Determination of Multiple Neurotransmitters through LC-MS/MS to Confirm the Therapeutic Effects of Althaea rosea Flower on TTX-Intoxicated Rats.

Tetrodotoxin (TTX) inhibits neurotransmission in animals, and there is no specific antidote. In clinical practice in China, Althaea rosea (A. rosea flower) extract has been used to treat TTX poisoning. In this work, the efficacy of the ethyl acetate fraction extract of A. rosea flower in treating TTX poisoning in rats was investigated. A high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to determine nine neurotransmitters in rat brain tissue, including γ-aminobutyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT), noradrenaline (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-3-acetic acid (5-HIAA), epinephrine (E), and tyramine (Tyn). The detoxifying effect of A. rosea flower was verified by comparing the changes in neurotransmitters' content in brain tissue before and after poisoning in rats. The assay was performed in multiple reaction monitoring mode. The quantification method was performed by plotting an internal-standard working curve with good linearity (R2 > 0.9941) and sensitivity. Analyte recoveries were 94.04-107.53% (RSD < 4.21%). Results indicated that the levels of 5-HT, DA, E, and NE in the brains of TTX-intoxicated rats decreased, whereas the levels of GABA, Tyn, and 5-HIAA showed an opposite trend, and HVA and DOPAC were not detected. The levels of all seven neurotransmitters returned to normal after the gavage administration of ethyl acetate extract of A. rosea flower to prove that the ethyl acetate extract of A. rosea flower had a therapeutic effect on TTX poisoning. The work provided new ideas for studies on TTX detoxification.

[Effect of Rehmanniae Radix on depression-like behavior and hippocampal monoamine neurotransmitters of chronic unpredictable mild stress model rats].

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.

Changes in metabolites level in internet-addicted adolescents through exercise.

BACKGROUND: Internet addiction has been particularly prevalent among adolescents in recent years. This type of addiction, similar to drug addiction, causes dependence and disturbance in brain reward pathways. Physical activity is one way to prevent and treat some types of addiction. Aerobic exercise affects the dopaminergic and serotonergic pathways and improves the side effects of addiction. In this study, we examined the effect of eight weeks of aerobic exercise on the dopamine metabolite including homovanillic acid, L-tryptophan and 5-hydroxy indole acetic acid, which are precursors and the final product of serotonin metabolism in adolescent boys with internet addiction. MATERIALS AND METHODS: Twenty-nine healthy non-internet addicted and internet-addicted boys were selected and divided into three groups: exercising internet-addicted (G1), internet-addicted (G2), and healthy non-internet addicted (G3) groups. Before and after aerobic exercise, 24-h urine samples were collected, and the target metabolites were analyzed spectrophotometrically. RESULTS: Results showed that for pre-exercise subjects, there was a significant difference in the homovanillic acid levels in G3 as compared to G1 and G2. For post-exercise cases, the changes were significant in G1 in comparison to G2 and for G2 in comparison G3. For pre-exercise subjects, there was a significant difference in the L-tryptophan level in G3 and G1, as well as G3 and G2. CONCLUSION: It can be concluded that Aerobic exercise can improve the dopaminergic system that is disrupted by internet addiction.

Saffron Extract-Induced Improvement of Depressive-Like Behavior in Mice Is Associated with Modulation of Monoaminergic Neurotransmission.

Depressive disorders represent a major public health concern and display a continuously rising prevalence. Importantly, a large proportion of patients develops aversive side effects and/or does not respond properly to conventional antidepressants. These issues highlight the need to identify further therapeutic strategies, including nutritional approaches using natural plant extracts with known beneficial impacts on health. In that context, growing evidence suggests that saffron could be a particularly promising candidate. This preclinical study aimed therefore to test its antidepressant-like properties in mice and to decipher the underlying mechanisms by focusing on monoaminergic neurotransmission, due to its strong implication in mood disorders. For this purpose, the behavioral and neurobiochemical impact of a saffron extract, Safr'Inside™ (6.5 mg/kg per os) was measured in naïve mice. Saffron extract reduced depressive-like behavior in the forced swim test. This behavioral improvement was associated with neurobiological modifications, particularly changes in serotonergic and dopaminergic neurotransmission, suggesting that Safr'Inside™ may share common targets with conventional pharmacological antidepressants. This study provides useful information on the therapeutic relevance of nutritional interventions with saffron extracts to improve management of mood disorders.

