Identification of Traditional Chinese Medicine Seahorses Using Graphene Oxide-based Fluorescent Sensing Technology / 中国实验方剂学杂志

ObjectiveTo establish a method for seahorse identification based on graphene oxide fluorescence sensing technology， and to provide a new research idea for identification of traditional Chinese medicine. MethodThe fluorophore FAM was labeled at the 5' end of the specificity upstream primer Ja-F of Hippocampus japonicus as the nucleic acid probe FAM-ssDNA （single strand DNA）. The recognition site of RNA polymerase Ⅱ was added to its specific downstream primer Ja-R as Ja-R1. The seahorse samples were amplified with Ja-F/Ja-R1 primers， and the ssDNA of H. japonicus was obtained by reverse transcription of the amplification products using vitro transcription method. The 20 μL nucleic acid probe FAM-ssDNA （500 nmol·L-1） was incubated at 90 ℃ for 5 min， and was gradually cooled to room temperature. Different volume of graphene oxide solution （100 mg·L-1） and Tris hydroxymethyl amino methane HCl （Tris-HCl） buffer （50 mmol·L-1） were added into each probe solution to make a final reaction volume of 1 mL. The fluorescence intensity of each sample was measured after mixing and placing different times at room temperature away from the light. So that the most appropriate graphene oxide concentration and reaction time were screened for constructing the best nucleic acid probe-graphene oxide biosensor. Adding probe complementary sequence FAM-ssDNA-match solution into the nucleic acid probe-graphene oxide solution， the fluorescence intensity of the reaction mixture was measured after being placed different times at room temperature. Therefore， the optimal reaction time of fluorescence recovery was screened and the feasibility of the sensor was tested. The sensitivity was detected via adding ddH2O as the blank control and different concentration H. japonicus ssDNA into each nucleic acid probe-graphene oxide solution， respectively. Finally， the commercial hippocampal were identified using the optimal experimental condition， and the feasibility of this method for the identification of Chinese medicinal materials was verified. ResultThe fluorescence of 1 mL reaction mixture including 10 nmol·L-1 nucleic acid probe FAM-ssDNA and 12 mg·L-1 go solution for 20 min at room temperature away from the light could be completely quenched. Feasibility test of the biosensor showed that when probe complementary sequence FAM-ssDNA-match solution （final concentration 90 nmol·L-1） was added to the biosensor solution and reacted 1 h reaction at room temperature， the fluorescence signal was significantly enhanced. Sensitivity test showed that the minimum concentration of ssDNA detected by this method was about 10 mg·L-1. This method was used to detect commercial seahorses， and only H. japonicus samples had obvious fluorescence signal. ConclusionThe graphene oxide-based fluorescent sensing technology could be used for zoological origin survey of commercial hippocampus.

Mechanism of seahorse ameliorating depression-like behavioral and pathological changes caused by chronic stress in zebrafish / 中国药理学通报

Aim To investigate the effect of marine herbal seahorse on chronic unpredictable mild stress ( CUMS ) -induced depression-like model in zebrafish. Methods Adult zebrafish were divided into control, Stress,Stress + low dose (Stress +0.044% SH) and Stress + high dose (Stress +0. 22% SH) seahorse intervention groups, and depression-like behavior was identified by novel tank test (NTT), cortisol, interleukin ( IL )-6 and interferon (IFN )-γ levels were detected by ELISA. The levels of dopamine (DA) ,norepinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyin-doleacetic acid (5-HIAA) were determined by high performance liquid chromatography. The mRNA expression levels of tryptophan hydroxylase(TPH)-2 and 5-HT2A receptor were measured by real-time quantitative PCR. Results Compared with the control group, the Stress group showed significantly longer latency to reach the top in NTT, significantly reduced number of transfers to the top region and top residence time, significantly increased levels of cortisol and IL-6, IFN-γ protein, significantly reduced levels of DA and 5-HT in brain as well as increased metabolism rate of 5-HT, while 5-HT2A mRNA expression was up-regulated and TPH2 mRNA expression was down-regulated. In contrast, low-dose seahorse intervention effectively reduced anxiety, decreased cortisol and IL-6 and IFN-γ concentrations, increased monoamine neurotransmitter levels and reversed dysregulation of the 5-HT ergic system in CUMS zebrafish. Conclusion Seahorse may exert an-tidepressant effects through anti-inflammation and mod¬ulation of monoamine neurotransmitter levels.

