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Graft-versus-host disease (GVHD), an immunological disorder that arises from donor T cell activation through recognition of host alloantigens, is the major limitation in the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Traditional immunosuppressive agents can relieve GVHD, but they induce serious side effects. It is highly required to explore alternative therapeutic strategy. Human amniotic epithelial stem cells (hAESCs) were recently considered as an ideal source for cell therapy with special immune regulatory property. In this study, we evaluated the therapeutic role of hAESCs in the treatment of GVHD, based on our previous developed cGMP-grade hAESCs product. Humanized mouse model of acute GVHD (aGVHD) was established by injection of huPBMCs via the tail vein. For prevention or treatment of aGVHD, hAESCs were injected to the mice on day -1 or on day 7 post-PBMC infusion, respectively. We showed that hAESCs infusion significantly alleviated the disease phenotype, increased the survival rate of aGVHD mice, and ameliorated pathological injuries in aGVHD target organs. We demonstrated that hAESCs directly induced CD4+ T cell polarization, in which Th1 and Th17 subsets were downregulated, and Treg subset was elevated. Correspondingly, the levels of a series of pro-inflammatory cytokines were reduced while the levels of the anti-inflammatory cytokines were upregulated in the presence of hAESCs. We found that hAESCs regulated CD4+ subset polarization in a paracrine mode, in which TGFß and PGE2 were selectively secreted to mediate Treg elevation and Th1/Th17 inhibition, respectively. In addition, transplanted hAESCs preserved the graft-versus-leukemia (GVL) effect by inhibiting leukemia cell growth. More intriguingly, hAESCs infusion in HSCT patients displayed potential anti-GVHD effect with no safety concerns and confirmed the immunoregulatory mechanisms in the preclinical study. We conclude that hAESCs infusion is a promising therapeutic strategy for post-HSCT GVHD without compromising the GVL effect. The clinical trial was registered at www.clinicaltrials.gov as #NCT03764228.
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Objective To explore the clinical efficacy of vaginal hysterectomy and uterine scar repair in the treatment of type Ⅱ cesare-an scar pregnancy. Methods A total of 157 patients with type Ⅱ cesarean scar pregnancy (CSP) admitted to our obstetrics and gynecology department of our hospital from July 2016 to July 2017 were selected as research subjects. They were divided into ultrasound curettage group (n =87) and vaginal repair group(n =70) according to different surgical methods. The surgical bleeding volume, operative time, hospital stay and human chorionic gonadotropin (β-HCG) level before and after operation,the time of blood β-HCG returning to normal level,hemoglobin (HB) level before and after operation,menstrual recovery time,stress response, inflammatory response and surgical complications were compared between the two groups. Results The intraoperative blood loss volume, operative time and hospital slay in ultrasound curettage group were lower than those in vaginal repair group,the difference was significant(P <0.05). The time of blood β-HCG returning to normal level in vaginal repair group after operation was shorter than that in ultrasound curettage group, the difference was significant (P < 0. 05). There was no significant difference in the HB level between the two groups(P>0.05). The menstrual recovery time in vaginal repair group was shorter than that in ultrasound curettage group,the difference was significant(P <0. 05). The levels of postoperative epinephrine (E) and Cortisol (Cor) in vaginal repair group after operation were lower than those in ultrasound curettage group, while the level of thyroid stimulating hormone (TSH) was higher than that in ultrasound curettage group,the difference was significant(P <0.05). The levels of IL-2, IL-6 and IL-8 in vaginal repair group after operation were lower than those in ultrasound curettage group while the level of C-reactive protein (CRP) was higher than that in ultrasound curettage group,the difference was significant(P <0.05). The incidence rate of postoperative complications in ultrasound curettage group was higher than that in vaginal repair group(P <0. 05). Conclusion Vaginal repair is more beneficial to shortern the time of blood β-HCG returning to normal level and menstrual recovery time, promote the inflammatory factor and hormone recovery to a normal level, and reduce the incidence of complication.