Antioxidant and Biological Activities of Hydroxytyrosol and Homovanillic Alcohol Obtained from Olive Mill Wastewaters of Extra-Virgin Olive Oil Production.

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

Probiotics and fructo-oligosaccharide intervention modulate the microbiota-gut brain axis to improve autism spectrum reducing also the hyper-serotonergic state and the dopamine metabolism disorder.

The prevalence of autism spectrum disorders (ASD) is increasing, but its etiology remains elusive and hence an effective treatment is not available. Previous research conducted on animal models suggests that microbiota-gut-brain axis may contribute to ASD pathology and more human research is needed. This study was divided into two stages,.At the discovery stage, we compared the differences in gut microbiota profiles (using 16S rRNA sequencing), fecal SCFAs (using GC-MS) and plasma neurotransmitters (using UHPLC-MS/MS) of 26 children with ASD and 24 normal children. All 26 children with ASD participated in the intervention stage, and we measured the gut microbiota profiles, SCFAs and neurotransmitters before and after probiotics + FOS (n = 16) or placebo supplementation (n = 10). We found that gut microbiota was in a state of dysbiosis and significantly lower levels of Bifidobacteriales and Bifidobacterium longum were observed at the discovery stage in children with ASD. An increase in beneficial bacteria (Bifidobacteriales and B. longum) and suppression of suspected pathogenic bacteria (Clostridium) emerged after probiotics + FOS intervention, with significant reduction in the severity of autism and gastrointestinal symptoms. Compared to children in the control group, significantly lower levels of acetic acid, propionic acid and butyric acid were found, and a hyperserotonergic state (increased serotonin) and dopamine metabolism disorder (decreased homovanillic acid) were observed in children with ASD. Interestingly, the above SCFAs in children with autism significantly elevated after probiotics + FOS intervention and approached those in the control group. In addition, our data demonstrated that decreased serotonin and increased homovanillic acid emerged after probiotics + FOS intervention. However, the above-mentioned changes did not appear in the placebo group for ASD children. Probiotics + FOS intervention can modulate gut microbiota, SCFAs and serotonin in association with improved ASD symptoms, including a hyper-serotonergic state and dopamine metabolism disorder.

Total glucosides of paeony (TGP) extracted from Radix Paeoniae Alba exerts neuroprotective effects in MPTP-induced experimental parkinsonism by regulating the cAMP/PKA/CREB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The totally-amounted glucosides of paeony (TGP), which are made up of paeoniflorin, albiflorin, oxypaeoniflorin as well as benzoylpaeoniflorin, constitute the Baishao' actively-working component extracted from Radix Paeonia alba employed in conventional oriental medicine aiming to treat cerebrovascular disorders, such as Parkinson's disease. However, its pharmacologic mechanism is not clear. AIM OF THE STUDY: The initial investigation was made on TGP's neuroprotective effects on PD of the mouse model based on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) as well as the identification of potential involvement of a molecular signaling pathway. MATERIALS AND METHODS: The evaluation of the behavioral damage as well as neurotoxicity in mice was made through MPTP. Spontaneous motor activity test, as well as a test of Rota-rod on mice was employed for the measurement of bradykinesia symptom. Additionally, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) works as the determiner of the main monoamine neurotransmitters dopamine (DA) along with its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) as well as homovanillic acid (HVA) based on mouse hippocampus connected with the anti-Parkinson's disease like effect of TGP. Besides, the measurement of the effects of TGP treatment on the expressions level of TH, DAT, a-synuclein, p-CREBS133 as well as apoptosis influence was made with the help of western-blot assay with apoptosis-related markers such as Bax and Bcl-2. RESULTS: The results showed that TGP treatment lessened the behavior-based loss shown "in the spontaneous motor activity as well as the potential of falling to rotarod test". In addition, we found that pretreatment with TGP markedly improved motor coordination, striatal dopamine and its metabolite levels. Furthermore, pretreatment of TGP conducted the protection for dopaminergic neurons with the prevented MPTP-induced reductions within the tyrosine hydroxylase (TH), substantia nigra dopaminergic transporter (DAT), as well as increasing α-synuclein protein levels with transformed dopamine catabolism as well as inhibited dopamine turnover. Besides, TGP treatment helped reversed apoptosis signaling molecules Bcl-2/Bax' reduction; meanwhile improving p-CREBS133 the factor of growth signaling in the substantia nigra' decrease. CONCLUSION: These results suggested that TGP can enhance dopaminergic neuron's cell survival in the SNpc in virtue of the activated cAMP/PKA/CREB factor of growth on inhibiting the pathway of second messenger apoptosis as well. In conclusion, the current findings indicate TGP is expected to be a new cure for PD.

