Effect of chemical interaction between oleic acid and L-Arginine on oral perception, as a function of polymorphisms of CD36 and OBPIIa and genetic ability to taste 6-n-propylthiouracil.

Oral sensitivity to fats varies in individuals influencing nutritional status and health. Variations in oleic acid perception are associated with CD36 and odorant binding protein (OBPIIa) polymorphisms, and 6-n-propylthiouracil (PROP) sensitivity, which is mediated by TAS2R38 receptor. L-Arginine (L-Arg) supplementation was shown to modify the perception of the five taste qualities. Here we analyzed the effect of three concentrations (5, 10, 15 mmol/L) of L-Arg on oral perception of oleic acid in forty-six subjects classified for PROP taster status and genotyped for TAS2R38, CD36 and OBPIIa polymorphisms. L-Arg supplementation was effective in increasing the perceived intensity of oleic acid in most subjects. The lowest concentration was the most effective, especially in PROP non-tasters or medium tasters, and in subjects with at least an allele A in CD36 and OBPIIa loci. Density Functional Theory (DFT) calculations were exploited to characterize the chemical interaction between L-Arg and oleic acid, showing that a stable 1:1 oleate·ArgH+ adduct can be formed, stabilized by a pair of hydrogen bonds. Results indicate that L-Arg, acting as a 'carrier' of fatty acids in saliva, can selectively modify taste response, and suggest that it may to be used in personalized dietetic strategies to optimize eating behaviors and health.

Improvement of implantation potential in mouse blastocysts derived from IVF by combined treatment with prolactin, epidermal growth factor and 4-hydroxyestradiol.

STUDY QUESTION: Can supplementation of medium with prolactin (PRL), epidermal growth factor (EGF) and 4-hydroxyestradiol (4-OH-E2) prior to embryo transfer improve implantation potential in mouse blastocysts derived from IVF? SUMMARY ANSWER: Combined treatment with PRL, EGF and 4-OH-E2 improves mouse blastocyst implantation rates, while alone, each factor is ineffective. WHAT IS KNOWN ALREADY: Blastocyst dormancy during delayed implantation caused by ovariectomy is maintained by continued progesterone treatment in mice, and estrogen injection rapidly activates blastocysts to implantation-induced status in vivo. While the expression of many proteins is upregulated in implantation-induced blastocysts, selective proteolysis by proteasomes, such as estrogen receptor α (ESR1), occurs in implantation-induced blastocysts to achieve implantation-competent status. It is worth evaluating the proteins expressed during these periods to identify humoral factors that might improve the implantation potential of IVF-derived blastocysts because the poor quality of embryos obtained by IVF is one of the major causes of implantation failure. STUDY DESIGN, SIZE, DURATION: Superovulated oocytes from ICR mice were fertilized with spermatozoa and then cultured in vitro in potassium simplex optimized medium (KSOM) without phenol red (KSOM-P) for 90-96 h. Blastocysts were treated with PRL (10 or 20 mIU/mL), EGF (5 or 10 ng/mL) or 4-OH-E2 (1 or 10 nM) in KSOM-P for 24 h. PARTICIPANTS/MATERIALS, SETTING, METHODS: Levels of breast cancer 1 (BRCA1), EGF receptor (EGFR, also known as ERBB1), ERBB4, tubulointerstitial nephritis antigen-like 1 (TINAGL1) and ESR1 protein were examined with immunohistochemical analysis using immunofluorescence methods and confocal laser scanning microscopy. For embryo transfer, six blastocysts were suspended in HEPES-buffered KSOM-P medium and transferred into the uteri of recipient mice on the morning of Day 4 (0900-1000 h) of pseudopregnancy (Day 1 = vaginal plug). The number of implantation sites was then recorded on Day 6 using the blue dye method. MAIN RESULTS AND THE ROLE OF CHANCE: PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the trophectoderm (TE). While PRL treatment resulted in an increase in EGFR, EGF increased both EGFR and ERBB4 in the blastocyst TE. TINAGL1 in the TE was enhanced by 4-OH-E2, which also increased localization of this protein to the basement membrane. Treatment with PRL, EGF or 4-OH-E2 alone did not improve blastocyst implantation rates. Combined treatment with PRL, EGF and 4-OH-E2 resulted in increased levels of EGFR, ERBB4, TINAGL1 and BRCA1 in the TE, whereas ESR1 was not upregulated in the treated blastocysts. Furthermore, combined treatment with PRL, EGF and 4-OH-E2 improved blastocyst implantation rates versus control (P = 0.009). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: Our studies were carried out in a mouse model, and the conclusions were drawn from limited results obtained from one species. Whether the increase in EGFR, ERBB4 and TINAGL1 protein in the TE improves implantation potential of blastocysts needs to be further studied experimentally by assessing other expressed proteins. The influence of combined supplementation in vitro of PRL, EGF and 4-OH-E2 on implantation also requires further examination and optimization in human blastocysts before it can be considered for clinical use in ART. WIDER IMPLICATIONS OF THE FINDINGS: Enhanced implantation potential by combined treatment with PRL, EGF and 4-OH-E2 appears to result in the upregulation of at least two distinct mechanisms, namely signaling via EGF receptors and basement membrane formation during the peri-implantation period in mice. While PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the TE, treatment with each alone did not improve blastocyst implantation. Therefore, BRCA1 protein appears to be unnecessary for the attachment reaction in blastocysts in mice Combined supplementation of PRL, EGF and 4-OH-E2 might also be of relevance for embryo transfer of human IVF-derived blastocysts for ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported in part by the JSPS KAKENHI [Grant numbers 22580316 and 25450390 (to H.M.)] and the Joint Research Project of Japan-U.S. Cooperative Science Program (to H.M.). The authors have no conflict of interest to declare.

