EGb761对LPS诱导THP-1细胞释放HMGB1蛋白表达的调节

目的:探讨EGb761对LPS诱导THP-1细胞释放HMGB1蛋白表达的调节,为EGb761的临床运用提供可行的依据。方法:LPS(1μg/m L)诱导不同时间后,western blotting检测THP-1细胞上清液中HMGB1蛋白含量变化及不同浓度EGb761对LPS诱导THP-1细胞释放HMGB1蛋白的表达和NF-κB的活性;酶联免疫吸附法(ELISA)检测细胞中IL-1β、IL-6、TNF-α的含量。共聚焦显微镜观察EGb761对LPS诱导THP-1细胞释放HMGB1蛋白核转位变化。结果:(1)LPS组IL-1β、IL-6、TNF-α的含量在刺激6-12 h后明显高于空白对照组,而EGb761+LPS组IL-1β、IL-6、TNF-α的含量均显著低于LPS组(P0.05)。(2)EGb761处理LPS诱导THP-1细胞6 h后细胞上清液NF-κB活性表达量较空白对照组低,随着处理时间延长至12 h,NF-κB的活性表达量呈明显下降趋势(P0.05)。(3)LPS诱导THP-1细胞18 h后,细胞上清液中HMGB1蛋白含量呈明显升高趋势(P0.05)。(4)不同浓度EGb761对LPS诱导THP-1细胞18 h后,HMGB1蛋白含量较空白对照组有下降趋势,HMGB1蛋白含量随着EGB761浓度增加至100μg/m L呈下降趋势并呈浓度依赖效应(P0.05)。(5)LPS诱导THP-1细胞后,在共聚焦显微镜下可见胞浆中大量HMGB1蛋白标记分布,而EGb761+LPS共同诱导THP-1细胞后胞浆中可见少量HMGB1蛋白分布。结论:LPS可诱导THP-1细胞IL-1β、IL-6、TNF-α表达增多及NF-κB活化,导致HMGB1蛋白表达增多及核转位,而EGB761能抑制THP-1细胞IL-1β、IL-6、TNF-α表达及NF-κB活化,调节HMGB1蛋白的表达及核转位。     

EGb 761 protects liver mitochondria against injury induced by in vitro anoxia/reoxygenation.

The present study investigated the protective effects of Ginkgo biloba extract (EGb 761) on rat liver mitochondrial damage induced by in vitro anoxia/reoxygenation. Anoxia/reoxygenation was known to impair respiratory activities and mitochondrial oxidative phosphorylation efficiency. ADP/O (2.57 +/- 0.11) decreased after anoxia/reoxygenation (1.75 +/- 0.09, p < .01), as well as state 3 and uncoupled respiration (-20%, p < .01), but state 4 respiration increased (p < .01). EGb 761 (50-200 microg/ml) had no effect on mitochondrial functions before anoxia, but had a specific dose-dependent protective effect after anoxia/reoxygenation. When mitochondria were incubated with 200 microg/ml EGb 761, they showed an increase in ADP/O (2.09 +/- 0.14, p < .05) and a decrease in state 4 respiration (-22%) after anoxia/reoxygenation. In EPR spin-trapping measurement, EGb 761 decreased the EPR signal of superoxide anion produced during reoxygenation. In conclusion, EGb 761 specially protects mitochondrial ATP synthesis against anoxia/reoxygenation injury by scavenging the superoxide anion generated by mitochondria.     

EGb761, a Ginkgo biloba extract, is effective against atherosclerosis in vitro, and in a rat model of type 2 diabetes

Background

EGb761, a standardized Ginkgo biloba extract, has antioxidant and antiplatelet aggregation and thus might protect against atherosclerosis. However, molecular and functional properties of EGb761 and its major subcomponents have not been well characterized. We investigated the effect of EGb761 and its major subcomponents (bilobalide, kaemferol, and quercetin) on preventing atherosclerosis in vitro, and in a rat model of type 2 diabetes.

