Pregnane X receptor mediated-transcription regulation of CYP3A by glycyrrhizin: a possible mechanism for its hepatoprotective property against lithocholic acid-induced injury.

Licorice (LE) has been commonly used in traditional Chinese medicine (TCM) for over 4000 years to reconcile various drugs and for hepatic disorders. Glycyrrhizin is the main bioactive component isolated from LE herbs. In the present study we examined the effects of glycyrrhizin on pregnane X receptor (PXR)-mediated CYP3A expression and its hepatoprotective activity. Treatment of HepG2 cells with glycyrrhizin resulted in marked increase in both CYP3A4 mRNA and protein levels. The transcriptional activation of the CYP3A4 gene through glycyrrhizin is PXR-dependent, as shown in transient transfection experiments. Glycyrrhizin activates the DNA-binding capacity of the PXR for the CYP3A4 element responding to xenobiotic signals, as measured by the electrophoretic-mobility shift assay (EMSA). These results indicate that the induction of the hepatic CYP3A4 by glycyrrhizin is mediated through the activation of PXR. The next aim of the current study was to determine whether the activation of PXR and induction of CYP3A by glycyrrhizin prevents hepatotoxicity during cholestasis as a mechanism of hepatoprotection. Mice were pretreated with glycyrrhizin prior to induction of intrahepatic cholestasis using lithocholic acid (LCA). Pre-treatment with glycyrrhizin, as well as the PXR activator pregnenolone 16α-carbontrile (PCN), prevents the increase in plasma ALT and AST activity, multifocal necrosis and prevents an increase in a level of serum LCA level in mice, as compared with the results in the mice treated with LCA alone. Activation of the PXR by glycyrrhizin results in induction of CYP3A11 (CYP3A4 for human) expression and inhibition of CYP7A1 through an increase in small heterodimer partner (SHP) expression. Glycyrrhizin regulates the expression of the gene mentioned above to prevent toxic accumulation of bile acids in the liver and it also protects mouse livers from the harmful effects of LCA. In conclusion, PXR-mediated effects on CYP3A and CYP7A may contribute to the hepatoprotective property of glycyrrhizin against LCA-induced liver injury.

Impact of biological gender and soluble guanylate cyclase stimulation on renal recovery after relief of unilateral ureteral obstruction.

PURPOSE: Gender difference and nitric oxide deficiency contribute to the progression of many chronic kidney diseases. In a model of unilateral ureteral obstruction relief we analyzed the impact of biological gender and nitric oxide/cyclic guanosine monophosphate signaling stimulation on renal disease severity and restoration. MATERIALS AND METHODS: Female and male rats underwent sham surgery or unilateral ureteral obstruction. After 5-day unilateral ureteral obstruction female and male rats were assigned to obstruction relief alone or obstruction relief plus 7-day treatment with the soluble guanylate cyclase stimulator BAY 41-8543. RESULTS: Compared to male rats with obstruction relief renal disease was less severe in female rats, which had significantly less tubulointerstitial matrix accumulation and tubular atrophy. In each gender group α1 and ß1-soluble guanylate cyclase was comparably and significantly increased but female rats produced significantly more cyclic guanosine monophosphate after treatment with the soluble guanylate cyclase stimulator. In each group BAY 41-8543 treatment was associated with significant amelioration of renal matrix protein expansion, macrophage infiltration, tubular apoptosis and atrophy. CONCLUSIONS: Female gender is protective for unilateral ureteral obstruction relief. This was linked to higher sensitivity of the soluble guanylate cyclase enzyme and cyclic guanosine monophosphate production in response to BAY 41-8543. In these female and male rats enhancing the signaling of nitric oxide/cyclic guanosine monophosphate with BAY 41-8543 significantly accelerated the restoration of renal architecture after obstruction relief and largely ameliorated the differences in disease severity due to the gender disparity.

Low-dose endotoxin induces inflammation by selectively removing nuclear receptors and activating CCAAT/enhancer-binding protein δ.