Targeting arachidonic acid pathway to prevent programmed hypertension in maternal fructose-fed male adult rat offspring.

Hypertension can be programmed in response to nutritional insults in early life. Maternal high-fructose (HF) intake induced programmed hypertension in adult male offspring, which is associated with renal programming and arachidonic acid metabolism pathway. We examined whether early treatment with a soluble epoxide hydrolase (SEH) inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) or 15-Deoxy-Δ12,14-prostagandin J2 (15dPGJ2) can prevent HF-induced programmed hypertension. Pregnant Sprague Dawley rats received regular chow or chow supplemented with fructose (60% diet by weight) during the whole period of pregnancy and lactation. Four groups of male offspring were studied: control, HF, HF+AUDA and HF+15dPGJ2. In HF+AUDA group, mother rats received AUDA 25 mg/L in drinking water during lactation. In the HF+15dPGJ2 group, male offspring received 15dPGJ2 1.5 mg/kg body weight by subcutaneous injection once daily for 1 week after birth. Rats were sacrificed at 12 weeks of age. Maternal HF-induced programmed hypertension is associated with increased renal protein level of SEH and oxidative stress, which early AUDA therapy prevents. Comparison of AUDA and 15dPGJ2 treatments demonstrated that AUDA was more effective in preventing HF-induced programmed hypertension. AUDA therapy increases angiotensin converting enzyme-2 (ACE2) protein levels and PGE2 levels in adult offspring kidney exposed to maternal HF. 15dPGJ2 therapy increases plasma asymmetric dimethylarginine (ADMA) levels and decreases L-arginine-to-ADMA ratio. Better understanding of the impact of arachidonic acid pathway, especially inhibition of SEH, on renal programming may aid in developing reprogramming strategy to prevent programmed hypertension in children exposed to antenatal HF intake.

Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.

Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH2, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 µM in the LAD2 cell assay.

Epigenetic induction of epithelial to mesenchymal transition by LCN2 mediates metastasis and tumorigenesis, which is abrogated by NF-κB inhibitor BRM270 in a xenograft model of lung adenocarcinoma.