Mechanism of Yiqi Jiedu Prescriptions in Protection of Mitochondria in PC12 Cells Against Hypoxia Injury Based on Energy Metabolism / 中国实验方剂学杂志

ObjectiveTo establish an evaluation method for mitochondrial energy metabolism with Seahorse analyzer and investigate the protective effect of Yiqi Jiedu prescriptions （YQ） on mitochondria in rat adrenal pheochromocytoma （PC12） cells against hypoxia injury. MethodThe PC12 cell injury model was induced in vitro using hypoxic chambers. Five groups were set up， ie， a control group， a model group （model）， high- （25 µmol·L-1）， medium- （5 µmol·L-1） and low-dose （1 µmol·L-1） YQ groups， and a positive drug trimetazidine （TMZ） group， with three replicate wells in each group. The experiment was repeated three times. The established method for energy metabolism analysis was used to assay the activity of mitochondrial complex in cells and screen the optimal dosing concentration. Subsequently， the YQ group and modified YQ groups were set up， and the aerobic respiration and glycolysis function were assayed by the Seahorse analyzer. According to the non-mitochondrial oxygen consumption， proton leakage， basal respiration， maximum respiration， ATP production， and potentially improved respiration， the effects of modified YQ groups on the aerobic respiration of mitochondria damaged by hypoxia were evaluated by principal component analysis （PCA） and variable importance in projection （VIP）. The expression of cytochrome C， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） was detected by Western blot. ResultCompared with the groups of other concentrations， the optimal dosing concentration of carbonyl cyanide-4 （trifluoromethoxy）phenylhydrazone （FCCP） was 2 µmol·L-1. Compared with the model group， the medium-dose YQ group showed enhanced mitochondrial complex activity （P<0.05）. The YQ groups were superior to the model group in improvement （P<0.01）. The combination of ginsenoside and geniposide showed the optimal effect among the modified YQ groups （P<0.01）. VIP analysis revealed that for the improvement of mitochondrial respiratory function， the contribution of geniposide in YQ was the greatest. Compared with the model group， the high-dose YQ group displayed reduced leakage of mitochondrial cytochrome C （P<0.01）， decreased expression of Bax protein （P<0.01）， and increased expression of Bcl-2 protein （P<0.05， P<0.01）. ConclusionA cellular， high-throughput quantitative evaluation method for mitochondrial energy metabolism was established， which demonstrated that YQ could significantly improve the impaired mitochondrial energy metabolism in PC12 cells damaged by hypoxia， and the underlying mechanism might be related to the protection against mitochondrial apoptosis.

Impaired tricarboxylic acid cycle flux and mitochondrial aerobic respiration during isoproterenol induced myocardial ischemia is rescued by bilobalide / 药物分析学报

There is an urgent need to elucidate the pathogenesis of myocardial ischemia (MI) and potential drug treatments.Here,the anti-MI mechanism and material basis of Ginkgo biloba L.extract (GBE) were studied from the perspective of energy metabolism flux regulation.Metabolic flux analysis (MFA) was performed to investigate energy metabolism flux disorder and the regulatory nodes of GBE components in isoproterenol (ISO)-induced ischemia-like cardiomyocytes.It showed that[U-13C]glucose derived m+2 isotopologues from the upstream tricarboxylic acid (TCA) cycle metabolites were markedly accu-mulated in ISO-injured cardiomyocytes,but the opposite was seen for the downstream metabolites,while their total cellular concentrations were increased.This indicates a blockage of carbon flow from glycolysis and enhanced anaplerosis from other carbon sources.A Seahorse test was used to screen for GBE components with regulatory effects on mitochondrial aerobic respiratory dysfunction.It showed that bilobalide protected against impaired mitochondrial aerobic respiration.MFA also showed that bilobalide significantly modulated the TCA cycle flux,reduced abnormal metabolite accumulation,and balanced the demand of different carbon sources.Western blotting and PCR analysis showed that bilobalide decreased the enhanced expression of key metabolic enzymes in injured cells.Bilobalide's efficacy was verified by in vivo experiments in rats.This is the first report to show that bilobalide,the active ingredient of GBE,protects against MI by rescuing impaired TCA cycle flux.This provides a new mechanism and potential drug treatment for MI.It also shows the potential of MFA/Seahorse combi-nation as a powerful strategy for pharmacological research on herbal medicine.

