Vesicular nanocarrier based treatment of skin fungal infections: Potential and emerging trends in nanoscale pharmacotherapy

Occurrence of skin fungal infections is increasing nowadays and their presence is more prominent in patients suffering from immunocompromised diseases like AIDS. Skin fungal infections are a major cause of visits by patients to dermatology clinics. Although, a large number of antifungal agents are available for treatment of skin fungal infections, but, their toxic profile and physicochemical characteristics reduce therapeutic outcome. When these antifungal agents are delivered topically using conventional formulations like creams and gels, they may cause various side effects like redness, burning, and swelling at the site of application. Therefore, various vesicular formulations(phospholipid based or non phospholipid based) have been explored by pharmaceutical scientists to treat skin fungal infections topically. Vesicular formulation explored for the purpose are liposomes, ethosomes, transfersomes, transethosomes, niosomes, spanlastics, oleic acid vesicles, and nanoparticles.These formulations show various advantages like bioavailability enhancement of bioactives,high skin permeation power, no side effects at application site, dosing frequency reduction,and sustained drug release. Therefore, in the present article, we have discussed about the utility of various vesicular nanocarrier systems to treat skin fungal infections.

Novel Vesicular Aqueous Two-Phase Systems

The interesting phenopmena of two aqueous phases coexisting in dilute aqueous solutions of sodium undecenoate-dodecyltrimethylammo brodride ndxture and sodium laurate-dodecyltrimethylammonium brondde mixture were investigated. Vesicles existing in both phases were shown by TEM images. The vesicles are dispersed in lower phase and flocculated in upper phase. Multilamellar structure of vesicles was found in the upper phase of sodium laurate-dodecyltrimethylammonium bromide system.

Vesicular glutamate transporter-immunoreactivities in the vestibular nuclear complex of rat

Objective Aims to delineate the distribution profile of three isoforms of vesicular glutamate transporter （VGluT）, viz. VGluT1-3, and their cellular localization within vestibular nuclear complex （VNC）. Methods Brain sections from normal Sprague-Dawley rats were processed immunohistochemically for VGluT detection, employing avidinbiotinylated peroxidase complex method with 3-3＇-diaminobenzidine （DAB） as chromogen. Results The whole VNC expressed all of the three transporters that were observed to be localized to the fiber endings. Compared with VGluT1 and VGluT3, VGluT2 demonstrated a relatively homogeneous distribution, with much higher density in VNC. VGluT3 displayed the highest density in lateral vestibular nucleus and group X, contrasting with the sparse immunostained puncta within vestibular medial and inferior nuclei. Conclusion Glutamtatergic pathways participate in the processing of vestibular signals within VNC mainly through the re-uptake of glutamate into synaptic vesicles by VGluT1 and 2, whereas VGluT3 may play a similar role mainly in areas other than medial and inferior nuclei of VNC.

Regulatory role of sorting nexin 5 in protein stability and vesicular targeting of vesicular acetylcholine transporter to synaptic vesicle-like vesicles in PC12 cells

Accurate targeting of vesicular acetylcholine transporter(VAChT)to synaptic vesicles(SVs)is indispensable for efficient cholinergic transmission.Previous studies have suggested that the dileucine motif within the C-terminus of the transporter is sufficient for its targeting to SVs.However,the cytosolic machinery underlying specific regulation of VAChT trafficking and targeting to SVs is still unclear.Here we used the C-terminus of VAChT as a bait in a yeast two-hybrid screen to identify sorting nexin 5(SNX5)as its novel interacting protein.SNX5 was detected in the SVs enriched LP2 subcellular fraction of rat brain homogenate and showed strong colocalization with VAChT in both brain sections and PC12 cells.Binding assays suggested that the C-terminal domain of VAChT can interact with both BAR and PX domain of SNX5.Depletion of SNX5 enhanced the degradation of VAChT and the process was mediated through the lysosomal pathway.More importantly,we found that,in PC12 cells,the depletion of SNX5 expression significantly decreased the synaptic vesicle-like vesicles(SVLVs)localization of VAChT.Therefore,the results suggest that SNX5 is a novel regulator for both stability and SV targeting of VAChT.

