CN118389663A - Application of seventy-ingredient pearl pills in regulation of cognitive dysfunction and research method and application of molecular mechanism of seventy-ingredient pearl pills - Google Patents
Application of seventy-ingredient pearl pills in regulation of cognitive dysfunction and research method and application of molecular mechanism of seventy-ingredient pearl pills Download PDFInfo
- Publication number
- CN118389663A CN118389663A CN202410435243.1A CN202410435243A CN118389663A CN 118389663 A CN118389663 A CN 118389663A CN 202410435243 A CN202410435243 A CN 202410435243A CN 118389663 A CN118389663 A CN 118389663A
- Authority
- CN
- China
- Prior art keywords
- seventy
- pearl
- pills
- ingredient
- cognitive dysfunction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000006187 pill Substances 0.000 title claims abstract description 88
- 239000004615 ingredient Substances 0.000 title claims abstract description 46
- 208000010877 cognitive disease Diseases 0.000 title claims abstract description 27
- 230000033228 biological regulation Effects 0.000 title claims abstract description 17
- 238000000034 method Methods 0.000 title claims abstract description 13
- 230000009456 molecular mechanism Effects 0.000 title claims abstract description 7
- 238000011160 research Methods 0.000 title abstract description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 52
- 239000003814 drug Substances 0.000 claims abstract description 24
- 230000000971 hippocampal effect Effects 0.000 claims abstract description 17
- 101150048336 Flt1 gene Proteins 0.000 claims abstract description 16
- 210000001320 hippocampus Anatomy 0.000 claims abstract description 15
- 230000001105 regulatory effect Effects 0.000 claims abstract description 14
- 229940079593 drug Drugs 0.000 claims abstract description 12
- 102100030684 Sphingosine-1-phosphate phosphatase 1 Human genes 0.000 claims abstract description 10
- 101710168942 Sphingosine-1-phosphate phosphatase 1 Proteins 0.000 claims abstract description 10
- 230000001965 increasing effect Effects 0.000 claims abstract description 9
- 238000012216 screening Methods 0.000 claims abstract description 9
- 101150049876 EDN1 gene Proteins 0.000 claims abstract description 5
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 3
- 101100540419 Danio rerio kdrl gene Proteins 0.000 claims description 14
- 101150088608 Kdr gene Proteins 0.000 claims description 14
- 238000010171 animal model Methods 0.000 claims description 13
- 238000012163 sequencing technique Methods 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 11
- -1 tek Proteins 0.000 claims description 9
- 238000003012 network analysis Methods 0.000 claims description 6
- 101100381481 Caenorhabditis elegans baz-2 gene Proteins 0.000 claims description 5
- 101100372762 Rattus norvegicus Flt1 gene Proteins 0.000 claims description 5
- 238000013115 immunohistochemical detection Methods 0.000 claims description 3
- 230000002103 transcriptional effect Effects 0.000 claims description 3
- 239000007850 fluorescent dye Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 238000011835 investigation Methods 0.000 claims 4
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 25
- 230000000694 effects Effects 0.000 abstract description 9
- 230000007246 mechanism Effects 0.000 abstract description 9
- 241000699666 Mus <mouse, genus> Species 0.000 abstract description 5
- 206010012601 diabetes mellitus Diseases 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 3
- 230000003287 optical effect Effects 0.000 abstract description 3
- 230000002452 interceptive effect Effects 0.000 abstract 1
- 230000006916 protein interaction Effects 0.000 abstract 1
- 238000011830 transgenic mouse model Methods 0.000 abstract 1
- 230000037361 pathway Effects 0.000 description 26
- 230000003993 interaction Effects 0.000 description 17
- 238000010201 enrichment analysis Methods 0.000 description 13
- 230000028327 secretion Effects 0.000 description 12
- 210000000225 synapse Anatomy 0.000 description 11
- 102100028255 Renin Human genes 0.000 description 10
- 108090000783 Renin Proteins 0.000 description 10
- 210000001947 dentate gyrus Anatomy 0.000 description 10
- 239000002671 adjuvant Substances 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 230000002060 circadian Effects 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 230000028956 calcium-mediated signaling Effects 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000002858 neurotransmitter agent Substances 0.000 description 5
- 230000000144 pharmacologic effect Effects 0.000 description 5
- 230000019491 signal transduction Effects 0.000 description 5
- 230000007730 Akt signaling Effects 0.000 description 4
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 4
- 230000004156 Wnt signaling pathway Effects 0.000 description 4
- 230000011496 cAMP-mediated signaling Effects 0.000 description 4