Methods and Results

EGb761 (100 and 200 mg/kg) or normal saline (control) were administered to Otsuka Long-Evans Tokushima Fatty rats, an obese insulin-resistant rat model, for 6 weeks (from 3 weeks before to 3 weeks after carotid artery injury). Immunohistochemical staining was performed to investigate cell proliferation and apoptosis in the injured arteries. Cell migration, caspase-3 activity and DNA fragmentation, monocyte adhesion, and ICAM-1/VCAM-1 levels were explored in vitro. Treatment with EGb761 dose-dependently reduced intima-media ratio, proliferation of vascular smooth muscle cells (VSMCs) and induced greater apoptosis than the controls. Proliferation and migration of VSMCs in vitro were also decreased by the treatment of EGb761. Glucose homeostasis and circulating adiponectin levels were improved, and plasma hsCRP concentrations were decreased in the treatment groups. Caspase-3 activity and DNA fragmentation increased while monocyte adhesion and ICAM-1/VCAM-1 levels decreased significantly. Among subcomponents of EGb761, kaemferol and quercetin reduced VSMC migration and increased caspase activity.

Conclusions

EGb761 has a protective role in the development of atherosclerosis and is a potential therapeutic agent for preventing atherosclerosis.     

Different effects of the constituents of EGb761 on apoptosis in rat cerebellar granule cells induced by hydroxyl radicals

The present study was conducted to evaluate the different effects of the constituents of EGb761 (Ginkgo biloba Extract) on apoptosis in cerebellar granule cells induced by hydroxyl radicals. The total flavonoid component of EGb761, two pure EGb761 components (rutin and quercetin), and a mixture of flavonoids and terpenes protected cerebellar granule cells from oxidative damage and apoptosis induced by hydroxyl radicals. ESR(electron spin resonance) results showed that the IC50 of the flavonoids for scavenging hydroxyl radicals was almost the same as that of EGb761, even though flavonoids make up only 24% of EGb761, implying that other constituents of EGb761 besides flavonoids can scavenge hydroxyl radicals. Total terpenes of EGb761 did not protect against apoptosis. Flavonoids and terpenes did not show a synergistic effect in this regard. Terpenes did not scavenge hydroxyl radicals directly, which might be related to their "cage-like" structures.     

EGb761对血管性痴呆大鼠海马突触可塑性的影响

目的：探讨银杏叶提取物（EGb761）对血管性痴呆（VD）模型大鼠海马突触可塑性的影响。方法：Morris水迷宫检测大鼠空间学习记忆能力;电生理学方法在体记录大鼠海马长时程增强。结果：各时间点模型组大鼠的逃逸潜伏期（EL）均较假手术组明显延长（P〈0.01）,药物组各亚组大鼠的EL均显著短于模型组（P〈0.01）,但仍长于假手术组（P〈0.01,P〈0.05）。模型组各亚组大鼠长时程增强（LTP）诱导率显著低于假手术组和药物组（P〈0.01）。模型组大鼠各时间点群发峰电位（PS）的相对幅值明显低于假手术组和药物组（P〈0.01,P〈0.05）。假手术组、模型组和药物组各时间点的PS潜伏期无显著差别。结论：VD模型大鼠长时间存在空间学习记忆障碍,EGb761能促进VD模型大鼠海马病理性突触可塑性的恢复,这可能是其促智作用的重要机制。     

Protective effect of rat pancreatic progenitors cells expressing Pdx1 and nestin on islets survival and function in vitro and in vivo

