Glucosamine Enhancement of Learning and Memory Functions by Promoting Fibroblast Growth Factor 21 Production.

Fibroblast growth factor 21 (FGF21) plays a crucial role in metabolism and brain function. Glucosamine (GLN) has been recognized for its diverse beneficial effects. This study aimed to elucidate the modulation of FGF21 production by GLN and its impact on learning and memory functions. Using both in vivo and in vitro models, we investigated the effects of GLN on mice fed with a normal diet or high-fat diet and on mouse HT22 hippocampal cells, STHdhQ7/Q7 striatal cells, and rat primary cortical neurons challenged with GLN. Our results indicated that GLN promotes learning and memory functions in mice and upregulates FGF21 expression in the hippocampus, cortex, and striatum, as well as in HT22 cells, STHdhQ7/Q7 cells, and cortical neurons. In animals receiving GLN together with an FGF21 receptor FGFR1 inhibitor (PD173074), the GLN-enhanced learning and memory functions and induction of FGF21 production in the hippocampus were significantly attenuated. While exploring the underlying molecular mechanisms, the potential involvement of NF-κB, Akt, p38, JNK, PKA, and PPARα in HT22 and NF-κB, Akt, p38, and PPARα in STHdhQ7/Q7 were noted; GLN was able to mediate the activation of p65, Akt, p38, and CREB in HT22 and p65, Akt, and p38 in STHdhQ7/Q7 cells. Our accumulated findings suggest that GLN may increase learning and memory functions by inducing FGF21 production in the brain. This induction appears to be mediated, at least in part, through GLN's activation of the NF-κB, Akt, p38, and PKA/CREB pathways.

The impact of low-intensity extracorporeal shock waves on testicular spermatogenesis demonstrated in a rat model.

BACKGROUND: In rodent models, low-intensity extracorporeal shock wave therapy has been shown to negatively impact semen concentration after treatment on the penis, implying that the reproductive system in close proximity may be indirectly affected by this modality. We hypothesized that shock waves are detrimental to spermatogenesis, and the aim of this study was to evaluate the effect of shock waves on spermatogenesis after direct shockwave treatment on testes using different energy settings. METHODS: Twenty-five male Sprague Dawley rats, 8 weeks old, were divided into five groups, including one control group and four treatment groups each treated using shock waves of different intensities. All rats in the treatment groups received 2000 shocks on the left testis twice a week for 4 weeks, with shock wave intensity and frequency varied by treatment group: 0.1 mJ/mm 2 at 4 Hz for Group A, 0.15 mJ/mm 2 at 4 Hz for Group B, 0.35 mJ/mm 2 at 4 Hz for Group C, and 0.55mJ/mm 2 at 3 Hz for Group D. At the end of the experiment, sperm collected from the epididymis was evaluated for concentration and motility. Testicular spermatogenesis, the apoptotic index of germ cells, and the expression of a meiotic-specific gene were also analyzed. RESULTS: The treatment group receiving shock wave intensity at 0.55 mJ/mm 2 showed a significant decrease in sperm concentration, motility, and Johnsen score as compared to other groups. The apoptotic index of spermatogenic cells increased as the intensity of the shock wave treatment escalated, and reach a statistically significant difference at 4 weeks posttreatment. Treating testes with intensity levels of 0.55 mJ/mm 2 at 3 Hz interfere with the quality or quantity of spermatogenesis and also increases in spermatogenic cell apoptosis, whereas the expression of the SYCP3 gene significantly decreased after treatment with intensity levels of 0.10 mJ/mm 2 , 0.15 mJ/mm 2 , and 0.35 mJ/mm 2 at 4 Hz. CONCLUSION: Treating testes with intensity levels of 0.55 mJ/mm 2 at 3 Hz interfere with the quality or quantity of spermatogenesis and also increases spermatogenic cell apoptosis, whereas the expression of the SYCP3 gene significantly decreased after treatment with intensity levels of 0.10 mJ/mm 2 , 0.15 mJ/mm 2 , and 0.35 mJ/mm 2 at 4 Hz.

Novel regulation of PKC-induced inflammation by Akt and protein phosphatase 2A in ovarian granulosa cells.

