Isocryptolepine, an indoloquinoline alkaloid from Cryptolepis sanguinolenta promotes LDL uptake in HepG2 cells

ABSTRACT About 31 percent of deaths worldwide result from atherosclerotic cardiovascular disease. Hyperlipidemia remains the major risk factor for this disease and therefore, it is necessary to identify antihyperlipidemic compounds for drug development. The crude ethanolic extract of Cryptolepis sanguinolenta (Lindl.) Schltr., Apocynaceae, has demonstrated antihyperlipidemic properties. However, the chemical constituents responsible for this action are unknown. Hence, to identify chemical constituent(s) of C. sanguinolenta with anti-hyperlipidemic effect, five indoloquinoline alkaloids were isolated and evaluated in 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate labeled low density lipoprotein uptake assay using HepG2 cells. The minor alkaloid, isocryptolepine, showed strong activity in promoting low lipid lipoprotein uptake by 1.85-fold. Isocryptolepine may, therefore, serve as a lead compound for future studies in the development of novel antihyperlipidemic drugs.

Role of vagus nerve on Diethylnitrosamine-induced hepatocellular carcinoma in rats / 中华肝胆外科杂志

Objective To study the role of vagusism on diethytlnitrosamine (DEN)-induced hepatocellular carcinoma in rats and to explore its mechanism.Methods The rat vagus nerve was labeled with 1,1'-dioctadecyl-3,3,33 -tetramethylindocarbocyanine perchlorate (DiI) tracer.The rats were divided into three groups:cancer induced rat models (cancer-induced group,n =20),cancer-induced and vagal detachment models (combined group,n =20) and normal rats (negative group,n =20),HE staining was performed on the liver tissue biopsied from the three groups of rats.The pathological grading score was evaluated and statistically analyzed.Immunohistochemistry (IHC) staing was used to determine the expression of vagal neurotransmitters and related receptors in liver tissue.Results The optimal concentration (1 mg/ml) and fixation duration with formalin (4 days) of DiI tracer marker vagal nerve demonstrated superior distribution and density of vagus nerve in the liver tissue.The incidence of hepatocellular carcinoma was significantly higher in the cancer-induced group (75％) than combined group (15％),the difference was statistically significant (P ＜ 0.05).IHC staining results of acetllcholine showed a significant difference between cancerinduced group (9.10) and combined group (2.30) or negative group (2.95),the difference was statistically significant (P ＜ 0.05).The M1 receptor expression was significantly higher in cancer-induced group (6.00) than combined group (2.30) and negative group (2.55),the differences were statistically significant (P ＜ 0.05).There was no significant difference of M2 receptor expression among the cancer-induced group (1.30),combined group (1.25) and negative group (1.35) (P ＞ 0.05).The M3 receptor expression detected with IHC staining shown there were statistical differences among cancer-induced group (8.95),combined group (6.30) and negative group (3.60) (all P ＜ 0.05).Conclusion The vagus nerve may play an important role in the development of DEN-induced hepatocellular carcinoma in rats.

The use of the software JST-XRD for identification of crystalline phases in pharmaceutical raw materials and tablets / Uso do software JST-XRD para identifcação de fases cristalinas em insumos farmacêuticos e medicamentos

Study of polymorphism is of great importance for the pharmaceutical industry once polymorphs may display different physicochemical properties, which, in turn, may result in stability differences that can bring problems for the manufacturing stages and the quality of fnal products. Although research on organic polymorphs has greatly increased in the last decades, it still does not cover all needs for the pharmaceutical market. Techniques such as spectroscopy in the infrared region, nuclear magnetic resonance, thermal analysis, X-ray diffraction, etc., can be used to identify polymorphism. The polymorphism is a property of the crystalline solid state, and can be evaluated by X-ray diffraction once each polymorph exhibits one specifc X-ray diffraction pattern. The JST-XRD program is a tool designed to help the identifcation of crystalline phases (including polymorphs) present in pharmaceutical ingredients and tablets by using X-ray diffraction data obtained from scientifc articles and patents. This paper presents new implementations for the JST-XRD and describes its use in the analysis of active pharmaceutical ingredient and marketed tablets of norfloxacin, mebendazole and atorvastatin calcium. By the means of comparison, JSTXRD allowed identifying the crystalline phases in the diffraction patterns of the analyzed drugs, showing the program suitability for polymorphism research, pre-formulation and quality control in pharmaceutical industries. JST-XRD can also be used for educational purposes in undergraduate and graduate programs in order to show the potentiality of X-ray powder diffraction in polymorphism analysis.(AU)

