High-performance Liquid Chromatography-electrochemical Detection Research Articles

Sub-chronic nicotine exposure influences methamphetamine self-administration and dopamine overflow in a sex-and genotype-dependent manner in humanized CHRNA6 3'-UTR SNP (rs2304297) adolescent rats.

Introduction: The rewarding effects of drugs of abuse are associated with the dopaminergic system in the limbic circuitry. Nicotine exposure during adolescence is linked to increased use of drugs of abuse with nicotine and methamphetamine (METH) commonly used together. Nicotine acts on neuronal nicotinic acetylcholine receptor (nAChR) systems, critical for reward processing and drug reinforcement, while METH leads to a higher dopamine (DA) efflux in brain reward regions. A human single nucleotide polymorphism (SNP) in the 3'-untranslated region (UTR) of the α6 nicotinic receptor subunit gene (CHRNA6, rs2304297), has been linked with tobacco/nicotine and general substance use during adolescence. Using CRISPR-Cas9 genomic engineering, our lab recapitulated the CHRNA6 3'UTRC123G SNP, generating α6CC and α6GG allele carriers in Sprague Dawley rats. We hypothesized the CHRNA6 3'UTRC123G SNP would sex- and genotype-dependently enhance nicotine-induced METH self-administration as well as nicotine-induced DA overflow in the nucleus accumbens shell of adolescent α6GG and α6CC carriers. Methods: Adolescent male and female rats underwent a 4-day sub-chronic, low-dose (0.03mg/kg/0.1mL, x2) nicotine pretreatment paradigm to assess intravenous METH (0.02mg/kg/0.1mL) self-administration as well as nicotine- and METH (0.02mg/kg/0.1mL)-induced DA overflow in the nucleus accumbens shell (NAcS) using in vivo microdialysis coupled with high-performance liquid-chromatography-electrochemical detection (HPLC-ECD). Results: Nicotine pretreatment sex- and genotype-dependently enhanced subsequent METH self-administration in adolescent CHRNA6 3'UTRC123G SNP rats. Further nicotine and METH-induced DA overflow is observed in α6CC females as compared to α6GG females, with METH-induced DA overflow enhanced in α6GG males when compared to α6CC males. Conclusion: These findings demonstrate that the CHRNA6 3'-UTRC123G SNP can sex- and genotype-dependently impact adolescent nicotine-induced effects on METH self-administration and stimulant-induced DA overflow in reward regions of the brain.

NAc-DBS corrects depression-like behaviors in CUMS mouse model via disinhibition of DA neurons in the VTA.

Major depressive disorder (MDD) is characterized by diverse debilitating symptoms that include loss of motivation and anhedonia. If multiple medications, psychotherapy, and electroconvulsive therapy fail in some patients with MDD, their condition is then termed treatment-resistant depression (TRD). MDD can be associated with abnormalities in the reward-system-dopaminergic mesolimbic pathway, in which the nucleus accumbens (NAc) and ventral tegmental area (VTA) play major roles. Deep brain stimulation (DBS) applied to the NAc alleviates the depressive symptoms of MDD. However, the mechanism underlying the effects of this DBS has remained elusive. In this study, using the chronic unpredictable mild stress (CUMS) mouse model, we investigated the behavioral and neurobiological effects of NAc-DBS on the multidimensional depression-like phenotypes induced by CUMS by integrating behavioral, in vivo microdialysis coupled with high-performance liquid chromatography-electrochemical detector (HPLC-ECD), calcium imaging, pharmacological, and genetic manipulation methods in freely moving mice. We found that long-term and repeated, but not single, NAc-DBS induced robust antidepressant responses in CUMS mice. Moreover, even a single trial NAc-DBS led to the elevation of the γ-aminobutyric acid (GABA) neurotransmitter, accompanied by the increase in dopamine (DA) neuron activity in the VTA. Both the inhibition of the GABAA receptor activity and knockdown of the GABAA-α1 gene in VTA-GABA neurons blocked the antidepressant effect of NAc-DBS in CUMS mice. Our results showed that NAc-DBS could disinhibit VTA-DA neurons by regulating the level of GABA and the activity of VTA-GABA in the VTA and could finally correct the depression-like behaviors in the CUMS mouse model.

The Development and Full Validation of a Novel Liquid Chromatography Electrochemical Detection Method for Simultaneous Determination of Nine Catecholamines in Rat Brain.