Tumor initiating cancer stem-like cells (TICSCs) have recently become the object of intensive study. Human-Lipocalin-2 (hLCN2) acts as a biomarker for cancers. The aim of the present study was to explore new insights regarding the potential role of LCN2 in inducing epithelial to mesenchymal transition (EMT) by transfecting LCN2 into CD133+-A549-TICSCs and its cross-talk with the NF-κB signaling pathway in adenocarcinoma of the lung. Furthermore, EMT was confirmed by transcriptomic analysis, immunoblotting and immunocyto/histochemical analyses. Tumorigenesis and metastasis were confirmed by molecular therapeutics tracer 2DG infrared optical probe in BALB/cSIc-nude mice. It was observed that the CD133+-expressing-LCN2-A549 TICSCs population increased in adenocarcinoma of the lung compared to the normal lung tissue. The expressions of genes involved in stemness, adhesion, motility and drug efflux was higher in these cells than in their non-LCN2 expressing counterparts. The present study revealed that elevated expression of LCN2 significantly induced metastasis via EMT. Overexpression of LCN2 significantly increased stemness and tumor metastasis by modulating NF-κB cellular signaling. BRM270, a novel inhibitor of NF-κB plays a significant role in the EMT reversal. BRM270, a naturaceutical induces cell shrinkage, karyorrhexis and programmed cell death (PCD) which were observed by Hoechst 33342 staining while flow cytometry analysis showed significant (P<0.05) decrease in cell population from G0-G1 phases. Also, 2DG guided in vivo model revealed that BRRM270 significantly (P<0.0003) reduced tumor metastasis and increased percent survival in real-time with complete resection. An elaborate study on the novel concept with respect to linking of naturaceutics as selective and potential anticancer agent that eliminates the elevated LCN2 induced EMT and tumor dissemination through cooperation with the NF-κB signaling as the baseline data for the planning of new therapeutic strategies was conducted for the first time. Our results also illustrate a molecular mechanistic approach for 2DG-guided molecular imaging-based cancer therapy using BRM270 as a novel cancer therapeutic drug to enhance the effect of doxorubicin (Dox)-resistant LCN2 induced metastasis of solid tumors in nude mice.

Growth inhibitory effect of polyunsaturated fatty acids (PUFAs) on colon cancer cells via their growth inhibitory metabolites and fatty acid composition changes.

BACKGROUND: Colorectal cancer is common. Polyunsaturated fatty acids (PUFAs) exert growth-inhibitory and pro-apoptotic effects on colon cancer cells. Metabolites of PUFAs such as prostaglandins (PGs), leukotrienes (LTs) and lipoxins (LXs) play a significant role in colon cancer. METHODS: Human colon cancer LoVo and RKO cells were cultured with different concentration of PUFAs and 5-fluorouracil (5-FU) in vitro. Cell morphological changes, fatty acid composition, formation of PGE2, LTB4 and LXA4 and expression of COX-2, ALOX5, PGD synthase (PGDS), microsomal prostaglandin E synthase (mPGES) were assessed in LoVo and RKO cells when supplemented with PUFAs and 5-FU. RESULTS: PUFAs and 5-FU inhibited growth of LoVo and RKO cells to the same extent at the doses used and produced significant alterations in their shape. As expected, higher concentrations of supplemented PUFAs were noted in the cells compared to control. LA, GLA, AA, ALA and EPA supplementation to LoVo cells suppressed production of PGE2, LTB4,and ALOX5, mPGES expression, but enhanced that of LXA4; whereas DHA enhanced PGE2 and LXA4 synthesis but decreased LTB4 formation and COX-2, ALOX5, mPGES expression. In contrast, 5-FU enhanced formation of PGE2, LTB4 and mPGES expression, but suppressed LXA4 synthesis and COX-2 expression. PGE2, LTB4 synthesis and ALOX5 expression was suppressed by LA, GLA, ALA and DHA; whereas AA, EPA and 5-FU enhanced PGE2 but paradoxically AA decreased and EPA and 5-FU enhanced LTB4 synthesis in RKO cells. All the PUFAs tested enhanced, while 5-FU decreased LXA4 formation in RKO cells; whereas GLA, AA, and 5-FU augmented while LA, ALA, EPA and DHA enhanced COX-2 expression in RKO cells. CONCLUSIONS: Tumoricidal action of PUFAs on colorectal LoVo and RKO cancer cells in vitro was associated with increased formation of LXA4, decreased synthesis of PGE2 and LTB4 and suppressed expression of COX-2, ALOX5, mPGES, whereas 5-FU produced contrasting actions on these indices.

A temperature induced lipocalin gene from Medicago falcata (MfTIL1) confers tolerance to cold and oxidative stress.