Effects of antimalarial drugs on mitochondrial respiration in erythrocytic stages of Plasmodium falciparum 3D7 / 中国临床药理学与治疗学

AIM: The Seahorse XFe96 analyzer was used to evaluate the effects of thirteen types of international first-line antimalarial drugs in six categories on the mitochondrial electron transport chain (ETC) of Plasmodium falciparum 3D7 (P. falciparum 3D7). METHODS: The antimalarial activity of in vitro drugs acting on P. falciparum 3D7 was evaluated using the three-day inhibition method and SYBR Green I fluorescence analysis method. MACS technology was used to separate and purify P. falciparum 3D7. The mitochondrial oxygen consumption rate (OCR) of Seahorse XF analysis system was used to characterize the bioenergy of P. falciparum 3D7 mitochondria at different times to investigate the effects of antimalarial drugs on mitochondrial aerobic respiration of Plasmodium falciparum. RESULTS: The results of flow cytometry showed that the Plasmodium of trophozoite stages was enriched successfully. The results of in vitro antimalarial activity evaluation showed that, except for the antimalarial drug proguanil (Pro), the other twelve antimalarial drugs were all of the nmol/L level against P. falciparum 3D7. The results of the mitochondrial aerobic respiration showed that the five concentrations of dihydroartemisinin (DHA) and chloroquine (CQ) (0.4, 1, 5, 10, 50×IC

Multiplex PCR to simultaneous identification of five traditional Chinese medicinal seahorses / 中国中药杂志

Seahorse is one the most commonly used medicinal animal in China. Five species of Hippocampus are recorded as seahorse in the Chinese Pharmacopoeia. Because of the rapid decrease, several other Hippocampus species are often adulterants as medicinal seahorse in the herbal market, which compromise clinical efficacy and pose threat to endangered seahorse species conversation. Herein, a multiplex polymerase chain reaction (mPCR) method was developed to identify the biological sources of medicinal seahorses.Based on the sequences of mitochondrial DNA, five specific primers for Hippocampus trimaculatus, H. kelloggi, H. kuda, H. histrix and H. mohnikei (H. japonicus)were designed, respectively. Multiplex PCR yields the products of 155, 222, 292, 352, 458 bp amplicons in the present of DNA templates of H. kuda, H. mohnikei, H. kelloggi, H. histrix and H. trimaculatus, respectively. This multiplex PCR method which electrophoresis migration of different lengths of DNA bands allowed simultaneous identification of all the five medicinal seahorses in a single assay. It showed that this multiplex PCR assay is useful for the simultaneous identification the biological sources of complex multi-source samples, which could provide a useful tool for the quality control of seahorses.

Relationship between mitochondrial dysfunction, oxidative stress and diabetic retinopathy / 国际眼科杂志(Guoji Yanke Zazhi)

As one of the serious complications of diabetes, diabetic retinopathy( DR) has become a main eye disease which causes blindness. The occurrence and development of DR is related to many factors. The pathogenesis is complicated, and the mechanism has not been clear. Early data suggest that the occurrence and development of DR has relations with many factors such as blood sugar level, diabetes duration and the environment. Among the factors, mitochondrial dysfunction and oxidative stress is the important mechanisms of DR and has become research focus in recent years. Consequences of mitochondrial dysfunction within cells include elevation of the rate of reactive oxygen species( ROS) production due to damage of electron transport chain proteins, mitochondrial DNA ( mtDNA ) damage, and loss of metabolic capacity. Clear understanding on the mechanism of mitochondrial functional change under high sugar level and oxidative stress response in the occurrence and development of DR is of great significance on prevention and cure of DR. ln this article, the development of mitochondrial metabolism and oxidative stress of DR is reviewed.

