Study On The Effect Of Coumarin On Improving Learning And Memory And Its Pharmacokinetics

Alzheimer’s disease(AD),a common neurodegenerative disease in the elderly,is the main cause of dementia and its incidence rate is increasing rapidly.The China Brain Project has listed AD as one of the three major neurological and psychiatric diseases of great concern.The risk factors for the onset of AD are diversified and the mechanism is complicated,which is still unclear and poses great difficulties for its treatment.At present,the clinical treatments of AD are symptomatic and do not decelerate or prevent the pathological progression,and have many adverse effects.Therefore,it is urgent to explore new therapeutic drugs for AD.The discovery of new drugs from natural products has great potential.Current studies have shown that a variety of stilbenes have potential anti-AD effects.Cajaninstilbene acid(CSA),a main bioactive stilbene from pigeon pea[Cajanus cajan(L.)Millsp.]leaves,possesses many bioactivities including neuroprotective effects.Therefore,it is hypothesized that CSA could be a potential therapeutic agent for AD.In new drug research and development,pharmacokinetic studies can evaluate the kinetic characteristics of drug candidates early,improve the rate of success,reduce costs and risks,and obtain the most effective therapeutic agents.At present,there is no research report about the enhancing effect of CSA on learning and memory at home and abroad,and there are only two literature about its pharmacokinetics.Therefore,this study intends to use different animal models to evaluate the pharmacological effects of CSA on learning and memory,then explores the underlying mechanism from multiple levels,and finally investigates its pharmacokinetics and the mechanisms of absorption and transport at the animal and cell levels.The completed research contents mainly have the following three aspects:1.Study on the pharmacological effects of CSA on learning and memory impairmentFirstly,a model of learning and memory impairment in mice induced by Aβ oligomers was established.Then,the established model of intracerebral injection of Aβ oligomers and chronic sleep deprivation model were used to evaluate the pharmacological effects of CSA on learning and memory impairment by using the open field,object location recognition,Morris water maze and passive avoidance tests.The results indicate that bilateral injection of Aβ1-42 oligomers(100 pmol/mouse)into the hippocampal CA1 regions could successfully establish the model of learning and memory impairment.In the intracerebral injection of Aβ oligomers model and chronic sleep deprivation model,CSA administration(7.5,15 and 30 mg/kg)by gavage did not affect the locomotor activity of model mice,but improved the short-term discrimination for the objects’ locations,rescued the impairment of learning and memory ability in water maze tests,and enhanced the passive avoidance ability in the training and consolidation trials of step-through tests.2.Study on the mechanism of CSA on learning and memory impairmentFirstly,a liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of tryptophan(Trp)and its metabolites(kynurenine,kynurenic acid,3-hydroxykynurenine,5-hydroxytryptamine and 5-hydroxyindoleacetic acid),glutamic acid(Glu)and γ-aminobutyric acid(GABA)in mice serum and the hippocampus.Then,the mechanism of CSA on learning and memory impairment induced by Aβ oligomers was explored from multiple levels,mainly including monitoring the status of neurons,astrocytes and microglia and the content of Aβ in hippocampus by Nissl staining and immunofluorescence staining;using established analytical methods to analyze the levels of Trp and its metabolites,Glu and GABA;determining the expression levels of GluNl and GluN2B receptors and downstream PKA/CREB/BDNF/TrkB signaling pathways by Western blot.The results indicate that the established analytical method had good specificity,linearity,sensitivity and stability,and could be used to compare the different contents of analytes in different experimental groups.The learning and memory enhancing effects of CSA could be mediated by clearing Aβ oligomers,preventing astrocyte reactivity and microglial activation,maintaining normal levels of Trp metabolism,reversing Glu and GABA dyshomeostasis,inhibiting excessive expression of GluN2B and upregulating the PKA/CREB/BDNF/TrkB signaling pathway in the hippocampus.3.Study on the pharmacokinetics of CSAFirstly,the main metabolites of CSA in rats were identified and the analytical method in biological samples was established.Then,the established analytical method was applied to study the pharmacokinetics and excretion pathways of CSA and its main metabolites in rats.Finally,the intestinal absorption and transport mechanisms of CSA were studied using the Caco-2 cell monolayer model.The results indicate that there were mainly five kinds of CSA metabolites(M1-M5)in plasma,bile and urine,namely CSA-3-O-glucuronide,CSA-2-COO-glucuronide,6,12-dihydroxy CSA,3-hydroxy-5-methoxystilbene-3-O-glucuronide and 6-hydroxy CSA-3-O-glucuronide,suggesting that glucuronidation and oxidation were the main metabolic pathways.The established analytical method had good specificity,linearity,sensitivity,precision,accuracy and stability,and could be used for in vivo pharmacokinetic study of CSA.CSA could be rapidly absorbed after oral administration,and the absolute oral bioavailability was 44.36%.CSA was mainly metabolized to CSA-3-O-glucuronide via the first-pass effect to limit its oral bioavailability,and the enterohepatic circulation,extravascular distribution,and renal reabsorption characteristics of CSA delayed its elimination and prolonged the action time.CSA and its metabolites M1-M5 were excreted predominantly through the biliary route with 78%of the CSA dose.Only about 0.6%of the CSA dose was excreted from bile and urine in its parent form,while the total excretion rate of M1-M5 was 81%,indicating that CSA was widely metabolized in the body to produce a large number of metabolites.CSA was transported through Caco-2 cell monolayer via passive diffusion and was well absorbed.In summary,CSA could rescue learning and memory impairment in different animal models.Its enhancing effects on learning and memory were mediated by clearing Aβoligomers,preventing astrocytic and microglial activation,maintaining normal levels of Trp metabolism,reversing Glu and GABA dyshomeostasis,inhibiting excessive expression of GluN2B and upregulating the PKA/CREB/BDNF/TrkB signaling pathway in the hippocampus.CSA could be rapidly absorbed by passive diffusion and slowly eliminated with pharmacokinetic characteristics including first-pass effect,enterohepatic circulation,extravascular distribution,and renal reabsorption.The main metabolic pathways of CSA were glucuronidation and oxidation,and CSA and its metabolites were excreted predominantly through the biliary route.This study enables us to understand the cognitive enhancing effects of CSA and its pharmacokinetics,provides the possibility for CSA to be a potential therapeutic agent for AD,and offers a reference for the development and utilization of stilbenes.