Systemic L-ornithine supplementation specifically increases ovarian putrescine levels during ovulation in mice†.

In all mammalian species examined thus far, the ovaries produce a burst of ornithine decarboxylase (ODC) and putrescine during ovulation or after application of human chorionic gonadotropin (hCG). Aged mice have significantly reduced levels of this periovulatory ODC and putrescine rise. Putrescine supplementation, in vitro during oocyte maturation or in mouse drinking water during the periovulatory period, reduces egg aneuploidies and embryo resorption, improving fertility of aged mice. These studies suggest that periovulatory putrescine supplementation may be a simple and effective therapy for reproductive aging for women. However, putrescine supplementation is expected to increase widespread tissue putrescine levels, raising concerns of nonspecific and unwanted side effects. Given that ODC is highly expressed in the ovaries during ovulation but otherwise exhibits low activity in most tissues, we hypothesized that periovulatory supplementation of L-ornithine, the substrate of ODC, might be suitable for delivering putrescine specifically to the ovaries. In this study, we have demonstrated that systemic application of L-ornithine via oral gavage or subcutaneous injection increased ovarian putrescine levels; the increase was restricted to animals that had been injected with hCG. Furthermore, L-ornithine specifically increased ovarian putrescine levels without affecting putrescine levels in any other tissues. However, our attempts to improve fertility of aged mice through L-ornithine supplementation in mouse drinking water produced either no effects (1% L-ornithine) or negative impact on fertility (4% ornithine). Our results suggest that it might not be feasible to achieve fertility-enhancing ovarian putrescine levels via L-ornithine supplementation in drinking water without encountering undesired consequences of high dose of exogenous L-ornithine.

In Situ Autophagy Disruption Generator for Cancer Theranostics.

Cancer remains a serious clinical disease awaiting new effective treatment strategies. Autophagy modulation has emerged as a novel and promising pharmacologic target critical to future drug development and anti-cancer therapy applications. Herein, we constructed an in situ autophagy disruption generator to break the balance of autophagy flow for tumor-targeting therapy. Hollow mesoporous manganese trioxide (Mn2O3) nanoparticles (NPs) were synthesized and conjugated with hyaluronic acid (HA) to form tumor-targeting drug carriers. Then, traditional autophagy inhibitor hydroxychloroquine (HCQ) was loaded into the hollow core of HA-Mn2O3, to form a multifunctional theranostics platform (HA-Mn2O3/HCQ). This nanoplatform displayed specific localization and retention in lysosomes after entering tumor cells. The synchronous release of HCQ and manganese ion (Mn2+) induced lysosomal alkalization and osmotic pressure elevation. Significantly greater lysosomal deacidification and autophagy blockade effect emerged after treatment by this nanoplatform, with in vitro tumor inhibition rate of 92.2%. Imaging experiment proved that it could selectively deliver HCQ to tumor sites and further degrade to realize simultaneous release of Mn2+ and HCQ. Micromorphological and immunofluorescence analysis demonstrated that in situ high concentrations of these two substances would achieve effective autophagy blockade. Pharmacodynamics test showed that this nanogenerator displayed the best therapeutic efficacy with 5.08-fold tumor inhibition ratio compared with the HCQ group. Moreover, the generated Mn2+ can be used as T1 contrast agent for visualizing tumor lesions and monitoring therapeutic effects. Overall, the as-made multifunctional drug-delivery system might provide a promising platform for cancer theranostics upon in situ autophagy disruption.

The Efficacy of Eye Movement Desensitization and Reprocessing in Children and Adults Who Have Experienced Complex Childhood Trauma: A Systematic Review of Randomized Controlled Trials.

Background: Survivors of complex childhood trauma (CT) such as sexual abuse show poorer outcomes compared to single event trauma survivors. A growing number of studies investigate Eye Movement Desensitization and Reprocessing (EMDR) treatment for posttraumatic stress disorder (PTSD), but no systematic reviews have focused on EMDR treatment for CT as an intervention for both adults and children. This study therefore systematically reviewed all randomized controlled trials (RCTs) evaluating the effect of EMDR on PTSD symptoms in adults and children exposed to CT. Methods: Databases including PubMed, Web of Science, and PsycINFO were searched in October 2017. Randomized controlled trials which recruited adult and children with experience of CT, which compared EMDR to alternative treatments or control conditions, and which measured PTSD symptoms were included. Study methodology quality was evaluated with Platinum Standard scale. Results: Six eligible RCTs of 251 participants were included in this systematic review. The results indicated that EMDR was associated with reductions in PTSD symptoms, depression and/or anxiety both post-treatment and at follow-up compared with all other alternative therapies (cognitive behavior therapy, individual/group therapy and fluoxetine) and control treatment (pill placebo, active listening, EMDR delayed treatment, and treatment as usual). However, studies suffered from significant heterogeneity in study populations, length of EMDR treatment, length of follow-up, comparison groups, and outcome measures. One study had a high risk of bias. Discussion: This systematic review suggests that there is growing evidence to support the clinical efficacy of EMDR in treating CT in both children and adults. However, conclusions are limited by the small number of heterogenous trials. Further RCTs with standardized methodologies, as well as studies addressing real world challenges in treating CT are required.