Anxiolytic Effect of Cedrol on Behavior and Brain Neurotransmitter Levels of Female Mice.

Cedrol has been reported to be effective in reducing anxiety of male mice. The limited application of females in animal models of anxiety makes it difficult to systematically investigate new drug substitutes with potential anxiolytic activity. In the present study, we investigated the behavioral response of female ICR mice to cedrol after intraperitoneal (i.p.) administration using the elevated plus maze (EPM) and the light-dark box (LDB) test, followed by determination of neurochemical changes in brain. The data suggested that cedrol at dose of 1200-1600 mg·kg-1 exhibited anxiolytic activity on the female mice, as reflected by greater percentage of entries into the open arms and time spent in the open arms in the EPM, and greater transitions between chambers and percentage of time spent in the light chamber in the LDB. Cedrol increased the level of 5-hydroxytryptamine (5-HT), decreased the level of dopamine (DA), reduced the ratio of 5-hydroxyindoleacetic acid (5-HIAA)/5-HT and increased the ratio of 3,4-dihydroxyphenyl acetic acid (DOPAC)/DA, compared with the control group, indicative of an anxiolytic-like effect on female mice via the 5-HTnergic or DAnergic pathways.

Hypothalamus - Response to early paracetamol exposure in male rats offspring.

One of the reasons for using paracetamol during pregnancy is fever. The brain structure responsible for maintaining proper body temperature, but also for controlling some endocrine aspects is hypothalamus. In this study we examined the effect of early pretreatment of paracetamol on hypothalamic neurotransmission in rats' offspring. We used two-month old rats previously exposed to paracetamol at doses of 5 (P5) and 15 mg/kg (P15) during gestational development and next postnatally. The concentration of monoamines, their metabolites and amino acids in hypothalamus was chromatographically determined. The results of biochemical analysis were compared with the Control animals (Con). We found differences between groups in the concentration of main noradrenaline metabolite in hypothalamus. The control group had significantly higher level of 3-methoxy-4-hydroxyphenylglycol (MHPG) compared with rats exposed to paracetamol (F(2,27) = 7.96, p < 0.005). Simultaneously the level of dopamine (DA) (F(2,27) = 4.33, p < 0.05) and its metabolite - homovanillic acid (HVA) (F(2,27) = 17.03, p < 0.005) was increased in the hypothalamus of animals treated with lower dose of the drug. Biochemical analyses show an increase in 3,4-dihydroxyphenyl acetic acid (DOPAC) concentration in P5 group compared to the control rats and group treated with higher dose of paracetamol (F(2,27) = 7.37, p < 0.005). In the hypothalamus significant decrease of glutamic acid concentration was also observed in the group treated with paracetamol at dose of 5 mg. These results demonstrated that paracetamol had a significant effect on dopaminergic and noradrenergic neurotransmission and changed the concentration of glutamic acid in hypothalamus - heat-regulating center and important element of hypothalamic-pituitary- gonadal axis.

Anxiolytic effect of a novel 9,10-dihydrophenanthrene, juncuenin H, is associated with metabolic changes in cortical serotonin/dopamine levels in mice.

Two novel phenanthrenoids, juncuenin H (1) and dijuncuenin B (2), together with eight known phenanthrenoids, effusol (3), dehydroeffusol (4), juncusol (5), dehydrojuncusol (6), juncuenin B (7), dehydrojuncuenin B (8), juncuenin A (9), and dehydrojuncuenin A (10), were isolated from the underground parts of Juncus setchuenensis. The structures of the compounds were determined by 1D and 2D NMR and mass spectroscopy. The anxiolytic activities of compounds 1, 6, 9, and 10 were evaluated. In order to explore the mechanisms underlying their anxiolytic activities, the levels of serotonin (5-HT), dopamine (DA), and their metabolites in the cerebral cortex and hippocampus of mice treated with compound 1 were determined by quantitative mass spectrometry. The mice treated with compound 1 had significantly lower levels of 5-HT, 3-methoxytyramine (3-MT), 5-hydroxyindole-3-acetic acid (5-HIAA), homovanillic acid (HVA), and 3, 4-dihydroxyphenylacetic acid (DOPAC) in the cerebral cortex than those of the vehicle control-treated mice. The levels of HVA and 5-HIAA in the hippocampus were also significantly lower in the mice treated with compound 1 than in the control group mice. These results suggest that the metabolic changes, reflected in the levels of DA and/or 5-HT, may contribute to the anxiolytic activity of the phenanthrenoids studied herein.

