PURPOSE: Our previous study demonstrated that persimmon (Diospyros kaki Thumb.) at different stages of ripening provided different protective effects against high-fat/cholesterol diet (HFD)-induced dyslipidemia in rats. In this study, we compared the metabolites profile and gene expressions related to triglyceride (TG)/cholesterol metabolism in vitro and in vivo after treating with persimmon water extracts (PWE) or tannin-enriched persimmon concentrate (TEP). METHODS: Primary and secondary metabolites in test materials were determined by GC-TOF/MS, UHPLC-LTQ-ESI-IT-MS/MS, and UPLC-Q-TOF-MS. The expression of genes related to TG and cholesterol metabolism were determined by RT-PCR both in HepG2 cells stimulated by oleic acid/palmitic acid and in liver tissues obtained from Wistar rats fed with HFD and PWE at 0, 150, 300, and 600 mg/d (experiment I) or TEP at 0, 7, 14, and 28 mg/d (experiment II) by oral gavage for 9 weeks. RESULTS: PLS-DA analysis and heatmap analysis demonstrated significantly differential profiling of metabolites of PWE and TEP according to processing of persimmon powder. In vitro, TEP showed similar hypolipidemic effects as PWE, but significantly enhanced hypocholesterolemic effects compared to PWE in sterol regulatory element-binding protein 2 (SREBP2), HMG-CoA reductase (HMGCR), proprotein convertase subtilisin/kexin type 9 (PCSK9), cholesterol 7α-hydroxylase (CYP7A1), and low density lipoprotein receptor (LDLR) gene expression. Consistently, TEP and PWE showed similar hypolipidemic capacity in vivo, but significantly enhanced hypocholesterolemic capacity in terms of SREBP2, HMGCR, and bile salt export pump (BSEP) gene expression. CONCLUSION: These results suggest that column extraction after hot water extraction may be a good strategy to enhance tannins and long-chain fatty acid amides, which might cause stimulation of hypocholesterolemic actions through downregulation of cholesterol biosynthesis gene expression and upregulation of LDL receptor gene expression.
Amides ; Animals ; Bile ; Cholesterol ; Diet ; Diospyros* ; Down-Regulation ; Dyslipidemias ; Gene Expression ; Hep G2 Cells ; In Vitro Techniques* ; Liver ; Metabolism ; Oxidoreductases ; Proprotein Convertases ; Rats ; Rats, Wistar ; Receptors, LDL ; Tannins ; Triglycerides ; Up-Regulation ; Water

BACKGROUND/AIMS: There has been controversy about the role of Toll-like receptor 2 (TLR2) in renal injury following ureteric obstruction. Although inhibition of the renin angiotensin system (RAS) reduces TLR2 expression in mice, the exact relationship between TLR2 and RAS is not known. The aim of this study was to determine whether the RAS modulates TLR2. METHODS: We used 8-week-old male wild type (WT) and TLR2-knockout (KO) mice on a C57Bl/6 background. Unilateral ureteral obstruction (UUO) was induced by complete ligation of the left ureter. Angiotensin (Ang) II (1,000 ng/kg/min) and the direct renin inhibitor aliskiren (25 mg/kg/day) were administrated to mice using an osmotic minipump. Molecular and histologic evaluations were performed. RESULTS: Ang II infusion increased mRNA expression of TLR2 in WT mouse kidneys (p < 0.05). The expression of renin mRNA in TLR2-KO UUO kidneys was significantly higher than that in WT UUO kidneys (p < 0.05). There were no differences in tissue injury score or mRNA expression of monocyte chemotactic protein 1 (MCP-1), osteopontin (OPN), or transforming growth factor beta (TGF-beta) between TLR2-KO UUO and WT UUO kidneys. However, aliskiren decreased the tissue injury score and mRNA expression of TLR2, MCP-1, OPN, and TGF-beta in WT UUO kidneys (p < 0.05). Aliskiren-treated TLR2-KO UUO kidneys showed less kidney injury than aliskiren-treated WT UUO kidneys. CONCLUSIONS: TLR2 deletion induced activation of the RAS in UUO kidneys. Moreover, inhibition of both RAS and TLR2 had an additive ameliorative effect on UUO injury of the kidney.
Amides/*pharmacology ; Angiotensin II/pharmacology ; Animals ; Disease Models, Animal ; Fibrosis ; Fumarates/*pharmacology ; Kidney/*drug effects/metabolism/pathology ; Male ; Mice, Inbred C57BL ; Mice, Knockout ; Nephritis, Interstitial/genetics/metabolism/pathology/*prevention & control ; RNA, Messenger/genetics/metabolism ; Renin/*antagonists & inhibitors/metabolism ; Renin-Angiotensin System/*drug effects ; Toll-Like Receptor 2/deficiency/drug effects/genetics/*metabolism ; Ureteral Obstruction/*drug therapy/genetics/metabolism/pathology

OBJECTIVETo investigate the role of non-receptor tyrosine kinase Tec in the production of TNF-α and IL-1β from macrophages induced by LPS and its related mechanism.

