Selective ischemic-hemisphere targeting Ginkgolide B liposomes with improved solubility and therapeutic efficacy for cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion injury(CI/RI)remains the main cause of disability and death in stroke patients due to lack of effective therapeutic strategies.One of the main issues related to CI/RI treatment is the presence of the blood-brain barrier(BBB),which affects the intracerebral delivery of drugs.Ginkgolide B(GB),a major bioactive component in commercially available products of Ginkgo biloba,has been shown significance in CI/RI treatment by regulating inflammatory pathways,oxidative damage,and metabolic disturbance,and seems to be a candidate for stroke recovery.However,limited by its poor hydrophilicity and lipophilicity,the development of GB preparations with good solubility,stability,and the ability to cross the BBB remains a challenge.Herein,we propose a combinatorial strategy by conjugating GB with highly lipophilic docosahexaenoic acid(DHA)to obtain a covalent complex GB-DHA,which can not only enhance the pharmacological effect of GB,but can also be encapsulated in liposomes stably.The amount of finally constructed Lipo@GB-DHA targeting to ischemic hemisphere was validated 2.2 times that of free solution in middle cerebral artery occlusion(MCAO)rats.Compared to the marketed ginkgolide injection,Lipo@GB-DHA significantly reduced infarct volume with better neurobehavioral recovery in MCAO rats after being intravenously administered both at 2 h and 6 h post-reperfusion.Low levels of reactive oxygen species(ROS)and high neuron survival in vitro was maintained via Lipo@GB-DHA treatment,while microglia in the ischemic brain were polarized from the pro-inflammatory M1 phenotype to the tissue-repairing M2 phenotype,which modulate neuroinflammatory and angiogenesis.In addition,Lipo@GB-DHA inhibited neuronal apoptosis via regulating the apoptotic pathway and maintained homeostasis by activating the autophagy pathway.Thus,transforming GB into a lipophilic complex and loading it into liposomes provides a promising nanomedicine strategy with excellent CI/RI therapeutic efficacy and industrialization prospects.

Effects of ginkgolide B on neuronal discharges in paraventricular nucleus of rat hypothalamic slices

Objective To study the central role of ginkgolide B （BN52021） in regulating cardiovascular function of nerve center by examining the effects of ginkgolide B on the electrical activity of rat paraventricular nucleus （PVN） neurons in hypothalamic slice preparation and to elucidate the mechanism involved. Methods Extracellular single-unit discharge recording technique. Results （1） In response to the application of ginkgolide t3 （0.1, 1, 10 μmol/L; n = 27） into the perfusate for 2 rain, the spontaneous discharge rates （SDR） of 26 （26/27, 96.30%） neurons were significantly decreased in a dose-dependent manner. （2） Pretreatment with L-glutamate （L-Glu, 0.2 mmol/L） led to a marked increase in the SDR of all 8 （100%） neurons in an epileptiform pattern. The increased discharges were suppressed significantly after ginkgolide B （1 μmol/L） was applied into the perfusate for 2 min. （3） In 8 neurons, perfusion of the selective L-type calcium channel agonist, Bay K 8644 （0.1 μmol/L）, induced a significant increase in the discharge rates of 8 （8/8, 100%） neurons, while ginkgolide B （1μmol/L） applied into the perfusate, could inhibit the discharges of 8 （100%） neurons. （4） In 8 neurons, the broad potassium channels blocker, tetraethylammonium （TEA, 1 mmol/L） completely blocked the inhibitory effect of ginkgolide B （1 μmol/L）. Conclusion These results suggest that ginkgolide B can inhibit the electrical activity of paraventricular neurons. The inhibitory effect may be related to the blockade of L-type voltage-activated calcium channel and potentially concerned with delayed rectifier potassium channel （KDR）.