Subclinical levels of circulating endotoxin are associated with the pathogenesis of diverse human inflammatory diseases, by mildly inducing the expression of proinflammatory mediators. In this study, we examined the molecular mechanism responsible for the effect of low-dose LPS in macrophages. In contrast to high-dose LPS, which activates NF-κB and induces the robust expression of proinflammatory mediators, we observed that low-dose LPS failed to activate NF-κB. Instead, it selectively activated C/EBPδ and removed nuclear repressors, including peroxisome proliferator-activated receptor α and retinoic acid receptor α, enabling a mild and leaky expression of proinflammatory mediators. The effect of low-dose LPS required IRAK-1, which interacts with and acts upstream of IκB kinase ε to contribute to LPS-mediated induction of C/EBPδ and proinflammatory mediators. Additionally, mice fed a high-fat diet acquired elevated levels of endotoxin and proinflammatory mediators in an IRAK-1-dependent fashion. Taken together, these data reveal a distinct pathway preferentially used by low-dose endotoxin in initiating low-grade inflammation.

Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells.

HepaRG cells possess the unique property to differentiate in vitro and to express various functions of mature hepatocytes, including the major cytochromes P450 (P450s). In the present study, we carefully analyzed mRNA expression and activity of the major P450s and their responsiveness to three prototypical inducers, phenobarbital, rifampicin, and omeprazole, in differentiated HepaRG cell cultures over a 4-week period after low and high seeding. Only minor differences were observed in P450 activities when measured by two cocktails of probe substrates, probably related to the choice and/or concentration of substrates. Similar results were obtained from the two cell seeding conditions. Expression and activities of several P450s were dimethyl sulfoxide-dependent. However, basal P450 expression and activities as well as their responsiveness to the prototypical inducers were well maintained over the 4-week period, and a good correlation was observed between transcript levels and corresponding activities. Thus, CYP1A2, CYP2B6, and CYP3A4 were found to accurately respond to their respective prototypical inducers, i.e., omeprazole, phenobarbital, and rifampicin. Likewise, basal expression of several phase II enzymes, transporters, and nuclear receptors, and response to inducers were also well preserved. More genes were found to be induced in HepaRG cells than in primary human hepatocytes, and no marked variation was noticed between the different passages. Taken together, these data support the conclusion that HepaRG cells represent a promising surrogate to primary human hepatocytes for xenobiotic metabolism and toxicity studies.

Low retinol levels differentially modulate bile salt-induced expression of human and mouse hepatic bile salt transporters.

UNLABELLED: The farnesoid X receptor/retinoid X receptor-alpha (FXR/RXRalpha) complex regulates bile salt homeostasis, in part by modulating transcription of the bile salt export pump (BSEP/ABCB11) and small heterodimer partner (SHP/NR0B2). FXR is activated by bile salts, RXRalpha by the vitamin A derivative 9-cis retinoic acid (9cRA). Cholestasis is associated with vitamin A malabsorption. Therefore, we evaluated the role of vitamin A/9cRA in the expression of human and mouse bile salt export pump (hBSEP/mBsep), small heterodimer partner (hSHP/mShp), and mouse sodium-dependent taurocholate co-transporting polypeptide (mNtcp). HBSEP and hSHP transcription were analyzed in FXR/RXRalpha-transfected HepG2 cells exposed to chenodeoxycholic acid (CDCA) and/or 9cRA. BSEP promoter activity was determined by luciferase reporter assays, DNA-binding of FXR and RXRalpha by pull-down assays. Serum bile salt levels and hepatic expression of Bsep, Shp, and Ntcp were determined in vitamin A-deficient (VAD)/cholic acid (CA)-fed C57BL/6J mice. Results indicated that 9cRA strongly repressed the CDCA-induced BSEP transcription in HepG2 cells, whereas it super-induced SHP transcription; 9cRA reduced DNA-binding of FXR and RXRalpha. The 9cRA repressed the CDCA-induced BSEP promoter activity irrespective of the exact sequence of the FXR-binding site. In vivo, highest Bsep messenger RNA (mRNA), and protein expression was observed in CA-fed VAD mice. Shp transcription was highest in CA-fed vitamin A-sufficient mice. Ntcp protein expression was strongly reduced in CA-fed VAD mice, whereas mRNA levels were normal. CA-fed control and VAD mice had similarly increased serum bile salt levels. CONCLUSION: We showed that 9cRA has opposite effects on bile salt-activated transcription of FXR/RXRalpha target genes. Vitamin A deficiency in CA-fed mice leads to high BSEP expression. Clearance of serum bile salts may, however, be limited because of post-transcriptional reduction of Ntcp. The molecular effects of vitamin A supplementation during cholestasis need further analysis to predict a therapeutic effect.