PKC-mediated inflammation is important in ovarian physiology. The roles of Akt and protein phosphatase 2A (PP2A) in PKC-mediated inflammation in ovarian granulosa cells (GCs) remain mostly unclear. PKC activator phorbol 12-myristate 13-acetate induced the Akt phosphorylation in rat primary GCs but reduced the Akt phosphorylation in KGN human GCs. In rat GCs, an inhibitory effect of PI3K inhibitor wortmannin and a stimulatory effect of Akt activator SC79 on PKC-induced cyclooxygenase-2 (COX-2)/PGE2production were noted; wortmannin and SC79 acted oppositely in human GCs. In rat GCs, PP2A inhibitor okadaic acid further enhanced the PKC-mediated promoter activation and elevation of mRNA and protein levels of the COX-2 gene, whereas PP2A activator sodium selenate attenuated the PKC-mediated COX-2 expression and promoter activation. PKC activation did not affect PP2A phosphorylation, but okadaic acid indeed augmented the PKC-induced NF-κB nuclear translocation. Thus, PP2A appears to act as a negative modulator in PKC-mediated cellular inflammation in rat GCs, at least in part due to its attenuating effect on the PKC-induced NF-κB activation.

Glucosamine Enhancement of BDNF Expression and Animal Cognitive Function.

Brain-derived neurotrophic factor (BDNF) is an important factor for memory consolidation and cognitive function. Protein kinase A (PKA) signaling interacts significantly with BDNF-provoked downstream signaling. Glucosamine (GLN), a common dietary supplement, has been demonstrated to perform a variety of beneficial physiological functions. In the current study, an in vivo model of 7-week-old C57BL/6 mice receiving daily intraperitoneal injection of GLN (0, 3, 10 and 30 mg/animal) was subjected to the novel object recognition test in order to determine cognitive performance. GLN significantly increased cognitive function. In the hippocampus GLN elevated tissue cAMP concentrations and CREB phosphorylation, and upregulated the expression of BDNF, CREB5 and the BDNF receptor TrkB, but it reduced PDE4B expression. With the in vitro model in the HT22 hippocampal cell line, GLN exposure significantly increased protein and mRNA levels of BDNF and CREB5 and induced cAMP responsive element (CRE) reporter activity; the GLN-mediated BDNF expression and CRE reporter induction were suppressed by PKA inhibitor H89. Our current findings suggest that GLN can exert a cognition-enhancing function and this may act at least in part by upregulating the BDNF levels via a cAMP/PKA/CREB-dependent pathway.

Glucosamine regulation of fibroblast growth factor 21 expression in liver and adipose tissues.

Obesity is associated with metabolic disorders. Fibroblast growth factor 21 (FGF21) has been recognized as important in metabolism. Glucosamine (GLN) has been demonstrated to perform diverse beneficial functions. This study aimed to reveal whether and how GLN would modulate FGF21 production in relation to metabolism. With in vivo model of normal diet (ND) and high-fat diet (HFD) mice receiving GLN injection and in vitro model of mouse AML12 liver cells and differentiated 3T3L1 adipocytes challenged with GLN, GLN appeared to improve the glucose metabolism in HFD and ND mice and to elevate FGF21 protein expression in HFD liver and to increase both FGF21 protein and mRNA levels in WAT from HFD and ND mice and it also upregulated FGF21 expression in both AML12 and differentiated 3T3L1 cells. By using inhibitors against various signaling pathways, p38, Akt, NF-κB, and PKA appeared potentially involved in GLN-mediated FGF21 production in AML12 cells; GLN was able to mediate activation of NF-κB, p38 or PKA/CREB signaling. Our accumulated findings suggest that GLN may potentially improve the metabolic performance by inducing FGF21 production in liver and adipose tissues and such induction in liver cells may act in part due to GLN induction of the NF-κB, p38 and PKA pathways.

Regulation of the regulator of G protein signaling 2 expression and cellular localization by PKA and PKC pathways in mouse granulosa cells.

