Prostaglandin D2 and its analog, 11d-11m-PGD2, added during the differentiation phase contribute to adipogenic program inhibition in 3T3-L1 cells.

We previously reported that prostaglandin (PG)D2 and its isosteric analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), promote adipogenesis in 3T3-L1 cells during the maturation phase. Focusing on the differentiation phase, although both PGs inhibited adipogenesis, this effect was canceled out by PGI2 and PGJ2 derivatives. Thus, PGD2 and 11d-11m-PGD2 play different roles during the phases, but do not affect PGI2- and PGJ2-derivative-induced adipogenesis.

Skatole regulates intestinal epithelial cellular functions through activating aryl hydrocarbon receptors and p38.

Intestinal bacteria produce skatole (3-methylindole) from tryptophan in dietary proteins and ingesting large quantities of animal protein is associated with increased fecal skatole concentrations. Although possibly associated with disrupted intestinal homeostasis, the influence of skatole on intestinal epithelial cellular function has not been characterized in detail. The present study aimed to determine whether skatole induces intestinal epithelial cell (IEC) dysfunction. We found that skatole dose-dependently caused IEC death and time-dependently induced IEC apoptosis. Since skatole directly interacts with aryl hydrocarbon receptors (AhR), we investigated whether these receptors influence the skatole-induced death of IEC. In addition to increased AhR transcriptional activity induced by skatole, the AhR antagonist CH223191 partially suppressed of skatole-induced IEC death. Extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) are mitogen-activated protein kinases (MAPK) induced by skatole. None of them were repressed by CH223191, whereas the p38 inhibitor SB203580 promoted skatole-induced IEC death. These findings together indicated that skatole induces both AhR-dependent activation pathways and the AhR-independent activation of p38, consequently regulating the amount of IEC death. Accumulating evidence indicates that consuming large amounts of animal protein is associated with the pathogenesis and progression of inflammatory bowel diseases (IBD). Thus, intestinal skatole production induced by large amounts of dietary animal protein might be associated via IEC death with intestinal pathologies such as IBD.

Comparison of pro-adipogenic effects between prostaglandin (PG) D2 and its stable, isosteric analogue, 11-deoxy-11-methylene-PGD2, during the maturation phase of cultured adipocytes.

Prostaglandin (PG) D2 is relatively unstable and dehydrated non-enzymatically into PGJ2 derivatives, which are known to serve as pro-adipogenic factors by activating peroxisome proliferator-activated receptor (PPAR) γ, a master regulator of adipogenesis. 11-Deoxy-11-methylene-PGD2 (11d-11m-PGD2) is a novel, chemically stable, isosteric analogue of PGD2 in which the 11-keto group is replaced by an exocyclic methylene. Here we attempted to investigate pro-adipogenic effects of PGD2 and 11d-11m-PGD2 and to compare the difference in their ways during the maturation phase of cultured adipocytes. The dose-dependent study showed that 11d-11m-PGD2 was significantly more potent than natural PGD2 to stimulate the storage of fats suppressed in the presence of indomethacin, a cyclooxygenase inhibitor. These pro-adipogenic effects were caused by the up-regulation of adipogenesis as evident with higher gene expression levels of adipogenesis markers. Analysis of transcript levels revealed the enhanced gene expression of two subtypes of cell-surface membrane receptors for PGD2, namely the prostanoid DP1 and DP2 (chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2)) receptors together with lipocalin-type PGD synthase during the maturation phase. Specific agonists for DP1, CRTH2, and PPARγ were appreciably effective to rescue adipogenesis attenuated by indomethacin. The action of PGD2 was attenuated by specific antagonists for DP1 and PPARγ. By contrast, the effect of 11d-11m-PGD2 was more potently interfered by a selective antagonist for CRTH2 than that for DP1 while PPARγ antagonist GW9662 had almost no inhibitory effects. These results suggest that PGD2 exerts its pro-adipogenic effect principally through the mediation of DP1 and PPARγ, whereas the stimulatory effect of 11d-11m-PGD2 on adipogenesis occurs preferentially by the interaction with CRTH2.

Pretreatment of cultured preadipocytes with arachidonic acid during the differentiation phase without a cAMP-elevating agent enhances fat storage after the maturation phase.