Mitochondrial Membranes Restitution Proceeds via Vesicular Import from ER and Cytosol. Counterparts’ Resemblances and Variances in Mitochondria and Golgi Pathways

The processes of mitochondrial restitution are controlled by nuclear genes that encode proteins synthesized in ER and cytosol and delivered as organelle- and membrane-specific transport vesicles. The analysis of the transporters recovered from inner mitochondrial space (Mitosol) revealed that the ER-synthesized mitochondria-specific transport vesicles consist of two carriers, one remaining in outer mitochondrial membrane (OMM), and the other that transfers specific membrane segments to the inner mitochondrial membrane (IMM). The ER-assembled and IMM-committed membrane segments, while first integrated into OMM, undergo intra-mitochondrial lipid modification reflected in the synthesis of cardiolipin (CL) and inversion into Mitosol with load of IMM associated cytosolic proteins. Then, the CL-bedecked vesicles are released from OMM to Mitosol and upon contact with IMM fuse with the membrane, and the release of cytosolic cargo ensues. While ER-assembled mitochondria-specific transport vesicles fuse with OMM with the aid of the cytosolic, phosphatidylglycerol (PG)-specific phospholipase A2 (PLA2), the Mitosol-contained CL-specific PLA guides vesicles fusion with IMM. The described path of translocation of the membrane segments and the cytosol synthesized proteins into the designated mitochondrial compartments sustains growth and identity of OMM, IMM, maintains protein delivery for intra-mitochondrial lipid and protein modification in Mitosol, and ensures conformity of the cytosolic proteins cargo delivered to matrix.

Endocytosis and Vesicular Transport of Plasmid DNA in Cells During Electric Field-Mediated Gene Delivery

Pulsed electric field has been used widely as a nonviral approach to improving gene delivery in basic and translational research[1-2].The technique has been called electrotransfection(ET),electroporation,electrogene transfer,and gene electroinjection in the literature [1,3].It has a great potential to improve clinical treatment of diseases through delivery of vaccines and therapeutic genes,genome and epigenome editing,and generation of human induced pluripotent stem cells for tissue engineering[1-3].During ET,extracellular transport of plasmid DNA(pDNA)relies on electrophoresis,which is critical for applications in vivo.However,mechanisms of intracellular transport remain to be understood.The lack of understanding has hindered the translation of ET technology to the clinic.It is well known that pulsed electric field can generate transient hydrophilic pores in the plasma membrane(i.e.,electroporation)that permit membrane-impermeant molecules to enter cells.Although the pores have yet to be visualized directly under a microscope,the electric field-induced membrane permeabilization has been demonstrated through experimental measurements of electrical conductance of synthetic lipid membranes and plasma membranes,direct observation of fluorescent markers crossing the membranes facing both cathode and anode,and numerical simulations of the membrane permeabilization[1,3].Results from the simulations have predicted that the cutoff size of the pores is on the order of a few hundred nanometers,and the lifetime of the pores that are larger than 100 nm is on the order of 10 msec.Although these data provide a solid evidence of the membrane permeabilization,recent studies have demonstrated that the generation of the pores is insufficient for ET[1,4].The reasons are as follows.First,the lifetime of the pores is several orders of magnitude shorter than the time scale for pDNA uptake,which is on the order of 10 min.Second,complex formation between pDNA and plasma membrane is a necessary condition for successful gene transfer.Third,inhibition of clathrin mediated endocytosis or Rac-1 dependent micropinocytosis can reduce the amount of pDNA internalized by cells [1].Finally,we demonstrate that few pDNA molecules can be observed in the cytosol that are not associated with the intracellular vesicles[5],suggesting that pDNA uptake is mediated by endocytosis.In addition to the internalization,ET requires the pDNA in the cytoplasm to reach the nucleus.To understand mechanisms of intracellular trafficking of pDNA,we have examined time-dependent pDNA distributions in cells,quantitatively determined percentages of pDNA molecules associated with different endocytic compartments using transmission electron microscopy(TEM),and investigated different approaches to facilitate cytoplasmic transport and nuclear entry of pDNA.Our data have shown that electrotransfected pDNA is located in different vesicular ultrastructures at or near the plasma membrane at10 min post application of electric pulses[5].In the hard-to-transfect cells(e.g.,4T1),pDNA penetration from the cell surface is less active,and the total number of vesicular structures associated with pDNA is low,compared to those in the easyto-transfect cells(e.g.,COS7).Our data have also shown that macropinocytosis is the most common pathway shared by all types of cells.To investigate how improve pDNA transport in cells,we have photochemically treated cells to non-specifically induce pDNA escape from intracellular vesicles,or blocked endosome and autophagic vacuole maturation through treatment of cells with Bafilomycin Al,an inhibitor of vacuolar H+ATPase.Our data demonstrate that both treatments can lead to reduction of ET efficiency although the treatment for inducing endosomal escape can enhance poly-L-lysine mediated gene delivery.These data suggest that the vesicles play an important role in protecting the naked pDNA during intracellular trafficking.The nuclear envelope is another major barrier to ET.To facilitate the nuclear entry,we have examined three different approaches.One is to synchronize the nuclear envelope breakdown(NEBD)prior to ET;the second approach is to pre-treat cells with a nuclear pore dilating agent(i.e.,trans-1,2-cyclohexanediol);and the third one is to incorporate a nuclear targeting sequence(NTS)(i.e.,SV40)into the pDNA.Our data have shown that the synchronization of the NEBD can significantly improve the ET efficiency without compromising the cell viability.The nuclear pore dilation can improve the ET as well but the dilating agent is cytotoxic.The incorporation of NTS into pDNA can improve the gene delivery efficiency but the improvement is cell-type dependent,suggesting that the NTS has to be screened and optimized for the cells of interest.In summary,the transient pores in the plasma membrane induced by the electric pulses will enable cellular uptake of membrane-impermeant molecules up to the size of small proteins.Larger molecules(e.g.,pDNA)have to be internalized via endocytic processes triggered by the pulsed electric field.Within the cells,pDNA transport is mediated by vesicles and can be blocked by non-specific escape from vesicles or inhibition of vesicle maturation.The nuclear entry of pDNA can be enhanced,without compromising cell viability,through the use of the NTS or the synchronization of the NEBD.