To maintain islets survival and function is critical in successful pancreatic transplantation. Pancreatic progenitors cells (PPCs) with lineage potentials, giving rise to exocrine, endocrine, and duct cells, reside in developing and adult pancreas. As tissue-specific stem cells, they can produce pancreatic tissue-specific matrix factors to promote islets survival and function. The aim of our research was to investigate the protective effect of rat pancreatic?Cduodenal homeobox 1 (Pdx1)⁺/nestin⁺ PPCs on islets. In vitro, co-culturing islets with Pdx1⁺/nestin⁺ PPCs prolonged the former survival from 7 to 14?days. Furthermore, with high glucose (300.8?mg/dl) stimuli, the yield of insulin in co-cultures was significantly higher than that in control group (single islets group). In vivo, co-transplanting islets and Pdx1⁺/nestin⁺ PPCs for 3?days, the blood glucose of diabetic rat was significantly decreased to normal level and sustained for 2?weeks. Without Pdx1⁺/nestin⁺ PPCs in islets transplantation, hyperglycemia was reversed at day 7 and recovered at day 15. Pathology analysis showed that islets had remnants in co-transplantation at day 21, as complete graft rejection in alone islets transplantation. Our study showed that Pdx1⁺/nestin⁺ PPCs displayed the ability of preserving islets viability and function in vitro and prolonging their survival in vivo.     

Impact of Ginkgo Biloba Extract EGb 761 on ischemia/reperfusion - induced oxidative stress products formation in rat forebrain

Dysbalance in reactive oxygen/nitrogen species is involved in the pathogenesis of cerebral ischemia/reperfusion injury (IRI). Ginkgo biloba extract (Egb 761) pre-treatment was used to observe potential antioxidant/neuroprotective effect after global ischemia/reperfusion. Egb 761 significantly decreased the level of lipoperoxidation (LPO) in rat forebrain total membrane fraction (homogenate) induced by in vitro oxidative stress (Fe(2+)+H(2)O(2)). In animals subjected to four-vessel global ischemia for 15 min and 2-24 h reperfusion the EGb pretreatment slightly decreased LPO in forebrain homogenate. However, as detected in EGb treated group, the LPO-induced lysine conjugates are attenuated in comparison to non-treated IRI animals. EGb significantly improved parameters which indicate forebrain protein oxidative damage after IRI. The intensity of tryptophane fluorescence was increased by the 18.2% comparing to non-treated IRI group and bityrosine fluorescence was significantly decreased in ischemic (21%) and 24 h reperfused (15.9%) group in comparison non-treated IRI group. In addition, the level of total free SH- groups in pre-treated animals was significantly higher comparing to non-treated animals. Our results indicate that extract of EGb 761 has potent antioxidant activity and could play a role to attenuate the IRI-induced oxidative protein modification and lipoperoxidation in the neuroprotective process.     

Innate antiviral immunity of human PBLs and immunoregulatory activity of EGb 761

Background

The aim of the studies was to examine the potential immunoregulatory activity of Ginkgo biloba extract (EGb 761) on cytokine production, one of the mechanisms of innate antiviral immunity, by human peripheral blood leukocytes (PBLs) ex vivo.

Methodology

PBLs isolated from healthy blood donors were treated with different, nontoxic concentrations of EGb 761. Levels of different cytokines (TNF-α, IFN-α, IFN-Γ, IL-10 and IL-12), important in innate immunity development, were determined by ELISA.

Results

EGb 761, apart from strengthening of antiviral response, showed a differential impact on cytokine production by human PBLs ex vivo. It decreased the level of TNF-α and IFN-α but strongly increased the level of IFN-γ in PBLs stimulated by vesicular stomatitis virus (VSV) and non-stimulated PBLs. The extract reduced the production of IL-10 and IL-12 by human PBLs. The results were discussed and compared with previously published findings on the activity of the synthetic drug donepezil.

Conclusions

According to the results from the present study and our previous investigations, we report immunoregulatory activity of EGb 761 on different cytokine production by human PBLs ex vivo, which indicates the possibility of using the drug for the treatment of many immune deficiencies or infectious diseases through strengthening of innate immunity reactions.     