O estudo do polimorfsmo é de grande importância na indústria farmacêutica porque os polimorfos podem apresentar diferentes propriedades físico-químicas, podendo resultar em diferenças na estabilidade e desse modo causar problemas nas etapas de manufatura e no produto fnal. Embora a pesquisa de moléculas orgânicas que apresentam polimorfsmo tenha aumentado bastante nas últimas décadas, ainda não contempla todas as necessidades do mercado farmacêutico. Para a identifcação de polimorfsmo podem ser utilizadas técnicas como espectroscopia na região do infravermelho, ressonância nuclear magnética, análise térmica (DSC), difração de raios X, etc. O polimorfsmo, por ser uma propriedade do estado sólido e cristalino, pode ser avaliado através da difração de raios X, já que cada polimorfo apresenta um padrão de difração de raios X único. O programa JST-XRD é uma ferramenta projetada para auxiliar a identifcação de fases cristalinas, incluindo polimorfos, presentes em insumos farmacêuticos e comprimidos, usando dados de difração de raios X obtidos em artigos científcos e patentes. Esse trabalho apresenta novas implementações no JST-XRD e descreve seu uso na análise de amostras de princípio ativo e comprimidos comerciais de norfloxacino, mebendazol e atorvastatina cálcica. Através das comparações realizadas, JSTXRD permitiu identifcar todas as fases cristalinas dos difratogramas dos fármacos analisados, mostrando que o programa é adequado para pesquisa em polimorfsmo; na pré-formulação e controle de qualidade em indústrias farmacêuticas, assim como para uso didático em cursos de graduação e pós-graduação a fm de mostrar as potencialidades da difração de raios X na análise de polimorfsmo.(AU)

Effects of chronic alcohol exposure on hippocampus and cerebral cortex neurons in mice / 药学学报

This study was performed to investigate the changes of the number, morphology and ultrastructure of the central nervous system of mice during the long-term alcohol exposure. Mice at 60 days in age were used to establish the long-term alcohol exposure model. The structure of the central nervous system, such as nuclear antigen, dendritic spines and synapses, were labeled by the methods of immunocytochemistry and DiI (1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethy lindocarbocyanine perchlorate) scattering. The results showed that prolonged alcohol exposure could promote apoptosis of nerve cells in the central nervous system, and inhibit the proliferation of neural stem cells, which reduced the number of nerve cells in the central nervous system. Long-term ethanol exposure can also lead to a decrease in the density of dendritic spines of neuron, a smaller number of synapses (connections between nerve cells), and some changes in synaptic ultrastructure. The density of nerve cells and their dendritic spines, as well as the changes of synaptic ultrastructure, suggest that the function of nerve cells may be low.

Descripción de un brote epidémico de toxoplasmosis aguda en pacientes inmunocompetentes miembros de las fuerzas militares de Colombia durante operaciones de selva / Description of an epidemic outbreak of acute toxoplasmosis in immunocompetent patients from Colombian Armed Forces during jungle operations

La toxoplasmosis es una de las infecciones oportunistas más frecuentes en los pacientes infectados con el virus de inmunodeficiencia humana (VIH), mientras que, en los pacientes inmunocompetentes, la infección es sintomática sólo en 10% a 20% de los casos, generalmente con un comportamiento benigno y autolimitado. En la última década se han informado casos graves de compromiso visceral por toxoplasmosis aguda en pacientes inmunocompetentes. En este artículo se presenta un brote epidémico causado por Toxoplasma gondii en personal militar durante el desarrollo de operaciones de selva en el área general de La Macarena, Meta, Colombia. Los 18 casos reportados se confirmaron mediante inmunofluorescencia indirecta (IFI) de anticuerpos IgG anti-toxoplasma, al obtener títulos iguales o superiores a 1:1.024 (valor negativo inferior a 1:16). Los síntomas más importantes en estos pacientes fueron fiebre prolongada, adenopatías y compromiso pulmonar y gastrointestinal. Un paciente desarrolló compromiso miopericárdico grave. Todos los pacientes se recuperaron después de tratamiento con pirimetamina/sulfodaxina y clindamicina durante tres semanas. Una hipótesis para la presentación del brote epidémico es el consumo de agua contaminada con ooquistes y, probablemente, la seriedad del compromiso puede atribuirse a una cepa silvestre del parásito, tal como se ha descrito en otros casos reportados en la literatura, aunque en nuestro caso en particular, no se pudo realizar el aislamiento y tipificación de las cepas involucradas.