Metabolomic analysis of mouse prefrontal cortex reveals upregulated analytes during wakefulness compared to sleep.

By identifying endogenous molecules in brain extracellular fluid metabolomics can provide insight into the regulatory mechanisms and functions of sleep. Here we studied how the cortical metabolome changes during sleep, sleep deprivation and spontaneous wakefulness. Mice were implanted with electrodes for chronic sleep/wake recording and with microdialysis probes targeting prefrontal and primary motor cortex. Metabolites were measured using ultra performance liquid chromatography-high resolution mass spectrometry. Sleep/wake changes in metabolites were evaluated using partial least squares discriminant analysis, linear mixed effects model analysis of variance, and machine-learning algorithms. More than 30 known metabolites were reliably detected in most samples. When used by a logistic regression classifier, the profile of these metabolites across sleep, spontaneous wake, and enforced wake was sufficient to assign mice to their correct experimental group (pair-wise) in 80-100% of cases. Eleven of these metabolites showed significantly higher levels in awake than in sleeping mice. Some changes extend previous findings (glutamate, homovanillic acid, lactate, pyruvate, tryptophan, uridine), while others are novel (D-gluconate, N-acetyl-beta-alanine, N-acetylglutamine, orotate, succinate/methylmalonate). The upregulation of the de novo pyrimidine pathway, gluconate shunt and aerobic glycolysis may reflect a wake-dependent need to promote the synthesis of many essential components, from nucleic acids to synaptic membranes.

Neurochemical and behavioral effects of Nigella sativa and Olea europaea oil in rats.

OBJECTIVES: In the last few decades, therapeutic uses of medicinal compounds present in food as a normal constituent has risen substantially, largely because of their fewer side effects and adequate efficacy. This study is designed to investigate a role of brain serotonin (5-HT) and dopamine (DA) in the potential nootropic, anxiolytic, and other beneficial effects of Nigella sativa (NS) and Olea europaea (OE) oil in rat models. METHODS: Animals were treated with NS and OE oil orally at doses of 0.1 ml/kg and 0.25 ml/kg for 5 weeks. Food intake and body weight change, anxiety-like effects in elevated plus maze and activity in a novel and familiar environment were monitored weekly. Effects on learning and memory after 5 weeks treatment were monitored using Morris water maze test. Neurochemical analysis was carried using HPLC-ECD method. RESULTS: NS and OE oil administration enhanced learning and memory in Morris water maze test and the effects were greater in NS than OE oil-treated animals. Low dose of OE oil increased exploration in an open field, higher dose of OE oil and both doses of NS oil produced no consistent effect on open field exploration. Effects of both oils on anxiety-like behavior, food and water intake, and activity in activity box were either not consistent or did not occur. The treatment increased homovanillic acid (HVA). 5-HT levels increased in high dose of NS oil and low dose of OE oil-treated groups. Low dose NS oil decreased 5-HT. DISCUSSION: The present study suggests that active components in NS and OE oil may prove useful in treating impaired cognition. OE oil may produce psychostimulant-like effect. Modulation of DA and serotonin neurotransmission seems important in the pharmacological effect of these oils.

Effects of growth hormone-releasing hormone gene targeted ablation on ghrelin-induced feeding.