Analysis of area-specific expression patterns of RORbeta, ER81 and Nurr1 mRNAs in rat neocortex by double in situ hybridization and cortical box method.

BACKGROUND: The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space. PRINCIPAL FINDINGS: Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal. CONCLUSIONS/SIGNIFICANCE: The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.

Expression of zebrafish ROR alpha gene in cerebellar-like structures.

Mouse genetic studies have identified several genes involved in cerebellar development. The mouse mutants staggerer and lurcher are functionally deficient for the retinoid-related orphan receptor alpha (ROR alpha) and glutamate receptor delta2 (Grid2) genes, respectively, and they show similar functional and developmental abnormalities in the cerebellum. Here, we report the cloning and expression pattern of zebrafish ROR alpha orthologues rora1 and rora2, and compare their expression pattern with that of grid2. Expression of rora1 and rora2 is initiated at late gastrula and pharyngula stages, respectively. Both rora1 and rora2 are spatially expressed in the retina and tectum. Expression of rora2 was further observed in the cerebellum, as reported for mammalian ROR alpha. In the adult brain, rora2 and grid2 are coexpressed in brain regions, designated as cerebellar-like structures. These observations suggest an evolutionarily conserved function of ROR alpha orthologues in the vertebrate brain.

[Effects of soybean isoflavones on expression levels of osteoprotegerin and osteoprotegerin ligand mRNAs in bone tissues of ovariectomized rats].

OBJECTIVE: To investigate the effects of soybean isoflavones (SI) on expression levels of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) mRNAs in bone tissues of ovariectomized rats, and to discuss the mechanism of soybean isoflavaones in preventing and treating postmenopausal osteoporosis (PMO). METHODS: Thirty adult SD rats were randomly divided into 3 groups: sham-operation group, untreated group and SI-treated group. The rats in the last two groups were bilaterally ovariectomized. The bone density of L(3) to L(6) vertebrae was detected after 12-week intervention. Total RNA was extracted from femur head and the expression levels of OPG and OPGL mRNAs were examined by real-time quantitative polymerase chain reaction. RESULTS: SI could increase the bone density of lumbar vertebrae in ovariectomized rats, up-regulate the expression level of OPG mRNA and down-regulate the ratio of OPGL mRNA/OPG mRNA, but exert no significant effect on the expression of OPGL mRNA. CONCLUSION: The therapeutic effects of SI on PMO may be related to regulating the expression levels of OPG and OPGL mRNAs, and the ratio of OPGL mRNA/OPG mRNA.

[Effects of kangfengshi granules on expressions of osteoprotegerin, RANKL and M-CSF in bone tissues of rats with collagen-induced arthritis].

OBJECTIVE: To observe the effects of Kangfengshi Granules (KFSG) on expressions of the mRNAs of osteoprotegerin (OPG), receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) in bone tissues of rats with collagen-induced arthritis. METHODS: Forty SD rats were randomly divided into four groups: normal control group, untreated group, cyclosporine A (CsA)-treated group and KFSG-treated group. Except the rats in the normal control group, all the other rats received subcutaneous injection of collagen II to establish collagen-induced arthritis (CIA) models. Then the rats in each group were fed normal saline or corresponding drugs for four weeks. Total RNA was extracted from carpal and digital bones. The expressions of OPG, RANKL and M-CSF mRNAs were examined by real-time PCR. RESULTS: The total incidence of arthritis induced by collagen II in the rats was approximately 90%. The expression levels of RANKL and M-CSF mRNAs and the RANKL mRNA/OPG mRNA ratio in the untreated group, KFSG-treated group and CsA-treated group were all significantly higher than those in the normal control group, while the expression levels of OPG mRNA in those three groups were significantly lower than that in the normal control group. The expression level of OPG mRNA in the KFSG-treated group was obviously higher while the expression level of M-CSF mRNA and the RANKL mRNA/OPG mRNA ratio in the same group were both lower as compared with those in the untreated group. CONCLUSION: The molecular mechanism of effects of KFSG on bone erosion and destruction induced by rheumatoid arthritis is closely correlated with up-regulating the expression of OPG mRNA, down-regulating the expression of M-CSF mRNA and RANKL mRNA/OPG mRNA ratio.