Selegiline potentiates the effects of EGb 761 in response to ischemic brain injury

We evaluated whether combined treatment with selegiline, a selective MAO-B inhibitor, and EGb 761, a standard extract of Ginkgo biloba, has synergistic effects against ischemic reperfusion injury (IRI) in gerbils. Interestingly, we observed that pretreatment with EGb 761 significantly attenuated selegiline-induced hyperactivity. This finding paralleled striatal fos-related antigen immunoreactivity (FRA-IR) in mice. Four minutes of bilateral carotid artery occlusion caused substantial cell loss in the CA1 of the hippocampus 5 days post-ischemic insult. Pretreatment with EGb 761, with or without selegiline, significantly attenuated this neuronal loss. Combined treatment with EGb 761 plus selegiline was more efficacious in preventing this loss. Synaptosomal formations of protein carbonyl, lipid peroxidation (malondialdehyde (MDA) + 4-hydroxyalkenal (4-HDA)), and reactive oxygen species (ROS) in the hippocampus remained elevated 5 days post-ischemic insult. The antioxidant effects appeared to be most significant in the group treated with EGb 761 plus selegiline. This combined treatment produced more significant attenuation of IRI-induced alterations in intramitochondrial calcium accumulation, the mitochondrial transmembrane potential, and mitochondrial Mn-superoxide dismutase-like immunoreactivity (Mn-SOD-IR) than either treatment alone. Our results suggest that co-administration of EGb 761 and selegiline produces significant neuroprotective effects via suppression of oxidative stress and mitochondrial dysfunction without affecting neurological function.     

Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements, taken for its multivalent properties. In this study, dosage effects of EGb761 on hydrogen peroxide (H₂O₂)-induced apoptosis of human neuroblastoma SH-SY5Y cells were investigated. It was found that H₂O₂-induced apoptotic cell death in SH-SY5Y cells, which was revealed in DNA fragmentation, mitochondrial membrane potential depolarization, and activation of Akt, c-Jun N-terminal kinases (JNK) and caspase 3. Low doses of EGb761 (50–100 μg/ml) inhibited H₂O₂-induced cell apoptosis via inactivation of Akt, JNK and caspase 3 while high doses of EGb761 (250–500 μg/ml) enhanced H₂O₂ toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments revealed that H₂O₂ decreased intracellular GSH content, which was also inhibited by low concentrations of EGb761 but enhanced after high concentrations of EGb761 treatment. This further suggests to us that dosage effects of EGb761 on apoptotic signaling proteins may be correlated with regulation of cell redox state. Therefore, treatment dosage may be one of the vital factors that determine the specific action of EGb761 on oxidative stress-induced cell apoptosis. To understand the mechanisms of dosage effects of EGb761 may have important clinical implications.     

Cardiovascular autonomic neuropathy in spontaneously diabetic rats with and without application of EGb 761

Cardiovascular autonomic neuropathy causes abnormalities in the diabetic heart with various clinical sequelae, including exercise intolerance, arrhythmias and painless myocardial infarction. Little is known about (ultra)structural alterations of the myocardial nervous network. On the assumption that this diabetes-specific neuropathy develops due to permanently increased oxidative stress by liberation of oxygen-free radicals, adjuvant application of antioxidative therapeutics appears promising in preventing or delaying long-term diabetic complications. We have investigated the effects of Ginkgo biloba extract (EGb 761), a radical scavenger, against diabetes-induced myocardial nervous damage in spontaneously diabetic BioBreeding/Ottawa Karlsburg (BB/OK) rats. Morphological and morphometric parameters were evaluated by electron microscopy. We used immunohistochemistry to investigate protein expression of protein gene product 9.5, S100 protein, and thyroxin hydroxylase as a neuronal marker. Alterations of cardiac sympathetic activity were measured using the in vivo 123I-metaiodobenzyl-guanidine imaging, and the immunofluorescent labeling of beta1-adrenergic receptors and adenylate cyclase. Our results revealed that A) Diabetes results in slight to moderate ultrastructural alterations (hydrops, disintegration of substructure) of autonomic nerve fibers and related Schwann cells in untreated BB diabetic rats; B) Cardiac sympathetic integrity and activity is impaired due to alterations in the presynaptic nerve terminals and the postsynaptic ?1-AR-AC coupling system; C) Pre-treatment of diabetic myocardium with EGb results in an improvement of most of these parameters compared to unprotected myocardium. In conclusion, EGb may act as a potent therapeutic adjuvant in diabetics with respect to cardiovascular autonomic neuropathy, which may contribute to the prevention of late complications in diabetes.     