Chemical neurotransmission, managed by neurotransmitters, has a crucial role in brain processes such as fear, memory, learning, and pain, or neuropathology such as schizophrenia, epilepsy, anxiety/depression, and Parkinson's disease. The measurement of these compounds is used to elucidate the disease mechanisms and evaluate the outcomes of therapeutic interventions. However, this can be quite difficult because of various matrix effects and the problems of chromatographic separation of analysts. In the current study; for the first time, an optimized and fully validated high-performance liquid chromatography-electrochemical detection (HPLC-EC) method according to Food and Drug Administration and European Medicines Agency Bioanalytical Validation Guidance was developed for the simultaneous analysis of nine neurotransmitter compounds, namely dopamine, homovanilic acid, vanilmandelic acid, serotonin (SER), 5-hydroxyindole-3-acetic acid, 4-hydroxy-3-methoxyphenylglycol, norepinephrine, 3,4 dihydroxyphenylacetic acid, and 3-methoxytyramine and simultaneously determined in rat brain samples. Separation was achieved with 150 mm x 4.6 mm, 2.6 μm Kinetex F5 (Phenomenex, USA) column isocratically, and analysis was carried out by HPLC equipped with a DECADE II EC detector. The method exhibited good selectivity, and the correlation coefficient values for each analyte's calibration curves were > 0.99. The detection and quantification limits ranged from 0.01 to 0.03 ng/mL and 3.04 to 9.13 ng/mL, respectively. The stability of the analyses and method robustness were also examined in detail in the study, and the obtained results are presented statistically. The developed and fully validated method has been successfully applied to actual rat brain samples, and important results have been obtained. In the rat brain sample analysis, the lowest number of SER and the highest amount of noradrenaline were found.

Zhi-Zi-Hou-Po decoction alleviates depressive-like behavior and promotes hippocampal neurogenesis in chronic unpredictable mild stress induced mice via activating the BDNF/TrkB/CREB pathway

Ethnopharmacological relevanceZhi-Zi-Hou-Po decoction (ZZHP), a traditional Chinese medicine (TCM) classic recipe, has been extensively applied for the remedy of depression. However, the underlying mechanism of ZZHP hasn't been fully elucidated and it needs to be further clarified. Aim of studyThe aim of the study is to uncover the mechanisms of ZZHP's effect on depression. Materials and methodsC57BL/6 mice were employed to establish Chronic Unpredictable Mild Stress (CUMS) models. Behavioral tests were conducted for evaluating the antidepressant effects of ZZHP. Then, the monoamine neurotransmitters in the hippocampus through High Performance Liquid Chromatography Electrochemical Detection (HPLC-ECD) were utilized to assess the effect of ZZHP on the maintenance of monoamine neurotransmitter homeostasis. Immunofluorescence staining and Golgi staining were detected to analyze the effects of ZZHP on neuroplasticity in the hippocampus. Western Blot (WB) was utilized to examine the effects of ZZHP on BDNF/TrkB/CREB pathways. Finally, behavioral tests, WB and immunofluorescence staining were repeated after TrkB receptor antagonist was added to further confirm the underlying mechanism. ResultsOur results shown that ZZHP attenuated depressive-like symptoms in CUMS mice. Moreover, ZZHP remarkably reversed the reduction and maintained the homeostasis of monoamine neurotransmitters in the hippocampus. Simultaneously, ZZHP protected neuronal synaptic plasticity and promoted hippocampal neurogenesis. Furthermore, ZZHP stimulated the BDNF/TrkB/CREB pathway in the hippocampus. The addition of TrkB receptor antagonist inhibited the antidepressant effects of ZZHP, suggesting that ZZHP could not work without triggering the BDNF/TrkB/CREB pathway. ConclusionThis study demonstrates that ZZHP can alleviate depressive-like behavior and promote hippocampal neurogenesis in CUMS mice via activating the BDNF/TrkB/CREB pathway.

Leukocyte 8-hydroxy-2'-deoxyguanosine as an oxidative stress marker to predict cardiovascular events and death in chronic hemodialysis patients.

Hemodialysis patients have a markedly increased risk of cardiovascular (CV) morbidity and mortality. Oxidative stress plays a pathogenic role in the progression of atherosclerosis and CV disease among chronic hemodialysis patients. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) content in leukocyte deoxyribonucleic acid (DNA) has been shown as a sensitive and well-known biomarker of oxidant-induced DNA damage in chronic hemodialysis patients. We conducted a retrospective cohort study to investigate the association of leukocyte 8-OHdG and CV events and deaths in patients of chronic hemodialysis. In this study, 217 chronic hemodialysis patients were recruited from 2016 to 2021. The 8-OHdG content of leukocyte DNA was measured by a high-performance liquid chromatography electrochemical detection method. Study outcomes were CV events as well as CV and all-cause deaths. The patients were followed until May 2021. The median follow-up period was 34.8 months. At the end of May 2021, 57 first CV events and 89 all-CV events occurred. Among the first and all CV events, 17 (29.8%) and 32 (36.0%) were fatal, respectively. Multivariate Cox regression analysis showed per 1/10 5 dG increment in leukocyte 8-OHdG values increased risk of CV events (adjusted hazard ratio [aHR], 1.19; 95% CI, 1.10-1.41; p < 0.001), CV death (aHR, 1.27; 95% CI, 1.03-1.72; p = 0.034), and all-cause death (aHR, 1.11; 95% CI, 1.01-1.30; p = 0.038). This is the first study to demonstrate that oxidative stress assessed by 8-OHdG levels of leukocyte DNA predicted CV events as well as CV and all-cause deaths among chronic hemodialysis patients.

