阅读说明：本技术 一组表达受药物dl-PHPB调节的大脑蛋白 (Group of brain proteins with expression regulated by drug dl-PHPB ) 是由 高洪伟 孙英妮 于 2019-08-19 设计创作，主要内容包括：本发明公开了一组表达受药物dl-PHPB调节的大脑蛋白,dl-PHPB处理可显著改变AD模型小鼠大脑皮层和海马组织中如下一组蛋白的表达：histidine triad nucleotide-binding protein 1、fatty acid-binding protein、pyruvate dehydrogenase E1 component subunitα(PDHE1α)、peroxiredoxin-6、cathepsin B、isocitrate dehydrogenase、dihydropyrimidinase-related protein 2(DRP-2)、dihydrolipoamide succinyltransferase、dihydropyrimidinase-related protein 5(DRP-5)、peptidyl-prolyl cis-trans isomerase NIMA-interacting 1(Pin 1)、voltage-dependent anion-selective channel protein 1(VDAC1)、carbonic anhydrase 2、malate dehydrogenase、α-enolase、cofilin-2。(The invention discloses a group of cerebral proteins of which the expression is regulated by a medicament dl-PHPB, and the expression of the following group of proteins in cerebral cortex and hippocampal tissues of an AD model mouse can be obviously changed by the treatment of the dl-PHPB: 13 enzyme nutrient binding protein1, fat acid binding protein, pyroltate dehydrogenase E1component repair alpha (PDHE1 alpha), peroxiredoxin-6, cathepsin B, isocitrate dehydrogenase, dihydropyridonase-related protein2(DRP-2), dihydrolipoamide succinyltransferase, dihydropyridonase-related protein5(DRP-5), peptidyl-prolyl cis-protein interaction 1(Pin1), volume-dependent interaction-selective protein AC 1(VD 1), carbonamide 2, mammalian dehydrogenase-related protein alpha, and polypeptide-protein-2.)

1. A group of brain proteins with expression regulated by a drug dl-PHPB is characterized in that: dl-PHPB treatment significantly altered the expression of the following group of proteins in the cerebral cortex and hippocampal tissues of AD model mice: 13 enzyme nucleotide-binding protein1, fat acid-binding protein, pyrolate dehydrogenase E1component repair alpha (PDHE1 alpha), peroxiredoxin-6, cathespin B, isocitrate dehydrogenase, dihydropyridonase-related protein2(DRP-2), dihydrolipoamide succinyltransferase, dihydropyridonase-related protein5(DRP-5), peptidyl-prolyst-related protein MA-interacting1(Pin1), volume-dependent mutation-selective protein1(VD 1), carboxylic acid AC, hydrogel-derived dehydrogenase, collagen-derived protein2, and collagen-derived protein 362.

Technical Field

The invention belongs to the technical field of medicine, and relates to influence of dl-PHPB on expression of cerebral cortex and hippocampal histones with cognitive function impairment.

background

Alzheimer's Disease (AD), commonly known as senile dementia, is an age-related neurodegenerative disease, the pathogenesis of which is currently unknown, and there are several hypotheses: neurotransmitter deficits, inflammation, free radical damage, amyloid neurotoxic effects, hormone deficiency, apoptosis, and the like. In addition, environmental factors such as culture, education, smoking, brain trauma, history of heavy metal exposure, pregnancy at low age, and viral infections all increase the risk of developing AD. At present, the treatment of AD is mainly based on drugs, including acetylcholine, cholinesterase inhibitors, acetylcholine release-promoting drugs, anti-inflammatory drugs, estrogen drugs, antioxidant drugs, brain metabolism activators, and the like. These drugs only provide temporary relief of symptoms in AD patients, but do not slow or block the progression of the disease process.