Temperature-induced lipocalins (TIL) are plasmalemma-localized proteins and responsive to environmental stresses. Physiological functions of MfTIL1 from Medicago sativa subsp. falcata (L.) Arcang. (hereafter falcata), a forage legume with cold and drought tolerance, were investigated in this study. MfTIL1 expression was greatly induced by 4-96 h of cold treatment, while transcript levels of the orthologs in Medicago truncatula, a model legume plant with lower cold tolerance than falcata, were reduced or not altered within 48-96 h. MfTIL1 expression was not responsive to dehydration and salinity. Compared to the wild type, transgenic tobacco plants overexpressing MfTIL1 had lower temperature (LT50) that resulted in 50 % lethal and elevated survival rate in response to freezing, elevated F v/F m and decreased ion leakage after treatments with chilling, high light and methyl viologen (MV). H2O2 and O2 (-) were less accumulated in transgenic plants than in the wild type after treatments with chilling, high light and MV, while antioxidant enzyme activities showed no difference between the two types of plants prior to or following treatments. Higher transcript levels of NtDREB3 and NtDREB4 genes were observed in transgenic plants than in the wild type under non-stressed conditions, but higher transcript levels of NtDREB1, NtDREB2, NtDREB4 and NtCOR15a genes under chilling conditions. It is suggested that MfTIL1 plays an important role in plant tolerance to cold and oxidative stress through promoted scavenging of reactive oxygen species and up-regulating expression of multiple cold responsive genes.

Protective Effects of Quercetin Against HgCl2-Induced Nephrotoxicity in Sprague-Dawley Rats.

Mercury is a well-known environmental pollutant that can cause nephropathic diseases, including acute kidney injury (AKI). Although quercetin (QC), a natural flavonoid, has been reported to have medicinal properties, its potential protective effects against mercury-induced AKI have not been evaluated. In this study, the protective effect of QC against mercury-induced AKI was investigated using biochemical parameters, new protein-based urinary biomarkers, and a histopathological approach. A 250 mg/kg dose of QC was administered orally to Sprague-Dawley male rats for 3 days before administration of mercury chloride (HgCl2). All animals were sacrificed at 24 h after HgCl2 treatment, and biomarkers associated with nephrotoxicity were measured. Our data showed that QC absolutely prevented HgCl2-induced AKI, as indicated by biochemical parameters such as blood urea nitrogen (BUN) and serum creatinine (sCr). In particular, QC markedly decreased the accumulation of Hg in the kidney. Urinary excretion of protein-based biomarkers, including clusterin, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), monocyte chemoattractant protein-1 (MCP-1), tissue inhibitor of metalloproteinases 1 (TIMP-1), and vascular endothelial growth factor (VEGF) in response to HgCl2 administration were significantly decreased by QC pretreatment relative to that in the HgCl2-treated group. Furthermore, urinary excretion of metallothionein and Hg were significantly elevated by QC pretreatment. Histopathological examination indicated that QC protected against HgCl2-induced proximal tubular damage in the kidney. A TUNEL assay indicated that QC pretreatment significantly reduced apoptotic cell death in the kidney. The administration of QC provided significant protective effects against mercury-induced AKI.

Inhibition of hematopoietic prostaglandin D2 synthase (H-PGDS) by an alkaloid extract from Combretum molle.

BACKGROUND: Hematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH2 to PGD2 which mediates inflammatory responses. The inhibition of H-PGDS is of importance in alleviating damage to tissues due to unwarranted synthesis of PGD2. Combretum molle has been used in African ethno medicinal practices and has been shown to reduce fever and pain. The effect of C. molle alkaloid extract on H-PGDS was thus, investigated. METHODS: H-PGDS was expressed in Escherichia coli XL1-Blue cells and purified using nickel immobilized metal affinity chromatography. The effect of C. molle alkaloid extract on H-PGDS activity was determined with 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. The effect of C. molle alkaloid extract with time on H-PGDS was determined. The mechanism of inhibition was then investigated using CDNB and glutathione (GSH) as substrates. RESULTS: A specific activity of 24 µmol/mg/min was obtained after H-PGDS had been purified. The alkaloid extract exhibited a 70% inhibition on H-PGDS with an IC50 of 13.7 µg/ml. C. molle alkaloid extract showed an uncompetitive inhibition of H-PGDS with Ki = 41 µg/ml towards GSH, and non-competitive inhibition towards CDNB with Ki = 7.7 µg/ml and Ki' = 9.2 µg/ml. CONCLUSION: The data shows that C. molle alkaloid extract is a potent inhibitor of H-PGDS. This study thus supports the traditional use of the plant for inflammation.