Loganin improves chronic unpredictable mild stress-induced depressive-like behaviors and neurochemical dysfunction.

Cornus officinalis Sieb. et Zucc., is a valuable herb commonly used in Chinese medicine clinics. Loganin is a major iridoid glycoside obtained from the traditional Chinese herb Corni Fructus. Loganin, which has been shown to improve depression-like behavior in mice exposed to acute stress, is probably a potential antidepressant candidate. Loganin was evaluated for its effect on chronic unpredictable mild stress (CUMS) induced depressive-like mice, and its action mechanisms were explored. ICR mice were subjected to the CUMS stimulation method to induce depression. The therapeutic effect of loganin on depressive-like behavior was evaluated by a series of behavioral tests such as sucrose preference test (SPT), forced swim test (FST), tail suspension test (TST) and open-field test (OFT). In addition, the serum levels of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) were measured using ELISA. The levels of monoamine neurotransmitters were detected by high performance liquid chromatography-electrochemical detection (HPLC-ECD). The levels of brain-derived neurotrophic factor (BDNF) in the hippocampus were measured using western blot analysis. The results showed that CUMS induced depressive-like behaviors in mice, as indicated by behavioral tests. Administration of loganin increased the sucrose preference in SPT, as well as decreased the immobility time in FST and TST. Loganin could also improve food intake, and increased crossing times in the OFT. In mechanism, loganin restored the secretion of monoamine neurotransmitters, ACTH and CORT to normal levels. In addition, loganin elevated the expression of BDNF in the hippocampus. In conclusion, loganin exerts antidepressant-like effects in CUMS model mice through modulating monoamine neurotransmitters, ACTH, CORT and BDNF. Loganin effectively ameliorated depressive-like symptoms in CUMS-exposed mice by increasing 5-hydroxytryptamine (5-HT) and dopamine (DA) levels, alleviating hypothalamic-pituitary-adrenal (HPA) axis dysfunction, and increasing BDNF expression. In conclusion, the findings of the current study extensive evidence for the application of loganin in stress-associated disorders, specifically targeting depression.

Filter blot method: A simple method for measuring 3-nitrotyrosine in proteins of atmospheric particulate matter

Air pollutants, such as nitrogen dioxide (NO2), ozone (O3), and particulate matter (PM), have been epidemiologically reported to contribute to the onset and exacerbation of asthma. We have previously shown that several proteins in atmospheric PM are allergenic in mouse asthma models and that these proteins are nitrated by atmospheric NO2 and O3 in chemical reactions. Based on these results, the amount of 3-nitrotyrosine (3-NT) in atmospheric PM could be an air pollution marker integrating NO2, O3, and PM. We established a method to measure 3-NT by high-performance liquid chromatography electrochemical detection (HPLC-ECD). Although this method is accurate, it requires a filter treatment process, which is time-consuming and costly for an environmental monitoring tool, in which many samples are measured simultaneously. Therefore, in this study, we investigated a simple immunoblotting method in which atmospheric PM proteins were directly transferred to a polyvinylidene fluoride (PVDF) membrane and measured using an anti-3-NT antibody (the filter blot method). The 3-NT value obtained from this method was significantly correlated (r = 0.809, p < 0.001) with that of the HPLC-ECD method, with a detection power of 0.1 μg/mL for tyrosine nitrated bovine serum albumin equivalents. Multiple regression analysis using the filter blot method showed that the amount of 3-NT in atmospheric PM was significantly associated with the published environmental measurements of O3 and PM in the region. Therefore, the filter blot method may be useful for the environmental monitoring of 3-NT in atmospheric PM.

Profiling of phenolic compounds in domestic and imported extra virgin olive oils in China by high performance liquid chromatography-electrochemical detection

In this study, a high performance liquid chromatography-electrochemical detection (HPLC-ECD) method has been developed for the sensitive determination of 22 phenolic compounds in extra virgin olive oil (EVOO). Chromatographic separation was performed on an Eclipse XDB-C18 column with a gradient methanol/aqueous phosphoric acid elution, and the ECD potential was set at 1.0 V in oxidative mode. Under the optimal conditions, good linearity was obtained for 22 phenolic compounds with coefficients of determination (R2) higher than 0.9971 in the range of 0.005–20.0 μg/mL. The limits of detection (LODs, S/N = 3) for 22 phenolic compounds were in the range of 0.4–6.7 μg/kg by ECD. Satisfactory recoveries ranging from 80.1% to 109.3% were obtained for spiked EVOO samples with relative standard deviations (RSDs) less than 5.4%. The proposed HPLC-ECD method featuring excellent sensitivity and selectivity has been applied to the quantification of phenolic compounds in 61 EVOO samples from seven countries. Good discrimination was achieved between domestic and imported EVOOs based on multivariate statistical approach and their phenolic profiles. These results provide promising perspectives for the use of HPLC-ECD combined with chemometrics for the origin identification of EVOOs.