Anesthetic induction and recovery of Hippocampus reidi exposed to the essential oil of Lippia alba

The aim of this study was to identify the times of anesthetic induction and recovery in slender seahorses (Hippocampus reidi) that were exposed to the essential oil of Lippia alba (EO), as well as the efficacy of EO as a stress-reducing agent in the transport of this species. Slender seahorses were placed in 1-L aquaria containing different concentrations of EO (0, 10, 20, 50, 150, 300 and 450 µL L-1), and after induction, fish were transferred to aquaria that were free of anesthetic to evaluate their recovery time. In an additional experiment, slender seahorses were transported in plastic bags with 15 µL L-1 of EO for 4 or 24 h. The increased concentration of EO proportionally decreased the time required for the induction of anesthesia. EO treatment (15 µL L-1) inhibited the increase in blood glucose levels that was provoked by transportation for 4 or 24 h. Transportation for 24 h also decreased the number of lymphocytes and increased the neutrophil count, and these effects were avoided with the addition of EO to the water. These results demonstrate that EO was effective as an anesthetic at concentrations of 10-20 µL L-1 for slight sedation and transport and at 150 µL L-1 for deep anesthesia in the slender seahorse.

O objetivo deste estudo foi identificar os tempos da indução e recuperação anestésica em cavalos marinhos (Hippocampus reidi) expostos ao óleo essencial de Lippia alba (OE), assim como a eficácia do OE como um agente redutor de estresse no transporte desta espécie. Os cavalos marinhos foram colocados em aquários contendo 1 litro de água e diferentes concentrações de OE (0, 10, 20, 50, 150, 300 e 450 µL L-1), após a indução, os peixes foram transferidos à aquários livre de anestésico para avaliar o tempo de recuperação. Em um outro experimento os cavalos marinhos foram transportados em sacos plásticos contendo 15 µL L-1 do OE por 4 ou 24h. A concentração crescente do OE diminuiu proporcionalmente o tempo exigido para a indução da anestesia. O óleo essencial (15 µL L-1) inibiu o aumento nos níveis de glicose sanguínea provocada pelo transporte por 4 ou 24 h. O transporte por 24 h igualmente diminuiu o número de linfócitos e aumentou o número de neutrófilos, estas alterações foram evitadas com o uso do OE na água. Estes resultados demonstram que no cavalo marinho o OE é eficaz para a sedação e transporte na faixa de 10-20 µL L-1 e para a anestesia profunda recomenda-se 150 µL L-1.

Field studies of a Brazilian seahorse population, Hippocampus reidi Ginsburg, 1933

This study was carried out to fill the gaps that remain under Hippocampus reidi biology. Analysis of variations of sex ratio, density, breeding season, distribution and home range of a population of the endangered Brazilian seahorse H. reidi from a rocky shore on Araçatiba beach, Ilha Grande, Brazil were carried out. Araçatiba beach is a tourist Environmental Protected Area, suffering antropic pressure. A fixed population of H. reidi was studied, where al lthe individuals were visually tagged and sex, reproductive state and location on site were identified from December 2002 to November 2004. A total of 20 individuals were visually tagged with a mean density of 0.18 m-2. Sex ratios were skewed, with more females than males. All the males brooded during 13 months and presented smaller home range than the females during the breeding season. The highest densities were found on shallowest areas.

