Preparation Of Plasma Exosomes As Drug Carrier And Study On Synergistic Effect Of Plasma Exosomes Loaded Ginkgolide B On Improving Cognitive Function In Dementia Model

Objective Dementia is an acquired syndrome in people with normal consciousness with comprehensive cognitive impairment.Dementia varies according to its pathogenesis,Alzheime’s disease(AD),vascular dementia(VD)and mixed dementia.Currently,mainly based on clinical diagnosis,pathological biopsy and autopsy are important ways of diagnosis.AD,a degenerative central nervous system disease,has progressed slowly.As patients grow older,they gradually develop cognitive impairment,language reduction,and memory loss.The insoluble neurofibrillary tangles(NFTs)are formed by hyperphosphorylated Tau self-folding,resulting in normal neuronal dysfunction.Therefore,inhibition of GSK-3β mediated Tau protein phosphorylation may help improve AD cognitive function.VD refers to cognitive dysfunction syndrome caused by cerebral hemorrhage and cerebral ischemia.Therefore,there is an urgent need for a better understanding of the pathogenesis of vascular dementia,explore effective neuroprotective agents to treat VD.Ginkgobalide B(GB)as the main active ingredient of Ginkgo biloba extract,by reducing neuroinflammation,neuropathies such as increased cerebral blood flow and increased learning and memory,is recognized as a natural neuroprotective agent.Exosomes(Exo)as a vesicle,40-200 nm,in diameter carrying genetic material and functional proteins associated with target cells,thereby regulating the expression of target genes or proteins and the function of receptor cells,with low immunogenicity and biocompatibility.This paper obtained plasma exoplasma(Pla-Exo)derived from plasma sources.Pla-Exo as a novel drug carrier,examined its delivery and loading capacity for GB,and analyzed the improved effect of Pla-Exo on learning and memory impairment in AD mice and VD rats.To investigate the dual therapeutic effect of Pla-Exo drug loading and its own function.Methods Pla-Exo was extracted by ultracentrifugation.The characteristic protein,particle size and surface morphology of Pla-Exo were characterized by Western Blot,nanometer particle size analyzer and atomic force microscope(AFM)respectively.This paper establishes two dementia models,AD model and VD model,respectively to explore the effect of Pla-Exo on learning and memory disorders.First,in the AD,the permeability of Pla-Exo to BBB was evaluated in vitro by using a blood-brain barrier(BBB)model of brain microvascular endothelial cells(h CMEC/D3),the fluorescence intensity of Di I labeled Pla-Exo in the brain,overall evaluation of Pla-Exo brain targeting ability.For the neuroprotective effects of Pla-Exo on damaged neuronal cells,cell viability was evaluated by the method of MTT reaction with tetramethyl azole salt,Okadaic acid(OA)was then injected into the lateral ventricle of C57/BL6 mice building AD models,daily intraperitoneal injection of 200 μL Pla-Exo(1.5 mg/m L)for 7days.Detection of apoptosis of hippocampal neurons by immunofluorescence staining;to evaluate the effect of Pla-Exo on the cognition of OA mice and to detect the learning ability of AD mice in each group by Morris water maze(MWM)behavior test;to investigate the effect of Pla-Exo on GSK-3β mediated tau phosphorylation,the mechanism of immunofluorescence staining and Western Blot in brain tissue was demonstrated.And then in the VD,to confirm whether GB has potential neuroprotective effects through TLR4/NF-κB pathways.MTT determination of cell viability in human neuroblastoma cells(SH-SY5Y)after oxygen-glucose deprivation(OGD)stimulation,TUNEL detection of apoptosis,through Western Blot experiments and to determine whether GB neuroprotective effects are associated with Toll-like receptor 4(TLR4)mediated regulation of inflammatory NF-κB signaling in OGD cells,and we used extra Lipopolysaccharides(LPS)treatment of OGD cells,and enhanced TLR4expression;VD models were prepared in vivo through permanent bilateral common carotid artery occlusion(BCCAO)in rats,intraperitoneal injection of GB(1 mg/m L)for 14 consecutive days,open field test was performed to determine motor activity in rats,Y-maze test and water maze were used to study the working and learning memory ability of BCCAO rats;the hippocampal tissue was evaluated by Nissl’s staining(Nissl)and Neu N immunofluorescence staining to assess BCCAO induced neuronal damage,Western Blot evaluate TLR4/NF-κB mediated neuroinflammatory responses in BCCAO rats.The drug loading efficiency of GB-Pla-Exo ginkgolide B was investigated by ultraviolet spectrophotometer(UV).The GB content in rat brain was quantitatively analyzed by HPLC method.For 14 consecutive days intraperitoneal injection(I.P.)of GB-Pla-Exo,open field experiment and water maze to explore whether the learning and memory ability of BCCAO rats can be further improved.Results Pla-Exo is successfully extracted.The particles are spherical,uniform distribution,the particle size is uniform.In the AD model,the results of the blood-brain barrier(BBB)model and in vivo imaging experiments show Pla-Exo with good brain targeting ability to freely cross BBB,and be transferred to the hippocampal site of mice.And in MTT,Pla-Exo reverse OA induced cell damage by increasing cell viability.On 12 h,24 h and 48 h,the survival rate of Pla-Exo treated OA cells was 94.3 %,72.3 % and 60.2 %,respectively.Fluorescence staining of living neuron cells(Neu N)showed that,compared to OA group,the Neu N fluorescence intensity of the hippocampus(CA1 、 CA3 and DG)was enhanced in the Pla-Exo group.Pla-Exo group improved the swimming behavior of AD mice in the water maze,increased the number of crossing the platform and the percentage of the platform quadrant and reduced the latency.Western Blot and Neu N immunofluorescence staining showed that,Pla-Exo enhanced GSK-3β(p-Ser9)expression,reduced tau protein(p-Ser396)activity,and then enhance the survival rate of neurons in AD mice,reduced cognitive impairment.And in the VD model,MTT results show that,cell survival rate was86.53 % in GB group.Meanwhile,during the TUNEL experiment,after GB,fewer TUNEL positive cells,effectively inhibit cell injury and death.Western blot shows,GB reduces TLR4 and activates NF-κB inflammatory signaling pathways in SH-SY5 Y cells.In open field experiments,GB improve the motor activity of BCCAO rats;escape latency and correct response times were reduced in the Yelectric maze,the escape latency of the search platform was reduced in the water maze test.The number of Nissl bodies in GB group increased.Immunofluorescence showed that the fluorescence intensity of Neu N increased.Western Blot showed that GB decreased the expression of TNF-α and NF-κB related proteins and reduced neuroinflammation in BCCAO rats.Through UV detection Pla-Exo for GB encapsulation efficiency of about 38.93 %,after HPLC analysis,Pla-Exo increased the cumulative content of GB in the rat brain.By open field experiment and water maze results,the motor activity and memory ability of GB-Pla-Exo group were improved.Conclusions The Pla-Exo prepared in this paper is a kind of nanoscale vesicles with good stability and uniform particle size,which can freely cross the BBB into the hippocampus,inhibit GSK-3β mediated hyperphosphorylation of Tau proteins,and improve the survival rate of neurons.The cognitive dysfunction of OA induced AD mice was alleviated.Pla-Exo,as a new drug carrier delivery system,successfully loaded GB and delivered to the brain,increased the accumulation of GB in the brain,further played a GB role in reducing TLR4/NF-κB mediated neuroinflammation,and significantly improved the learning and memory ability of VD rats.