Measurement of Tetrahydrobiopterin in Animal Tissue Samples by HPLC with Electrochemical Detection-Protocol Optimization and Pitfalls.

Tetrahydrobiopterin (BH4) is an essential cofactor of all nitric oxide synthase isoforms, thus determination of BH4 levels can provide important mechanistic insight into diseases. We established a protocol for high-performance liquid chromatography/electrochemical detection (HPLC/ECD)-based determination of BH4 in tissue samples. We first determined the optimal storage and work-up conditions for authentic BH4 and its oxidation product dihydrobiopterin (BH2) under various conditions (pH, temperature, presence of antioxidants, metal chelators, and storage time). We then applied optimized protocols for detection of BH4 in tissues of septic (induced by lipopolysaccharide [LPS]) rats. BH4 standards in HCl are stabilized by addition of 1,4-dithioerythritol (DTE) and diethylenetriaminepentaacetic acid (DTPA), while HCl was sufficient for BH2 standard stabilization. Overnight storage of BH4 standard solutions at room temperature in HCl without antioxidants caused complete loss of BH4 and the formation of BH2. We further optimized the protocol to separate ascorbate and the BH4 tissue sample and found a significant increase in BH4 in the heart and kidney as well as higher BH4 levels by trend in the brain of septic rats compared to control rats. These findings correspond to reports on augmented nitric oxide and BH4 levels in both animals and patients with septic shock.

Reversal of temperature responses to methylone mediated through bi-directional fecal microbiota transplantation between hyperthermic tolerant and naïve rats

ABSTRACT The synthetic cathinone (“bath salt”) methylone induces a hyperthermia response and with chronic administration tolerance to this hyperthermia has been reported. The microbiome-gut-brain axis has been implicated in multiple bodily systems and pathologies, and intentional manipulation of the gut-microbiome has yielded clinically significant results. Here, we examined the effects of bi-directional Fecal Microbiota Transplantation (FMT) between methylone-induced hyperthermic tolerant (MHT) and methylone-naïve (MN) rats. Rats treated with methylone once per week developed tolerance to methylone-induced hyperthermia by the fourth week. Once tolerant, daily bi-directional FMT between the two groups were performed for seven days prior to the next methylone treatment. The FMT abated the developed tolerance in the MHT group. When treated with methylone for the first time following FMT, recipient MN rats displayed significant tolerance to hyperthermia despite it being their initial drug treatment. Post-FMT, MHT rats displayed elevations in norepinephrine and expression of UCP1, UCP3 and TGR5 in brown adipose tissue, with reductions in expression of TGR5 and UCP3 in skeletal muscle. The pre- and post-FMT methylone tolerance phenotypes of transplant recipients are concurrent with changes in the relative abundance of several classes of Proteobacteria, most evident for Gammaproteobacteria and Alphaproteobacteria. MHT recipients demonstrated a marked increase in the relative proportion of the Firmicutes class Erysipelotrichia. These findings suggest that transplantation of gut-microbiomes can confer phenotypic responses to a drug and that the microbiome may be playing a major role in sympathomimetic-mediated hyperthermia. Abbreviations: 3,4-methylenedioxymethamphetamine (MDMA); methylone-induced hyperthermic tolerant (MHT); methylone-naïve (MN); fecal microbiota transplantation (FMT); uncoupling protein (UCP); subcutaneous (sc); intraperitoneal (ip); brown adipose tissue (BAT); skeletal muscle (SKM); sympathetic nervous system (SNS); norepinephrine (NE); quantitative PCR (qRT-PCR); quantification cycle (Cq); High Performance Liquid Chromatography-Electrochemical Detection (HPLC-EC); amplicon sequence variants (ASVs); principal coordinates analysis (PCoA); permutational multivariate analysis (PERMANOVA)

Cornuside ameliorates cognitive impairments in scopolamine induced AD mice: Involvement of neurotransmitter and oxidative stress