Chemopreventive and chemotherapeutic effect of dietary supplementation of vitamin D on cholangiocarcinoma in a Chemical-Induced animal model.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer. Vitamin D, a pro-hormone, is getting popular due to its hormone-like functions after converted to its active form, 1α,25(OH)2D3. Here, we show that dietary supplementation with 6 IU/g of vitamin D greatly suppressed ICC initiation and progression without apparent toxicity in a chemically induced rat model. Microarray analysis of rat ICC tissues showed vitamin D supplementation modulated the expressions of several unique genes, including lipocalin 2 (Lcn2), confirmed by RT-qPCR and immunohistochemical (IHC) staining. Further, 53 of 80 human ICC specimens (66%) exhibited high LCN2 expression and LCN2 knockdown in SNU308 cells decreased cell growth and migration, suggesting LCN2 be an oncogene in human ICC. As human ICC SNU1079 cells were treated by 1α,25(OH)2D3, LCN2 expression and cell proliferation were attenuated. The downregulation of LCN2 expression was blunted when vitamin D receptor (VDR) was knocked down, implicating that the in vivo Lcn2 downregulation is a direct consequence of vitamin D supplementation Our results support the prevailing concept that vitamin D status is negatively associated with cancer incidence and mortality and suggest LCN2 may be a potential target against ICC. Further studies of application of vitamin D or its analog against ICC are warranted.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

Reactive astrocytes secrete lcn2 to promote neuron death.

Glial reaction is a common feature of neurodegenerative diseases. Recent studies have suggested that reactive astrocytes gain neurotoxic properties, but exactly how reactive astrocytes contribute to neurotoxicity remains to be determined. Here, we identify lipocalin 2 (lcn2) as an inducible factor that is secreted by reactive astrocytes and that is selectively toxic to neurons. We show that lcn2 is induced in reactive astrocytes in transgenic rats with neuronal expression of mutant human TAR DNA-binding protein 43 (TDP-43) or RNA-binding protein fused in sarcoma (FUS). Therefore, lcn2 is induced in activated astrocytes in response to neurodegeneration, but its induction is independent of TDP-43 or FUS expression in astrocytes. We found that synthetic lcn2 is cytotoxic to primary neurons in a dose-dependent manner, but is innocuous to astrocytes, microglia, and oligodendrocytes. Lcn2 toxicity is increased in neurons that express a disease gene, such as mutant FUS or TDP-43. Conditioned medium from rat brain slice cultures with neuronal expression of mutant TDP-43 contains abundant lcn2 and is toxic to primary neurons as well as neurons in cultured brain slice from WT rats. Partial depletion of lcn2 by immunoprecipitation reduced conditioned medium-mediated neurotoxicity. Our data indicate that reactive astrocytes secrete lcn2, which is a potent neurotoxic mediator.

Cloning, monoclonal antibody production, and bodily distribution pattern of a bovine lipocalin.

A bovine lipocalin, previously identified as a putative odorant-binding protein in bovine colostrum (bcOBP), was cloned and expressed, and its monoclonal antibody was established. bcOBP was constantly secreted into milk on day of parturition until at least 10 d postpartum at a concentration of 181±39 µg/L. Besides milk, bcOBP occurred in the nasal mucus, saliva, amniotic fluid, vaginal discharge, and blood plasma. Despite its low concentration, the distribution pattern and the finding that bcOBP harbored a characteristic sequence motif, CxxxC, which is conserved among insect and mammal pheromone binding proteins, suggest that bcOBP functions as a pheromone carrier. The presence of bcOBP in the plasma at varied concentrations depending on the lactation period does not exclude the possibility that bcOBP is secreted into milk from the blood. Cross-reactivity of the monoclonal antibody indicated presence of proteins homologous to bcOBP in the colostrum of farm animals of Cetartiodactyla.

Stable expression of lipocalin-type prostaglandin D synthase in cultured preadipocytes impairs adipogenesis program independently of endogenous prostanoids.

