of PPAR and Interference with Endoplasmic Reticulum-Associated Degradation.

Vitamin D has been identified as an innate anti-hepatitis C virus (HCV) agent but the possible mechanisms for this issue remain unclear. Here, we clarified the mechanisms of calcitriol-mediated inhibition of HCV infection. Calcitriol partially inhibited HCV infection, nitric oxide (NO) release and lipid accumulation in Huh7.5 human hepatoma cells via the activation of vitamin D receptor (VDR). When cells were pretreated with the activators of peroxisome proliferator-activated receptor (PPAR)-α (Wy14643) and -γ (Ly171883), the calcitriol-mediated HCV suppression was reversed. Otherwise, three individual stimulators of PPAR-α/ß/γ blocked the activation of VDR. PPAR-ß (linoleic acid) reversed the inhibition of NO release, whereas PPAR-γ (Ly171883) reversed the inhibitions of NO release and lipid accumulation in the presence of calcitriol. The calcitriol-mediated viral suppression, inhibition of NO release and activation of VDR were partially blocked by an inhibitor of endoplasmic reticulum-associated degradation (ERAD), kifunensine. Furthermore, calcitriol blocked the HCV-induced expressions of apolipoprotein J and 78 kDa glucose-regulated protein, which was restored by pretreatment of kifunensine. These results indicated that the calcitriol-mediated HCV suppression was associated with the activation of VDR, interference with ERAD process, as well as blockades of PPAR, lipid accumulation and nitrative stress.

Changes of dopamine content and cell proliferation by dexamethsone via pituitary adenylate cyclase-activating polypeptide in PC12 cell.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous neuropeptide observed in adrenal gland and sympathetic ganglia to regulate catecholamine synthesis and release. Both PACAP and glucocorticoid showed the activity to elevate catecholamine level through the stimulation of biosynthesis. However, the relationship of glucocorticoid and PACAP for this action is still unclear. Thus, alterations of gene expression, dopamine (DA) content, and cell proliferation in rat pheochromocytoma PC12 cells are employed as indicators to clarify this relationship in the present study. From the analysis of RT-PCR, the mRNA level of PACAP was observed to be raised by dexamethasone (DEX) and this action was blocked in cells treated with RU486 (mifepristone), a glucocorticoid receptor (GR) antagonist, or actinomycin D, a transcriptional inhibitor. An increase of DA content by HPLC analysis and/or cell proliferation identified by MTT assay by DEX was also observed which could be inhibited by PACAP (6-38) at concentration sufficient to block PACAP type 1 (PAC1) receptor. These results suggest that PACAP is involved in DEX-induced DA biosynthesis and cell proliferation in PC12 cells.

Differential Ca2+ mobilization and mast cell degranulation by FcεRI- and GPCR-mediated signaling.

Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca2+ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca2+ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca2+ levels, respectively. Histamine release and Ca2+ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca2+ increase. FcεRI-induced Ca2+ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca2+ release was both necessary and sufficient for mast cell degranulation, extracellular Ca2+ influx enhanced the process. Furthermore, cytosolic Ca2+ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca2+ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca2+ signaling in regulating histamine degranulation.

Cytokines and serotonin transporter in patients with major depression.

Altered cytokine secretion as a mechanism in the etiology of depression is still obscure. The serotonin transporter (5-HTT) may play an important role in the termination of serotonergic neurotransmission by serotonin (5-HT) uptaking into presynaptic neurons and representing as an initial action site for selective 5-HTT reuptake inhibitors (SSRI). In our study, we evaluated whether cytokines and 5-HTT acted as biological markers for depression. Blood samples were collected from 42 participants. The differences in cytokine and 5-HTT mRNA expressions of leukocytes were assessed between the patients with major depression (n=20) and the healthy controls (n=22), along with the measurements prior and after treatment with a SSRI, fluoxetine, for 3 months in the follow-up patient group (n=8). The results revealed that the mRNA expressions of IL-1beta, IL-6, IFNgamma, TNFalpha, and 5-HTT were higher in the depressed patients than those of the healthy controls. The higher level of mRNA expressions of IFNgamma and 5-HTT diminished after fluoxetine treatment. Furthermore, we found a positive correlation between 5-HTT and cytokines mRNA expressions in total participants, which suggested that pro-inflammatory cytokines and 5-HTT might play critical roles in the pathogenesis of major depression and that their levels were affected by chronic treatment with 5-HTT inhibitors.

Fluoxetine regulates cell growth inhibition of interferon-α.

Fluoxetine, a well-known anti-depression agent, may act as a chemosensitizer to assist and promote cancer therapy. However, how fluoxetine regulates cellular signaling to enhance cellular responses against tumor cell growth remains unclear. In the present study, addition of fluoxetine promoted growth inhibition of interferon-alpha (IFN-α) in human bladder carcinoma cells but not in normal uroepithelial cells through lessening the IFN-α-induced apoptosis but switching to cause G1 arrest, and maintaining the IFN-α-mediated reduction in G2/M phase. Activations and signal transducer and transactivator (STAT)-1 and peroxisome proliferator-activated receptor alpha (PPAR-α) were involved in this process. Chemical inhibitions of STAT-1 or PPAR-α partially rescued bladder carcinoma cells from IFN-α-mediated growth inhibition via blockades of G1 arrest, cyclin D1 reduction, p53 downregulation and p27 upregulation in the presence of fluoxetine. However, the functions of both proteins were not involved in the control of fluoxetine over apoptosis and maintained the declined G2/M phase of IFN-α. These results indicated that activation of PPAR-α and STAT-1 participated, at least in part, in growth inhibition of IFN-α in the presence of fluoxetine.