Este estudo foi realizado com o objetivo de preencher algumas lacunas sobre a biologia natural de Hippocampus reidi. Foram analisadas as variações na proporção sexual e densidade, período reprodutivo, distribuição e área de deslocamento de uma população do cavalo marinho brasileiro ameaçado de extinção Hippocampus reidi de um costão rochoso da praia de Araçatiba, Ilha Grande, Brasil. A praia de Araçatiba é uma Área de Proteção Ambiental turística, a qual sofre com a pressão antropogênica. Foi estudada uma população fixa de H. reidi, sendo que todos os indivíduos foram marcados visualmente e foram determinados o sexo, o período reprodutivo e a localização na área de novembro de 2002 a novembro de 2004. Um total de 20 indivíduos foram marcados com uma densidade média na área de 0,18 m-2. A proporção sexual variou de forma alternada com maior número de fêmeas que de machos. Durante 13 meses todos os machos encontrados estavam incubando. Os cavalos marinhos apresentaram área de deslocamento menor que as fêmeas durante o período reprodutivo. As maiores densidades foram encontradas em áreas mais rasas da área de estudo.

Morphological variation in the seahorse vertebral system / Variaciones morfológicas en el sistema vertebral del caballo de mar

The vertebral system in Hippocampus hippocampus is highly specialised because of the vertical locomotion and tail prehensility. The vertebral elements represent a special case of morphological changes, being the metameric structures organised along a natural functional series. We investigated the shape changes along the vertebral spine in H. hippocampus through geometric morphometrics, in order to describe functional and structural patterns. Actually, the dorso-ventral tail bending ability in the genus Hippocampus is one of the most impressive morphological modifications in the evolutionary history of fishes. Vertebrae were analysed using a 2D configuration from the left lateral view. The variation along the vertebral series suggests the identification of cervical, abdominal, dorsal, and caudal groups. The first three (cervical) elements and the lOth (supra-dorsal) structure show peculiar morphologies and local adaptations, associated with neck angulation and dorsal fin muscles, respectively. The vertebral size decreases from the anteriormost element backward, with some local variation at the dorsal area. Major changes are related to allometric variation at the neural región. The caudal elements are characterised by a marked size decrease, with consequent allometric shape changes involving the rotation of the posterior vertebral opening. This allometric trajectory leads to a natural ventral bending of the tail, promoting its prehensile function. This morphological survey suggests an interesting structural network between posture, locomotion, and vertebral anatomy, underlying the main functional changes in the Hippocampus biomechanics. Geometric morphometrics is rather suitable to approach metameric studies in terms of serial variation and functional adaptations.

El sistema vertebral en Hippocampus hippocampus es altamente especializado, debido a la locomoción vertical y la cola prehensible. Los elementos vertebrales representan un caso especial de cambios morfológicos, siendo estructuras metaméricas organizadas a lo largo de una serie funcional natural. Se investigaron los cambios en la forma, a lo largo de la columna vertebral en H. hippocampus a través de morfometría geométrica, con el fin de describir sus patrones funcionales y estructurales. Actualmente, la capacidad de flexión dorso-ventral de la cola en el género Hippocampus es una de las más impresionantes modificaciones morfológicas en la historia evolutiva de los peces. Las vértebras se analizaron mediante una configuración 2D de la vista lateral izquierda. La variación a lo largo de la serie vertebral sugiere la identificación de grupos cervicales, abdominales, dorsales, caudales. Los tres primeros elementos (cervicales) y la 10ª (supra-dorsal) muestran una estructura morfológica peculiar y adaptaciones locales, asociadas con la angulación del cuello y los músculos de la aleta dorsal, respectivamente. El tamaño vertebral disminuye desde elemento más anterior hacia atrás, con algunas variaciones locales en la zona dorsal. Los principales cambios están relacionados con la variación alométrica en la región neural. Los elementos caudales se caracterizan por una marcada disminución de tamaño, con los consiguientes cambios en la formas aloméricas de rotación de la abertura vertebral posterior. Esta trayectoria alométrica conduce a una flexión ventral natural de la cola, promoviendo su función prensil. Este estudio morfológico sugiere una interesante red estructural entre postura, locomoción y la anatomía vertebral junto a los principales cambios funcionales en la biomecánica de Hippocampus. La morfometría geométrica es bastante adecuada para los estudios con enfoques metaméricos, en términos de variación de serie y adaptaciones funcionales.