G protein-coupled receptor (GPCR) activation-mediated PKA and PKC pathways have been recognized to be important in ovarian physiology. Expression of regulator of G-protein signaling 2 (RGS2) has been reported in ovarian granulosa cells. The detailed mechanisms in PKA- and PKC-regulated RGS2 expression and cellular translocation in granulosa cells remain mostly unclear. PKA activator 8-bromo-cAMP and PKC activator phorbol-12, 13-didecanoate appeared to rapidly elevate both protein and mRNA levels and promoter activation of RGS2 gene. Two consensus Sp1 elements within the shortest 78 bp fragment of RGS2 promoter sequence were essential for the full responsiveness to PKA and PKC. PKC activation appeared to increase the RGS2 translocation from nucleus to cytosol. PKA- and PKC-mediated RGS2 transcription in a Sp-1-dependent manner and a PKC-mediated RGS2 intracellular translocation were noted in granulosa cells.

Novel augmentation by bufalin of protein kinase C-induced cyclooxygenase-2 and IL-8 production in human breast cancer cells.

Cyclooxygenase-2 (COX-2) and IL-8 are two inflammatory mediators induced by protein kinase C (PKC) via various stimuli. Both contribute significantly to cancer progression. Bufalin, a major active component of the traditional Chinese medicine Chan Su, is known to induce apoptosis in various cancer cells. This study clarifies the role and mechanism of bufalin action during PKC regulation of COX-2/IL-8 expression and investigates the associated impact on breast cancer. Using MB-231 breast cancer cells, bufalin augments PKC induction of COX-2/IL-8 at both the protein and mRNA levels, and the production of prostaglandin E2 (PGE2) and IL-8. The MAPK and NF-κB pathways are involved in both the PKC-mediated and bufalin-promoted PKC regulation of COX-2/IL-8 production. Bufalin increases PKC-induced MAPKs phosphorylation and NF-κB nuclear translocation. PGE2 stimulates the proliferation/migration of breast cancer cells. Furthermore, PKC-induced matrix metalloproteinase 3 expression is enhanced by bufalin. Bufalin significantly enhances breast cancer xenograft growth, which is accompanied by an elevation in COX-2/IL-8 expression. In conclusion, bufalin seems to promote the inflammatory response in vitro and in vivo, and this occurs, at least in part, by targeting the MAPK and NF-κB pathways, which then enhances the growth of breast cancer cells.

Growth Hormone Releasing Peptide-2 Attenuation of Protein Kinase C-Induced Inflammation in Human Ovarian Granulosa Cells.

Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two important inflammatory mediators in ovulation. Ghrelin may modulate inflammatory signaling via growth hormone secretagogue receptors. We investigated the role of ghrelin in KGN human ovarian granulosa cells using protein kinase C (PKC) activator phorbol 12, 13-didecanoate (PDD) and synthetic ghrelin analog growth hormone releasing peptide-2 (GHRP-2). GHRP-2 attenuated PDD-induced expression of protein and mRNA, the promoter activity of COX-2 and IL-8 genes, and the secretion of prostaglandin E2 (PGE2) and IL-8. GHRP-2 promoted the degradation of PDD-induced COX-2 and IL-8 proteins with the involvement of proteasomal and lysosomal pathways. PDD-mediated COX-2 production acts via the p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways; PDD-mediated IL-8 production acts via the p38, JNK and ERK pathways. GHRP-2 reduced the PDD-induced phosphorylation of p38 and JNK and activator protein 1 (AP-1) reporter activation and PDD-induced NF-κB nuclear translocation and reporter activation. The inhibitors of mitogen-activated protein kinase phosphatase-1 (MKP-1) and protein phosphatase 2 (PP2A) reduced the inhibitory effect of GHRP-2 on PDD-induced COX-2 and IL-8 expression. Our findings demonstrate an anti-inflammatory role for ghrelin (GHRP-2) in PKC-mediated inflammation of granulosa cells, at least in part, due to its inhibitory effect on PKC-induced activation of p38, JNK and NF-κB, possibly by targeting to MKP-1 and PP2A.

Novel effects of the cyclooxygenase-2-selective inhibitor NS-398 on IL-1ß-induced cyclooxygenase-2 and IL-8 expression in human ovarian granulosa cells.