2- (alpha-Hydroxypentyl) benzoate (Potasium 2- (1-Hydroxypentyl) -benzoate, dl-PHPB) is a precursor of butylphthalide (3-n-butylphthalide, dl-NBP), a clinical anti-ischemic stroke drug. dl-PHPB can be converted into dl-NBP by biological enzyme system to exert drug effect in vivo. dl-NBP is a new anti-ischemic cerebral apoplexy drug approved by the State food and drug administration in 2002, and can improve cerebral infarction volume in an ischemic area of a cerebral apoplexy patient, improve cerebral edema caused by ischemia, improve energy metabolism in the ischemic area, cerebral pia microcirculation disturbance and the like. But dl-NBP is a high-boiling point oily substance which cannot be prepared into an effective injection form and is not suitable for serious patients who cannot take orally, the lactone ring-opening product dl-PHPB has good water solubility and can be prepared into various dosage forms, and the dl-PHPB is solid crystals which can be purified after recrystallization, is easy to prepare in large quantities and is convenient to store. dl-PHPB as a novel candidate drug for resisting cerebral ischemia can improve cerebral blood flow in an ischemic area, inhibit the generation of free radicals, improve the activity of choline acetyltransferase and reduce the activation of glial cells, and the action links are closely related to the occurrence of AD. Earlier studies show that dl-PHPB has obvious improvement effect on learning and memory of chronic cerebral hypoperfusion rats, Abeta dementia-causing mice and APP/PS1 transgenic mice. In order to further research the anti-AD action mechanism of dl-PHPB and define the anti-AD action target of dl-PHPB, the influence of dl-PHPB on the expression of cerebral cortex and hippocampal tissue protein of cognitive impairment is observed by using a pharmacogenomic method.

Proteomics is a research object, mainly studying the composition of proteins in cells and their activity rules on the whole level, and studying the life activity rules and important physiological and pathological phenomena on the life essence level. Since the mechanism of action of drugs is directly or indirectly related to proteins, proteomics is of great significance in drug research. The pharmacogenomics mainly refers to the research on the effect of drugs on diseases on the protein expression level by applying a proteomics method. By comparing the expression difference of proteome of model cells or tissues before and after the treatment of the pharmaceutically active compound and identifying the proteins with corresponding changes, the mechanism of action of the drug can be revealed and new drugs can be screened; by comparing and analyzing the drug-resistant and drug-sensitive pathogenic bacterium proteome, the pathogenic bacterium drug-resistant mechanism and the design of new therapeutic drugs can be revealed. In addition, the pharmaproteomics can also be used for discovering new drug target proteins and carrying out toxicology research of drugs and the like.

The development of the proteomics technology provides possibility for the discovery of the anti-AD action target of the dl-PHPB and the exploration of the action mechanism. Therefore, an APP/PS1 double-transgenic mouse is selected as an AD model, from the proteome perspective, the two-dimensional gel electrophoresis technology, the mass spectrometry technology and other proteomics means are adopted, the difference change of the expression of cortex and hippocampal tissue proteins of the AD model mouse before and after dl-PHPB intervention is researched, and the possible action mechanism and action target point of dl-PHPB anti-AD are clarified on the protein expression level. Memantine is an NMDA receptor antagonist and donepezil is a cholinesterase inhibitor, which are currently the commonly used drugs in the clinic for the treatment of AD. In the research, the expression conditions of the differential proteins in the brain tissues of the transgenic mice after the administration of memantine and donepezil are also considered, so that whether the pharmacological action targets of dl-PHPB and the existing positive drugs are similar or not is compared, and a new drug target which can be used for designing a new drug is found.

