OBJECTIVETo screen the active component of Wuzhuyutang (WZYT, Evodiae prescription) and investigate the regulatory effects of the components in WZYT on the TPH2 promoter, and to explore the possible molecular mechanism of WZYT on migraine.

METHODBy transfecting a TPH2 promoter regulating Red Fluorescent Protein expressing plasmid into PC12 cell, the global fluorescence intensities and calculations of fluorescent cells after components treatment were statistically evaluated.

RESULTDifferent regulatory effects of different components in WZYT with different concentrations on TPH2 promoter were observed.

CONCLUSIONTPH2 promoter drove Red Fluorescent Protein expressing cell line can be used as system screening components targeting TPH2 promoter activity. The possible mechanism of WZYT on migraine may due to its stimulating effects on TPH2 promoter, and promote the synthesis and release of 5-HT in cerebral.

Animals ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Evodia ; chemistry ; Humans ; Migraine Disorders ; drug therapy ; enzymology ; genetics ; PC12 Cells ; Promoter Regions, Genetic ; drug effects ; Rats ; Tryptophan Hydroxylase ; genetics ; metabolism

This study was conducted to evaluate the effects of vardenafil (Levitra), a phosphodiesterase-5 (PDE-5) inhibitor, on cell proliferation in the hippocampal dentate gyrus and on 5-hyroxytryptamine (5-HT, serotonin) synthesis and tryptophan hydroxylase (TPH) expression in the rat dorsal raphe nucleus. Male Sprague-Dawley rats were divided into 6 groups (n=5 in each group): a control group, a 0.5 mg/kg-1 day vardenafil-treated group, a 1 mg/kg-1 day vardenafil-treated group, a 2 mg/kg-1 day vardenafil-treated group, a 1 mg/kg-3 day vardenafil-treated group, and a 1 mg/kg-7 day vardenafil-treated group. 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry was then performed to evaluate cell proliferation in the dentate gyrus. In addition, 5-HT and TPH immunohistochemistry was conducted to evaluate serotonin expression in the dorsal raphe. The results revealed that treatment with vardenafil increased cell proliferation in the dentate gyrus and enhanced 5-HT synthesis and TPH expression in the dorsal raphe in a dose- and duration-dependent manner. The findings demonstrate that the increasing effect of vardenafil on cell proliferation is closely associated with the enhancing effect of vardenafil on serotonin expression under normal conditions.
Animals ; Cell Proliferation/*drug effects ; *Dentate Gyrus/cytology/drug effects/metabolism ; Imidazoles/*pharmacology ; Male ; Phosphodiesterase Inhibitors/*pharmacology ; Piperazines/*pharmacology ; *Raphe Nuclei/cytology/drug effects/metabolism ; Rats ; Rats, Sprague-Dawley ; Serotonin/*biosynthesis ; Sulfones/pharmacology ; Triazines/pharmacology ; Tryptophan Hydroxylase/metabolism

Phenylketonuria is metabolic disorder that results from a deficiency of the hepatic phenylalanine hydroxylase. But among patients with hyperphenylalaninemia, the defect resides in one of the enzymes necessary for production or recycling of tetrahydrobiopterin (BH4). The reduction of BH4 affects not only phenylalanine metabolism but also formation of the tyrosine related neurotransmitter, dopamin and tryptophan related neurotransmitter, serotonin. Administration of L-dopa and 5-hydroxytriptophan seems to be the most effective treatment and may prevent irreversible neurologic damage if started early in life in hyperphenylalaninemia due to deficiency of cofactor BH4. Therefore, all patients with PKU and hyperphenylalaninemia should be tested for BH4 deficiency as early as passible. So we measured reduced forms of biopterin in urines of 19 phenylketonuria patients by Funkushima and Nixon method and 13 of PKU patients measured dihydropterin reductase (DHPR) in white blood cells by modified Narisawa method. We could not find abnormal pterin patterns of cofactor BH4 and normal value of DHPR. All Korean 19 PKU children were classic PKU. A missense mutation has been identified in the phenylalanine hydroxylase (PAH) gene of 16 Koran PKU patients. 5 mutations (IVS4, Y204 C, R243Q, Y356 X, R413 P) have been identified. The frequency of these mutations was found to be 50% of PKU alleles. The IVS4 mutation had a high frequency in Korea and southern China, due to the result of the founder effect and genetic drift. the R413 P mutation, which may have originated in the regions surrounding the Baikal, expanded to northen China and Japan. We were not able to find Caucasian mutations in Korean ptiets. PKU mutations occured after racial divergence between Caucasian and Mongoloids. We observed that PKU patients with Y 204 C and R413 P mutations showed mild mild clinical phenotype but IVS4 mutation had severe mental retardation. the establishment of genotype will therefore aid in the prediction of clinical phenotypes in patients with this disease. So, pterin and DHPR measurement and DNA analsis will be useful for prognosis and proper treatment of PKU patients.
Alleles ; Biopterin ; Child ; China ; DNA* ; Founder Effect ; Genetic Drift ; Genotype ; Humans ; Intellectual Disability ; Japan ; Korea ; Leukocytes ; Levodopa ; Metabolism ; Mutation, Missense ; Neurotransmitter Agents ; Oxidoreductases ; Phenotype ; Phenylalanine ; Phenylalanine Hydroxylase ; Phenylketonurias ; Prognosis ; Recycling ; Reference Values ; Serotonin ; Tryptophan ; Tyrosine

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.
3,4-Dihydroxyphenylacetic Acid/pharmacology* ; Animals ; Antidepressive Agents/therapeutic use* ; Chromatography, Liquid ; Depression/drug therapy* ; Disease Models, Animal ; Dopamine ; Eosine Yellowish-(YS)/pharmacology* ; Hematoxylin/pharmacology* ; Hippocampus/metabolism* ; Homovanillic Acid/pharmacology* ; Hydroxyindoleacetic Acid/metabolism* ; Methoxyhydroxyphenylglycol/pharmacology* ; Monoamine Oxidase/metabolism* ; Neurotransmitter Agents/metabolism* ; Norepinephrine/pharmacology* ; Plant Extracts ; Rats ; Rehmannia/chemistry* ; Serotonin/metabolism* ; Serotonin Plasma Membrane Transport Proteins/pharmacology* ; Stress, Psychological/metabolism* ; Tandem Mass Spectrometry ; Tryptophan Hydroxylase/metabolism*