[Effect of Herba Epimedii flavone on the osteoblasts metabolism in vitro].

OBJECTIVE: To explore the effect of Herba Epimedii flavone (HEF) on the osteoblast metabolism in vitro. METHOD: Osteoblast were obtained from new born rat calvaria by digestive enzymes. MTF, PNPP and RT-PCR were used to observe the proliferation, activity of ALP and mRNA expression of OPG and RANKL of cultured osteoblasts in vitro. RESULT: It was found that HEF had the effect on stimulating cell proliferation, activity of ALP and the mRNA expression of OPG of cultured osteoblasts (P < 0.01, P < 0.05). CONCLUSION: HEF can promote the proliferation, the differentiation and the expression of OPG mRNA of the osteoblasts cultured in vitro.

Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis.

OBJECTIVE: Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG). METHODS: We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting. RESULTS: Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression. CONCLUSION: The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.

Low-energy irradiation stimulates formation of osteoclast-like cells via RANK expression in vitro.

Low-energy laser irradiation (Ga-Al-As semiconductor laser, output 50 mW) was applied to rat osteoclast precursor cells for 1, 3, 6, or 10 min at 24-h intervals during the culture period. The number of tartrate-resistant acid phosphatase positive multinucleate cells was increased by approximately 1.3-fold in the 3- and 6-min irradiation groups. Further, osteoclasts appeared on day 2 in the laser irradiation groups but not until day 3 in the control groups. In immunohistochemical staining for receptor activator of NF-kappaB (RANK), the laser irradiation groups showed significantly greater amounts of staining in comparison with the control group on days 2 and 3. Reverse transcription-polymerase chain reaction results also showed that the expressions of RANK were upregulated. In the pit formation assay, resorption pits were significantly more abundant in the laser irradiation groups than in the controls. The present results suggest that low-energy laser irradiation facilitates differentiation and activation of osteoclasts via RANK expression.

Evaluation of human liver slices and reporter gene assays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans.

PURPOSE: The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. METHODS: The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). RESULTS: In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values. CONCLUSIONS: Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers.

The expression of the CAR gene and inducibility of CYP2B protein in the liver of male Wistar rats treated with phenobarbital (PB) and triphenyldioxane (TPD) were investigated. To clarify the role of phosphorylation/dephosphorylation in these processes, rats were treated with inhibitors of Ca(2+)/calmodulin-dependent kinase II (W7) or protein phosphatases PP1 and PP2A (OA) before induction. Constitutive expression of the CAR gene in livers of untreated rats was detected by multiplex RT-PCR. Treatment with W7 resulted in a 2.8-fold induction of CAR gene expression, whereas OA led to a 2.4-fold decrease of the mRNA level. The same results were obtained for CYP2B genes expression, which were increased by W7 treatment (two-fold) and decreased by OA (2.3-fold). PB-induction did not lead to significant alteration in the level of CAR gene expression, although CYP2B genes expression was enhanced two-fold over control values. TPD caused a two-fold increase of both CAR and CYP2B mRNA levels. Both inducers reduced the effects of inhibitors on CAR gene expression. Results of EMSA showed that PB, TPD or W7 alone induced formation of complexes of NR1 with nuclear proteins. Appearance of the complexes correlated with an increase in CYP2B expression, and their intensities were modulated by the protein kinase inhibitors. Thus, our results demonstrate that constitutive expressions of CAR as well as CYP2B during induction are regulated by phosphorylation/dephosphorylation processes.

Areca nut extracts modulated expression of alkaline phosphatase and receptor activator of nuclear factor kappaB ligand in osteoblasts.