Mechanism and dose-effect of Ginkgolide B on severe acute pancreatitis of rats

AIM:To determine the optimal dosage and mechanism of Ginkgolide B(BN52021) on severe acute pancreatitis(SAP) of rats.METHODS:Seventy male Wistar rats were randomly divided into seven groups(10 for each group).Shamoperation group(SO),SAP model group(SAP),dimethyl sulfoxide(DMSO) contrast group(DMSO),and groups treated with 2.5 mg/kg BN52021(BN1),5 mg/kg BN52021(BN2),10 mg/kg BN52021(BN3),and 20 μg/kg Sandostatin(SS).The SAP model was established in Wistar rats by injecting 5% sodium taurocholate retrogradely into the common bilio-pancreatic duct.The rats of SO,DMSO and BN52021 were injected with 0.9% NaCl,0.5% DMSO and BN52021 through femoral vein 15 min after the operation.The SS group was injected with Sandostatin subcutaneously.All rats were anaesthetized at 6 h after operation,and venous blood was collected to determine the levels of serum amylase and phospholipase A2(PLA2),and pancreas tissue was harvested and stained.RESULTS:There was no significant difference between the SAP and DMSO groups in serum amylase level,PLA2,ascites and pathologic score,but significant difference was found in SAP/DMSO groups compared with those in SO group(P < 0.05) and the levels of serum amylase,PLA2,ascites,and pathologic score were lower in the BN1,BN2,BN3 and SS groups than in the SAP and DMSO groups(P < 0.05).However,among BN1,BN2,BN3 and SS groups,BN2 had the best effect in decreasing the levels of serum amylase and PLA2(P < 0.05).Expression of platelet activating factor(PAF) receptor(PAFR) mRNA and protein showed no significant difference between the SAP and DMSO groups,or among BN1,BN2,BN3 and SS groups,but there was remarkable difference between SAP/DMSO group and SO group(P < 0.05),and expression of PAFR mRNA and protein was higher in the BN1,BN2,BN3 and SS groups than in the SAP and DMSO groups(P < 0.05).PAFR expression was observed in the nucleus and cytoplasm of pancreatic islet cells in Wistar rats by immunohistochemistry.CONCLUSION:By iv injection,5 mg/kg of BN52021 is the optimal dosage for SAP rats.BN52021 may inhibit the interaction/binding of PAF with PAFR.

Anti-atherosclerotic effects and molecular targets of ginkgolide B from Ginkgo biloba

Bioactive compounds derived from herbal medicinal plants modulate various therapeutic targets and signaling pathways associated with cardiovascular diseases(CVDs),the world’s primary cause of death.Ginkgo biloba,a well-known traditional Chinese medicine with notable cardiovascular actions,has been used as a cardio-and cerebrovascular therapeutic drug and nutraceutical in Asian countries for centuries.Preclinical studies have shown that ginkgolide B,a bioactive component in Ginkgo biloba,can ameliorate atherosclerosis in cultured vascular cells and disease models.Of clinical relevance,several clinical trials are ongoing or being completed to examine the efficacy and safety of ginkgolide B-related drug preparations in the prevention of cerebrovascular diseases,such as ischemia stroke.Here,we present a comprehensive review of the pharmacological activities,pharmacokinetic characteristics,and mechanisms of action of ginkgolide B in atherosclerosis prevention and therapy.We highlight new molecular targets of ginkgolide B,including nicotinamide adenine dinucleotide phosphate oxidases(NADPH oxidase),lectin-like oxidized LDL receptor-1(LOX-1),sirtuin 1(SIRT1),platelet-activating factor(PAF),proprotein convertase subtilisin/kexin type 9(PCSK9)and others.Finally,we provide an overview and discussion of the therapeutic potential of ginkgolide B and highlight the future perspective of developing ginkgolide B as an effective therapeutic agent for treating atherosclerosis.

Pharmacological action and mechanisms of ginkgolide B

Objective To review the recent research progress in pharmacological actions and mechanisms of ginkgolide B. Data sources Information included in this article was identified by searching of PUBMED （1987-2006） online resources using the key terms ＂ginkgolide B＂, ＂platelet activating factor＂, and ＂pharmacological＂. Study selection Mainly original milestone articles and critical reviews written by major pioneer investigators of the field were selected. Results The key issues related to the pharmacological actions and mechanisms of ginkgolide B were summarized. The ginkgolide B possesses a number of beneficial effects such as anti-inflammatory, anti-allergic, antioxidant, and neuroprotective effects. Meantime, their mechaniams were discussed. Conclusions The Ginkgolide B is the most potent antagonist of platelet activating factor （PAF） and exhibits therapeutic action in a variety of diseases mainly by the PAF receptor.

Isolation and Preparative Purification for Ginkgolides A and B

In this paper a simple preparative method for isolation and purification of ginkgolides A and B was developed,As starting material,a commercially available standardized ginkgo extract (EGb761,containing 24% flavonoid and 6% terpene trilactones) was used,After a pretreatment step,optimized by the uniform design method ,the concentrated intermediate extract with high content of GA and gb(+90%) was separated into the individual terpenes by preparative liquid chromatography eluted with petroleum ether-ethylacetate,Analysis of products was carried out by means of HPLC-ELSD(evaporative light -scattering detector),The results show that ginkgolides A and B are obtained in higher yield and better purity.