Disclosure of Invention

The invention aims to provide a group of brain proteins with expression regulated by a medicament dl-PHPB. The technical scheme adopted by the invention is that an APP/PS1 double-transgenic mouse is selected as an AD model, and from the proteome angle, proteomics means such as a two-dimensional gel electrophoresis technology and a mass spectrometry technology (LC-MS/MS) are adopted to research the difference change of the whole proteome of cortex and hippocampus tissues of the AD model mouse before and after dl-PHPB intervention. The method is characterized in that: dl-PHPB can significantly change the expression of the following group of proteins in cerebral cortex and hippocampal tissues of a cognition-impaired mouse: 13 nutrient nucleotide-binding protein1, fat acid-binding protein, pyrolate dehydrogenase E1component repair alpha (PDHE1 alpha), peroxiredoxin-6, cathepsin B, isocitrate dehydrogenase, dihydropyridonase-related protein2(DRP-2), dihydrolipoamide succinyltransferase, dihydropyridonase-related protein5(DRP-5), peptidyl-prolysaccharide NIMA-interacting1(Pin1), volume-dependent amino-selective protein1(VDAC1), carbonitrile dehydrogenase 2, mammalian dehydrogenase, alpha-2, and so forth.

Detailed Description

the present invention will be described in detail with reference to the following embodiments.

1. Laboratory animal

Male B6C3-Tg (APPsw, PSEN1dE9)85Dbo/J (APP/PS1) transgenic mice, age-matched Wild Type (WT) mice, purchased from Jackson Lab, USA and bred, raised and genotyped at the Nanjing university model animal center. When the mice are raised to 9 months of age, the mice are transported to an experimental center in a clean-grade package, one cage is placed every 3-5 mice, the room temperature is kept at 24 +/-1 ℃, the humidity is 50-60%, the illumination is carried out at 8:00-20:00 every day, the light is closed at 8: 00-next day at 20:00, and the mice can freely eat and drink water.

2. Experimental methods

Animals are randomly divided into 5 groups, each group comprises 8-10 animals, and the animals are respectively a control group (WT mouse), a model group (APP/PS1 mouse), a dl-PHPB treatment group (APP/PS1 mouse), a memantine treatment group (APP/PS1 mouse) and a donepezil treatment group (APP/PS1 mouse), the treatment group mice are gavaged with dl-PHPB (30mg/kg), memantine (30mg/kg) and donepezil (30mg/kg) every day according to the group, the control group and the model group mice are fed with deionized water with the same volume every day, and after 90 days of continuous gavage, the Morris water maze is adopted to test learning memory and memory retention capacity of mouse positioning navigation, space search and the like. The animals were then sacrificed and the material was taken.

the Morris water maze method is as follows: the experiment is carried out in a sound-proof, non-direct-light and dark room, and various experimental conditions such as the position of a pool, the brightness of the room and the like are kept unchanged. The water pool is filled with tap water in advance, the water depth is 20cm, the water surface is 1cm higher than the platform surface (the platform can not be seen by the mouse), because the mouse is black, for the convenience of recording, about 500g of milk powder is added into the water pool and is uniformly stirred, so that the water in the water pool is milky, the water temperature is controlled to be 21 +/-2 ℃, and the platform is positioned in the center of the second quadrant. The experiment is carried out for 5 days, water entry points are randomly selected every day, the experiment is carried out for 4 times, the heads of the mice enter the water towards the wall of the pool, meanwhile, the camera starts to record, the mice which find the platform stay on the platform for 30s, the mice which do not find the platform are led to the platform after 60s, and the mice are placed for 30s to guide the mice to learn and memorize. The data acquisition and the image analysis are completed by an automatic image monitoring and processing system. At the end of the experiment, the escape latency, swimming distance and speed of each group of animals reaching the platform were recorded.

The proteome analysis and mass spectrometry identification methods are as follows:

2.1 preparation of Total protein of cortex and Hippocampus

The whole brain was quickly removed after the mice were sacrificed by decapitation, and the hippocampus and cortex were rapidly separated in an ice bath. Adding 300 mul of precooled protein lysate into each mouse hippocampus, and preparing tissue homogenate by adopting an ultrasonic disruption method (60w, 6s multiplied by 4 times, 30s interval); 1ml of pre-cooled protein lysate was added to each mouse cortex and a tissue homogenate was prepared by electrokinetic homogenization (12,000g, 6s × 4 times, 30s apart). Centrifugation (1,000g × 10min, 2 times) gave a supernatant as total protein, all at 4 ℃. And (4) determining the protein concentration by using a 2-D Quant Kit, subpackaging, and storing at-80 ℃ for later use.