Rosiglitazone regulates anti-inflammation and growth inhibition via PTEN.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonist has anti-inflammatory and anticancer properties. However, the mechanisms by which PPARγ agonist rosiglitazone interferes with inflammation and cancer via phosphatase and tensin homolog-(PTEN)-dependent pathway remain unclear. We found that lower doses (<25 µ M) of rosiglitazone significantly inhibited lipopolysaccharide-(LPS)-induced nitric oxide (NO) release (via inducible nitric oxide synthase, iNOS), prostaglandin E2 (PGE2) production (via cyclooxygenase-2, COX-2), and activation of Akt in RAW 264.7 murine macrophages. However, rosiglitazone did not inhibit the production of reactive oxygen species (ROS). In PTEN knockdown (shPTEN) cells exposed to LPS, rosiglitazone did not inhibit NO release, PGE2 production, and activation of Akt. These cells had elevated basal levels of iNOS, COX-2, and ROS. However, higher doses (25-100 µ M) of rosiglitazone, without LPS stimulation, did not block NO release and PGE2 productions, but they inhibited p38 MAPK phosphorylation and blocked ROS generation in shPTEN cells. In addition, rosiglitazone caused G1 arrest and reduced the number of cells in S + G2/M phase, leading to growth inhibition. These results indicate that the anti-inflammatory property of rosiglitazone is related to regulation of PTEN independent of inhibition on ROS production. However, rosiglitazone affected the dependence of PTEN-deficient cell growth on ROS.

Fluoxetine a novel anti-hepatitis C virus agent via ROS-, JNK-, and PPARß/γ-dependent pathways.

More than 20% of chronic hepatitis C (CHC) patients receiving interferon-alpha (IFN-α)-based anti-hepatitis C virus (HCV) therapy experienced significant depression, which was relieved by treatment with fluoxetine. However, whether and how fluoxetine affected directly the anti-HCV therapy remained unclear. Here, we demonstrated that fluoxetine inhibited HCV infection and blocked the production of reactive oxygen species (ROS) and lipid accumulation in Huh7.5 cells. Fluoxetine facilitated the IFN-α-mediated antiviral actions via activations of signal transducer and activator of transcription (STAT)-1 and c-Jun amino-terminal kinases (JNK). Alternatively, fluoxetine elevated peroxisome proliferator-activated receptor (PPAR) response element activity under HCV infection. The inhibitory effects of fluoxetine on HCV infection and lipid accumulation, but not production of ROS, were partially reversed by the PPAR-ß, -γ, and JNK antagonists. Furthermore, fluoxetine intervention to the IFN-α-2b regimen facilitated to reduce HCV titer and alanine transaminase level for CHC patients. Therefore, fluoxetine intervention to the IFN-α-2b regimen improved the efficacy of anti-HCV treatment, which might be related to blockades of ROS generation and lipid accumulation and activation of host antiviral JNK/STAT-1 and PPARß/γ signals.

Glycogen synthase kinase-3ß regulates anti-inflammatory property of fluoxetine.

A selective serotonin reuptake inhibitor fluoxetine not only is widely used in the treatment of depression but also has an anti-inflammatory property. Glycogen synthase kinase-3beta (GSK-3ß) is a vital factor in the inflammation process. How fluoxetine interferes with inflammation via a GSK-3ß-dependent pathway remains unclear. The aim of this study is to investigate the effects of fluoxetine on lipopolysaccharide (LPS)-induced inflammation. Results showed that fluoxetine decreased mortality rate of the mice. It also inhibited LPS-induced release of nitric oxide (NO) and prostaglandin E2 (PGE2) in serum and RAW264.7 murine macrophages and expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Upon LPS stimulation, fluoxetine caused a delay but increased in the phosphorylated levels of GSK-3ß (ser9), whereas it did not affect LPS-induced activation of mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS). Fluoxetine in combination with phosphatidylinositol 3-kinases/Akt inhibitors (LY294002 and Wortmannin) did not have a synergistic inhibition on LPS-induced NO release and PGE2 production. In addition, peroxisome proliferator-activated receptor γ (PPARγ) antagonist GW9622 showed no reverse effects of this inhibition of fluoxetine. GSK-3ß knockdown blocked the inhibitory effects of fluoxetine on LPS-induced iNOS/NO release and COX-2/PGE2 production. These results indicated that GSK-3ß regulated anti-inflammatory property of fluoxetine. However, Akt activation, ROS generation, and altered PPARγ activity were not involved in this inhibition of fluoxetine.