Arachidonic acid (AA) and the related prostanoids exert complex effects on the adipocyte differentiation depending on the culture conditions and life stages. Here, we investigated the effect of the pretreatment of cultured 3T3-L1 preadipocytes with exogenous AA during the differentiation phase without 3-isobutyl-1-methylxanthine (IBMX), a cAMP-elevating agent, on the storage of fats after the maturation phase. This pretreatment with AA stimulated appreciably adipogenesis after the maturation phase as evident with the up-regulated gene expression of adipogenic markers. The stimulatory effect of the pretreatment with AA was attenuated by the co-incubation with each of cyclooxygenase (COX) inhibitors. Among exogenous prostanoids and related compounds, the pretreatment with MRE-269, a selective agonist of the IP receptor for prostaglandin (PG) I2, strikingly stimulated the storage of fats in adipocytes. The gene expression analysis of arachidonate COX pathway revealed that the transcript levels of inducible COX-2, membrane-bound PGE synthase-1, and PGF synthase declined more greatly in cultured preadipocytes treated with AA. By contrast, the expression levels of COX-1, cytosolic PGE synthase, and PGI synthase remained constitutive. The treatment of cultured preadipocytes with AA resulted in the decreased synthesis of PGE2 and PGF2α serving as anti-adipogenic PGs although the biosynthesis of pro-adipogenic PGI2 was up-regulated during the differentiation phase. Moreover, the gene expression levels of EP4 and FP, the respective prostanoid receptors for PGE2 and PGF2α, were gradually suppressed by the supplementation with AA, whereas that of IP for PGI2 remained relatively constant. Collectively, these results suggest the predominant role of endogenous PGI2 in the stimulatory effect of the pretreatment of cultured preadipoccytes with AA during the differentiation phase without IBMX on adipogenesis after the maturation phase.

The tyrosine-sorting motif of the vacuolar sorting receptor VSR4 from Arabidopsis thaliana, which is involved in the interaction between VSR4 and AP1M2, µ1-adaptin type 2 of clathrin adaptor complex 1 subunits, participates in the post-Golgi sorting of VSR4.

µ1-Adaptin of adaptor protein (AP) 1 complex, AP1M, is generally accepted to load cargo proteins into clathrin-coated vesicles (CCVs) at the trans-Golgi network through its binding to cargo-recognition sequences (CRSs). Plant vacuolar-sorting receptors (VSRs) function in sorting vacuolar proteins, which are reportedly mediated by CCV. We herein investigated the involvement of CRSs of Arabidopsis thaliana VSR4 in the sorting of VSR4. The results obtained showed the increased localization of VSR4 at the plasma membrane or vacuoles by mutations in CRSs including the tyrosine-sorting motif YMPL or acidic dileucine-like motif EIRAIM, respectively. Interaction analysis using the bimolecular fluorescence complementation (BiFC) system, V10-BiFC, which we developed, indicated an interaction between VSR4 and AP1M2, AP1M type 2, which was attenuated by a YMPL mutation, but not influenced by an EIRAIM mutation. These results demonstrated the significance of the recognition of YMPL in VSR4 by AP1M2 for the post-Golgi sorting of VSR4.

New Gateway-compatible vectors for a high-throughput protein-protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis.

Protein-protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth ß-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC-CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.

Generation of monoclonal antibody against 6-Keto PGF1α and development of ELISA for its quantification in culture medium.

Prostacyclin or prostaglandin I2 (PGI2), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI2 in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF1α, a stable PGI2 metabolite, and its quantification to determine the role of PGI2 in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF1α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF1α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF1α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI2 regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI2 in maintaining homeostasis and adipocyte differentiation.

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.

Arachidonic Acid Added during the Differentiation Phase of 3T3-L1 Cells Exerts Anti-Adipogenic Effect by Reducing the Effects of Pro-Adipogenic Prostaglandins.

A linoleic acid (LA) metabolite arachidonic acid (AA) added to 3T3-L1 cells is reported to suppress adipogenesis. The purpose of the present study aimed to clarify the effects of AA added during the differentiation phase, including adipogenesis, the types of prostaglandins (PG)s produced, and the crosstalk between AA and the PGs produced. Adipogenesis was inhibited by AA added, while LA did not. When AA was added, increased PGE2 and PGF2α production, unchanged Δ12-PGJ2 production, and reduced PGI2 production were observed. Since the decreased PGI2 production was reflected in decreased CCAAT/enhancer-binding protein-ß (C/EBPß) and C/EBPδ expression, we expected that the coexistence of PGI2 with AA would suppress the anti-adipogenic effects of AA. However, the coexistence of PGI2 with AA did not attenuate the anti-adipogenic effects of AA. In addition, the results were similar when Δ12-PGJ2 coexisted with AA. Taken together, these results indicated that the metabolism of ingested LA to AA is necessary to inhibit adipogenesis and that exposure of AA to adipocytes during only the differentiation phase is sufficient. As further mechanisms for suppressing adipogenesis, AA was found not only to increase PGE2 and PGF2α and decrease PGI2 production but also to abrogate the pro-adipogenic effects of PGI2 and Δ12-PGJ2.