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Δ3,2-Hydroxybakuchiol is isolated from Psoralea corylifolia (L.), which has therapeutic applications in traditional Chinese medicine. Our previous studies have showed that Δ3,2-hydroxybakuchiol inhibited the decreased activity of reserpinized mice, suggestive of its antidepressive potential. In this study, we explored the antidepressant profile of Δ3,2-hydroxybakuchiol in various rodent models and its possible monoamine-modulating mechanism. Δ3,2-Hydroxybakuchiol significantly reduced immobility time of mice in forced swim test and tail suspension test. Δ3,2-Hydroxybakuchiol also significantly increased sucrose consumption in chronic unpredictable mild stress (CUMS) rat model. Furthermore, isotope uptake study showed that Δ3,2-hydroxybakuchiol inhibited the activity of human dopamine transporter (DAT) and norepinephrine transporter (NET) in transporter-overexpressing pheochromocytoma (PC12) cells with IC50 values similar to the potency of bupropion. Microdialysis showed that Δ3,2-hydroxybakuchiol increased dopamine and norepinephrine concentration in rat striatum. In summary, Δ3,2-hydroxybakuchiol exerts antidepressant effects on various types of depression models through a possible mechanism of monoamine transporter inhibition.
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Human amniotic epithelial cells (hAECs), derived from the innermost layer of the term placenta closest to the fetus, have been shown to be potential seed cells for allogeneic cell therapy. Previous studies have shown a certain therapeutic effect of hAECs. However, no appropriate isolation and culture system for hAECs has been developed for clinical applications. In the present study, we established a serum-free protocol for hAEC isolation and cultivation, in which better cell growth was observed compared with that in a traditional culture system with serum. In addition to specific expression of cell surface markers (CD29, CD166 and CD90), characterization of the biological features of hAECs revealed expression of the pluripotent markers SSEA4, OCT4 and NANOG, which was greater than that in human mesenchymal stem cells, whereas very low levels of HLA-DR and HLA-DQ were detected, suggesting the weak immunogenicity of hAECs. Intriguingly, CD90+ hAECs were identified as a unique population with a powerful immunoregulatory capacity. In a systemic safety evaluation, intravenous administration of hAEC did not result in hemolytic, allergy, toxicity issues or, more importantly, tumorigenicity. Finally, the therapeutic effect of hAECs was demonstrated in mice with radiation-induced damage. The results revealed a novel function of hAECs in systemic injury recovery. Therefore, the current study provides an applicable and safe strategy for hAEC cell therapy administration in the clinical setting.
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Amnios/citología , Células Epiteliales , Trasplante de Células Madre , Animales , Pruebas de Carcinogenicidad , Células Cultivadas , Medio de Cultivo Libre de Suero , Citocinas/metabolismo , Células Epiteliales/fisiología , Células Epiteliales/trasplante , Femenino , Cobayas , Humanos , Masculino , Ratones Endogámicos ICR , Ratones Endogámicos NOD , Ratones SCID , Embarazo , Cultivo Primario de Células , Traumatismos Experimentales por Radiación/terapia , Ratas Sprague-Dawley , Antígenos Thy-1/metabolismoRESUMEN
OBJECTIVE: To establish the method for cotransferring human A20 gene and human heme oxygenase-1 (HO-1) gene into the isolated rat islets using lentiviral transfection system, and to study the protective effect of A20 and HO-1 protein against the apoptosis induced by cycloheximide (CHX) and TNF-α, and finally to explore the underlying mechanism. METHOD: The A20 gene and HO-1 gene were cloned and inserted into the lentiviral transfection system. The efficacy of gene transfer was measured by the intensity of the enhanced green fluorescent protein (EGFP) fluorescence-positive islets. Western blot was applied to verify the expression of the A20 and HO-1 genes. To induce apoptosis in vitro, the isolated islets were treated with CHX+TNF-α, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the fluorescence-activated cell sorting (FACS) methods were used to evaluate the apoptosis of the islet cells and Western blot was used to detect caspase-3 activation. RESULT: (1) A20 and HO-1 genes were introduced into the isolated islets by lentiviral transfection, both of the genes were highly expressed in the islets after 96 hours culture detected by Western blot method. (2) The insulin levels in the cell culture medium from A20 and/or HO-1 transgenic islets were significantly higher than that in non-transgenic controls (P < 0.01). (3)After CHX + TNF-alpha treatment, the cell culture medium insulin concentration in the A20 gene transfected group [(93.58 ± 4.12)µg/ml], HO-1 gene transfected group [(88.98 ± 4.77) µg/ml ] and A20/HO-1 co-transfected group [(103.33 ± 3.16) µg/ml] were significantly higher than that in the EGFP group [(9.03 ± 0.65) µg/ml ] and the control group [(8.86 ± 0.38) µg/ml] (P < 0.001). Minimum expression level of the activated caspase-3 was found in the A20/HO-1 co-transfected group. CONCLUSION: The lentiviral gene transfer system was an efficient and stable gene transfer vector, the over-expressed A20 and HO-1 protein delivered via lentivirus could preserve rats' islets function and act against the apoptosis induced by CHX and TNF-α.