Eukaryotic Expression and Activity Analysis of Capsid Gene of Swine Vesicular Disease Virus HK/70

The capsid protein precursor （P1）, which plays a major role for the generation of polypeptides of swine vesicular disease virus （SVDV）, was cloned from SVDV HK/70 strain into the retroviral vector pBABE puro and expressed in the mammalian cell line PK15 through the retroviral expression system. The activity of recombinant protein to induce immune response was evaluated in guinea pigs. IFA and Western Blot were used to detect the recombinant protein expression. The results showed that the recombinant protein could be recognized by SVDV positive serum, and animal test showed SVDV-specific antibodies. All of those results indicate that a retroviral-based vaccine carrying the capsid protein precursor （P1） of SVD is able to be expressed in the eukaryotic cell and elicites strong SVDV-specific immune responses in guinea pigs.

Diagnosis and Control of Swine Vesicular Stomatitis

Swine vesicular stomatitis is an acute, febrile, highly contagious zoonotic diseases caused by vesicular stomatitis virus （VSV）. Disease in affected pigs was typically characterized by vesicular lesions on the mouth, snout, oral mucosa, coronet and interdigital skin. The disease is endemic in the Americas, Europe and Africa and other places. As the foreign trade of animals and animal products increase, the risk of swine vesicular stomatitis spreading into our country also will be increased. Therefore, the accurate and timely diagnosis and control of swine vesicular stomatitis are critical and necessary.

Development and Preliminary Application of Double Antibody Sandwich ELISA for Detection of Swine Vesicular Disease Virus

[Objective] The aim of this study was to develop an indirect sandwich EUSA method for detection of swine vesicular disease virus （SVDV）. [Method] High titer serum against SVDV was prepared respectively by inoculated rabbits and guinea pigs with purified virus. To develop an indirect sandwich ELISA, the optimum concentrations of capture antibody, detection antibody, enzyme conjugate and standard antigen were determined using block titration, and positive threshold value was also determined. The specificity, sensitivity and repeatability of the developed IELISA were evaluated using cross-reaction test, comparison test and intra-assay repeated test. In addition, standard samples and clinical samples were detected by this method. [ Result] The best working conditions of the developed ELISA are as follows： capture antibody, 1：400; detection antibody, 1 ： 200; enzyme conjugate, 1 ： 8 000; and standard antigen, 1 ： 4. The positive threshold value was found to be 0.20. For the detection by the developed EUSA, no cross-reaction with foot and mouth disease was observed. The developed ELISA had close sensitivity with ELISA recommended by the World Organisation for Animal Health （OIE） but had sensitivity 2 -4 times higher than that of reverse indirect hernagglutination test. In addition, the developed method also had good reproducibility, and the detection results of standard samples were in line withtheir own background. All the 36 clinical samples were negative in the developed ELISA. [ Conclusion] The developed indirect sandwich ELISA can be used for diagnosis of swine vesicular disease.