Effects of Ginseng Fruit Saponins on Serotonin System in Sprague-Dawley Rats with Myocardial Infarction, Depression, and Myocardial Infarction Complicated with Depression.

BACKGROUND: Our previous studies have demonstrated that the levels of 5-hydroxytryptamine (5-HT) and 5-HT 2A receptor (5-HT2AR) in serum and platelet were associated with depression and myocardial infarction (MI), and pretreatment with ginseng fruit saponins (GFS) before MI and depression had an effect on the 5-HT system. In this study, the effects of GFS on the 5-HT system in the Sprague-Dawley (SD) rats with MI, depression, and MI + depression were evaluated. METHODS: A total of eighty SD rats were allocated to four groups: MI, depression, MI + depression, and control groups (n = 20 in each group). Each group included two subgroups (n = 10 in each subgroup): Saline treatment subgroup and GFS treatment subgroup. The levels of 5-HT, 5-HT2AR, and serotonin transporter (SERT) were quantified in serum, platelet lysate, and brain tissue through the enzyme-linked immunosorbent assay method, respectively. RESULTS: Compared with those in the saline treatment subgroups, the levels of 5-HT in serum and platelet lysate statistically significantly increased in the GFS treatment subgroups of MI, depression, and MI + depression groups (serum: all P = 0.000; platelet lysate: P = 0.002, 0.000, 0.000, respectively). However, the 5-HT levels in brain homogenate significantly decreased in the GFS treatment subgroups compared with those in the saline treatment subgroups in MI and depression groups (P = 0.025 and 0.044 respectively), and no significant difference was observed between saline and GFS treatment subgroups in MI + depression group (P = 0.663). Compared with that in GFS treatment subgroup of control group, the 5-HT2AR levels in the platelet lysate significantly decreased in GFS treatment subgroups of MI, depression, and MI + depression groups (all P = 0.000). Compared to those in the saline treatment subgroups, the serum SERT levels significantly decreased in the GFS treatment subgroups in MI, depression, and MI + depression groups (P = 0.009, 0.038, and P = 0.001, respectively), while the SERT levels of platelet lysate significantly decreased in GFS treatment subgroup of MI group (P = 0.000), significantly increased in GFS treatment subgroup of depression group (P = 0.019), and slightly changed in GFS treatment subgroup of MI + depression group (P = 0.219). No significant changes for SERT levels in brain homogenate could be found between the saline and GFS treatment subgroups in MI, depression, and MI + depression groups (P = 0.421, 0.076 and P = 0.642). CONCLUSIONS: This study indicated that GFS might inhibit the reuptake of 5-HT from serum to platelet according to decreased 5-HT2AR in platelet and SERT in serum and platelet. The change of 5-HT in serum after GFS treatment was inconsistent with that in the brain. It seemed that GFS could not pass through the blood-brain barrier to affect the central serotonergic system.

[Effect of two Pi deficiency syndrome models on the configuration and function of the skeletal muscle in mice].

OBJECTIVE: To observe the relation between Pi deficiency syndrome (PDS) and the configuration and functions of extensor digitorum longus (EDL)and soleus (SOL). METHODS: Totally 36 ICR mice were randomly divided into 3 groups according to weight matching principle, the control group, the exhausted group, and the rhubarb group, 12 in each group. Two PDS models were established by either purgation with rhubarb diarrhea (as Group A) or exhausted swimming plus sleep deprivation (as Group B).The cross sectional area (CSA) of type I and II fibers of extensor digitorum longus (EDL) and soleus (SOL), relative proportions of type I and II fibers were measured by m-ATPase histochemical method. The isotonic contraction and the maximum tetanus contraction of EDL and SOL were detected by PowerLab system. RESULTS: Compared with the control group, the body weight, body temperature, and the general health condition of PDS model rats obviously decreased; the spleen index and the thymus index were also lower; the maximal isotonic contraction and the maximum tetanus contraction obviously decreased; the cross section areas of EDL and SOL were reduced with loosely arranged cells. In EDL, the proportion of type I fibers was added and the proportion of type II fibers was lowered. In SOL, there was no change in the proportion of type I and type II fibers. CONCLUSIONS: EDL and SOL were obviously atrophied in the two PDS model mice. The type I fibers of SOL was more significantly atrophied in Group B.

A comparative study on the pharmacokinetics of a traditional Chinese herbal preparation with the single herb extracts in rats by LC-MS/MS method.