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Apoptosis/efectos de los fármacos , Proteínas de Unión al ADN/genética , Hemo-Oxigenasa 1/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Islotes Pancreáticos/fisiología , Proteínas Nucleares/genética , Transfección/métodos , Animales , Caspasa 3/metabolismo , Línea Celular , Proteínas de Unión al ADN/metabolismo , Femenino , Citometría de Flujo , Vectores Genéticos , Hemo-Oxigenasa 1/metabolismo , Humanos , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Lentivirus/genética , Masculino , Proteínas Nucleares/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Increased attention has been paid to the role of oxidant/antioxidant imbalance in neurodegenerative process and pharmaceutical neuroprotective interventions. Food-derived compound luteolin possesses multitarget actions including reactive oxygen species (ROS)-scavenging activity in cultured human endothelial cells or permanent immature rat oligodendrocytes. This study aims to elucidate whether luteolin has a neuroprotective tendency toward ROS-insulted neural cells. The present results showed that luteolin, isolated from the ripe seed of Perilla frutescens (L.) Britt., markedly reversed hydrogen peroxide-induced cytotoxicity in primary culture cortical neurons but not in cultured human neuroblastoma cells. Upon the ROS-insulted primary neurons, luteolin concentration-dependently enhanced neuronal cell survival with efficacy higher than and potency similar to vitamin E. Additionally, luteolin significantly attenuated the increase in ROS production and prevented the decreases in activities of mitochondria, catalase, and glutathione in ROS-insulted primary neurons. Thus, luteolin functions by neuroprotection possibly through a rebalancing of pro-oxidant-antioxidant status. This agent points to possible interventions for preventing neurodegenerative diseases such as cerebral ischemia, Parkinson's disease, and Alzheimer's disease, as well as for improving brain aging.
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Supervivencia Celular/efectos de los fármacos , Depuradores de Radicales Libres , Luteolina/farmacología , Neuronas/metabolismo , Neuronas/patología , Perilla/química , Especies Reactivas de Oxígeno/metabolismo , Animales , Antioxidantes , Células Cultivadas , Evaluación Preclínica de Medicamentos , Humanos , Luteolina/aislamiento & purificación , Enfermedades Neurodegenerativas/prevención & control , Fármacos Neuroprotectores , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Human amniotic epithelial cells (HAECs), which have characteristics of both embryonic and pluripotent stem cells, are therefore a candidate in cell therapy without creating legal or ethical problems. In the present study, we aimed to investigate the effects of intracerebroventricular transplantation of HAECs on doubly transgenic mice of Alzheimer's disease (AD) coexpressing presenilin-1 (PS1) and mutant Sweden amyloid precursor protein (APPswe) genes. METHODS: The offspring mice genotypes were detected using PCR identification of APPswe and PS1 gene. The doubly transgenic (TG) mice (n = 20) and wild-type (WT) mice (n = 20) were randomly divided into two groups respectively: the transplantation group treated with HAECs and the control group with phosphate buffered saline. Six radial arm water maze test was used to assess the spatial memory in the TG and WT mice. Amyloid plaques and neurofibrillary tangles were analyzed using congo red and acid-silver methenamine staining respectively. Immunofluorescence cytochemistry was used to track the survival of HAECs. Immunohistochemistry was used to determine the expression of octamer-binding protein 4 (Oct-4) and Nanog in the HAECs. High performance liquid chromatography was used to measure acetylcholine in hippocampus. The density of cholinergic neurons in basal forebrain and nerve fibers in hippocampus was measured using acetylcholinesterase staining. RESULTS: Amyloid deposition occurred in hippocampus and frontal cortex in the double TG mice aged 8 months, but not in WT mice. The results also showed that transplanted HAECs can survive for at least 8 weeks and migrate to the third ventricle without immune rejection. The graft HAECs can also express the specific marker Oct-4 and Nanog of stem cell. Compared with the control group, transplantation of HAECs can not only significantly improve the spatial memory of the TG mice, but also increase acetylcholine concentration and the number of hippocampal cholinergic neurites. CONCLUSIONS: These results demonstrate that intracerebroventricular transplantation of HAECs can improve the spatial memory of the double TG mice. The higher content of acetylcholine in hippocampus released by more survived cholinergic neurites is one of the causes of this improvement.