Toxoplasmosis is a common opportunistic infection in patients infected with HIV/AIDS while in immunocompetent patients this infection causes symptoms only in 10% to 20% of the cases, generally with a benign and autoresolutive course. In the last decade some severe cases with visceral involvement has been reported in immunocompetent patients, though they were isolated or recovered during years. We present the first Colombian report of an epidemic outbreak caused by Toxoplasma gondii in military personnel deployed to rural areas located at La Macarena, Meta, Colombia. All 18 cases were confirmed by IgG indirect immunofluorescence (IFI) with titers higher than 1:1,024 (normal range: 1:16). Their main symptoms were fever, adenopathies and pulmonary and gastrointestinal compromise. One patient developed severe myocardial compromise. All the patients recovered after treatment with pyrimetamine/sulfadoxine and clyndamicin for 3 weeks. A possible hypothesis for this outbreak was the consumption of contaminated water with oocysts, and probably the severity of the compromise could be elicited by a “wild” strain of the parasite as it is reported in the literature. Unfortunately, it was impossible to isolate and identify the specific strain.

Experimental study of tendon-bone healing using bone mesenchymal stem cells in a bone tunnel and their labeling and tracing / 中国矫形外科杂志

[Objective]To evaluate the effect of bone mesenchymal stem cells(BMSCs) on healing of tendon-bone junction in a bone tunnel and to provide experimental evidence of labeling and tracing the stem cell.[Method]BMSCs from rats were harvested by adherent separation screening method and labeled by SPIO and DiI.Thirty-nine 8-week-old rats were randomly divided into two groups,21 rats injected with double-labeled BMSCs and Pluronic F-127 as experimental group and 18 rats injected with Pluronic F-127 alone as control group.In each group,biomechanical analysis was carried out to assess the effect of healing at 2,4 and 8 weeks.The transplanted BMSCs were traced by fluorescence microscope at 2,4,8 weeks and 7.0T MR instantly,3 and 7days after operation.[Result]BMSCs were labeled effectively by SPIO and DiI.DiI-labeled positive cells were observed between tendon and bone with fluorescence microscope at 2,4 and 8 weeks.No significant signal changes of tendon-bone interface could be observed by 7.0 T MR.Biomechanically,maximum pullout load had no statistical difference between experimental group and control group at 2 weeks.At 4 and 8 weeks,the experimental group had significantly higher maximum pullout load compared with control group(P

INTRODUCTION OF DiI DIOLISTIC ASSAY TO LABEL THE NERVOUS CELL AND GLIA / 解剖学报

Objective The diolistic assay has been modified to make it simpler and more efficient in labeling neurons and glia. Methods CNS neurons and glial cells were labeled with DiI diolistic assay in fixed tissue and living brain slices of C57/B6J mice. Results The method allowed the visualization of the fine structure of neurons and glia including synaptic structures such as dendritic spines. Conclusion With the method, the labeling efficacy of cell's fine structure is improved, making it preferable for the analysis of dendritic spine. In addition, the ability to label the living neuron and glia will extend its application vastly.

The DiI Staining of Interneuron Dendrites in Kainate-Treated Hippocampal Slice Culture / 대한소아신경학회지

PURPOSE: Excitotoxic injury of the dendrites of inhibitory interneurons could lead to decrease in their synaptic activation, and explain subsequent local circuit hyperexcitability and epilepsy. A hallmark of dendrotoxicity at least in principal neurons of the hippocampus and cortex are focal or varicose swellings of dendritic abors. This research was designed to study morphologic changes of interneuron dendrite in kainate-treated hippocampal slice culture overtime. METHODS: Cultures aged 15-16 Equivalent Postnatal Days (EPD) were exposed to 10 microM kainic acid (KA), and analyzed at 0, 8, 24, 48, 72 hours after transient (1 hour) KA exposure. Neuronal injury was determined by morphologic changes of interneuron dendrites in area CA1 of DiI stained sections. RESULTS: 1) Transient (1 hour) exposure of hippocampal explant cultures to KA produced marked focal swellings of the dendrites of DiI stained interneurons in a highly reproducible manner. 2) The presence of focal swellings was reversible with kainate washout. The dendrites of KA treated explants were no longer beaded at 8, 24, 48, 72 hours after KA exposure. 3) There was no significant difference in the thickness of dendrites in DiI stained interneuron among 8hr, 24hr, 48hr and 72hr recovery group, compared with control group. CONCLUSION: The presence of focal swellings was reversible with kainate washout, and was not accompanied by interneuronal cell death.