Ethnopharmacological relevanceCornus officinalis Sieb. et Zucc., traditional Chinese medicine, has been widely used in the treatment of dementia. Cornel iridoid glycosides of Cornus officinalis is therapeutic to Alzheimer's disease (AD), while its pharmacodynamic material basis is not clear. Cornuside, an iridoid glycoside extracted from of Cornus officinalis Sieb. et Zucc, might be a potential anti-AD candidate. Aim of the studyCornuside was evaluated for its effect on scopolamine induced AD mice, and its action mechanisms were explored. Materials and methodsICR mice were administered with 1 mg/kg scopolamine intraperitoneally to induce amnesia. The therapeutic effect of cornuside of cognitive function was evaluated via series of behavioral tests, including Morris water maze test, step-through test and step-down test. In addition, specific enzyme reaction tests were used to detect the content of acetylcholine (ACh) and malondialdehyde (MDA), as well as the activities of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), choline acetyltransferase (ChAT), superoxide dismutase (SOD), catalase (CAT), monoamine oxidase (MAO) in the brain. The levels of monoamine neurotransmitters were detected by high performance liquid chromatography-electrochemical detection (HPLC-ECD). ResultsCornuside ameliorated the spatial memory impairment in Morris water maze test and cognitive disruption in step-through and step-down test. Furthermore, cornuside improved the level of ACh by reducing the activities of AChE and BuChE, and increasing the activity of ChAT in hippocampus. Cornuside also increased the levels of monoamine neurotransmitters by inhibiting MAO activity in hippocampus and cortex. In addition, cornuside attenuated MDA by enhancing the activities of SOD and CAT in hippocampus and cortex. ConclusionCornuside improved cognitive dysfunction induced by scopolamine in behavioral tests. The mechanisms of cornuside were further investigated from the aspects of neurotransmitters and oxidative stress. Cornuside could inhibit oxidative stress and neurotransmitter hydrolases, increase ACh and monoamine neurotransmitters, which finally contributed to its therapeutic effect on scopolamine induced amnesia.

The Concentration of 8-Hydroxy-2'-Deoxyguanosine in Plasma During the Menstrual Cycle in Young Japanese Women.

Background: The exact pathophysiology of premenstrual syndrome (PMS) and premenstrual dysphoric disorder (PMDD) is still unknown. This study aimed to investigate the concentration of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in plasma in relation to the menstrual cycle and the severity of premenstrual symptoms in young Japanese women.Methods: The study included 21 healthy Japanese women 19–24 years of age. Fourteen women had no or mild PMS [PMS (−)], while five women had moderate to severe PMS and two women exhibited PMDD [PMS (+)]. The concentration of 8-OHdG in plasma was measured by means of high-performance liquid chromatography–electrochemical detector. The Center for Epidemiologic Studies Depression (CES-D) scale was used to evaluate the depressive tendency.Results: The concentration of 8-OHdG before menstruation was significantly higher than that after menstruation in total subjects (p = 0.04). In the PMS (+) group, the 8-OHdG concentration before menstruation was higher than that after menstruation (p = 0.02). Moreover, the PMS (+) group showed a higher 8-OHdG concentration compared with the PMS (−) group before menstruation (p < 0.01), as well as higher CES-D scores compared with the PMS (−) group both before and after menstruation (p < 0.01).Conclusions: These results suggested that the oxidation of DNA occurred before menstruation in PMS. The depression was associated with PMS symptoms both before and after menstruation in patients with PMS. Oxidation of DNA due to oxidative stress and depression in PMS patients may be involved in the pathogenesis of PMS. Clinical Trial Registration number 15-02-011.

Effects of Lactiplantibacillus plantarum PS128 on alleviating canine aggression and separation anxiety

Certain strains of Lactiplantibacillus were found to have a positive impact on host neuromodulation through the gut-brain axis and thus ameliorate emotional and behavioral problems. A number of researches have been performed on humans, mice and rats; however, studies on dogs are limited. Forty-five dogs with behavioral problems were enrolled in this study, including aggression (n = 22), separation anxiety (n = 15), compulsive disorder (n = 7) and unclassifiable inappropriate behavior (n = 1, excessive barking). The behavioral diagnosis was made based on the primary behavioral consultation questionnaires and the careful interrogations at interviews. Lactiplantibacillus plantarum PS128 (PS128) probiotic was administered to these physically healthy dogs with behavioral problems over a course of two weeks to determine the probiotic effectiveness on canine behaviors. Dogs were evaluated and scored using the Evaluation of Dog’s Emotional and cognitive Disorders (EDED) scale and the Canine Behavioral Checklist (CBC) questionnaire at the visits before and after the probiotic treatment. Plasma serotonin levels were measured using high-performance liquid chromatography- electrochemical detection (HPLC-ECD) and the serotonin turnover ratios (5-HIAA/5-HT) were compared pre- and post-treatment. The results showed that the general behavioral stability was improved, and the problems of aggression and separation anxiety were ameliorated after treatment. A significant decrease in 5-HIAA/5-HT ratio was observed in dogs with separation anxiety, suggesting a serotonin-related mechanism. These results proved that PS128 was beneficial for emotional stabilization, which might be useful as a therapeutic supplement for canine aggression and separation anxiety.