2.2 dimensional electrophoresis

2.2.1 isoelectric focusing

The loading of Silver dye is 120 mug/strip and the loading of Blue Silver colloid test dye is 1 mg/strip. The loaded protein and the hydration solution are fully mixed, the total volume is 350 mul, an IPG adhesive tape groove is added, a pH 3-10NL, 18cm dry adhesive tape is used, the adhesive surface is covered downwards, the covering solution is added and then is placed on an IPG phor electrode plate, the hydration and the focusing processes are carried out at 20 ℃, and the parameters are as follows:

Immobiline dry strip IEF parameters for IPGphor isoelectric focusing system.

2.2.2 Balancing

after the completion of the isoelectric focusing, the IPG strip was placed in 10ml of equilibration solution A (6M Urea, 30% Glycerol, 2% SDS, 50mM Tris-HCl pH 8.8, 0.002% Bromophenol blue, 0.2% DTT added immediately before use) and equilibration solution B (6M Urea, 30% Glycerol, 2% SDS, 50mM Tris-HCl pH 8.8, 0.002% Bromophenol blue, 2.5% Iodoacetamide added immediately before use) in sequence and equilibrated for 15min each.

2.2.3SDS-PAGE

Second-phase vertical SDS-Polyacrylamide Gel Electrophoresis (PAGE), deionized water is quickly washed on the well-balanced IPG Gel strip, the well-balanced IPG Gel strip is moved to 12.5% uniform separation Gel of 3 with the size of 200X 1.0mm, the Gel is fixed by 1% agarose Gel sealing, Electrophoresis is carried out for 1h at constant power of 2W/Gel, and then Electrophoresis is carried out at constant power of 15W/Gel until the distance between the bromophenol blue front edge and the bottom edge is about 1.5 cm.

2.2.4 dyeing

(1) silver staining-detection of analytical gel by silver staining method, the detection steps are according to the specification of silver staining kit of GE healthcare company, the concrete steps are as follows:

The above steps were carried out at 20 ℃ with shaking.

(2) Test-detection of the preparative gel by a Blue Silver colloid test method comprises the following specific steps:

2.2.5 gel scanning and image analysis

The stained gel was scanned at 300bpi resolution using a Umax scanner and the images were spot-detected, background subtracted, two-dimensional corrected, spot paired, differential spot analyzed, screened, and data stored using ImageMaster 2D Platinum Software 7.0. The screening criteria for the difference points were: by comparing the relative gray values (i.e. volume% values) of the protein points, when the Ratio value is greater than or equal to 2, the protein with the change rate of 2 times or more than 2 times is the differential protein.

2.3 in-gel enzymatic hydrolysis and Mass Spectrometry (LC-MS/MS)