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Amnios/citología , Células Epiteliales/trasplante , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/terapia , Acetilcolina/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Células Epiteliales/citología , Genotipo , Hipocampo/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Trastornos de la Memoria/genética , Ratones , Ratones Transgénicos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reacción en Cadena de la Polimerasa , Presenilina-1/genética , Presenilina-1/metabolismoRESUMEN
Oxidative stress-induced neuronal cell death has been implicated in neurodegenerative diseases; one such disease is ischemic stroke. Using reactive oxygen species (ROS)-insulted primary neurons, we screened neuroprotectants with clinical potential and then, using ischemia/reperfusion (I/R) model, investigated the anti-ischemic potential of candidate neuroprotectants. Here, we showed that luteolin, isolated from the ripe fruit of Perilla frutescens (L.) Britt, exhibited a neuroprotective action upon the in vitro platform, thus serving as candidate for in vivo pharmacological evaluation. Liposome-encapsulated luteolin produced dramatic preventing effects on I/R-induced behavioral and histological injuries after a 13-day post-ischemic treatment. Furthermore, this phytochemical not only lowered the increased level of mitochondrial ROS but also substantially up-regulated the decreased activity of catalase and glutathione in I/R rat brains. Collectively, luteolin as a neuroprotectant acts by anti-ischemic activity likely through a rebalancing of pro-oxidant/antioxidant status. Its multitarget mechanisms implicate potential effectiveness for clinically treating ischemia stroke.
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Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Luteolina/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Daño por Reperfusión/prevención & control , Animales , Animales Recién Nacidos , Antioxidantes/metabolismo , Catalasa/metabolismo , Muerte Celular , Femenino , Glutatión/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Liposomas , Luteolina/administración & dosificación , Mitocondrias/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/etiologíaRESUMEN
We previously reported that safflower (Carthamus tinctorius L.) ethyl acetate extract (HE) possessed an inhibitory action on serotonin (5HT) uptake in Chinese hamster ovary (CHO) cells expressing 5HT transporter (SERT) (S6 cells). Here, HE was adopted to go through an activity-guided isolation, and then an ingredient with potent SERT inhibitory action was obtained, which was elucidated as N(1),N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine (CX), a new coumaroylspermidine analog, by using spectroscopic methods including extensive 1D- and 2D-NMR analyses. Preliminary pharmacological study demonstrated that CX was a potent SERT inhibitor.