Retrograde tracing study on neurons labeled by injecting rear-thigh muscles with DiI in mice / 第三军医大学学报

Objective To explore the fluorescent distribution, and the relation between fluorescent intensity and time after injecting rear-thigh muscles with DiI. Methods Sixty-five new born mice were equally and randomly divided into 13 groups. Fluorescent distribution and intensity were investigated at 2, 4, 6, 8, 12, 24 h, and 2, 4, 7, 14, 28 d and 2, 3 months after injecting the left posterior limb rear thigh muscles with 1.5 ?l 4 mg/ml DiI for one mouse. Results The labeling neurons were scattered from L2 level to S2 level and associated dorsal root ganglion (DRG), but the most were located at L4 to L6 section. The faint red fluorescence neurons were observed at dorsal root ganglion and cornu anterius medullae spinalis 6 h post-injection. The labeling neurons increased up to the 4th day. The fluorescent intensity enhanced gradually from 6 h to 24 h, then kept the intensity for 3 months. Conclusion It is a quick, precise, persistent method to trance and label the dorsal root ganglions sensory neuron and spinal motoneuron by injecting rear-thigh muscles with DiI, and the rate of labeling neurons can be improved by prolonging the tracing time properly. It is also provide basic data for clinical or experimental neuron label and location.

MORPHOLOGICAL STUDIES ON THE TECTAL CELLS PROJECTING TO NUCLEUS ROTUNDUS IN THE CHICK / 解剖学报

Objective To study the morphological characteristics of tectal cells in stratum griseum centrale(SGC) which project to nucleus rotundus(Rt) in chick. Methods Tectal cells projecting to the Rt were retrogradely labeled by using the injection or implantation of a small amount of carbocyanine fluorescent tracer(DiI) into Rt postmortemly in chicks.Results Labeled SGC cells were classified into four types according to the location of the soma and dendritic endings in the tectal layers.Type 1 cells of the SGC,whose somata were located in superficial part of the SGC,gave off dendritic endings to layer F.Type 2 cells in the SGC,whose somata were also located in superficial part of the SGC,gave off dendritic endings to layer D.Type 3 cells,whose somata were located in deep part of the SGC,gave off primary dendrites obliquely in layer H-J of SGFS.Type 4 SGC cells,whose somata were located in the deep part of the SGC,gave off dendrites horizontally and their dendrites were located within the SGC.The labeled dendrites of type I and 2 cells of the SGC formed bush-like or bifurcated endings extending horizontally in layer F and bottle brush endings vertically in layer D,respectively.The dendrites of type 3 and type 4 cells mainly formed free endings in their extending deep tectal layers.Conclusion The dendrites of superficial SGC cells(type 1 and type 2 cells) extend to retinorecipient tectal layers(layer F and layer D,respectively),having the shape of bush-like or bifurcated endings extending horizontally in layer F and bottle brush endings vertically in layer D,respectively corresponding to the shapes of terminals of optic nerves in these layers.The dendrites of deep SGC cells(type 3 and type 4 cells) do not extend to the retinorecipient tectal layers,mainly forming free endings in the tectal layers deeper than layer H.

EFFECT OF MONOCHROMATIC LIGHT ON CELL DENSITY AND CELL SIZE OF RETINAL GANGLION CELLS IN CHICKS / 解剖学报

Objective To study the effect of monochromatic light on the distribution of retinal ganglion cells in chicks. Methods Sixty One-day-old male broilers were reared under four light treatments,red(660nm),green(560nm),blue(480nm) and white(400-760nm) by using LED(light-emitting diodes) as light sources until the 49th day(n=15).Light intensity was 15 lux at the height of birds' heads and scheduled for 23hours of light and 1hour of darkness during the entire experiment.The retinas were stained by Nissl-staining and DiI-labeling.The retinal area,cell size and density in ganglion cell layer were estimated by image analysis. Results The retinal area and RGCs' number in the blue and green light groups were higher than that in the red and white light groups(10.35% and 17.07%,P