Prostaglandin D2 Added during the Differentiation of 3T3-L1 Cells Suppresses Adipogenesis via Dysfunction of D-Prostanoid Receptor P1 and P2.

We previously reported that the addition of prostaglandin, (PG)D2, and its chemically stable analog, 11-deoxy-11-methylene-PGD2 (11d-11m-PGD2), during the maturation phase of 3T3-L1 cells promotes adipogenesis. In the present study, we aimed to elucidate the effects of the addition of PGD2 or 11d-11m-PGD2 to 3T3-L1 cells during the differentiation phase on adipogenesis. We found that both PGD2 and 11d-11m-PGD2 suppressed adipogenesis through the downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, the latter suppressed adipogenesis more potently than PGD2, most likely because of its higher resistance to spontaneous transformation into PGJ2 derivatives. In addition, this anti-adipogenic effect was attenuated by the coexistence of an IP receptor agonist, suggesting that the effect depends on the intensity of the signaling from the IP receptor. The D-prostanoid receptors 1 (DP1) and 2 (DP2, also known as a chemoattractant receptor-homologous molecule expressed on Th2 cells) are receptors for PGD2. The inhibitory effects of PGD2 and 11d-11m-PGD2 on adipogenesis were slightly attenuated by a DP2 agonist. Furthermore, the addition of PGD2 and 11d-11m-PGD2 during the differentiation phase reduced the DP1 and DP2 expression during the maturation phase. Overall, these results indicated that the addition of PGD2 or 11d-11m-PGD2 during the differentiation phase suppresses adipogenesis via the dysfunction of DP1 and DP2. Therefore, unidentified receptor(s) for both molecules may be involved in the suppression of adipogenesis.

Eicosapentaenoic Acid Induces the Inhibition of Adipogenesis by Reducing the Effect of PPARγ Activator and Mediating PKA Activation and Increased COX-2 Expression in 3T3-L1 Cells at the Differentiation Stage.

Obesity has received increasing attention in recent years because it is a factor in the development of non-communicable diseases. The current study aimed to analyze how representative fatty acids (FAs) such as palmitic acid, stearic acid, oleic acid, α-linolenic acid (ALA), and eicosapentaenoic acid (EPA) affected adipogenesis when/if introduced at the differentiation stage of 3T3-L1 cell culture. These FAs are assumed to be potentially relevant to the progression or prevention of obesity. EPA added during the differentiation stage reduced intracellular triacylglycerol (TAG) accumulation, as well as the expression of the established adipocyte-specific marker genes, during the maturation stage. However, no other FAs inhibited intracellular TAG accumulation. Coexistence of Δ12-prostaglandin J2, a peroxisome proliferator-activated receptor γ activator, with EPA during the differentiation stage partially attenuated the inhibitory effect of EPA on intracellular TAG accumulation. EPA increased cyclooxygenase-2 (COX-2) expression and protein kinase A (PKA) activity at the differentiation stage, which could explain the inhibitory actions of EPA. Taken together, exposure of preadipocytes to EPA only during the differentiation stage may be sufficient to finally reduce the mass of white adipose tissue through increasing COX-2 expression and PKA activity.

Sustained expression of lipocalin-type prostaglandin D synthase in the antisense direction positively regulates adipogenesis in cloned cultured preadipocytes.

Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD(2) and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD(2) synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE(2) was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense L-PGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE(2) and PGF(2α), while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- Δ(12,14)-prostaglandin J(2). These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ(2) series.

15-Deoxy-Δ(12,14)-prostaglandin J(2) interferes inducible synthesis of prostaglandins E(2) and F(2α) that suppress subsequent adipogenesis program in cultured preadipocytes.

Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E(2) and PGF(2α) involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ(2) interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ(12)-PGJ(2) and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ(2). Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ(2) or PDTC although 15d-PGJ(2) alone has no stimulatory effect. Moreover, 15d-PGJ(2) did not block the inhibitory effects of exogenous PGE(2) and PGF(2α) on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ(2) can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes.

Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase.

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to J(2) series of PGs including 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2), which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ(12)-PGJ(2) has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ(12)-PGJ(2). According to the standard curve, the amount of Δ(12)-PGJ(2) can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ(2), while it only showed the cross-reaction of 28% with unstable PGJ(2). This immunological assay was applied to the determination of the endogenous formation of Δ(12)-PGJ(2) in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ(12)-PGJ(2) increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ(2). Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ(12)-PGJ(2) in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ(2) series at various doses on adipogenesis during the maturation phase. Although Δ(12)-PGJ(2) was slightly less potent than 15d-PGJ(2), each of these PGJ(2) series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ(12)-PGJ(2) contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ(2) during the maturation phase of cultured adipocytes.

Effects of polyphenols from seed shells of Japanese horse chestnut (Aesculus turbinata BLUME) on methotrexate-induced intestinal injury in rats.

The purpose of this study was to evaluate the effects of polyphenols from seed shells of Japanese horse chestnut (JHP) on methotrexate (MTX)-induced intestinal injury in rats. MTX application caused intestinal morphological injury and increase in malondialdehyde (MDA) levels, decrease in levels of glutathione (GSH) and glutathione peroxidase (GSH-Px) activities in small intestine. However, oral administration of JHP ameliorated MTX-induced intestinal injury and inhibited the increase in MDA and the decrease in GSH and GSH-Px activity in small intestine. In conclusion, our results indicated that oral administration of JHP alleviated MTX-induced intestinal injury through its antioxidant properties.

Anti-inflammatory and bioavailability studies on dietary 3,5,4'-trihydroxy-6,7-methylenedioxyflavone-O-glycosides and their aglycone from indigo leaves in a murine model of inflammatory bowel disease.

Persicaria tinctoria (Aiton) Spach, also called Polygonum tinctorium Lour., (family Polygonaceae) for indigo plant has been traditionally useful as a medicinal or edible plant with a variety of biological activities. Of these, much attention has been paid to their anti-inflammatory activities. We have recently demonstrated that indigo leaves contain high levels of flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) as an aglycone. In this study, we attempted to evaluate anti-inflammatory activities of TMF-O-glycosides and free TMF prepared from indigo leaves after extraction with hot water. Free TMF was found to appreciably down-regulate the gene expression of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6, inducible nitric oxide, and tumor necrosis factor-α in cultured macrophage cells stimulated with lipopolysaccharide while up-regulating the expression of anti-inflammatory IL-10. However, no study has been conducted regarding in vivo anti-inflammatory activities of TMF-O-glycosides and free TMF until now. Here, we assessed in vivo anti-inflammatory effects of these dietary compounds on ulcerative colitis in a murine model of inflammatory bowel disease by the induction with dextran sulfate sodium (DSS). Histological evaluation revealed that both TMF-O-glycosides and free TMF effectively protected against DSS-induced ulcerative colitis. The analysis of digested products by liquid chromatography and mass spectrometry led us to detect free TMF as a predominant metabolite in the feces of mice fed with TMF-O-glycosides. Moreover, free TMF was later detected as glucuronyl conjugates of TMF in the liver of mice fed with both fractions. These results indicate the effective digestion of TMF-O-glycosides and the subsequent absorption of free TMF in the gut of mice for exerting anti-inflammatory effects. Taken together, our findings suggest that dietary TMF-O-glycosides could be promising natural sources for the utilization as herbal medicine and nutraceuticals to expect in vivo anti-inflammatory activities.

Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms.

Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE(2) upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and troglitazone as peroxisome proliferator-activated receptor gamma (PPARgamma) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARgamma and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.

Up-regulation of adipogenesis in adipocytes expressing stably cyclooxygenase-2 in the antisense direction.