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Carthamus tinctorius/química , Ácidos Cumáricos/química , Inhibidores Selectivos de la Recaptación de Serotonina/química , Proteínas de Transporte de Serotonina en la Membrana Plasmática/química , Espermidina/análogos & derivados , Animales , Células CHO , Ácidos Cumáricos/aislamiento & purificación , Ácidos Cumáricos/farmacología , Cricetinae , Cricetulus , Espectroscopía de Resonancia Magnética , Conformación Molecular , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/aislamiento & purificación , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Espermidina/química , Espermidina/aislamiento & purificación , Espermidina/farmacologíaRESUMEN
Cynomorium songaricum Rupr. (SY) is a central nervous system-oriented herb material that has actions of anti-dementia, anti-epilepsy, and anti-stress. It is unclear whether SY would be biologically active in functionally regulating neurotransmitter transporters. Here, we assessed these potential actions using Chinese hamster ovary cells expressing gamma-aminobutyric acid (GABA) transporter (GAT-1), dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. G1, D8, N1, or S6 cells, respectively). It was shown that SY extracts, such as SYw, SYa, SYp, SYc, SYe, and SYb (SY water, ethanol, petroleum ether, chloroform, ethyl acetate, and n-butyl alcohol extract, respectively) increased dopamine/norepinephrine (DA/NE) uptake by corresponding D8/N1 cells and decreased gamma-aminobutyric acid/serotoin (GABA/5HT) uptake by corresponding G1/S6 cells; wherein, the potency or efficacy of SYc for up-regulating DA/NE uptake and that of SYb for inhibiting GABA/5HT uptake were relatively stronger. Additionally, GABA/5H-uptake inhibition by SY extracts were also seen in cortical synaptosomes, and DA/NE-uptake enhancement by SYc was dependent on the activity of DAT and NET. Thus, SY extracts especially SYc and SYb are novel neurotransmitter-transporter modulators functioning as DAT/NET activators and/or GAT-1/SERT inhibitors.
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Cynomorium/química , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Extractos Vegetales/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Serotonina/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Monoamine transporters playing major roles in regulating normal and abnormal synaptic activity are associated with various neuropsychological disorders. In spite of the discovery of a series of structurally different monoamine transporter antagonists for the therapy approach, no practical pharmaceutical can act as a transporter activator. Here, we isolated luteolin and apigenin from the fruit of Perilla frutescens (L.) Britt by using an activity-guided extraction technique, and proved that the two compounds possess actions of enhancing monoamine uptake either upon monoamine-transporter transgenic Chinese hamster ovary (CHO) cells or upon wild dopaminergic cell lines, with higher specificity for dopamine (DA) uptake than for norepinephrine (NE)- and serotonin (5HT)-uptake, as well as with more potency and greater efficacy for luteolin than for apigenin. Further, in the transgenic cells, the principal NE/DA uptake activation by luteolin was significantly prevented by respective transporter inhibitor, and the transmitter-uptake-enhancing action was independent of its ligands, which is in support of the compounds as monoamine transporter activators. Furthermore, luteolin evoked a marked disinhibition of cocaine-targeted effect in CHO cells overexpressing dopamine transporter. Thus, luteolin and apigenin function as monoamine transporter activators, which would improve several hypermonoaminergic neuropsychological disorders, especially cocaine dependence, through up-regulating monoamine transporter activity.
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Apigenina/farmacología , Luteolina/farmacología , Neuronas/efectos de los fármacos , Perilla frutescens/química , Extractos Vegetales/farmacología , Proteínas de Transporte Vesicular de Monoaminas/efectos de los fármacos , Animales , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Apigenina/aislamiento & purificación , Células CHO , Línea Celular , Cocaína/agonistas , Trastornos Relacionados con Cocaína/tratamiento farmacológico , Trastornos Relacionados con Cocaína/metabolismo , Trastornos Relacionados con Cocaína/fisiopatología , Cricetinae , Cricetulus , Dopamina/metabolismo , Inhibidores de Captación de Dopamina/agonistas , Luteolina/aislamiento & purificación , Ratones , Neuronas/metabolismo , Norepinefrina/metabolismo , Extractos Vegetales/aislamiento & purificación , Ratas , Serotonina/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Safflower, the dry flower of Carthamus tinctorius L., has long been applied for empirically treating cerebral ischemia and depression in traditional Chinese medicine. Pathogenesis of major depression involves monoaminergic transmission. The present study assessed whether safflower or its isolate would be effective in functionally regulating monoamine transporter using in vitro screening cell lines. We discovered that safflower insoluble fraction significantly inhibited serotonin uptake in Chinese hamster ovary cells stably expressing serotonin transporter (i.e. S6 cells). This fraction went through an activity-guided isolation and an active ingredient was obtained, which was subsequently elucidated as a novel coumaroylspermidine analog N(1),N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine using NMR techniques. Pharmacologically, this compound potently and selectively inhibited serotonin uptake in S6 cells or in synaptosomes, with IC(50) of 0.74+/-0.15 microM for S6 cells or 1.07+/-0.23 microM for synaptosomes and with a reversible competitive property for the 5HT-uptake inhibition. The potency of it for 5HT uptake was weaker than that of fluoxetine whereas efficacy generally similar for both. Animals treated with this testing compound showed a significant decrease in synaptosomal 5HT uptake capacity. Thus, N(1),N(5)-(Z)-N(10)-(E)-tri-p-coumaroylspermidine is a novel serotonin transporter inhibitor, which could improve neuropsychological disorders through regulating serotoninergic transmission.