METHODSRAW264.7 mononuclear-macrophages cultured in 6-well plates were divided into 4 groups according to the random number table, with 24 wells in each group. Cells in blank group were routinely cultured (cultured with DMEM medium containing 10% FBS) for 2 hours. Cells in LFM-A13 group were pretreated with 75 µmol/L Tec specific inhibitor LFM-A13 for 1 hour and then routinely cultured for 1 hour. Cells in LPS group were routinely cultured for 1 hour and then treated with 0.1 µg/mL LPS for 1 hour. Cells in LPS+LFM-A13 group were pretreated with 75 µmol/L LFM-A13 for 1 hour and then treated with 0.1 µg/mL LPS for 1 hour. The content of TNF-α and IL-1β in culture supernatant of cells was determined with ELISA. The mRNA expressions of TNF-α and IL-1β in cells were assayed with real-time fluorescent quantitative RT-PCR. The activity of intracellular Tec, p38 MAPK, and transforming growth factor activated kinase 1 (TAK1) was determined with Western blotting. Data were processed with one-way analysis of variance and LSD test.

RESULTSThe content of TNF-α and IL-1β in culture supernatant of cells in LFM-A13 group was close to that in blank group (with P values above 0.05). The mRNA expressions of TNF-α and IL-1β in the cells of LFM-A13 group were close to those of blank group (with P values above 0.05). The content of TNF-α and IL-1β in culture supernatant of cells in LPS group was respectively (1 213 ± 154) and (636 ± 90) pg/mL, which was higher than that in blank group [(330 ± 44) and (211 ± 31) pg/mL, with P values below 0.01]. The mRNA expressions of TNF-α and IL-1β in the cells of LPS group were respectively 1.57 ± 0.22 and 1.44 ± 0.24, which were significantly higher than those of blank group (1.00 ± 0.18 and 1.00 ± 0.19, with P values below 0.01). The content of TNF-α and IL-1β in culture supernatant of cells in LPS+LFM-A13 group was respectively (787 ± 109) and (453 ± 64) pg/mL, which was significantly lower than that in LPS group (with P values below 0.05). The mRNA expressions of TNF-α and IL-1β in the cells of LPS+LFM-A13 group were respectively 1.21 ± 0.15 and 1.21 ± 0.22, and they were significantly lower than those of LPS group (with P values below 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS+LFM-A13 group was close to that in blank group (with P values above 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS group was respectively 2.69 ± 0.41, 3.99 ± 0.65, and 2.07 ± 0.31, which was significantly higher than that in blank group (1.00 ± 0.17, 1.00 ± 0.16, and 1.00 ± 0.18, with P values below 0.01) and LPS+LFM-A13 group (1.02 ± 0.17, 1.18 ± 0.20, and 1.58 ± 0.28, P < 0.05 or P < 0.01).

CONCLUSIONSTec promotes the production and release of pro-inflammatory cytokines TNF-α and IL-1β from macrophages induced by LPS via TAK1-p38 MAPK signaling pathway.

Amides ; metabolism ; Cell Line ; Cytokines ; Interleukin-1beta ; metabolism ; secretion ; Lipopolysaccharides ; Macrophages ; metabolism ; Nitriles ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism ; secretion

Induced pluripotent stem cells (iPSCs) can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical research. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel approach to generating iPSCs. In this study, a new combination of small-molecule compounds (SMs) (sodium butyrate, A-83-01, CHIR99021, Y-27632) under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts (MEFs) in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs. The resulting cell lines resembled mouse embryonic stem (ES) cells with respect to proliferation rate, morphology, pluripotency-associated markers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcinogenicity and the use of exogenous reprogramming factors.
Amides ; pharmacology ; Animals ; Butyric Acid ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Extraembryonic Membranes ; cytology ; drug effects ; Folic Acid ; pharmacology ; Induced Pluripotent Stem Cells ; cytology ; drug effects ; Kruppel-Like Transcription Factors ; metabolism ; Mice ; Octamer Transcription Factor-3 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Pyrazoles ; pharmacology ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; SOXB1 Transcription Factors ; metabolism ; Thiocarbamates ; pharmacology ; Thiosemicarbazones