OBJECTIVES: Areca chewers have a higher prevalence of periodontal diseases than non-chewers. This study was to determine the possible effects of ripe areca nut extracts (rANE) on viability and gene expression of alkaline phosphatase (ALP), receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) in human osteoblasts. METHODS: The effects of rANE on cell viability of osteoblast-like MG63 cells were determined using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) that measures metabolic activity. Gene expression of ALP, RANKL and OPG was examined using reverse transcription-polymerase chain reaction. ALP activity and RANKL protein were further examined using substrate assay and confocal laser scanning microscopy, respectively. RESULTS: Relative viability was reduced to approximately 50% when 25 microg/ml of rANE was used. The expression of OPG mRNA in MG63 cells was not altered by rANE. However, decreased levels of mRNA and enzyme activity of ALP were observed. Moreover, the expressions of mRNA and protein of RANKL were stimulated by rANE in a dose-dependent manner. CONCLUSIONS: The rANE affected morphology and viability of osteoblasts. We also present novel evidence demonstrating that areca nut may compromise the periodontal health of areca chewers by suppression of ALP gene expression and elevation of RANKL gene expression in osteoblasts.

[Effect of Herba Epimedii Flavone on expression of OPG and RANKL in rat osteoblasts].

OBJECTIVE: To explore the effect of Herba Epimedii Flavone (HEF) on expression of OPG and RANKL in rat osteoblasts. METHODS: Osteoblasts were obtained from new born rat calvaria by digestive enzymes. The different HEF concentrations (0.1, 1, 10, 100 ng/ml)were added into the culture medium. Total mRNA was prepared from osteoblasts after 48 h and 96 h, and mRNA expression of OPG and RANKL were detected by RT-PCR. RESULTS: The mRNA expression of OPG in osteoblasts significantly increased compared with that of the control group, and the V(OPF)/V(RANKL) ratio was deceased markedly in the treatment of 10 ng/ml group after 48 h. After 96 h, the mRNA expression of OPG significantly increased and the V(OPG)/V(RANKL) ratio was also decreased in the treatment group compared with that of the control group. CONCLUSION: HEF suppress the differentiation and maturate of osteoblast. Osteoporosis can be prevented and cured by HEF.

Activation of PPAR gamma and delta by conjugated linoleic acid mediates protection from experimental inflammatory bowel disease.

BACKGROUND & AIMS: The molecular targets for the protective actions of conjugated linoleic acid (CLA) on experimental inflammatory bowel disease (IBD) are unknown. We used a loss-of-function approach to investigate whether CLA ameliorated colitis through a peroxisome proliferator-activated receptor gamma (PPAR gamma)-dependent mechanism. METHODS: The expression of PPAR gamma, delta, and their target genes in the colon of mice fed control or CLA-supplemented diets was assayed after a 7-day dextran sodium sulfate (DSS) challenge by quantitative real-time polymerase chain reaction (PCR). Additionally, nuclear factor-kappa B (NF-kappaB) p65 activation was quantified in the colon. To determine the involvement of PPAR gamma in the mechanism of action of CLA directly, specific deletions of PPAR gamma in the colon were performed in mice by using the Cre-lox recombination system. Colonic PPAR gamma null mice and wild-type littermates were fed either a CLA-supplemented or a control diet for 42 days and challenged with 2.5% DSS. The therapeutic efficacy of CLA also was examined by using the CD4 + CD45RB hi transfer colitis model. RESULTS: CLA induced PPAR gamma and delta, transcriptionally modulated PPAR gamma and delta-responsive gene clusters involved in lipid metabolism (uncoupling protein [UCP]1, UCP3, PPAR gamma coactivator 1alpha [PGC-1alpha], and CD36) and epithelial cell maturation (Gob-4 and Keratin 20). Additionally, CLA repressed tumor necrosis factor alpha (TNF-alpha) expression and NF-kappaB activation while inducing the immunoregulatory cytokine transforming growth factor beta 1 (TGF-beta 1 ). Clinically, CLA ameliorated DSS- and CD4 + -induced colitis. Loss of the PPAR gamma gene in the colon abrogated the beneficial effects of CLA in DSS colitis. CONCLUSIONS: Our studies provide molecular evidence in vivo, suggesting that CLA ameliorates colitis through a PPAR gamma-dependent mechanism.