Bushen Jieyu Tiaochong Formula reduces apoptosis of granulosa cells via the PERK-ATF4-CHOP signaling pathway in a rat model of polycystic ovary syndrome with chronic stress

Ethnopharmacological relevancePolycystic ovary syndrome (PCOS) is a common and complex endocrine disorder that is also an important cause of infertility. Adverse psychological stress can aggravate the occurrence and development of PCOS. Bushen Jieyu Tiaochong Formula (BJTF), a prescription of Traditional Chinese Medicine (TCM), has been used in the treatment of PCOS and shown to be effective in reducing negative emotion. However, the therapeutic mechanism has yet to be clearly elucidated. In the current study, we investigated the potential mechanism of action of BJTF. Aim of the studyTo investigate the role of PERK-ATF4-CHOP signaling in the molecular mechanisms that mediate the effects of BJTF in a rat model of PCOS, with chronic stress induced by letrozole and a chronic unpredictable mild stress (CUMS) paradigm. Materials and methodsIn addition to the normal control group, the PCOS combined with CUMS model rats were randomly assigned to a model group, a Diane-35 (ethinylestradiol 35 μg/cyproterone acetate 2 mg)-treated positive control group, or one of three BJTF-treated groups receiving a low, medium, or high dose. Behavioral testing, including the sucrose preference test and open field test, was conducted, and hematoxylin and eosin (H&E) staining was used to observe changes in the pathological morphology of ovarian tissue. Free testosterone (FT), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels in serum were quantified by enzyme-linked immunosorbent assays (ELISA). The hippocampal levels of norepinephrine (NE), 5-hydroxytryptamine/serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) were measured using high-performance liquid chromatography–electrochemical detection (HPLC-ECD). Apoptotic granulosa cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to detect the expression of glucose-regulated protein 78 (GRP78) and CHOP in the ovarian tissues. The expression levels of GRP78, CHOP, PERK, and ATF4 in ovarian tissues were also measured by western blotting. ResultsTreatment with either BJTF or Diane-35 ameliorated the abnormal cystic dilatation of follicles in the model rats and reduced the serum levels of FT and LH, and the LH/FSH ratio. BJTF treatment also attenuated chronic psychological stress-like behavior and regulated the expression and metabolism of cerebral monoamine neurotransmitters. The efficacy of BJTF was greater than that of Diane-35, with the optimal effects observed at the medium dose. BJTF also lowered the apoptotic index of ovarian granulosa cells and downregulated the expression of GRP78, CHOP, and ATF4. Although the expression level of PERK was not significantly altered by BJTF, the mean PERK expression level was the lowest in the medium-dose BJTF group. ConclusionsAdministration of BJTF has the therapeutic potential to promote the homeostasis of the reproductive endocrine environment and to restore follicular development and ovulation, possibly through the inhibition of the PERK-ATF4-CHOP signaling pathway, leading to downregulation of GRP78 expression to further delay ovarian granule cell apoptosis mediated by endoplasmic reticulum stress (ERS). Moreover, BJTF could improve behavioral performance by regulating cerebral monoamine neurotransmitters in this rat model. These findings provide a new perspective for treating PCOS related to psychological stress using TCM.

Evaluation of the phytochemicals and antioxidant activity of Lophatherum gracile Brongn based on chemical fingerprinting by HPLC with electrochemical detection.

A combinative method using high-performance liquid chromatography-electrochemical detection for fingerprinting and quantitative analysis was developed and successfully applied for the quality evaluation of Lophatherum gracile Brongn leaves collected from 21 geographical locations in China. In the fingerprint analysis, 18 common peaks were observed among the 21 samples, and 10 peaks were identified. Simultaneous quantification of the 10 components was conducted to interpret the variations in these compounds among the L. gracile Brongn leaves originating from different geographical locations. The correlation between the chromatograms and the antioxidant activities of the samples was further studied. The results indicated a linear correlation between the antioxidant activity and the total common peak areas of the fingerprints obtained by high-performance liquid chromatography-electrochemical detection. Importantly, it was found that high-performance liquid chromatography-electrochemical detection fingerprinting can not only determine the quantities of individual components present in such samples but also evaluate the antioxidant activities of the samples. The developed method is a valuable reference for the further study and development of L. gracile Brongn.

Effect of electroacupuncture preconditioning on the contents of dopamine and 5-hydroxytryptamine in lateral hypothalamus area and cerebellar fastigial nucleus of rats with myocardial ischemia-reperfusion injury