Effects of Ginkgo biloba extract (EGb 761) on learning and possible actions on aging

A study of the effect of Ginkgo biloba extract (EGb 761) has shown enhancing effects on training in adult and aged Swiss mice. An analysis of inbred mice has confirmed this sensitivity to EGb 761, but depending on the strains, with different effects at different ages. The most interesting results are related to improvements in performances observed with aged mice of the DBA/2J strain. The results obtained with inbred strains in the study of the mossy fibers of the hippocampus make it possible to suggest a link between the improvements in training and the histological structure of the hippocampus. This possibility, which can be confirmed by further studies, is presented here.     

Specific memory effects of Ginkgo biloba extract EGb 761 in middle-aged healthy volunteers

Introduction

Recent reviews showed that Ginkgo biloba extract EGb 761¹ is effective to enhance performance in patients with cognitive impairment (e.g., dementia). The aim of this study was to investigate the effects of EGb 761 on memory and the specificity of such effects on distinct memory functions in middle-aged healthy volunteers.

Methods

A total of 188 healthy subjects aged 45-56 years were randomised to receive EGb 761 (240 mg once daily) or placebo for 6 weeks. Outcome measures were the change in memory performance in a demanding standardised free recall paradigm (list of appointments) and a less demanding standardised recognition test (driving-route). Based on previous findings we predicted superiority of EGb 761 in recall testing. Specificity in effects was assessed by separating immediate vs. delayed and quantitative vs. qualitative free recall measures.

Results

After 6 weeks, EGb 761-treated subjects improved significantly in quantity of recall, i.e., the number of correctly recalled appointments (drug-placebo differences: p = 0.038 for immediate and p = 0.008 for delayed recall). Effects on qualitative recall performance (ratio of false to correct items) were similar (drug-placebo differences: p = 0.092 for immediate and p = 0.010 for delayed recall). No superiority of Ginkgo was evident in another everyday memory test which asked for recognition of a driving route (drug-placebo differences: p > 0.10). The incidence of adverse events was low and not significantly different between treatment groups.

Discussion

EGb 761 (240 mg once daily) improves free recall of appointments in middle-aged healthy volunteers, which requires high demands on self-initiated retrieval of learned material. This function is known to be sensitive to normal aging, i.e., reduced in healthy middle-aged subjects. No effects are seen in a less demanding everyday memory task which does not tap this critical function. This ties in with previous studies which found specific patterns of benefit from EGb 761 in demanding cognitive tasks.     

Mapping of rat hippocampal neurons with NeuN after ischemia/reperfusion and Ginkgo biloba extract (EGb 761) pretreatment

1. The neuroprotective effect of Ginkgo biloba extract (EGb 761) against transient forebrain ischemia following 7 days of reperfusion was studied in male Wistar rats after four-vessel occlusion for 20 min.2. NeuN, a neuronal specific nuclear protein was used for immunohistochemical detection of surviving pyramidal neurons in the hippocampus, as well as counterstaining with hematoxylin in the same sections for detection of neurons that underwent delayed neuronal death and for glial nuclei staining. GFAP immunohistochemistry was used for detection of astrocytes in the studied area of CA1 region.3. In the group of rats pretreated 7 days with Ginkgo biloba extract (EGb 761), following 20 min of ischemia and 7 days of reperfusion without EGb 761, increased number of NeuN immunoreactive cells were counted in the most vulnerable CA1 pyramidal layer of hippocampus. On the other hand, the group of rats with 7 days of EGb 761 pretreatment following 20 min of ischemia and 7 days of reperfusion with EGb 761 showed decreased number of surviving NeuN immunoreactive CA1 pyramidal cells in comparison with the first above-mentioned experimental group.4. Increased number of reactive astrocytes immunolabeled for GFAP (Glial fibrilary acidic protein) was observed in both experimental groups in the stratum oriens and stratum lacunosum and moleculare.5. Twenty minutes of ischemia is lethal for most population of CA1 pyramidal cell layer. Our results showed that prophylactic oral administration of Ginkgo biloba extract (EGb 761) in the dose 40 mg/kg/day during the 7 days protects the most vulnerable CA1 pyramidal cells against 20 min of ischemia.     