Lipopolysaccharide-induced cerebral inflammatory damage and the therapeutic effect of platelet activating factor receptor antoganist

Objective To investigate lipopolysaccharide （LPS） induced acute cerebral inflammatory damage and the therapeutic effect of ginkgolide B （BN52021）. Methods Thirty Sprague-Dawley rats were randomly divided into 3 groups （n = 10 for each group）： Control group, Model group and Treatment group （treated with BN52021）. LPS were injected into the fourth ventricle of rat to make a neuroinflammatory murine model. Morris water maze was used to detect the learning and memory ability of rats; changes of synapse number and subcellular ultrastructures were observed under a transmission electron microscope; OX-42 positive microglia in the brain was detected by immunohistochemical method. Results The average escape latency in the Treatment group were significantly shortened than that in the Model group; and the percentage of swimming distance traveled in platform quadrant accounting for total distance increased markedly. The rough endoplasmic reticulum and polyribosomes in the Treatment group were more than that in the Model group, but the number of synapses seemed to have no obvious change. The number of OX-42 positive microglia in the Treatment group decreased markedly than that in the Model group, and the grey density of OX-42-positive cells increased significantly. Conclusion LPS can induce inflammatory damages to the brain, but the damage could be antagonized by BN52021. Platelet activating factor receptor antagonist may offer an effective therapy for neurodegeneration diseases.

鲍曼不动杆菌通过活化血小板激活因子受体引起感染人支气管上皮细胞的氧化应激和细胞凋亡

目的探讨血小板激活因子受体(PAFR)在鲍曼不动杆菌(A.baumannii)感染人支气管上皮(HBE)细胞过程中的作用。方法 HBE细胞分为对照组、银杏内酯B(GB)处理组、A.baumannii感染组、A.baumannii感染联合GB处理组。使用临床分离的A.baumannii感染HBE细胞,GB处理组分别用1×10~3CFU/m L、1×10~5CFU/m L和1×10~7CFU/m L的A.baumannii感染HBE细胞,A.baumannii感染联合GB处理组则先用10μmol/L GB阻断PAFR活性而后进行1×10~3CFU/m L A.baumannii感染。采用Western blot法检测HBE细胞PAFR、磷酸化的Janus激酶1(p-JAK1)、磷酸化的信号转导子与转录激活子1(p-STAT1)的蛋白水平,使用CCK-8法检测细胞增殖能力,使用超氧化物歧化酶(SOD)和丙二醛(MDA)试剂盒检测各组细胞氧化应激水平;异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)染色结合流式细胞术检测细胞凋亡。结果与对照组相比,A.baumannii感染的HBE细胞中PAFR蛋白水平显著升高,细胞活力显著下降,细胞内MDA水平和细胞凋亡明显增加,p-JAK1和p-STAT1蛋白水平增加。结论 A.baumannii感染的HBE细胞PAFR活化,激活JAK1/STAT1信号通路促进HBE细胞的氧化应激和细胞凋亡。

Neuroprotective effects of Ginkgo biloba extract and GinkgoUde B against oxygen-glucose deprivation/reoxygenation and glucose injury in a new in vitro multicellular network model

Acute ischemic stroke （AIS）, as the third leading cause of death worldwide, is characterized by its high incidence, mortality rate, high incurred disability rate, and frequent reoccurrence. The neuroprotective effects of Ginkgo biloba extract （GBE） against several cerebral diseases have been reported in previous studies, but the underlying mechanisms of action are still unclear. Using a novel in vitro rat cortical capillary endothelial cell- astrocyte-neuron network model, we investigated the neuroprotective effects of GBE and one of its important constituents, Ginkgolide B （GB）, against oxygenglucose deprivation/reoxygenation and glucose （OGD/R） injury. In this model, rat cortical capillary endothelial cells, astrocytes, and neurons were cocultured so that they could be synchronously observed in the same system. Pretreatment with GBE or GB increased the neuron cell viability, ameliorated cell injury, and inhibited the cell apoptotic rate through Bax and Bcl-2 expression regulation after OGD/R injury. Furthermore, GBE or GB pretreatment enhanced the transendothelial electrical resistance of capillary endothelial monolayers, reduced the endothelial permeability coefficients for sodium fluorescein （Na-F）, and increased the expression levels of tight junction proteins, namely, ZO-1 and occludin, in endothelial cells. Results demonstrated the preventive effects of GBE on neuronal cell death and enhancement of the function of brain capillary endothelial monolayers after OGD/R injury in vitro; thus, GBE could be used as an effective neuroprotective agent for AIS/reperfusion, with GB as one of its significant constituents.