Adipocytes and the precursor cells express two types of cyclooxygenase (COX) isoforms that are involved in the biosynthesis of different types of prostaglandins (PGs) exerting opposite effects on adipogenesis. To evaluate the role of the inducible COX-2 isoform in the control of the differentiation and maturation of adipocytes, we employed an antisense technology to suppress specifically the expression of COX-2 in adipocytes. Cultured 3T3-L1 preadipocytes were transfected stably with a mammalian expression vector having the full-length cDNA encoding mouse COX-2 oriented in the antisense direction. The cloned transfectants with antisense COX-2 exhibited stable expression of antisense RNA for COX-2, which was accompanied by the suppressed expression of mRNA and protein levels of sense COX-2. However, almost no alteration in the expression of COX-1 was detected. The transfectants with antisense COX-2 showed significant decreases in the delayed synthesis of PGE(2) involving the inducible COX-2 in response to cell stimuli. By contrast, the immediate synthesis of PGE(2) associated with the constitutive COX-1 was not influenced appreciably. The stable expression of antisense mRNA of COX-2 resulted in significant stimulation of fat storage during the maturation phase without affecting the cell proliferation associated with the clonal expansion phase. The gene expression studies revealed higher expression levels of adipocyte-specific markers in the transfectants with antisense COX-2, indicating the mechanism that stimulates adipogenesis program. The up-regulation of fat storage was appreciably prevented by anti-adipogenic prostanoids, such as PGE(2) and PGF(2alpha), during the maturation phase. These results suggest that COX-2 is more preferentially involved in the generation of endogenous anti-adipogenic prostanoids during the maturation phase of adipocytes.

Constituents of flavonol O-glycosides and antioxidant activities of extracts from seeds, sprouts, and aerial parts of Polygonum tinctorium Lour.

Polygonum tinctorium Lour. (family Polygonaceae), known as indigo plant, has been useful as a medicinal or edible plant abundant in polyphenolic compounds. We have recently shown that flavonol O-glycosides with 3,5,4'-trihydroxy-6,7-methylenedioxyflavone (TMF) are predominant flavonoids in indigo leaves. However, no study has been performed regarding changes in the levels of flavonoid species during the germination and growth of indigo plant. Here, we attempted to determine the individual constituents of flavonol O-glycosides and the changes in their contents of the seeds, sprouts, and aerial parts. These results revealed that only the seeds predominantly contained flavonol O-(acetyl)-rhamnosides with quercetin or kaempferol as an aglycone. During the development of the sprouts and aerial parts, flavonol O-glycosides with TMF as an aglycone became mainly detectable and accounted for 79.4% and 74.9% of total flavonol O-glycosides from the extracts of aerial parts harvested in 2016 and 2017, respectively. Of the plant organs tested, the aerial parts exhibited the highest antioxidant activities concomitant with greatly increased levels of total polyphenols. Thus, we were able to conduct the identification and quantification of flavonol O-glycosides from the seeds, sprouts, and aerial parts of indigo plant and to evaluate antioxidant activities of their extracts. Taken together, our findings clearly provide the evidence that the aerial parts of indigo plant are a rich source of flavonol O-glycosides with TMF and exhibit much higher antioxidant activities, indicating the usefulness for the application to food and nutraceutical purposes.

In Vitro Anti-inflammatory and Antioxidant Activities of 3,5,4'-Trihydroxy-6,7-Methylenedioxyflavone-O-Glycosides and Their Aglycone from Leaves of Polygonum tinctorium Lour.

Polygonum tinctorium Lour (indigo plant) has been regarded as a useful medicinal plant for traditional herbal medicine. The polyphenolic fraction of indigo leaves exhibited anti-inflammatory activities as determined by the suppressed synthesis of nitric oxide (NO) in cultured RAW264 macrophage cells. The acid hydrolysate of the fraction showed much more potent effect than the unhydrolyzed one. In sharp contrast, those fractions of indigo stems had almost no effect. 3,5,4'-Trihydroxy-6,7-methylenedioxyflavone (TMF)-O-glycosides and tryptanthrin were detected exclusively in the extracts of the leaves. The isolated flavonol species were furthermore tested for their anti-inflammatory activities against the synthesis of NO and prostaglandin E2 in the cultured macrophage cells. More potent anti-inflammatory effects were recognized with different aglycones of flavonols than their flavonol O-glycosides. Although the inhibitory effects of TMF were less effective than those of tryptanthrin, the levels of flavonol O-glycosides with TMF were much more abundant than those of tryptanthrin in the leaves. Oral administration of the fraction containing flavonol O-glycosides with TMF into mice revealed the detection of free TMF in the blood circulation, indicating that the aglycone moiety can be cleaved by digestive enzymes and absorbed in the gut. Alternatively, the assay of hydrophilic oxygen radical absorbance capacity revealed that the isolated species of flavonol O-glycosides with TMF and their aglycone had appreciable antioxidant activities. Taken together, our findings suggest that the predominant flavonol O-glycosides with TMF as an aglycone could be promising natural agents for the application to herbal medicine, nutraceuticals, and food additives.