Population characteristics, space use and habitat associations of the seahorse Hippocampus reidi (Teleostei: Syngnathidae)

This paper provides a case study of a threatened seahorse species, Hippocampus reidi, highlighting the importance of using ecological information to assist conservation and management initiatives. Underwater visual sighting data (50 x 2m transect) gathered along the NE, SE and S portions of the Brazilian coast revealed an unequal distribution across localities, perhaps related to harvesting pressure, and a mean density of 0.026 ind.m-2. Our findings suggest some restricted spatial use by H. reidi, which was consistent with its estimated home range, and with re-sighting of specimens. Reproduction was recorded year-round, however productive peaks may exist. Components of habitat structure mostly used as anchoring points were mangrove plants, macroalgae, cnidarians, seagrass, sponges, and bryozoans. Conservation recommendations include: further characterization and mapping of habitats; assessment of availability and condition of microhabitats in selected areas, and studies on dispersal routes during initial life stages

Neste artigo apresenta-se um estudo de caso de uma espécie ameaçada de cavalo-marinho, Hippocampus reidi, enfatizando a importância de utilizar informações ecológicas nas ações de conservação e manejo. Dados de censo visual (transectos de 50 x 2m) obtidos nas porções NE, SE e S da costa brasileira revelaram uma distribuição heterogênea entre as localidades, talvez relacionada à pressão de coleta, e uma densidade média de 0.026 ind.m-2. Os resultados sugerem limitação no uso do espaço por H. reidi (consistente com a área vital estimada e com a re-avistagem de exemplares) e que a reprodução ocorre o ano inteiro, embora com possíveis picos. Os componentes da estrutura do habitat mais freqüentemente utilizados como pontos de ancoragem pelos cavalos-marinhos foram vegetação de mangue, macroalgas, cnidários, capim marinho e briozoários. Recomendações conservacionistas incluem caracterização adicional e mapeamento de habitats; avaliação da disponibilidade e condição dos microhabitats em áreas selecionadas, e estudos sobre rotas de dispersão durante as fases iniciais de vida

Fecundity, survival, and growth of the seahorse Hippocampus ingens (Pisces: Syngnathidae) under semi-controlled conditions

We studied fecundity, survival, and growth of the seahorse Hippocampus ingens under semi-controlled conditions. Three wild brood stock mature males of 14.8, 24.5, and 32.0 g released 1,598, 1,703, and 1,658 juveniles. Juvenile stocking densities of 12 were settled in 60-1 aquariums in groups of 1, 12, and 20 days old organisms. The rate of survival was 21.5, 61.9, and 59.0%, respectively, in 35 days. Juveniles were fed a mix diet of rotifers B. plicatilis and Artemia nauplii, then they were transferred to a cement tank of 100,000 1 at a density of 50/1,000 1 and fed with live adult Artemia for 60 days more. They grew from an average of 0.7, 1.5, and 2.7 to 4.5, 5.4, and 6.7 cm, respectively, in 95 days. The seawater temperature varied from 17 to 23 degrees C.

Estudiamos la fecundidad, supervivencia, y crecimiento del caballito de mar, Hippocampus ingens en condiciones semi-controladas. Tres machos reproductores silvestres de 14.8, 24.5 y 32.0 g produjeron 1 598, 1 703 y 1 658 jóvenes. La densidad utilizada fue de 12 jóvenes por acuario de 60 l. Se agruparon en 1, 12 y 20 días de nacidos por acuario. La supervivencia fue de 78.5, 38.1 y 41.0 % en 35 días. Se les alimentó con una mezcla de rotíferos B. plicatilis y nauplios de Artemia para después transferirlos a estanques de 100 000 l a una densidad de 50/1 000 l, donde se les alimentó con Artemia adulta durante 60 días más. Crecieron de un promedio de 0.7, 1.5, y 2.7 a 4.5, 5.4 y 6.7 cm, respectivamente, en 95 días. La temperatura del agua marina utilizada varió de 17 a 23 oC.

THE PREPARATION OF COMPOUND SEA-HORSE MEDICINAL LIQUOR AND ITS HEALTH PROTECTION EFFECT / 中国海洋药物

Based on a secret prescription(Dongyuan Jiajiu),Compound Sea-horse Medicinal Liquor (CSM