In vivo and in vitro effect of naloxone on prolactin response to TRH in rat

In adult male Wistar rats submitted to a standardized noise stress, intravenous TRH induced a prolactin (PRL) secretory response. Prior IV naloxone administration not only lowered plasma PRL levels in those stressed rats but abolished also the stimulatory action of TRH. This effect was further studied by superfusion experiments on enriched PRL cell suspensions (70% lactotrophs) from female adult Wistar rats. Naloxone kept unaffected the basal PRL secretion but lowered significantly that induced by TRH. These experiments suggest a dual effect of naloxone on rat PRL secretion, one exerted on central opioid receptors lowering stress-related increased basal PRL levels, the other inhibiting the TRH-dependent PRL secretion exerted at the lactotroph level itself.     

银杏叶提取物(EGb761)导致人红细胞溶血作用的研究

本文用不同剂量的EGb761在37℃的环境下对健康人的红细胞(RBC)进行处理,发现EGb761对RBC有损伤作用,主要表现为导致溶血和诱导细胞形变,且其作用大小与浓度和时间呈正相关.这一实验结果对长期大剂量服用EGb761及其它银杏叶提取物的人群具有警示意义.EGb761对RBC损伤作用的机制仍不清楚,有待于进一步的研究.     

Biphasic action of retinoids on gonadotropin receptor induction in rat granulosa cells in vitro

Vitamin A (retinol) has been held to be uniquely essential for normal vision and reproduction, all other functions being served by its metabolite retinoic acid. The inability of retinoic acid to maintain adequate serum progesterone is implicated as the cause of fetal resorption. The availability of lipoproteins is a major limiting factor in progesterone production and the ovarian expression of lipoprotein receptors is dependent on the action of luteinizing hormone (LH). Therefore, we investigated the effects of retinol and retinoic acid on LH receptor induction by ovarian cells in an attempt to determine the basis for the reported differences in the gonadal action of these two retinoids. Our results indicate that retinoic acid (10(-10) M) and retinol (10(-8) M) each synergistically enhance the ability of follicle stimulating hormone (FSH) to induce LH-receptors and to stimulate the formation of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone. However, at higher concentrations, both retinoids inhibited these effects of FSH. For every measured effect, retinoic acid was more potent than retinol. Since retinol is metabolized to retinoic acid in other tissues, these results suggest that retinoic acid may be the mediator of the action of retinol on the ovary and that retinol's unique effect on reproduction needs to be investigated further.     

Induction of glutathione synthesis in human keratinocytes by Ginkgo biloba extract (EGb761).

The objective of the present study was to characterize the action of Ginkgo biloba extract (EGb761) and its sub-fractions on glutathione homeostasis in a human keratinocyte cell culture model. Cells were incubated with EGb761, its purified flavonoid (quercetin, kaempferol, rutin) or terpenoids (gingkolides A, B, C, J, bilobalide) constituents or the vehicle for up to 72 hours. Incubation of keratinocytes with the purified flavonoids or terpenoids did not affect cellular GSH levels. However, EGb761 treatment (up to 200 microg/ml) resulted in a dose-dependent increase of cellular GSH. Western blot analysis of extracts from cells treated with EGb761 revealed increased levels of the catalytic subunit of gamma-glutamylcysteinyl synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis. The abundance of mRNA for the catalytic subunit (assayed by RT-PCR) was also increased by the treatment with EGb761. Increased levels of cellular GSH by EGb761 were also observed in other cell lines including those from human bladder and liver as well as in murine macrophages indicating that the induction of gamma-GCS mRNA, protein and GSH may be an ubiquitous effect of EGb761 in mammalian cells.