To observe the effect of electroacupuncture (EA) preconditioning at heart meridian acupoints on the contents of dopamine (DA) and 5-hydroxytryptamine (5-HT) in lateral hypothalamus area (LHA) and cerebellar fastigial nucleus (FN) in the rats with acute myocardial ischemia-reperfusion injury (MIRI), and explore the role and mechanism of LHA and FN in the effect of EA at heart meridian acupoints against acute MIRI. Sixty SD rats were randomly divided into a sham-operation group, a model group, an EA heart meridian group and an EA lung meridian group, 12 rats in each group, as well as an LHA plus heart meridian group (damage of bilateral LHA) and an FN plus heart meridian group (damage of bilateral FN), 6 rats in each one. Three days after nucleus destruction, EA was applied to "Shenmen" (HT 7) and "Tongli" (HT 5) in the EA heart meridian group, the LHA plus heart meridian group and the FN plus heart meridian group and EA was applied to "Taiyuan" (LU 9) and "Lieque" (LU 7) in the EA lung meridian group, with 1 V in stimulating voltage and 2 Hz in frequency, lasting 20 minutes each time, once a day, for consecutively 7 days before model replication. Except in the sham-operation group, MIRI rat models were duplicated by ligation of the left anterior descending branch of the coronary artery in the rest groups. Using Power lab physiological recorder, ST segment displacement value was recorded before modeling, 30 min after ligation and 120 min after reperfusion separately. The high performance liquid chromatography-electrochemical detection and analysis system was adopted to determine the contents of DA and 5-HT in LHA and FN dialysate after rat modeling in each group. In comparison of ST segment displacement value 30 min after ligation and 120 min after reperfusion among groups, the value in the model group was higher than that in the sham-operation group (P<0.01), those in the EA heart meridian group, the LHA plus heart meridian group, the FN plus heart meridian group and the EA lung meridian group were lower than those in the model group successively (P<0.01) and those in the EA heart meridian group were lower than those in the EA lung meridian group, the LHA plus heart meridian group and the FN plus heart meridian group successively (P<0.01). The contents of DA and 5-HT in FN and LHA in the model group were lower than those in the sham-operation group (P<0.01). Except in the groups with nucleus lesions, the contents of DA and 5-HT in FN and LHA of each intervention group were higher than those in the model group (P<0.01), the contents of DA and 5-HT in FN in the EA heart meridian group were higher than those in the EA lung meridian group and the LHA plus heart meridian group (P<0.01) and the content of 5-HT in LHA was higher than those in the EA lung meridian group and the FN plus heart meridian group (P<0.01) separately, the content of DA in LHA was higher than that in the EA lung meridian group (P<0.01). EA preconditioning at heart meridian acupoints can effectively alleviate myocardial injury in acute MIRI rats, during which, DA and 5-HT in LHA and FN may be the important material basis.

Disposable electrochemical flow cell with paper-based electrode assemble

Abstract High performance liquid chromatography-electrochemical detector (HPLC-EC) is employed as a reliable device for the detection of catecholamines in blood or urine samples without any time-consuming extraction procedure. However, there is a need for the construction of cheap and easy-to-operate electrode assemble systems embedded in HPLC-EC. In this study, we present a disposable and low-cost paper-based electrode (PBE) assemble utilizing a nitrocellulose membrane, which serves support material in flow-cell device for HPLC-EC. A custom-built flow cell was inventively designed as specific to the prepared PBEs via stereolithography 3D printing. To gain the electrochemical signal enhancement, the working electrode was modified with gold nanoparticles and carboxylated-multi-walled carbon nanotubes (MWCNTs). Here, we focused on the construction of the paper-based electrode assemble and characterization of the flow cell device performance. To demonstrate the functionality of the flow cell device, we analyzed common catecholamines from spiked biological fluids using the optimized experimental parameters. Amperometric detection of the catecholamines was carried out at the potential of + 0.5 V and linear range was found to be 0.5–20.0 μg/mL, with a limit of detection in the range of 4.0–15.0 ng/mL. The obtained results confirmed our disposable paper-based electrode assemble to be promising for the construction of an electrochemical platform for reliable and fast detection of electroactive species.

Brain Concentrations of Methylone and Its Metabolites after Systemic Methylone Administration: Relationship to Pharmacodynamic Effects.

3,4-Methylenedioxy-N-methylcathinone (methylone) is a new psychoactive substance with stimulant properties and potential for abuse. Despite its popularity, limited studies have examined relationships between brain concentrations of methylone, its metabolites, and pharmacodynamic effects. The goal of the present study was 2-fold: 1) to determine pharmacokinetics of methylone and its major metabolites-4-hydroxy-3-methoxy-N-methylcathinone (HMMC), 3,4-dihydroxy-N-methylcathinone (HHMC), and 3,4-methylenedioxycathinone (MDC)-in rat brain and plasma and 2) to relate brain pharmacokinetic parameters to pharmacodynamic effects including locomotor behavior and postmortem neurochemistry. Male Sprague-Dawley rats received subcutaneous methylone (6, 12, or 24 mg/kg) or saline vehicle (n = 16/dose), and subgroups were decapitated after 40 or 120 minutes. Plasma and prefrontal cortex were analyzed for concentrations of methylone and its metabolites by liquid chromatography-tandem mass spectrometry. Frontal cortex and dorsal striatum were analyzed for dopamine, 5-HT, and their metabolites by high-performance liquid chromatography-electrochemical detection. Brain and plasma concentrations of methylone and its metabolites rose with increasing methylone dose, but brain methylone and MDC concentrations were greater than dose-proportional. Brain-to-plasma ratios for methylone and MDC were ≥ 3 (range 3-12), whereas those for HHMC and HMMC were ≤ 0.2 (range 0.01-0.2). Locomotor activity score was positively correlated with brain methylone and MDC, whereas cortical 5-HT was negatively correlated with these analytes at 120 minutes. Our findings show that brain concentrations of methylone and MDC display nonlinear accumulation. Behavioral and neurochemical effects of systemically administered methylone are related to brain concentrations of methylone and MDC but not its hydroxylated metabolites, which do not effectively penetrate into the brain. SIGNIFICANCE STATEMENT: Behavioral and neurochemical effects of methylone are related to brain concentrations of methylone and its metabolite MDC but not its hydroxylated metabolites, 4-hydroxy-3-methoxy-N-methylcathinone and 3,4-dihydroxy-N-methylcathinone, which do not effectively penetrate into the brain. Methylone and MDC display nonlinear accumulation in the brain, which could cause untoward effects on serotonin neurons in vulnerable brain regions, including the frontal cortex.