The specific process is as follows: the bidirectional protein spots to be identified were cut into approximately 1mm3 micelles and transferred to 1.5ml Axygen EP tubes. Mu.l of destaining solution (100mM ammonium bicarbonate mixed with pure acetonitrile in a volume ratio of 1: 1) was added to the EP tube and placed on a vortex mixer and shaken for about 30min until most of the blue color had gone (this operation will be repeated depending on the specific destaining degree). The destaining solution was discarded and 500. mu.l of pure acetonitrile was added to the EP tube and dehydrated at room temperature with shaking until the gel particles were white by shrinkage. After the acetonitrile is volatilized, a proper amount of 20mM DTT is added into an EP tube, the mixture is placed in a water bath at the temperature of 56 ℃ for reduction for 30min, and after the mixture is cooled to the room temperature, 55mM iodoacetamide is added for alkylation reaction for 20min in the dark. After the reaction is finished, pure acetonitrile is added to dehydrate the colloidal particles again, and a proper amount of trypsin is added to cover the dehydrated colloidal particles so that the pancreatin solution is fully absorbed into the colloidal particles. The EP tubes were incubated overnight on a 37 ℃ incubator. The micelle after enzymolysis is extracted by 100 mul of peptide fragment extracting solution (5 percent formic acid and pure acetonitrile are mixed according to the volume ratio of 1: 2). The extract was concentrated to about 20. mu.l on a rotary drier. The concentrated peptide extract was analyzed by C18-HPLC coupled LC-MS LTQ. The generation of the "peak list" was done automatically by the EXTRACTMS program in the Xcalibur software package and only MS/MS maps with total ion current intensity higher than 500 were used to search the database NCBI RefSeq mouse, the retrieval software being the TurboSeQUEST algorithm in the Bioworks3.31 software package. The specific retrieval parameters are set as follows: i) the mass error of the parent ion fragment is 2.0 amu; ii) the mass error of the fragment of the daughter ion is 1.0 amu; iii) allowing up to 2 incomplete cleavage sites; iv) setting the fixed modification to a cysteine mercaptocarboxymethylation modification (mass increase of 57.02amu) and the variable modification to a methionine oxidation modification (mass increase of 15.99 amu). By screening the search results, peptides meeting the following requirements were considered correctly identified proteins: i) the Xcorr coefficients of the +1 valence, the +2 valence and the +3 valence peptide sections are respectively higher than 1.8, 2.2 and 3.0; ii) the delta correlation coefficient for the best-matching peptide fragment must be greater than 0.1; iii) Sp value must be higher than 500. The correctly identified protein needs to contain at least two different peptide stretches, and all b, y ion series of the secondary mass spectrum of the identified protein are verified manually.

3. And (4) counting and analyzing results:

All results are expressed as mean ± SEM data. The proteomics difference point analysis adopts statistical software of GE Healthcare company, and the result takes Ratio value more than or equal to 2 and Student ttest <0.05 as meaningful difference protein; other comparative experiments compared differences between groups using One-way analysis of variance (One-WayANOVA) combined with Dunnett's post hoc test, with p <0.05 considered significant differences.

The Morris water maze experiment result shows that the swimming speed of the mice has no obvious change among groups, which indicates that the health condition of the mice has no obvious difference among the groups. With the increase of training time, the latency of each group of mice for finding the hidden platform is gradually reduced, the latency of the control group of mice is reduced from 47.15 +/-2.99 s to 24.68 +/-2.79 s, and the latency of the model group of mice is reduced from 51.06 +/-2.53 s to 35.10 +/-2.35 s, so that the latency is remarkably prolonged compared with that of the wild type mice of the same age (p is less than 0.01), and the AD transgenic mice are suggested to have obvious learning and memory loss. After administration, the latency of the dl-PHPB treatment group is reduced to 26.16 +/-2.92 s, the latency of the memantine treatment group is reduced to 28.12 +/-2.25 s, and the latency of the donepezil treatment group is reduced to 29.03 +/-2.01 s, which indicates that the dl-PHPB has an obvious improvement effect on learning and memory loss of APP/PS1 transgenic mice.

By utilizing a pharmacogenomics method, an APP/PS1 double-transgenic mouse is taken as an AD animal model, and the change of the whole proteome in the cerebral cortex and the hippocampus tissues of the model mouse before and after the administration of dl-PHPB is analyzed by adopting a method of combining a two-dimensional gel electrophoresis technology and a mass spectrum technology (LC-MS/MS), so that a group of proteins with the expression influenced by the dl-PHPB is identified. The results are as follows: 13 nucleotide-binding protein1, fat acid-binding protein, phosphate dehydrogenase E1component repair, peroxiredoxin-6, cathepsin B, isocitrate dehydrogenase, dihydrofolate-related protein2(DRP-2), dihydrolipoamide succinyltransferase, dihydrofolate-related protein5(DRP-5), peptidyl-prolyscin NIMA-interacting1(Pin1), protein-dependent mutation-selected channel protein1 (AC VD 1), carbonic anhydride 2, protein dehydrogenase, alpha-2, and so on.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not intended to limit the present invention in any way, and all simple modifications, equivalent variations and modifications made to the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.