Ovulation is a critical inflammation-like event that is central to ovarian physiology. IL-1ß is an immediate early pro-inflammatory cytokine that regulates production of several other inflammatory mediators, such as cyclooxygenase 2 (COX)-2 and IL-8. NS-398 is a selective inhibitor of COX-2 bioactivity and thus this drug is able to mitigate the COX-2-mediated production of downstream prostaglandins and the subsequent inflammatory response. Here we have investigated the action of NS-398 using a human ovarian granulosa cell line, KGN, by exploring IL-1ß-regulated COX-2 and IL-8 expression. First, NS-398, instead of reducing inflammation, appeared to further enhance IL-1ß-mediated COX-2 and IL-8 production. Using selective inhibitors targeting various signaling molecules, MAPK and NF-κB pathways both seemed to be involved in the impact of NS-398 on IL-1ß-induced COX-2 and IL-8 expression. NS-398 also promoted IL-1ß-mediated NF-κB p65 nuclear translocation but had no effect on IL-1ß-activated MAPK phosphorylation. Flow cytometry analysis demonstrated that NS-398, in combination with IL-1ß, significantly enhanced cell cycle progression involving IL-8. Our findings demonstrate a clear pro-inflammatory function for NS-398 in the IL-1ß-mediated inflammatory response of granulosa cells, at least in part, owing to its augmenting effect on the IL-1ß-induced activation of NF-κB.

Statins are associated with a reduced risk of cholangiocarcinoma: a population-based case-control study.

AIMS: Cholangiocarcinoma (CCA) is the second most common primary liver cancer in the world. Due to the lack of effective treatments, the survival rate of CCA is low and it is usually considered difficult to diagnose early. To date, no effective strategies for the prevention of CCA have been developed. Statins are cholesterol-lowering agents which possess pleiotropic properties and the use of statins may reduce cancer risk. The aim of the study was to investigate the effect of statin use on the risk of CCA. METHODS: We used nationwide insurance data to perform a case-control study including 3174 CCA patients diagnosed in 2002-2011 and 3174 propensity score matched controls. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated to assess the association between CCA risk and statin use by type of statin and dose. RESULTS: Patients with CCA were slightly younger than controls with mean ages of 67.4 (SD 12.3) and 68.5 (SD 13.2) years (P = 0.001), respectively, and had less users of statins (22.7 vs. 26.5%, P < 0.001). The overall adjusted OR of statin use associated CCA was 0.80 (95% CI 0.71, 0.90) and lowered for those with longer medications. The OR ranged from 0.65 to 0.77. Stronger dose-response association was seen when using lovastatin. CONCLUSIONS: Statin use is associated with reduced risk of CCA and there is a dose-response relationship between the use of statins and risk of CCA.

Attenuation of LPS-induced cyclooxygenase-2 and inducible NO synthase expression by lysophosphatidic acid in macrophages.

LPS can activate the inflammatory cascades by inducing various inflammatory mediators, such as prostaglandin E(2) (PGE(2)) resulting from cyclooxygenase-2 (COX-2), and NO produced by inducible NO synthase (iNOS). Lysophosphatidic acid (LPA) has been demonstrated to participate in inflammation. This study aimed to clarify the impact and the involving mechanisms of LPA on LPS-incurred inflammation in macrophages. First, LPA appeared to attenuate LPS-induced protein and mRNA expression of COX-2 and iNOS genes, as well as production of PGE(2) and NO. By using selective inhibitors targeting various signaling players, the inhibitory G protein alpha subunit (Gα(i)) seemed to be involved in the effect of LPA; p38, ERK and NF-κB were involved in the LPS-mediated COX-2/PGE(2) pathway; and p38, JNK, phosphoinositide-3-kinase and NF-κB were involved in the LPS-mediated iNOS/NO pathway. LPA was able to diminish LPS-induced phosphorylation of p38 and Akt, as well as NF-κB p65 nuclear translocation. By utilization of inhibitors of COX-2 and iNOS, there appeared to be no modulation between the COX-2/PGE(2) and the iNOS/NO signaling pathways. Our findings demonstrate a clear anti-inflammatory role of LPA acting via Gα(i) in LPS-mediated inflammatory response in macrophages, owing, at least in part, to its suppressive effect on LPS-induced activation of p38, Akt and NF-κB.

Inhibition of PKC-Induced COX-2 and IL-8 Expression in Human Breast Cancer Cells by Glucosamine.

Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti-inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC-regulation of COX-2/IL-8 expression and the associated impact on breast cancer. In MCF-7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX-2 and IL-8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E(2) (PGE(2)) and IL-8. Glucosamine is able to promote COX-2 protein degradation in a calpain-dependent manner and IL-8 protein degradation in calpain-dependent and proteasome-dependent manners. The MAPK and NF-κB pathways are involved in PKC-induced COX-2 expression, but only the NF-κB pathway is involved in PKC-induced IL-8 expression. Glucosamine attenuates PKC-mediated IκBα phosphorylation, nuclear NF-κB translocation, and NF-κB reporter activation. Both PGE(2) and IL-8 promote cell proliferation and IL-8 induces cell migration; thus, glucosamine appears to suppress PKC-induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX-2 and IL-8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF-κB signaling pathway, resulting in an inhibition of breast cancer cell growth.

Glucosamine attenuates cigarette smoke-induced lung inflammation by inhibiting ROS-sensitive inflammatory signaling.

Cigarette smoking causes persistent lung inflammation that is mainly regulated by redox-sensitive pathways. We have reported that cigarette smoke (CS) activates a NADPH oxidase-dependent reactive oxygen species (ROS)-sensitive AMP-activated protein kinase (AMPK) signaling pathway leading to induction of lung inflammation. Glucosamine, a dietary supplement used to treat osteoarthritis, has antioxidant and anti-inflammatory properties. However, whether glucosamine has similar beneficial effects against CS-induced lung inflammation remains unclear. Using a murine model we show that chronic CS exposure for 4 weeks increased lung levels of 4-hydroxynonenal (an oxidative stress biomarker), phospho-AMPK, and macrophage inflammatory protein 2 and induced lung inflammation; all of these CS-induced events were suppressed by chronic treatment with glucosamine. Using human bronchial epithelial cells, we demonstrate that cigarette smoke extract (CSE) sequentially activated NADPH oxidase; increased intracellular levels of ROS; activated AMPK, mitogen-activated protein kinases (MAPKs), nuclear factor-κB (NF-κB), and signal transducer and activator of transcription proteins 3 (STAT3); and induced interleukin-8 (IL-8). Additionally, using a ROS scavenger, a siRNA that targets AMPK, and various pharmacological inhibitors, we identified the signaling cascade that leads to induction of IL-8 by CSE. All these CSE-induced events were inhibited by glucosamine pretreatment. Our findings suggest a novel role for glucosamine in alleviating the oxidative stress and lung inflammation induced by chronic CS exposure in vivo and in suppressing the CSE-induced IL-8 in vitro by inhibiting both the ROS-sensitive NADPH oxidase/AMPK/MAPK signaling pathway and the downstream transcriptional factors NF-κB and STAT3.

Attenuation of LPS-induced lung inflammation by glucosamine in rats.

Acute inflammation is often observed during acute lung injury (ALI) and acute respiratory distress syndrome. Glucosamine is known to act as an anti-inflammatory molecule. The effects of glucosamine on acute lung inflammation and its associated mechanisms remain unclear. The present study sought to address how glucosamine plays an anti-inflammatory role in acute lung inflammation in vivo and in vitro. Using the LPS intratracheal instillation-elicited rat lung inflammation model, we found that glucosamine attenuated pulmonary edema and polymorphonuclear leukocyte infiltration, as well as the production of TNF-α, IL-1ß, cytokine-induced neutrophil chemoattractant (CINC)-1, macrophage inflammatory protein (MIP)-2, and nitric oxide (NO) in the bronchoalveolar lavage fluid (BALF) and in the cultured medium of BALF cells. The expression of TNF-α, IL-1ß, IFN-γ, CINC-1, MIP-2, monocyte chemotactic protein-1, and inducible NO synthase (iNOS) in LPS-inflamed lung tissue was also suppressed by glucosamine. Using the rat alveolar epithelial cell line L2, we noted that the cytokine mixture (cytomix)-regulated production and mRNA expression of CINC-1 and MIP-2, NO production, the protein and mRNA expression of iNOS, iNOS mRNA stability, and iNOS promoter activity were all inhibited by glucosamine. Furthermore, glucosamine reduced LPS-mediated NF-κB signaling by decreasing IκB phosphorylation, p65 nuclear translocation, and NF-κB reporter activity. Overexpression of the p65 subunit restored the inhibitory action of glucosamine on cytomix-regulated NO production and iNOS expression. In conclusion, glucosamine appears to act as an anti-inflammatory molecule in LPS-induced lung inflammation, at least in part by targeting the NF-κB signaling pathway.