The identification of tyrosine-nitrated human serum albumin in airborne particulate matter from Japan

Atomospheric particulate matter (PM) has been accepted as a risk to human health worldwide. PM contains a variety of components, but it is not clear which components are harmful to human health. For example, there have been many reports on metals, but there have been only a few studies on proteins. Based on an analysis of serum antibodies in a mouse model of PM-induced asthma reported previously, we have been conducting research focusing on the proteins contained in PM. In the present study, we found for the first time that human serum albumin (HSA) is bound to PM. HSA was detected in total suspended particulate matter (TSP) and PM2.5, from various regions in Japan, during all seasons. The presence of HSA in PM was confirmed by proteomics analysis, amino acid analysis, immunoblotting and BD140, which emits fluorescence when bound to a specific HSA site. Moreover, we discovered that TSP-binding HSA was tyrosine-nitrated, one of a chemical modification via environmental atmospheric pollutants using high performance liquid chromatography-electrochemical detection. It was concluded that although the origin of HSA in PM was unclear, HSA is present in the airborne PM of Japan, partially modified for tyrosine nitration.

Exercise preconditioning ameliorates cognitive impairment and anxiety-like behavior via regulation of dopamine in ischemia rats

Cognitive impairment and anxiety are common health problems in acute ischemic stroke patients. Meanwhile, dopamine in the striatal brain region is significantly increased during the acute phase of cerebral ischemia. Besides, the studies shown that striatum and change of striatal dopamine are associated with learning and memory and anxiety. Further, physical exercise has been shown to improve neurocognitive and emotional function in animal models and patients with cerebral ischemia. However, the exact mechanism underlying this effect is unclear. The purpose of this research is to explore the effect of pre-ischemic voluntary wheel running on levels of striatal dopamine, cognition and anxiety in cerebral ischemia rats. Methods: 48 adult male Sprague-Dawley rats were enrolled in this study and divided randomly in following 6 groups: sham group (S group, n = 8), ischemia group (I group, n = 8), 1 week wheel running group (1R group), 4 weeks wheel running group (4R group), 1 week pre-ischemia wheel running group (1RI group, n = 8) and 4 weeks pre-ischemia wheel running group (4RI group, n = 8). After training, cerebral ischemia was induced by permanent bilateral common carotid artery ligation (2-VO) operation. Microdialysis was used to collect dialysates from the striatum immediately from 30 min before ischemia to 90 min after ischemia. High-performance liquid chromatography-electrochemical detection system (HPLC) was used to determine the content of dopamine in the dialysates. Passive avoidance and elevated plus maze test were used to test neurocognitive function 24 h after 2-VO cerebral ischemia. Results: As compare with the constant striatal dopamine level of S group, the striatal dopamine level in I group after ischemia showed a trend of rapid increasing and reached maximum value at the 20 min (P<0.001), then decreased gradually. The striatal dopamine level in 1RI and 4RI group showed the trend were similar to I group, but the increasing magnitude was attenuated. A comparison of the basal striatal dopamine level in 4 groups found that the basal dopamine level in 1RI and 4RI group were higher than S and I group (P<0.001). In passive avoidance task, the retention latency of I group was significantly shorter than S group (P<0.001), and the retention latency of the 1RI, 1R and 4R, 4RI group were longer than I group (P<0.001), there was no significant difference in S, 1RI, 1R, 4R and 4RI group (P>0.05). In elevated plus maze test, the time and entrance numbers of open arms in I group were significantly less than S group (P<0.05), but these indices were no significant difference in S, 1RI, 1R, 4RI and 4RI group. Conclusion: According to our results, 1 or 4 weeks pre-ischemia wheel running can significantly increase the basal dopamine level, attenuate the increase of striatal dopamine induced by cerebral ischemia and improve neurocognitive function in ischemia rats.