Lipocalin-type prostaglandin D synthase (L-PGDS) expressed preferentially in adipocytes is responsible for the synthesis of PGD(2) and its non-enzymatic dehydration products, PGJ(2) series, serving as pro-adipogenic factors. However, the role of L-PGDS in the regulation of adipogenesis is complex because of the occurrence of several derivatives from PGD(2) and their distinct receptor subtypes as well as other functions such as a transporter of lipophilic molecules. To manipulate the expression levels of L-PGDS in cultured adipocytes, cultured preadipogenic 3T3-L1 cells were transfected stably with a mammalian expression vector having cDNA encoding murine L-PGDS oriented in the sense direction. The isolated cloned stable transfectants with L-PGDS expressed higher levels of the transcript and protein levels of L-PGDS, and synthesized PGD(2) from exogenous arachidonic acid at significantly higher levels. By contrast, the synthesis of PGE(2) remained unchanged, indicating no influence on the reactions of cyclooxygenase (COX) and PGE synthase. Furthermore, the ability of those transfectants to synthesize Δ(12)-PGJ(2) increased more greatly during the maturation phase. The sustained expression of L-PGDS in cultured stable transfectants hampered the storage of fats during the maturation phase of adipocytes, which was accompanied by the reduced gene expression of adipocyte-specific markers reflecting the down-regulation of the adipogenesis program. The suppressed adipogenesis was not rescued by either exogenous aspirin or peroxisome proliferator-activated receptor γ (PPARγ) agonists including troglitazone and Δ(12)-PGJ(2). Taken together, the results indicate the negative regulation of the adipogenesis program by the enhanced expression of L-PGDS through a cellular mechanism involving the interference of the PPARγ signaling pathway without the contribution of endogenous pro-adipogenic prostanoids.

[Characteristics of changes in urinary NGAL, KIM-1 and IL-18 in Phytolaccae Radix-induced renal injury in rats and significance of combined detection].

OBJECTIVE: To explore the characteristics of changes in neutrophil gelatinase-associated lipocalcin (NGAL), kidney injury molecule-1 (KIM-1) and interleukin-18 (IL-18) in Phytolaccae Radix-induced kidney injury in rats and the significance of the combined detection. METHOD: Wistar rats were divided into three groups: high and low dose (crude drug 40, 20 g x kg(-1) x d(-1)) Phytolaccae Radix decoction groups and the control group, and orally administrated with distilled water or equal volume of Phytolaccae Radix decoction for 35 consecutive days. Their blood and urine samples were collected on day 7, 14, 21, 28, 35,42. The anatomical analysis was conducted for each group. The contents of serum total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CR) and urinary TP and ALB were detected-by means of biochemical analyzer. The concentrations of urinary NGAL, KIM-1 and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA). The morphological changes of renal pathology were observed by light or electron microscopy. The curve areas of various serum or urine indexes and the combined detection were compared by receiver operating characteristic curve (ROC curve). RESULT: Rats were given Phytolaccae Radix decoction at the doses of 40, 20 g crude drug/kg daily for 35 consecutive days to induce kidney injury characterized by the degeneration of renal tubular epithelial cell and protein cast. The injury was partially reversible during the recovery period. Compared with the control group, the content of serum BUN, CR and urinary TP in each dose group mostly showed a downward trend. On day 21, the content of urinary ALB obviously increased till the end of administration. The contents of urinary NGAL, KIM-1 and IL-18 began increasing on day 7. Since day 14, high and low dose groups showed significant difference (P<0.01). The high dose group even showed notable changes during the recovery period. According to ROC analysis, the curve areas of NGAL, KIM-1 and IL-18 were 0.846, 0.837 and 0.863 (P <0.01), respectively, much higher than that of BUN and CR. The area of the combined detection was up to 0.947. CONCLUSION: Urinary NGAL, IL-18 and KIM-1 could forecast and indicate the occurrence and development of renal injury to some degree, and show higher sensitivity and site specificity. The combined detection could further improve the test efficiency.

Selenoprotein-dependent up-regulation of hematopoietic prostaglandin D2 synthase in macrophages is mediated through the activation of peroxisome proliferator-activated receptor (PPAR) gamma.