Sphingosylphosphorylcholine (SPC) induces differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs) into smooth muscle-like cells expressing alpha-smooth muscle actin (alpha-SMA) via transforming growth factor-beta1/Smad2- and RhoA/Rho kinase-dependent mechanisms. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) have been known to have beneficial effects in the treatment of cardiovascular diseases. In the present study, we examined the effects of simvastatin on the SPC-induced alpha-SMA expression and Smad2 phosphorylation in hASCs. Simvastatin inhibited the SPC-induced alpha-SMA expression and sustained phosphorylation of Smad2 in hASCs. SPC treatment caused RhoA activation via a simvastatin-sensitive mechanism. The SPC-induced alpha-SMA expression and Smad2 phosphorylation were abrogated by pretreatment of the cells with the Rho kinase inhibitor Y27632 or overexpression of a dominant negative RhoA mutant. Furthermore, SPC induced secretion of TGF-beta1 and pretreatment with either Y27632 or simvastatin inhibited the SPC-induced TGF-beta1 secretion. These results suggest that simvastatin inhibits SPC-induced differentiation of hASCs into smooth muscle cells by attenuating the RhoA/Rho kinase-dependent activation of autocrine TGF-beta1/Smad2 signaling pathway.
Amides/pharmacology ; Blotting, Western ; Cell Differentiation/*drug effects ; Cells, Cultured ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunohistochemistry ; Mesenchymal Stem Cells/*cytology/*drug effects ; Myocytes, Smooth Muscle/*cytology/*drug effects ; Phosphorylcholine/*analogs & derivatives/pharmacology ; Pyridines/pharmacology ; Simvastatin/*pharmacology ; Sphingosine/*analogs & derivatives/pharmacology ; rhoA GTP-Binding Protein/antagonists & inhibitors/metabolism

It has been demonstrated by our previous research that 4-(2-acetoxybenzoylamino) butyramide derivatives exhibited good antiepileptic activities. In this paper, to explore the SAR and improve the antiepileptic activities of these derivatives, a series of novel 4-(2-acetoxybenzoylamino) butyramide heterocyclic compounds (5a-5n) were synthesized and biologically evaluated. Their structures were confirmed by 1H MNR, ESI-MS and elemental analysis. Pharmacological test in vivo showed that target compounds (5f, 5i-5n) displayed strong antiepileptic activities on 4-AP induced epilepsy in mice with ED50 values ranging from 0.3137 to 0.3604 mmol x kg(-1).
4-Aminopyridine ; Amides ; administration & dosage ; chemical synthesis ; chemistry ; therapeutic use ; Animals ; Anticonvulsants ; administration & dosage ; chemical synthesis ; chemistry ; therapeutic use ; Dose-Response Relationship, Drug ; Epilepsy ; chemically induced ; drug therapy ; Female ; Lethal Dose 50 ; Male ; Mice ; Molecular Structure ; Random Allocation ; Receptors, GABA ; metabolism ; Structure-Activity Relationship

BACKGROUNDGastric cancer (GC) is one of the most common types of cancer in the world. A change in the metabolism of lipids in tumor cells could lead to the pathogenesis of cancer. In this study, we investigated fatty acid and fatty acid amide metabolic perturbations associated with GC morbidity.

METHODSGas chromatography/mass spectrometry (GC/MS) was utilized to analyze fatty acids (FAs) and fatty acid amides (FAAs) of GC tissues and matched normal mucosae from 30 GC patients. Acquired lipid data was analyzed using non parametric Wilcoxon rank sum test to find the differential biomarkers for GC and diagnostic models for GC were established by using orthogonal partial least squares discriminant analysis (OPLS-DA).

RESULTSA total of 13 FAs and 4 FAAs were detected using GC/MS and 5 differential FAs as well as oleamide were identified with significant difference (P<0.05). The OPLS-DA model generated from lipid profile showed adequate discrimination of GC tissues from normal mucosae while the OPLS-DA model failed to separate GC specimens of different TNM stages. A total of 8 variables were obtained for their most contribution in the discriminating model (Variable importance in the projection (VIP) value>1.0), five of which were detected with significant difference (P<0.05).

CONCLUSIONSFA and FAA metabolic profiles have great potential in detecting GC and helping understand perturbations of lipid metabolism associated with GC morbidity.