Suppressive effect of Kanzo-bushi-to, a Kampo medicine, on collagen-induced arthritis.

Kanzo-bushi-to (KBT) is a traditional Japanese herbal medicine (Kampo medicine), which is used in Japan to treat rheumatoid arthritis. In the present study, we investigated the suppressive effect of KBT on collagen-induced arthritis (CIA) and further studied the underlying mechanism. CIA was induced in male DBA/1J mice by immunization with bovine type II collagen, followed by a booster injection 21 d later. KBT was given at a dose of 430 mg/kg/d from three days before the first immunization to the end of the experiment. KBT suppressed CIA development effectively and further protected focal bone erosion and bone destruction as evidenced by the reduced histological score. Histochemical examination revealed that KBT decreased TRAP-positive cells at the synovium-bone interface and at the sites of focal bone erosion, coincident with the findings that RANKL/OPG mRNA ratio was significantly reduced by KBT treatment. KBT also decreased mRNA levels of M-CSF and iNOS in joints and of iNOS in peritoneal macrophages. In conclusion, KBT prevented osteoclast generation by decreasing RANKL/OPG ratio and M-CSF mRNA levels, resulting in reduction in bone erosion and destruction. In addition, KBT has anti-inflammatory effect such as the suppression of iNOS expression in peritoneal macrophages and joints of CIA mice. These finding suggests that KBT is a potential new therapeutic agent for the treatment of RA.

[Effect of conjugated linoleic acid on PPAR gamma gene expression and serum leptin in obese rat].

OBJECTIVE: In order to study the effect of conjugated linoleic acid on PPAR gamma gene expression, serum leptin, blood glucose and blood lipid level. METHODS: Five groups of male Wistar rats were randomly selected and fed with different diets (basic diet, high-fat diet, high-fat with 0.75% CLA, high-fat with 1.50% CLA, and high-fat with 3.00% CLA) for 12 weeks. At the end of experiment, the rats were sacrificed, the ratio of lipids to body weight were calculated, and the blood glucose, serum lipids, serum leptin were measured. RT-PCR was used for measuring the expression of PPAR gamma mRNA in white adipose tissue of rats. RESULTS: It was shown that the supplement of CLA could decrease decreased blood glucose, serum triglyceride, cholesterol and leptin levels, and increased the expression of PPAR gamma in white adipose tissue of obese rat. CONCLUSION: CLA could decrease blood glucose, serum lipids, and decrease the levels of leptin possibly via activating peroxisome proliferator--activated receptor gamma (PPAR gamma), resulting in the improvement of leptin resistance of obese rat.

MT1 melatonin receptor mRNA tissular localization by PCR amplification.

OBJECTIVES: The pineal gland transduces photoperiodic informations to the neuroendocrine axis through the nocturnally melatonin secretion. This hormonal message plays a major role in the biological rhythm regulation. By autoradiography, more than 130 melatonin putative targets have been reported in the central nervous system (CNS) and in peripheral tissues. However, cross-species consensus concern only a few of them like the suprachiasmatic nuclei (SCN), the master circadian clock, and the pars tuberalis of the pituitary. Recently, MT1 melatonin receptor cDNA have been cloned in several mammals providing us with new tools to investigate its tissular location at the gene level. In the present study, we report a screening for MT1 mRNA by RT-PCR amplification of numerous tissue mRNA. METHOD: mRNA were extracted from a large variety of rat tissues. To semi-quantify the melatonin receptor mRNA expression level, each cDNA was amplified concomitantly with both beta-actin and MT1 specific primers. RESULTS: In central and peripheral tissues previously reported to bind melatonin, strong PCR signals were logically observed. More surprisingly, a vast majority of studied tissues express MT1 mRNA and then might be responsive to melatonin. CONCLUSION: Numerous biological functions express diurnal rhythmicity and internal-synchronization. As, most of them apparently do not receive any out-coming neuronal message from the SCN, endocrine communication was proposed to support biological rhythm synchronization. Our present data strengthen the idea that the nocturnally restricted melatonin secretion could be one internal zeitgeber that putatively distributes the endogenous circadian rhythmicity to all tissues expressing melatonin receptors.