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Carthamus tinctorius/química , Ácidos Cumáricos/aislamiento & purificación , Medicamentos Herbarios Chinos/aislamiento & purificación , Medicamentos Herbarios Chinos/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Espermidina/análogos & derivados , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Ácidos Cumáricos/farmacología , Cricetinae , Cricetulus , Fluoxetina/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismo , Espermidina/aislamiento & purificación , Espermidina/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Safflower (HH), the dry flower of Carthamus tinctorius L., has long been used to empirically treat neuropsychological disorders such as stroke and major depression in traditional Chinese medicine, and recently been proven effective for regulating levels of dopamine and serotonin in new-born rat brain. The present study assessed whether HH would be bioactive for functionally regulating monoamine transporters using in vitro drug-screening cell lines. Our current results showed that all solvent-extracted HH fractions, in different degrees, markedly increased both dopamine uptake by Chinese hamster ovary (CHO) cells stably expressing dopamine transporter (DAT) and norepinephrine uptake by CHO cells expressing norepinephrine transporter (NET), and also showed that chloroform (HC), ethyl acetate (HE), and n-butyl alcohol extract strikingly depressed serotonin uptake by CHO cells expressing serotonin transporter (SERT); wherein, the potencies of ethanol extract, HC, HE, and aqueous extract to up-regulate dopamine/norepinephrine uptake and potency of HE to inhibit serotonin uptake were relatively stronger. Further investigation revealed that the enhancement of dopamine/norepinephrine uptake by HC and HE was dependent of DAT/NET activity, and the HE-induced inhibition of serotonin uptake was typical of competition. Thus, HH extracts are novel monoamine transporter modulators functioning as DAT/NET activators and/or SERT inhibitors, and would likely improve neuropsychological disorders through regulating monoamine-transporter activity.
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Monoaminas Biogénicas/metabolismo , Transporte Biológico/efectos de los fármacos , Carthamus tinctorius , Extractos Vegetales/farmacología , Simportadores/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Femenino , FloresRESUMEN
Caulis Sinomenii (QFT) has analgesic, sedative, and anxiolytic-like actions, and is proven effective for improving drug dependence that is known to be associated with abnormal monoaminergic transmission. We assessed whether QFT would be biologically active in functionally regulating monoamine transporters using CHO cells expressing dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. D8, N1, or S6 cells, respectively). Here, we showed that its primary extracts, such as QA, QC, QE, QD, and QB (QFT ethanol, chloroform, ethyl acetate, alkaloid-free chloroform, and alkaloid-containing chloroform extract, respectively), and secondary extracts, such as QE-2, - 3, - 5, - 7, QD-1, - 2, - 3, - 4, - 5, and QB-1, - 2, - 3, - 4, - 5 (fractioned from QE, QD, and QB, respectively), in differing degrees, either increased DA/ NE uptake by corresponding D8/N1 cells or decreased 5HT uptake by S6 cells; wherein, QE-2, QD-3, and QE-7 were potent DA/NE uptake activators while both QE-7 and QB-5 were potent 5HT uptake inhibitors. Furthermore, the enhancement of DA/NE uptake was dependent of DAT/NET activity, and the inhibition of 5HT uptake was typical of competition. Thus, QFT extracts, especially QE-2 and QE-7 (both with stronger potencies), are novel monoamine transporter modulators functioning as DAT/ NET activators and/or SERT inhibitors, and would likely improve neuropsychological disorders through regulating monoamine transporters.