Cell cycle regulation by glucosamine in human pulmonary epithelial cells.

Airway epithelial cells play an important role against intruding pathogens. Glucosamine, a commonly used supplemental compound, has recently begun to be regarded as a potential anti-inflammatory molecule. This study aimed to uncover how glucosamine impacts on cellular proliferation in human alveolar epithelial cells (A549) and bronchial epithelial cells (HBECs). With trypan blue-exclusion assay, we observed that glucosamine (10, 20, 50 mM) caused a decrease in cell number at 24 and 48 h; with a flow cytometric analysis, we also noted an enhanced cell accumulation within the G(0)/G(1) phase at 24 h and induction of late apoptosis at 24 and 48 h by glucosamine (10, 20, 50 mM) in A549 cells and HBECs. Examination of phosphorylation in retinoblastoma (Rb) protein, we found an inhibitory effect by glucosamine at 20 and 50 mM. Glucosamine at 50 mM was demonstrated to elevate both the mRNA and protein expression of p53 and heme oxygenase-1 (HO-1), but also caused a reduction in p21 protein expression. In addition, glucosamine attenuated p21 protein stability via the proteasomal proteolytic pathway, as well as inducing p21 nuclear accumulation. Altogether, our results suggest that a high dose of glucosamine may inhibit cell proliferation through apoptosis and disturb cell cycle progression with a halt at G(0)/G(1) phase, and that this occurs, at least in part, by a reduction in Rb phosphorylation together with modulation of p21, p53 and HO-1 expression, and nuclear p21 accumulation.

Novel role of RGS2 in regulation of antioxidant homeostasis in neuronal cells.

Regulator of G-protein signaling protein (RGS)-2 is a modulator of anxiety and dysregulation of oxidative stress is implicated in anxiety. Also, RGS2 expression is reported to be induced by oxidative stress. Thus, if oxidative stress induces RGS2 expression and lack of RGS2 causes anxiety, then mechanisms that link RGS2 and oxidative stress potentially critical to anxiety must be revealed. Our study is the first to suggest role of RGS2 in regulation of enzymes involved in antioxidant defense namely glyoxalase-1 and glutathione reductase-1 via activation of p38 MAPK and PKC pathways in an Sp-1 dependent manner.

Novel role of AMP-activated protein kinase signaling in cigarette smoke induction of IL-8 in human lung epithelial cells and lung inflammation in mice.

Cigarette smoke (CS) increases chemokine production in lung epithelial cells (LECs), but the pathways involved are not completely understood. AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, may modulate inflammation. Here, we show that cigarette smoke extract sequentially activated NADPH oxidase; increased intracellular reactive oxygen species (ROS) level; activated AMPK, NF-κB, and STAT3; and induced interleukin 8 (IL-8) in human LECs. Inhibition of NADPH oxidase activation by apocynin or siRNA targeting p47(phox) (a subunit of NADPH oxidase) attenuated the increased intracellular ROS level, AMPK activation, and IL-8 induction. Removal of intracellular ROS by N-acetylcysteine reduced the AMPK activation and IL-8 induction. Prevention of AMPK activation by Compound C or AMPK siRNA lessened the activation of both NF-κB and STAT3 and the induction of IL-8. Abrogation of the activation of NF-κB and STAT3 by BAY11-7085 and AG490, respectively, attenuated the IL-8 induction. We additionally show that chronic CS exposure in mice promoted AMPK phosphorylation and expression of MIP-2α (an IL-8 homolog) in LECs and lungs, as well as lung inflammation, all of which were reduced by Compound C treatment. Thus, a novel NADPH oxidase-dependent, ROS-sensitive AMPK signaling is important for CS-induced IL-8 production in LECs and possibly lung inflammation.

α-Lipoic acid ameliorates foam cell formation via liver X receptor α-dependent upregulation of ATP-binding cassette transporters A1 and G1.