Impairment of growth hormone (GH) signaling has been associated with increased feeding and adiposity. The gastric hormone ghrelin, in addition to its GH-secretagogue effects, stimulates food intake after both central and peripheral administration. In the present study we further investigated the feeding regulatory role of the ghrelin-GH axis in a mouse model of isolated GH deficiency due to targeted ablation of the GH-releasing hormone (GHRH) gene [GHRH knockout (GHRHKO)]. We evaluated the effects of intracerebroventricular ghrelin administration on feeding behavior, related hypothalamic neuropeptides and neurotransmitters, and serum ghrelin levels in mice homozygous for GHRHKO allele (-/-) and heterozygous (+/-) control animals. Vehicle-treated GHRHKO mice showed increased food intake compared to heterozygotes, associated with increased circulating ghrelin levels. Moreover, -/- mice showed elevated hypothalamic levels of neuropeptide Y (NPY), agouti-related peptide (AgRP) mRNAs and norepinephrine (NE) and decreased corticotropin-releasing hormone (CRH) mRNA levels. Ghrelin treatment significantly augmented food intake in both genotypes, but the relative increase compared to vehicle-treated animals was higher in -/- than +/- mice. In the hypothalamus, ghrelin increased AgRP and decreased CRH gene expression only in heterozygous mice, while it induced a significant reduction in proopiomelanocortin (POMC) mRNA levels in -/- mice. Ghrelin treatment also decreased hypothalamic serotonin (5-hydroxytriptamine, 5-HT) and dopamine (DA) levels in both genotypes. Additionally, we observed increased DA metabolism induced by ghrelin in both genotypes. In conclusion, dysregulation of the ghrelin-GHRH-GH axis in GHRHKO mice could lead to increased feeding secondary to elevated circulating levels of ghrelin, and the obesogenic phenotype is likely mediated by elevated NPY and AgRP, and decreased CRH gene expression in the hypothalamus.

Effects of Fluorencarbonic Acid Derivative on the Levels of Monoamines and Their Metabolites in Brain Structures of Rats with Modeled Depression-Like State.

The effects of the new structural analogue of benactyzine, a derivative of fluorencarbonic acid, on monoamine levels in brain structures were studied in male Wistar rats with experimental depression. Depressive state in rats was modeled by single injection of reserpine (4 mg/kg). The concentrations of norepinephrine (NE), dopamine (DA), serotonin (5-HT), and their metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanilic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in the hypothalamus and striatum were measured by HPLC. It was found that preliminary treatment (30 days) with the derivative of fluorencarbonic acid prevented a decrease in monoamine level in the hypothalamus (NE, 5-HT, and 5-HIAA) and striatum (DA, 5-HT, and 5-HIAA). The neurochemical shifts (correction of 5-HT deficiency and stabilization of DA and NE levels) correlated with the high antidepressant activity of this agent observed in Porsolt forced swimming test.

Buckwheat bioactive compounds, their derived phenolic metabolites and their health benefits.

SCOPE: Buckwheat (BW) consumption has been associated with a broad range of health benefits: antioxidant, anti-inflammatory and anticancer. These beneficial effects have been partially related to the presence of flavonoids. However, some of these compounds (i.e., rutin and quercetin) are metabolized in the gastrointestinal tract generating derived phenolic metabolites. In this study, we investigated the biological activity of rutin (Ru), quercetin (Q) an their derived phenolic metabolites 3,4-dihydroxyphenylacetic acid (3,4-DHPAA), 3-hydroxyphenylacetic acid (3-HPAA), and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid, HVA). METHODS AND RESULTS: Q showed the highest antioxidant and reducing activity, and Ru the maximum chelating activity (85.33%). Antioxidant activity of 3,4-DHPAA was 5-fold higher than that of HVA, whereas their reducing activity was similar. The formation of methylglyoxal (MGO)-BSA and glucose-BSA (advanced glycation end products) was inhibited by Ru (98.5 and 92.7%), Q (95.6 and 89.1%) and 3,4-DHPPA (84.4.6 and 77.5%). Furthermore, Q (10-50 µM) and Ru (1-50 µM) downregulated the release of PGE2 , IL-8 and MCP-1, molecules involved in the inflammatory response, in IL1ß-inflamed myofibroblasts of colon CCD-18Co. CONCLUSION: This study suggests that BW phytochemicals and their phenolic metabolites may be responsible for the beneficial effects against chronic diseases attributed to BW consumption.

Medicinal plant Combretum leprosum mart ameliorates motor, biochemical and molecular alterations in a Parkinson's disease model induced by MPTP.