Molecular physiology of vesicular glutamate transporters in the digestive system

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Packaging and storage of glutamate into glutamatergic neuronal vesicles require ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. Three vesicular glutamate transporters (VGLUT1-3) have been recently identified from neuronal tissue where they play a key role to maintain the vesicular glutamate level. Recently, it has been demonstrated that glutamate signaling is also functional in peripheral neuronal and non-neuronal tissues, and occurs in sites of pituitary, adrenal, pineal glands, bone, GI tract, pancreas,skin, and testis. The glutamate receptors and VGLUTs in digestivesystem have been found in both neuronal and endocrinal cells. The glutamate signaling in the digestive system may have significant relevance to diabetes and GI tract motility disorders. This review will focus on the most recent update of molecular physiology of digestive VGLUTs.

Expression and Immunological Analysis of Capsid Protein Precursor of Swine Vesicular Disease Virus HK/70

VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

A Study on Antitoxic Role of Vesicular Monoamine Transporter 2 in Transgenic Chinese Hamster Overy Cells

Objective:To study the antitoxic role of vesicular monoamine transporter 2 (VMAT2) in transpgenic Chinese Hamster ovary(CHO) cell.Methods:With the technology of transgene from PC12 to CHO,MTT reduction assay was used to detect MPP^+ toxic effect on wild type CHO(wtCHO) and transgenic CHO.Meanwhile,the role of reserpine was also observed in MPP^+ toxic effects.Results:The sensitivity of transgenic CHO to MPP^+ was much less than that of wtCHO with 0.5 mmol/L MPP^+.Transgenic CHO had the same sensitivity as wtCHO if rotenone was given.WtCHO,by given reserpine alone,didn''''''''t change its sensitivity to MPP^+.Conclusions:VMAT2 has protective effect on transgenic CHO by transporting MPP^+ to vesicles.

Vesicular drug delivery systems for oral absorption enhancement

Oral administration is the most acceptable route of drug delivery at this stage due to its convenience,safety,and non-invasiveness.However,drugs given orally are exposed to a complex gastrointestinal environment,causing a tremendous challenge for their successful absorption into the circulation.Over the past decades,researchers have developed various novel pharmaceutical technologies to improve oral absorption,among which the vesicular drug delivery system(like liposomes,niosomes and transfersomes)has received extensive attention.Encouragingly,there have been several investigations confirming the improved effect of vesicular drug delivery systems on oral drug absorption.Nevertheless,the clinical translation of oral vesicular drug delivery systems has been less impressive than implied by the positive results,and few vesicular formulations for oral use have been marketed yet.Against this background,this article provides an overview of the current applications and challenges associated with the vesicular delivery systems available for oral drug delivery,specifically liposomes,niosomes,transfersomes,chitosomes and bilosomes.The composition,formation mechanism,drug delivery advantages and application cases of these carriers in oral drug delivery are summarized.The possible mechanisms by which vesicular carriers enhance oral drug absorption are analyzed in terms of the in vivo process of oral drugs.Further,the challenges that oral vesicular carriers now face,such as safety,undefined in vivo fate,and scale-up production,are summarized,while possible strategies to deal with them are indicated.By reviewing the aforementioned,it can facilitate a more comprehensive knowledge of vesicular systems that can be used for oral drug delivery,providing a theoretical basis and reference for the design of oral formulations.

Immunogenicity of mucosal COVID-19 vaccine candidates based on the highly attenuated vesicular stomatitis virus vector(VSV_(MT))in golden syrian hamster