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Extractos Vegetales/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sinomenium/química , Animales , Monoaminas Biogénicas/metabolismo , Transporte Biológico/efectos de los fármacos , Células CHO , Fraccionamiento Químico , Cricetinae , Cricetulus , Humanos , Ratones , Extractos Vegetales/farmacocinética , RatasRESUMEN
Common flowering quince (FQ) is the fruit of Chaenomeles speciosa (Sweet) Nakai. FQ-containing cocktails have been applied to the treatment of neuralgia, migraine, and depression in traditional Chinese medicine. The present study assessed whether FQ is effective in dopamine transporter (DAT) regulation and antiparkinsonism by utilizing in vitro and in vivo assays, respectively. FQ at concentrations of 1-1000 microg/ml concentration-dependently inhibited dopamine uptake by Chinese hamster ovary (CHO) cells stably expressing DAT (D8 cells) and by synaptosomes. FQ had a slight inhibitory action on norepinephrine uptake by CHO cells expressing the norepinephrine transporter and no inhibitory effect on gamma-aminobutyric acid (GABA) uptake by CHO cells expressing GABA transporter-1 or serotonin uptake by the serotonin transporter. A viability assay showed that FQ mitigated 1-methyl-4-phenylpyridinium-induced toxicity in D8 cells. Furthermore, in behavioral studies, FQ alleviated rotational behavior in 6-hydroxydopamine-treated rats and improved deficits in endurance performance in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Furthermore, immunohistochemistry revealed that FQ markedly reduced the loss of tyrosine hydroxylase-positive neurons in the substantia nigra in MPTP-treated mice. In summary, FQ is a selective, potent DAT inhibitor and has antiparkinsonian-like effects that are mediated possibly by DAT suppression. FQ has the potential to be further developed for Parkinson's disease treatment.
Asunto(s)
Antiparkinsonianos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/antagonistas & inhibidores , Rosaceae/química , 1-Metil-4-fenilpiridinio/toxicidad , Animales , Células CHO , Línea Celular , Cricetinae , Cricetulus , Dopamina/metabolismo , Dopaminérgicos/toxicidad , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/genética , Inhibidores de Captación de Dopamina/farmacología , Tractos Extrapiramidales/efectos de los fármacos , Frutas/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neostriado/efectos de los fármacos , Neostriado/metabolismo , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/genética , Proteínas de Transporte de Noradrenalina a través de la Membrana Plasmática/metabolismo , Organismos Modificados Genéticamente , Extractos Vegetales/farmacología , Equilibrio Postural/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismoRESUMEN
Monoamine transporters play key roles in controlling monoamine levels and modulating monoamine reuptake. The objective of the present study was to identify monoamine transporter inhibitors from herbal sources. We discovered that bakuchiol analogs isolated from Fructus Psoraleae inhibited monoamine transporter uptake to differing degrees. The bakuchiol analog, Delta3,2-hydroxybakuchiol was the most potent and efficacious reuptake blocker and was thus selected as the candidate target. Monoamine transporter inhibition by Delta3,2-hydroxybakuchiol was more selective for the dopamine transporter (DAT) (IC50=0.58+/-0.1 microM) and norepinephrine transporter (NET) (IC50=0.69+/-0.12 microM) than for the serotonin transporter (SERT) (IC50=312.02+/-56.69 microM). Delta3,2-Hydroxybakuchiol exhibited greater potency (pEC50 for DAT and NET) than bupropion and exhibited similar efficacy (E(max) for DAT and/or NET) to bupropion and GBR12,935. Pharmacokinetically, Delta3,2-hydroxybakuchiol competitively inhibited DAT and NET with partial reversibility and occupied cocaine binding sites. Moreover, Delta3,2-hydroxybakuchiol counteracted 1-methyl-4-phenylpyridinium-induced toxicity in cells expressing DAT with similar efficacy to GBR12,935. In vivo studies showed that Delta3,2-hydroxybakuchiol increased the activity of intact mice and improved the decreased activity of reserpinized mice. In the conditioned place preference test, preference scores in intact mice were unaffected by Delta3,2-hydroxybakuchiol treatment. Bakuchiol analogs, especially Delta3,2-hydroxybakuchiol, are monoamine transporter inhibitors involved in regulating dopaminergic and noradrenergic neurotransmission and may have represented potential pharmacotherapies for disorders such as Parkinson's disease, depression, and cocaine addiction.