ETHNOPHARMACOLOGICAL RELEVANCE: Combretum leprosum is a popular medicinal plant distributed in north and northeastern regions of Brazil. Many different parts of this plant are used in traditional medicine to treat several inflammatory diseases. Parkinson's disease (PD) is a disorder associated with inflammatory toxic factors and the treatments available provide merely a delay of the neurodegeneration. AIM OF THE STUDY: We investigated the potential neuroprotective properties of the C. leprosum ethanolic extract (C.l.EE) in a murine model of PD using the toxin 1-methyl-4 phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). MATERIALS AND METHODS: The mice were split into four groups: V/S (vehicle/saline), E/S (extract/saline), V/M (vehicle/MPTP) and E/M (extract/ MPTP). Mice received MPTP (30mg/kg, i.p.) or vehicle (10ml/kg, i.p.) once a day for 5 consecutive days and vehicle (10ml/kg) or C.l.EE (100mg/kg) orally by intra-gastric gavage (i.g.) during a 14-d period, starting 3 days before the first MPTP injection. All groups were assessed for behavioural impairments (amphetamine-induced locomotor activity and muscle strength), dopamine content in striatum using high performance liquid chromatography (HPLC), tyrosine hydroxylase (TH) and dopamine transporter (DAT) gene expressions using qPCR. RESULTS: Animals were injected with d-amphetamine (2mg/kg) and the activity was recorded. Amphetamine-induced hyperlocomotion was observed in all groups; however animals treated with MPTP showed exacerbated hyperlocomotion (approximately 3 fold increase compared to control groups). By contrast, mice treated with MPTP that received C.l.EE exhibited attenuation of the hyperlocomotion and did not differ from control groups. Muscle strength test pointed that C.l.EE strongly avoided muscular deficits caused by MPTP (approximately 2 fold increase compared to V/M group). Dopamine and its metabolites were measured in the striatum. The V/M group presented a dopamine reduction of 80%. On the other hand, the E/M group exhibited an increase in dopamine and its metabolites levels (approximately 3 fold increase compared to V/M group). Tyrosine hydroxylase (TH) and dopamine transporter (DAT) gene expressions were significantly reduced in the V/M group (60%). Conversely, C.l.EE treatment was able to increase the mRNA levels of those genes in the E/M group (approximately 2 fold for TH and DAT). CONCLUSIONS: These data show, for the first time, that C. leprosum ethanolic extract prevented motor and molecular changes induced by MPTP, and partially reverted dopamine deficit. Thus, our results demonstrate that C.l.EE has potential for the treatment and prevention of PD.

Anti-depressant effects of Gastrodia elata Blume and its compounds gastrodin and 4-hydroxybenzyl alcohol, via the monoaminergic system and neuronal cytoskeletal remodeling.

ETHNOPHARMACOLOGY RELEVANCE: Gastrodia elata Blume is a highly valuable traditional Chinese medicine used in the treatment of depression. However, compounds with antidepressant effects in water extracts of G. elata Bl. (WGE) have not been identified. The aims of this study were to determine the major antidepressant compound in WGE and to evaluate the antidepressant effects of WGE and its active compounds which involved the monoaminergic system and neuronal cytoskeletal remodeling. MATERIALS AND METHODS: Gastrodin (GAS) and 4-hydroxybenzyl alcohol (HBA) in WGE, were analyzed with high-performance liquid chromatography (HPLC)-ultraviolet detection. The forced swimming test (FST) was used to induce depression-like symptoms in 9 weeks old male Sprague-Dawley rats. The open field test (OFT) was used to measure anxiety after WGE, GAS, and HBA treatments. The levels of monoamine such as serotonin (5-HT), dopamine (DA), and their metabolites 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were measured using HPLC-electrochemical detection. Western blotting was used to examine the 5-HT1A receptor and the neuronal cytoskeleton remodeling-related proteins, Slit, dihydropyrimidinase-related protein 2 (DPYSL2, also called CRMP2), Ras homologous member A (RhoA), and profilin 1 (PFN1) in vivo. Slit1 expression was evaluated in Hs683 cell line after treated with WGE (0.5mg/mL), GAS (50, 100 and 100µM), and HBA (50, 100 and 100µM). RESULTS: Oral administration of WGE (500mg/kg bw), GAS (100mg/kg bw), and HBA (100mg/kg bw) exhibited the anti-depressant effect by significantly reducing the immobility time in FST, monoamine metabolism including the 5-HT to 5-HIAA in the hippocampus and DA to DOPAC and HVA ratios in the frontal cortex, amygdala, and hippocampus. In the hippocampus, the expression of the neuronal cytoskeleton remodeling-related negative regulators Slit1 and RhoA were significantly down-regulated. In addition, the positive regulators CRMP2 and PFN1 were significantly up-regulated following GAS, HBA, and WGE treatments. Moreover, WGE, GAS, and HBA were directly down-regulated Slit1 expression in Hs683 cells. CONCLUSION: WGE, GAS, and HBA exhibited potential anti-depressant effects in rats by decreasing monoamine metabolism and modulated cytoskeleton remodeling-related protein expression in the Slit-Robo pathway. These results suggest that WGE can be used as agent for depressive prevention.