COVID-19 is caused by coronavirus SARS-CoV-2.Current systemic vaccines generally pro-vide limited protection against viral replication and shedding within the airway.Recombinant VSV(rVSV)is an effective vector which inducing potent and comprehensive immunities.Currently,there are two clinical trials investigating COVID-19vaccines based on VSV vectors.These vaccines were developed with spike protein of WA1 which administrated intramuscularly.Although intranasal route is ideal for activating mucosal immunity with VSV vector,safety is of concern.Thus,a highly attenuated rVSV with three amino acids mutations in matrix protein(VSV_(MT))was developed to construct safe mucosal vaccines against multiple SARS-CoV-2 variants of concern.It demonstrated that spike protein mutant lacking 21 amino acids in its cytoplasmic domain could rescue rVSV efficiently.VSV_(MT) indicated improved safeness compared with wild-type VSV as the vector encoding SARS-CoV-2 spike protein.With a single-dosed intranasal inoculation of rVSV_(ΔGMT)-S_(Δ21),potent SARS-CoV-2specific neutraliza-tion antibodies could be stimulated in animals,particularly in term of mucosal and cellular immunity.Strikingly,the chimeric VSV encoding S_(Δ21) of Delta-variant can induce more potent immune responses compared with those encoding S_(Δ21) of Omicron-or WA1-strain.VSV_(MT) is a promising platform to develop a mucosal vaccine for countering COVID-19.

热休克蛋白27对水泡性口炎病毒体外增殖的调控作用

本研究旨在探究热休克蛋白27(heat shock protein 27, HSP27)对水泡性口炎病毒(vesicular stomatitis virus, VSV)体外增殖及VSV感染介导的维甲酸诱导基因I样受体家族(retinoid acid-inducible gene-I-like receptor, RLR)信号通路的作用及机制。利用实时荧光定量PCR、免疫印迹、免疫共沉淀、病毒滴度测定及免疫荧光等方法,首先检测VSV感染对HSP27 mRNA和蛋白表达的影响,进而验证过表达和干扰HSP27对VSV增殖的影响,进一步探究HSP27对VSV感染介导的RLR信号通路的影响,最后分析HSP27与RLR信号通路靶分子的相互作用及共定位情况。结果表明,VSV感染可促使HSP27基因转录和蛋白表达上调,稳定过表达和瞬时过表达HSP27均能显著抑制VSV的体外增殖;干扰宿主细胞中HSP27表达可促进VSV增殖。HSP27能够增强VSV及维甲酸诱导基因I蛋白(retinoic acid-inducible gene I protein, RIG-I)介导的IFN-β的产生及RIG-I的蛋白表达,而且HSP27与RIG-I相互作用并共定位于细胞质中。本研究揭示了HSP27靶向RIG-I上调其表达,增强RLR信号通路的转导,进而负调控VSV体外增殖,为深入揭示宿主因子HSP27在病毒感染中的作用机制奠定基础。

氮掺杂多孔碳材料阳极制备及其在微生物燃料电池上的应用

微生物燃料电池(MFCs)作为一种可以替代传统能源的生物电化学系统引起研究者的极大兴趣,其阳极材料的构造是目前的研究热点.本文从改善阳极材料表面物理化学性质的角度出发,用吐司作为多孔碳前驱体,三聚氰胺为氮源,直接烧制氮掺杂三维碳材料,并与不添加氮源的阳极材料和未改性的商用碳布进行比较.制备的掺氮NB1000阳极具有较大的比表面积(216.664 m^(2)·g^(-1))和优良的电导率.利用Geobacter和Shewanella混合菌落在微生物燃料电池(MFCs)中进行培养和性能评价,NB1000阳极的微生物燃料电池最大面功率密度为3049.714 mW·m^(-2),电流密度为7.4464 A·m^(-2),分别是普通碳布阳极的6.54倍和1.54倍.结果表明,NB1000作阳极的MFCs具有较高的功率密度,主要归因于阳极中引入氮掺杂,促进了产电微生物胞外电子传递过程所需的外膜c型细胞色素OmcA和MtrC的分泌.

Electroacupuncture Alleviates Motor Symptoms and Up-Regulates Vesicular Glutamatergic Transporter 1 Expression in the Subthalamic Nucleus in a Unilateral 6-Hydroxydopamine-Lesioned Hemi-Parkinsonian Rat Model