The plasticity of macrophages is evident from their dual role in inflammation and resolution of inflammation that are accompanied by changes in the transcriptome and metabolome. Along these lines, we have previously demonstrated that the micronutrient selenium increases macrophage production of arachidonic acid (AA)-derived anti-inflammatory 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) and decreases the proinflammatory PGE(2). Here, we hypothesized that selenium modulated the metabolism of AA by a differential regulation of various prostaglandin (PG) synthases favoring the production of PGD(2) metabolites, Δ(12)-PGJ(2) and 15d-PGJ(2). A dose-dependent increase in the expression of hematopoietic-PGD(2) synthase (H-PGDS) by selenium and a corresponding increase in Δ(12)-PGJ(2) and 15d-PGJ(2) in RAW264.7 macrophages and primary bone marrow-derived macrophages was observed. Studies with organic non-bioavailable forms of selenium and the genetic manipulation of cellular selenium incorporation machinery indicated that selenoproteins were necessary for H-PGDS expression and 15d-PGJ(2) production. Treatment of selenium-deficient macrophages with rosiglitazone, a peroxisome proliferator-activated receptor γ ligand, up-regulated H-PGDS. Furthermore, electrophoretic mobility shift assays indicated the presence of an active peroxisome proliferator-activated receptor-response element in murine Hpgds promoter suggesting a positive feedback mechanism of H-PGDS expression. Alternatively, the expression of nuclear factor-κB-dependent thromboxane synthase and microsomal PGE(2) synthase was down-regulated by selenium. Using a Friend virus infection model of murine leukemia, the onset of leukemia was observed only in selenium-deficient and indomethacin-treated selenium-supplemented mice but not in the selenium-supplemented group or those treated with 15d-PGJ(2). These results suggest the importance of selenium in the shunting of AA metabolism toward the production of PGD(2) metabolites, which may have clinical implications.

Cytoprotective effect of acetyl-L-carnitine evidenced by analysis of gene expression in the rat brain.

Acetyl-L-carnitine (ALC), the acetyl ester of L-carnitine, is a naturally occurring substance that when administered at supraphysiological concentrations is neuroprotective. ALC plays an essential role in intermediary and mitochondrial metabolism. It has also neurotrophic and antioxidant actions. ALC has demonstrated efficacy and high tolerability in the treatment of neuropathies of various etiologies, and it is a molecule of considerable interest for its clinical application in various neural disorders, such as Alzheimer's disease and painful neuropathies, although little is known regarding the effects of ALC on gene expression. Suppression subtractive hybridization methodology was used for the generation of subtracted complementary DNA libraries and the subsequent identification of differentially expressed transcripts in the rat brain after a chronic ALC treatment. In the present paper, we provide evidences for the up-regulation of the expression of prostaglandin D(2) synthase, brain-specific Na(+)-dependent inorganic phosphate transporter, and cytochrome b oxidase, bc1 complex induced in the rat brain by ALC. On the contrary, ALC treatment down-regulates the expression of the gene of ferritin-H. Altogether, these results suggest that ALC might play a cytoprotective role against various brain stressors.

Biochemical, functional, and pharmacological characterization of AT-56, an orally active and selective inhibitor of lipocalin-type prostaglandin D synthase.

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD(2) production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE(2) and PGF(2alpha) and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Anti-inflammatory effects and changes in prostaglandin patterns induced by 7beta-hydroxy-epiandrosterone in rats with colitis.

High dose levels of dehydroepiandrosterone and its 7-hydroxylated derivatives have been shown to reduce oxidative stress and inflammatory responses in dextran sodium sulfate (DSS)-induced colitis in rats. Another endogenous steroid, 7beta-hydroxy-epiandrosterone (7beta-hydroxy-EpiA) has been shown to exert neuroprotective effects at much smaller doses. Our aims were to evaluate whether 7beta-hydroxy-EpiA pre-treatment prevents DSS-induced colitis and to determine whether the effects involve changes in anti-inflammatory prostaglandin (PG) D(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) levels. Rats were administered 0.01, 0.1 and 1mg/kg 7beta-hydroxy-EpiA i.p. once a day for 7 days. Thereafter, colitis was induced by administration of 5% DSS in drinking water for 7 days. Levels of the PGs and the expression of cyclooxygenase (COX-2) and PG synthases were assessed during the course of the experiment. Administration of 7beta-hydroxy-EpiA caused a transient increase in COX-2 and PGE synthase expression within 6-15h and augmented colonic tissue levels of 15d-PGJ(2) levels starting at day 2. Treatment with DSS resulted in shortened colon length, depleted mucus in goblet cells and induced oxidative stress. COX-2 and mPGES-1 synthase expression were enhanced and accompanied by increased PGE(2), D(2) and 15d-PGJ(2) production. Although all dose levels of 7beta-hydroxy-EpiA reduced PGE(2) production, only the lowest dose (0.01mg/kg) of the steroid completely prevented colitis damage and tissue inflammation. 7beta-Hydroxy-EpiA pre-treatment prevents the occurrence of DSS-induced colitis through a shift from PGE(2) to PGD(2) production, associated with an early but transient increase in COX-2 expression and a sustained increase in the production of the anti-inflammatory prostaglandin 15d-PGJ(2).