The Er-Mu preparation (EMP) is a well-known traditional Chinese prescription that has been clinically employed for the treatment of asthma and bronchial inflammation for hundreds of years. Neomangiferin, mangiferin, peimine, peiminine, timosaponin BII and timosaponin AIII are the major active ingredients of EMP for their anti-inflammatory or anti-asthmatic effects. The aim of this study was to investigate the pharmacokinetics of the target compounds from the recipe of EMP and the single herb extracts of Anemarrhenae asphodeloides Bge. (ARR) and Fritillariae cirrhosae D.Don (FCB), and the influence of compatibility on the pharmacokinetics of the main active ingredients. The rats were randomly assigned to three groups and orally administered with the recipe of EMP and the single herb extracts of ARR and FCB, respectively. The concentrations of the target compounds in rat plasma were determined by an optimal liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) and multiple reaction monitoring (MRM) with a multi-switching monitoring mode coupled with simple protein precipitation method, and the main pharmacokinetic parameters were estimated. Significant differences (p<0.05) were found in the pharmacokinetic parameters of neomangiferin, mangiferin, peimine and peiminine between the single ARR or FCB extract and the combination treatment (p<0.05). The developed HPLC-ESI-MS method by switching positive and negative ESI sources in a single run was successfully applied to study the pharmacokinetics of six compounds in SD rat, which was powerful in terms of sensitivity, selectivity, time savings and solvent consumption in the quantitative analysis of complex herbal medicines. It was surmised that formula compatibility could significantly influence the pharmacokinetics of EMP and our study has preliminarily elucidated the priority in the compatible administration of EMP based on pharmacokinetic studies.

Determination of cnidilin and its two metabolites in rat bile and stool after oral administration by HPLC/electrospray ionization tandem mass spectrometry.

A simple, fast and sensitive method for the simultaneous determination of cnidilin and its two metabolites (M1 and M2) in rat bile and stool using HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) has been developed. The sample pretreatment was simple, because methanol was the only additive used for dilution of bile and ultrasound of stool. Pimpinellin was used as internal standard (IS). The separation was performed on a reverse phase C18 column with gradient elution consisting of 0.5‰ aqueous formic acid and methanol (containing 0.5‰ formic acid). The detection was in the multiple-reaction monitoring mode within 7 min. All the analytes were in accordance with the requirement of the validation of the method in vivo (linearity, precision, accuracy, limit of detection and limit of quantification). After oral administrating 24 mg/kg of the prototype drug cnidilin, M1 and M2 were determined in bile within 36 h, and in stool within 60 h. Cnidilin in bile was completely excreted in 24 h, and the main excretive amount of cnidilin was 80% in the first 6 h, but the drug recovery in bile within 24 h was <1.95%. In stool, the main excretive amount of cnidilin was 95.8% in the first 24 h, and the drug recovery within 48 h was lower than 1.48%.

Determination of cnidilin and its two metabolites in rat plasma by high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

Simple, fast, and sensitive HPLC coupled with an electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method for the simultaneous determination of cnidilin and its two metabolites, 3",8-methoxy-isoimperatorin (M1) and 5"-hydroxyl-8-methoxy-isoimperatorin (M2), in rat plasma has been developed. Pimpinellin was used as the internal standard and the total analysis time was 7 min. Methanol was the only reagent during the process of sample handling used as a protein precipitant. The analytes were separated on a reversed-phase C18 column with gradient elution consisting of 0.5% aqueous formic acid and methanol (containing 0.5% formic acid) in the multiple-reaction monitoring mode. Cnidilin, M1, M2, and the internal standard could be well ionized under positive electrospray ionization conditions. Full validation of the method (linearity, precision, accuracy, limit of detection, and limit of quantification) was carried out and the method was successfully employed in a metabolism study of cnidilin administered to rats at a dose of 24 mg/kg. The pharmacokinetic parameters of cnidilin determined after orally administrating the single coumarin to the rat were obtained and the mass fragmentation pathways for analysis are proposed in this article.

Simultaneous detection of flavonoids and phenolic acids in Herba Lysimachiae and Herba Desmodii Styracifolii using liquid chromatography tandem mass spectrometry.

A sensitive liquid chromatography and tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous analysis of 15 flavonoids and 3 phenolic acids in Herba Lysimachiae and Herba Desmodii Styracifolii. Separation was performed using a Diamonsil C(18) column, which was eluted with methanol (A) and 0.1‰ acetic acid (B). The gradient condition was as follows: 0-34 min, 20-34% A; 34-38 min, 34-95% A; maintained 95% A for the next 4 min. Analytes were detected using a hybrid quadrupole linear ion trap mass spectrometer that was equipped with an electrospray ionisation source in the negative ion and multiple-reaction monitoring modes. A full validation of the method was performed, including the linearity, precision, and accuracy, as well as limits of detection and quantification. The results indicated that the developed method was simple, sensitive and reliable. Furthermore, the method was successfully applied to differentiate 16 batches of Herba Lysimachiae and 21 batches of Herba Desmodii Styracifolii.