Asunto(s)
Monoaminas Biogénicas/metabolismo , Proteínas de Transporte de Neurotransmisores/antagonistas & inhibidores , Fenoles/farmacología , Psoralea/química , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Condicionamiento Clásico/efectos de los fármacos , Cricetinae , Cricetulus , Frutas/química , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Proteínas de Transporte de Neurotransmisores/genética , Fenoles/química , Fenoles/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Ratas , Ratas Sprague-Dawley , Sinaptosomas/efectos de los fármacos , Sinaptosomas/metabolismo , TransfecciónRESUMEN
OBJECTIVE: To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death. METHODS: a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated. RESULT: the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect). CONCLUSION: GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.
Asunto(s)
Células Epiteliales/efectos de los fármacos , Ganciclovir/farmacología , Cristalino/efectos de los fármacos , Lentivirus , Timidina Quinasa/farmacología , Línea Celular , Células Epiteliales/metabolismo , Genes Transgénicos Suicidas , Terapia Genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Cristalino/citología , Cristalino/metabolismo , Simplexvirus/genética , Timidina Quinasa/genética , TransfecciónRESUMEN
OBJECTIVE: To construct CMV-mediated Lentiviral vectors coexpressing EGFP and HSV-tk gene in order to establish a novel lentiviral vector platform for the suicide gene therapy of eye disease. METHODS: The restriction endonuclease and T4 DNA ligase were used to construct the vector plasmid. HSV-tk fragments from pcDNA3-HSV-tk were cloned into the site of lenti-internal ribosomal entry site (IRES)-EGFP to construct the bicistronic lenti-HSV-tk-EGFP vector. Human embryonic kidney 293T cells were co-transfected with the lentiviral vector (three plasmids) by calcium phosphate DNA precipitation. HLEC, HXO-Rb(44), SH-SY-5Y and Hela cells were transfected with viral production and the expression of EGFP was examined under fluorescent microscope after transfection. The expression of HSV-tk and EGFP was examined by RT-PCR. RESULTS: Lentivirus mediated stable integration and efficient expression of EGFP and TK genes in the cells tested. Coexpression of HSV-tk and EGFP in HLECs mediated by lentiviral vectors was confirmed by the result of RT-PCR. The transfection efficiency for HLECs was about 100% at MOI = 100, and kept the same level for at least 6 months. CONCLUSION: The bicistronic lentiviral vector platform carrying HSV-tk-EGFP is an efficient and stable gene transfer vector, it might be used for suicide genes therapy in the treatment some eye disease.
Asunto(s)
Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Lentivirus/genética , Timidina Quinasa/genética , Células Cultivadas , Células Epiteliales/citología , Genes Reporteros , Genes Transgénicos Suicidas , Humanos , Cristalino/citologíaRESUMEN
A petroleum ether extract (FP) from Fructus Psoraleae, seeds of Psoralea corylifolia L. (Leguminosae), was found to strongly inhibit dopamine (DA) uptake by dopamine transporter (DAT) heterogeneously expressed cells (D8 cells) and noradrenaline (NE) uptake by noradrenaline transporter (NET) heterogeneously expressed cells, which, however, had no effect on gamma-aminobutyric acid transporter heterogeneously expressed cells and serotonin transporter heterogeneously expressed cells at the concentration up to 100 microg/ml. These inhibitory effects were also confirmed by experiments on SK-N-SH cell line and synaptosomes from rats' brains. In addition, FP showed a significantly mitigating effect on 1-methyl-4-pyridinium induced injury of D8 cells. Meanwhile, FP dose-dependently reduced the binding of tritium-labeled cocaine analog (-)-2beta-carbomethoxy-3beta-(4-fluorophenyl) tropane to DAT of D8 cells, which suggests that FP may inhibit DAT activity in the same way as cocaine does. Behavioral study showed FP had a long-lasting stimulant effects on the activity of intact mice and reserpinized mice. So FP is proposed as a kind of DAT and NET inhibitor and may be involved in the process of regulating the DA and NE system, and FP or its unknown bioactive compounds may be developed into new medicines for disorders such as Parkinson's disease, depression, Attention Deficit Hyperactivity Disorder (ADHD) or cocaine addiction.