High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters, dopamine and serotonin, have been implicated in the pathophysiology of neuropsychiatric dysfunction in phenylketonuria (PKU). Increased brain phenylalanine concentration likely competitively inhibits the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate limiting steps in dopamine and serotonin synthesis respectively. Tetrahydrobiopterin (BH4) is a required cofactor for TH and TPH activity. Our hypothesis was that treatment of hyperphenylalaninemic Pah(enu2/enu2) mice, a model of human PKU, with sapropterin dihydrochloride, a synthetic form of BH4, would stimulate TH and TPH activities leading to improved dopamine and serotonin synthesis despite persistently elevated brain phenylalanine. Sapropterin (20, 40, or 100mg/kg body weight in 1% ascorbic acid) was administered daily for 4 days by oral gavage to Pah(enu2/enu2) mice followed by measurement of brain biopterin, phenylalanine, tyrosine, tryptophan and monoamine neurotransmitter content. A significant increase in brain biopterin content was detected only in mice that had received the highest sapropterin dose, 100mg/kg. Blood and brain phenylalanine concentrations were unchanged by sapropterin therapy. Sapropterin therapy also did not alter the absolute amounts of dopamine and serotonin in brain but was associated with increased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), dopamine and serotonin metabolites respectively, in both wild type and Pah(enu2/enu2) mice. Oral sapropterin therapy likely does not directly affect central nervous system monoamine synthesis in either wild type or hyperphenylalaninemic mice but may stimulate synaptic neurotransmitter release and subsequent metabolism.

Ursolic acid attenuates oxidative stress in nigrostriatal tissue and improves neurobehavioral activity in MPTP-induced Parkinsonian mouse model.

Parkinson's disease (PD) is characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc) region of brain. Oxidative stress and inflammation plays important role in the neurodegeneration and development of PD. Ursolic Acid (UA: 3ß-hydroxy-urs-12-en-28-oic acid) is a natural pentacyclic triterpenoid found in various medicinal plants. Its anti-inflammatory and antioxidant activity is a well-established fact. In this paper, the neuroprotective efficiency of UA in MPTP induced PD mouse model has been explored. For this purpose, we divided 30 mice into 5 different groups; first was control, second was MPTP-treated, third, fourth and fifth were different doses of UA viz., 5 mg/kg, 25 mg/kg, and 50 mg/kg body weight (wt) respectively, along with MPTP. After 21 days of treatment, different behavioral parameters and biochemical assays were conducted. Tyrosine hydroxylase (TH) immunostaining of SN dopaminergic neurons as well as HPLC quantification of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) were also performed. Our results proved that, UA improves behavioral deficits, restored altered dopamine level and protect dopaminergic neurons in the MPTP intoxicated mouse. Among three different doses, 25 mg/kg body wt was the most effective dose for the PD. This work reveals the potential of UA as a promising drug candidate for PD treatment.

Effects of Gypenosides on Dopaminergic Neuronal Cell Death in 6-Hydroxydopamine-lesioned Rat Model of Parkinson's Disease with Long-term L-DOPA Treatment

The goal of this study was to determine whether gypenosides (GPS) exert protective effects against dopaminergic neuronal cell death in a 6-hydroxydopamine (OHDA)-lesioned rat model of Parkinson's disease (PD) with or without long-term 3,4-dihydroxyphenylalanine (L-DOPA) treatment. Rats were injected with 6-OHDA in the substantia nigra to induce PD-like symptoms; 14 days after injection, groups of 6-OHDA-lesioned animals were treated for 21 days with GPS (25 or 50 mg/kg) and/or L-DOPA (20 mg/kg). Dopaminergic neuronal cell death was assessed by counting tyrosine hydroxylase (TH)-immunopositive cells in the substantia nigra and measuring levels of dopamine, norepinephrine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum. Dopaminergic neuronal cell death induced by 6-OHDA lesions was ameliorated by GPS treatment (50 mg/kg). L-DOPA treatment exacerbated 6-OHDA-induced dopaminergic neuronal cell death; however, these effects were partially reversed by GPS treatment (25 and 50 mg/kg). These results suggest that GPS treatment is protective against dopaminergic neuronal cell death in a 6-OHDA-lesioned rat model of PD with long-term L-DOPA treatment. Therefore, GPS may be useful as a phytotherapeutic agent for the treatment of PD.