Previous studies have shown that electroacupuncture（EA） promotes recovery of motor function in Parkinson＇s disease（PD）. However the mechanisms are not completely understood. Clinically, the subthalamic nucleus（STN） is a critical target for deep brain stimulation treatment of PD, and vesicular glutamate transporter 1（VGlu T1） plays an important role in the modulation of glutamate in the STN derived from the cortex. In this study,a 6-hydroxydopamine（6-OHDA）-lesioned rat model of PD was treated with 100 Hz EA for 4 weeks. Immunohistochemical analysis of tyrosine hydroxylase（TH） showed that EA treatment had no effect on TH expression in the ipsilateral striatum or substantia nigra pars compacta,though it alleviated several of the parkinsonian motor symptoms. Compared with the hemi-parkinsonian rats without EA treatment, the 100 Hz EA treatment significantly decreased apomorphine-induced rotation and increased the latency in the Rotarod test. Notably, the EA treatment reversed the 6-OHDA-induced down-regulation of VGlu T1 in the STN. The results demonstrated that EA alleviated motor symptoms and up-regulated VGlu T1 in the ipsilateral STN of hemi-parkinsonian rats, suggesting that up-regulation of VGlu T1 in the STN may be related to the effects of EA on parkinsonian motor symptoms via restoration of function in the cortico-STN pathway.

细胞外基质蛋白ABI3BP抗水疱性口炎病毒初步作用研究

目的探讨细胞外基质蛋白ABI3BP对水疱性口炎病毒(vesicular stomatitis virus,VSV)基因组复制和先天免疫信号通路的影响。方法在人皮肤成纤维细胞BJ-5ta中转染小干扰RNA(siRNA)敲低ABI3BP基因,建立ABI3BP基因缺失的VSV-GFP病毒感染细胞模型。通过鬼笔环肽细胞免疫荧光实验,检测敲低ABI3BP后细胞内肌动蛋白(F-actin)形态结构的变化。在ABI3BP基因缺失的VSV-GFP病毒感染细胞模型上,利用RT-qPCR方法检测细胞中病毒mRNA水平的变化。利用免疫印迹法(Western blotting)检测IRF3和TBK1磷酸化水平的变化。结果成功建立敲减ABI3BP基因的VSV-GFP感染的细胞模型。鬼笔环肽细胞免疫荧光染色表明,敲减ABI3BP基因后,细胞内F-actin的表达发生结构重排。采用不同剂量的病毒感染细胞,与对照组相比,ABI3BP敲低细胞中基因拷贝数分别增加2.5~3.5倍(P<0.01)和2.2~4倍(P<0.01),VSV-GFP病毒蛋白表达水平显著上调;在ABI3BP基因敲低的细胞模型上,VSV-GFP病毒感染导致Ⅰ型干扰素免疫通路关键免疫分子p-IRF3和p-TBK1蛋白磷酸化水平明显下调。结论本研究表明细胞外基质蛋白ABI3BP对维持细胞内F-actin纤维网状结构具有重要作用。ABI3BP缺失促进RNA病毒的复制,ABI3BP是I型干扰素通路激活的重要调控分子。

电针刺激耳穴对小鼠丧失可控应激诱发学习记忆损害行为的作用机制研究

目的:观察丧失可控应激是否会诱发学习记忆损害行为,探讨针刺治疗学习记忆损害行为的作用机制。方法:结合经典抑郁症动物模型——习得性无助(learned helplessness,LH)模型的范式并在此基础上进行了改进,即小鼠前3天能够通过学习控制斯金纳箱中的有效鼻触器来主动躲避足底电击;后3天有效鼻触器功能丧失,小鼠从而丧失了对足底电击的控制,建立丧失可控应激(loss of controllable stress,LOC)小鼠模型。同时,设计无足底电击的对照组(control of non-foot-shock,Control)。建模完成后,利用新物体识别箱和Morris水迷宫检测学习记忆行为的变化,并电针刺激耳穴,观察对学习记忆损害行为的改善。随后,利用高尔基染色观察腹侧前额叶(ventral prefrontal cortex,vPFC)神经元结构的变化,免疫组化染色观察vPFC脑区C-fos的表达,Western Blot检测I型囊泡谷氨酸转运体蛋白(vesicular glutamate transporter I,VGLUT1)、突触蛋白I(synapsin I,SYN-1)的表达及针刺后相应指标的变化。结果:LOC组小鼠在经历认知挫败后造成了学习记忆功能的损害;针刺能够改善其记忆的获取及巩固;vPFC谷氨酸能(glutamate,Glu)神经元显著激活,VGLUT1、突触小泡蛋白(synaptophysin,SYN)表达水平降低,且耳迷走神经刺激能够显著降低其活动水平,调控VGLUT1、SYN蛋白的表达。结论:耳穴刺激通过调控v PFC中神经元结构的变化及VGLUT1、SYN蛋白表达改善丧失可控应激诱发的学习记忆损害行为。