α-Lipoic acid (α-LA), a key cofactor in cellular energy metabolism, has protective activities in atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined whether α-LA affects foam cell formation and its underlying molecular mechanisms in murine macrophages. Treatment with α-LA markedly attenuated oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, which was due to increased cholesterol efflux. Additionally, α-LA treatment dose-dependently increased protein levels of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 but had no effect on the protein expression of SR-A, CD36, or SR-BI involved in cholesterol homeostasis. Furthermore, α-LA increased the mRNA expression of ABCA1 and ABCG1. The upregulation of ABCA1 and ABCG1 by α-LA depended on liver X receptor α (LXRα), as evidenced by an increase in the nuclear levels of LXRα and LXRE-mediated luciferase activity and its prevention of the expression of ABCA1 and ABCG1 after inhibition of LXRα activity by the pharmacological inhibitor geranylgeranyl pyrophosphate (GGPP) or knockdown of LXRα expression with small interfering RNA (siRNA). Consistently, α-LA-mediated suppression of oxLDL-induced lipid accumulation was abolished by GGPP or LXRα siRNA treatment. In conclusion, LXRα-dependent upregulation of ABCA1 and ABCG1 may mediate the beneficial effect of α-LA on foam cell formation.

EGb761 ameliorates the formation of foam cells by regulating the expression of SR-A and ABCA1: role of haem oxygenase-1.

AIMS: Accumulation of foam cells in the intima is a hallmark of early-stage atherosclerotic lesions. Ginkgo biloba extract (EGb761) has been reported to exert anti-oxidative and anti-inflammatory properties in atherosclerosis, yet the significance and the molecular mechanisms of action of EGb761 in the formation of macrophage foam cells are not fully understood. METHODS AND RESULTS: Treatment with EGb761 resulted in a dose-dependent decrease in oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, a consequence that was due to a decrease in cholesterol uptake and an increase in cholesterol efflux. Additionally, EGb761 significantly down-regulated the mRNA and protein expression of class A scavenger receptor (SR-A) by decreasing expression of activator protein 1 (AP-1); however, EGb761 increased the protein stability of ATP-binding cassette transporter A1 (ABCA1) by reducing calpain activity without affecting ABCA1 mRNA expression. Small interfering RNA (siRNA) targeting haem oxygenase-1 (HO-1) abolished the EGb761-induced protective effects on the expression of AP-1, SR-A, ABCA1, and calpain activity. Accordingly, EGb761-mediated suppression of lipid accumulation in foam cells was also abrogated by HO-1 siRNA. Moreover, the lesion size of atherosclerosis was smaller in EGb761-treated, apolipoprotein E-deficient mice compared with the vehicle-treated mice, and the expression of HO-1, SR-A, and ABCA1 in aortas was modulated similar to that observed in macrophages. CONCLUSION: These findings suggest that EGb761 confers a protection from the formation of foam cells by a novel HO-1-dependent regulation of cholesterol homeostasis in macrophages.

Glucosamine regulation of LPS-mediated inflammation in human bronchial epithelial cells.

Inflammation is a complex process involving cytokine production to regulate host defense cascades in order to clear pathogenic agents. Upregulation of inflammatory cytokines, such as IL-6 and IL-8 by bacteria infection, occurs in pulmonary tissues and has been demonstrated to be critical to the lung inflammatory response. Glucosamine, primarily identified as an anti-arthritis supplement, has been also regarded as a potential anti-inflammatory agent. Thus we hypothesized that lipopolysaccharide (LPS) would activate IL-6 and IL-8 expressions in human primary bronchial epithelial cells and glucosamine could attenuate such an effect. The RT-PCR, real-time PCR, and ELISA analyses demonstrated that LPS-induced mRNAs encoding IL-6 and IL-8 and the subsequent secretion of IL-6 and IL-8 were inhibited by glucosamine treatment. MTT, alamarBlue, and annexin V apoptosis assays all suggested that this inhibition effect was not due to a cytotoxic effect mediated by glucosamine. Using the inhibitors of the MAP kinases and NFkappaB, it was revealed that p38, JNK and ERK, as well as NFkappaB, are all involved in LPS-induced IL-8 secretion; however only p38 is involved in LPS-induced IL-6 secretion. Immunoblot analysis further demonstrated that LPS-mediated phosphorylation of JNK and ERK, but not the LPS-induced NFkappaB translocation, was inhibited by glucosamine. Altogether, our results indicate that glucosamine can potently suppress LPS-induced inflammatory cytokine expression, at least in part via attenuation of MAPK activation.