Simultaneous analysis of 11 main active components in Cirsium setosum based on HPLC-ESI-MS/MS and combined with statistical methods.

A novel method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 11 major active components including ten flavonoids and one phenolic acid in Cirsium setosum. Separation was performed on a reversed-phase C(18) column with gradient elution of methanol and 0.1‰ acetic acid (v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple-reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the assay was carried out including linearity, precision, accuracy, stability, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid, and specific. The 25 batches of C. setosum samples from different sources were first determined using the developed method and the total contents of 11 analytes ranged from 1717.460 to 23028.258 µg/g. Among them, the content of linarin was highest, and its mean value was 7340.967 µg/g. Principal component analysis and hierarchical clustering analysis were performed to differentiate and classify the samples, which is helpful for comprehensive evaluation of the quality of C. setosum.

Simultaneous determination and pharmacokinetic study of six flavonoids from Fructus Sophorae extract in rat plasma by LC-MS/MS.

In this study, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of six flavonoids including sophoricoside, genistin, genistein, rutin, quercetin and kaempferol in rat plasma after oral administration of Fructus Sophorae extract using sulfamethalazole as internal standard (IS). The plasma samples were pretreated and extracted by liquid-liquid extraction. Chromatographic separation was accomplished on a C(18) column with a simple linear gradient elution. The detection was accomplished by multiple-reaction monitoring (MRM) scanning after electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion pairs (m/z) for quantitation were 431.1/267.9 for sophoricoside and genistin, 269.0/133.0 for genistein, 609.2/300.0 for rutin, 301.0/150.9 for quercetin, 284.9/93.0 for kaempferol and 252.0/155.9 for IS. The total run time was 8.0 min. Full validation of the assay was implemented including specificity, linearity, accuracy, precision, recovery and matrix effect. This is the first report on determination of the major flavones in rat plasma after oral administration of Fructus Sophorae extract. The results provided a meaningful basis for the clinical application of this herb.

Simultaneous determination of nine components in Anemarrhena asphodeloides by liquid chromatography-tandem mass spectrometry combined with chemometric techniques.

A novel quantitative method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the important active constituents including four steroidal saponins, two xanthone glycosides, two isoflavonoids, and one anthraquinone in different parts of Anemarrhena asphodeloides from different habitats. Hierarchical clustering analysis and principal components analysis were performed to differentiate and classify the samples. The separation was performed on a C(18) column with acidified aqueous acetonitrile gradients. Quantification of the analytes was achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring scanning was employed with switching electrospray ion source polarity between positive and negative modes in a single run. The validation results of the method indicated that the method was simple, rapid, specific, and reliable. The results demonstrated that the quantitative difference in content of nine active compounds was useful not only for chemotaxonomy of many samples from different sources but also for the standardization and differentiation of many similar samples. Simultaneous quantification of bioactive components by HPLC-ESI-MS coupled with chemometric techniques would be a well-acceptable strategy to comprehensively control the quality of A. asphodeloides.

Ouabain at pathological concentrations might induce damage in human vascular endothelial cells.

AIM: To examine the time- and dose-dependent effects of ouabain on human umbilical vein endothelial cells (HUVEC) in vivo, and the changes in aortic endothelium and the different expression levels of Kv4.2 in vitro. METHODS: The proliferation of HUVEC and cell death were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, the incorporation of [3H]TdR, trypan blue staining, and lactate dehydrogenase (LDH) release. The response of endothelial cells to ouabain was explored with a complementary DNA microarray and a candidate gene was found. Ouabain-sensitive hypertensive rats were established by chronic administration of ouabain. Changes in the aortic endothelium were observed by electron microscopy, and the expression level of Kv4.2 in different animals was studied by using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Ouabain stimulated the proliferation of HUVEC at physiological concentrations (0.3-0.9 nmol/L). Ouabain at pathological concentrations (0.9-1.8 nmol/L) inhibited proliferation and induced cell death. mRNA profile analysis indicated that 340 genes were differentially expressed after ouabain treatment: 145 were upregulated, of which 6 were upregulated significantly, including KCND2 (encoding the potassium voltage-gated channel shal-related subfamily member 2). The upregulated genes were mainly related to cell metabolism and transcription. In ouabain-sensitive hypertensive rats, the aortic endothelium was damaged and Kv4.2 (coded by KCND2) was over-expressed. CONCLUSION: The physiological role of ouabain in HUVEC might involve the control of growth and metabolism. Ouabain at pathological concentrations might affect the structure and function of the vascular endothelium by modification of expression of the KCND2 gene, and participate vascular remodeling in hypertension.