Amides ; metabolism ; Fatty Acids ; metabolism ; Female ; Gas Chromatography-Mass Spectrometry ; Humans ; In Vitro Techniques ; Male ; Metabolic Diseases ; physiopathology ; Stomach Neoplasms ; metabolism ; pathology

The aim of the current study was to investigate the spectra in the different organs of the rats which died of massive hemorrhage; to explore their spectral changes 15 days postmortem and the best mathematical model with different band absorption ratio changes to postmortem interval(PMI); and to compare the spectral changes of different temperature. Thirty male Sprague-Dawley rats were sacrificed by cutting abdominal aorta, and the cadavers were divided equally and kept at 4 degrees C, 20 degrees C and 30 degrees C in the control chamber. From the same rat, seven different organs were sampled at intervals of 1-15 days postmortem, and then measured by Fourier transfom infrared (FTIR) spectrometer. Six mathematical model functions were explored. The absorbance of bands and band absorbance ratios of absorption peak in each organ showed a time-dependent increase or decrease, most band absorbance ratios remaining stable for 7-15 days postmortem. Cubic model functions of the various bands absorbance ratios against PMI showed a stronger related coefficient. The absorbance bands with obvious changes at 20 degrees C showed stabilized tendencies at 4 degrees C and significant changes at 30 degrees C within 15 days postmortem. In addition, FTIR spectroscopy revealed a time-dependent metabolic process, with potential of being used to estimate PMI during 7 days postmortem, which merits further investigation.
Amides/analysis* ; Animals ; Brain Chemistry ; Cadaver ; Forensic Pathology ; Hemorrhage/pathology* ; Liver/metabolism* ; Lung/metabolism* ; Male ; Models, Animal ; Models, Statistical ; Models, Theoretical ; Muscle, Skeletal/metabolism* ; Postmortem Changes ; Rats ; Rats, Sprague-Dawley ; Spectroscopy, Fourier Transform Infrared ; Temperature ; Time Factors

OBJECTIVE[corrected] To assess the effects of metallothionein on myocyte apoptosis and energy supply of isolated rabbit heart muscle during perfusion with ropivacaine..

METHODSSixty New Zealand white male rabbits were randomized into 3 equal groups. In group I, the rabbits received a intreaperitioneal injection of distilled water 24 h before isolation of the heart with perfusion by Langendoff model; in group II, distilled water was injected intreaperitioneally, and 24 h later the heart was isolated and perfused with Langendoff model and ropivacaine; in group III, 3.6% ZnSO(4) was injected intreaperitioneally and the isolated heart was perfused with Langendoff model and ropivacaine. The myocardial metallothionein content, myocyte apoptosis, and myocardial ATP, ADP and AMP content were detected.

RESULTSThe myocardial metallothionein content was significantly higher in group III than in the other two groups; the percent of myocyte apoptosis was the highest in group II, and was significantly higher in group III than in group I. The myocardial content of ATP was the highest in group I, and was significantly higher in group III than in group II.

CONCLUSIONMetallothionein can significantly inhibit myocyte apoptosis and alleviate energy supply disorder induced by ropivacaine.

Amides ; pharmacology ; Animals ; Apoptosis ; drug effects ; Energy Metabolism ; drug effects ; In Vitro Techniques ; Male ; Metallothionein ; pharmacology ; Myocardium ; cytology ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Perfusion ; Rabbits

OBJECTIVETo investigate of the regulatory effect of Rho-kinase pathway activation on angiotensin II (Ang II)-induced contraction of human airway smooth muscle cells (HASMCs) in vitro.

METHODSCultured primary HASMCs were divided into control group, AngII group, AngII + irbesartan group and AngII + Y-27632 group with corresponding treatment. AngII-induced contraction of HASMCs was evaluated using collagen gel lattices and observed morphologically using immunofluorescence assay. Western Blotting was significantly performed to examine the protein expression of Rho-kinase signal pathway.

RESULTSAngII-induced HASMC contraction was inhibited by treatments with irbesartan and Y-27632 as shown by gel contraction assay (P<0.001). Y-27632 treatment produced a stronger inhibitory effect than irbesartan on the expression of phosphorylated moesin, a substrate of Rho kinase (P<0.05).

CONCLUSIONAngII induces the contraction of HASMCs partially as a result of activation of Rho-kinase pathway.

Amides ; pharmacology ; Angiotensin II ; pharmacology ; Asthma ; physiopathology ; Biphenyl Compounds ; pharmacology ; Bronchi ; cytology ; Humans ; Muscle Contraction ; drug effects ; Muscle, Smooth ; cytology ; Primary Cell Culture ; Pyridines ; pharmacology ; Signal Transduction ; drug effects ; Tetrazoles ; pharmacology ; rho-Associated Kinases ; metabolism