- 230000012292 cell migration Effects 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000003371 gabaergic effect Effects 0.000 description 4
- 230000000848 glutamatergic effect Effects 0.000 description 4
- 101150063780 spp1 gene Proteins 0.000 description 4
- 230000032258 transport Effects 0.000 description 4
- 108091006146 Channels Proteins 0.000 description 3
- 101150027068 DEGS1 gene Proteins 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002055 immunohistochemical effect Effects 0.000 description 3
- 208000000230 African Trypanosomiasis Diseases 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 2
- 241000224489 Amoeba Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000004310 Ion Channels Human genes 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 206010057852 Nicotine dependence Diseases 0.000 description 2
- 102000037602 Platelet Endothelial Cell Adhesion Molecule-1 Human genes 0.000 description 2
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 2
- 208000025569 Tobacco Use disease Diseases 0.000 description 2
- 241000223105 Trypanosoma brucei Species 0.000 description 2
- 210000003050 axon Anatomy 0.000 description 2
- 230000004009 axon guidance Effects 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000000857 drug effect Effects 0.000 description 2
- 230000033822 gland development Effects 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 208000029080 human African trypanosomiasis Diseases 0.000 description 2
- 206010020871 hypertrophic cardiomyopathy Diseases 0.000 description 2
- 238000003364 immunohistochemistry Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000003061 melanogenesis Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 208000008013 morphine dependence Diseases 0.000 description 2
- 230000009644 regulation of neurotransmitter levels Effects 0.000 description 2
- 230000000862 serotonergic effect Effects 0.000 description 2
- 230000015607 signal release Effects 0.000 description 2
- 201000002612 sleeping sickness Diseases 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 210000002182 synaptic membrane Anatomy 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 230000025102 vascular smooth muscle contraction Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241001289435 Astragalus brachycalyx Species 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 208000007333 Brain Concussion Diseases 0.000 description 1
- 101150104494 CAV1 gene Proteins 0.000 description 1
- 101100368700 Caenorhabditis elegans tac-1 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical group [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 101150091877 Ccn2 gene Proteins 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 208000004929 Facial Paralysis Diseases 0.000 description 1
- 235000002917 Fraxinus ornus Nutrition 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102100027551 Ras-specific guanine nucleotide-releasing factor 1 Human genes 0.000 description 1
- 108050004793 Ras-specific guanine nucleotide-releasing factor 1 Proteins 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 208000036826 VIIth nerve paralysis Diseases 0.000 description 1
- 102000013814 Wnt Human genes 0.000 description 1
- 230000007059 acute toxicity Effects 0.000 description 1
- 231100000403 acute toxicity Toxicity 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003705 background correction Methods 0.000 description 1
- 230000003542 behavioural effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000001914 calming effect Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000027288 circadian rhythm Effects 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002996 emotional effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 210000001661 hippocampal ca3 region Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000008035 nerve activity Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000003227 neuromodulating effect Effects 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000003068 pathway analysis Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003518 presynaptic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035082 regulation of cellular response to growth factor stimulus Effects 0.000 description 1
- 230000012481 regulation of membrane potential Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- 102000042565 transient receptor (TC 1.A.4) family Human genes 0.000 description 1
- 108091053409 transient receptor (TC 1.A.4) family Proteins 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000032636 vascular process in circulatory system Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses application of seventy-ingredient pearl pills in cognitive dysfunction regulation and a research method and application of molecular mechanisms thereof, and belongs to the technical field of biological medicines. The invention adopts db/db transgenic mouse model to observe action mechanism of the seventy pearl pills (without zotai) with original medicine for interfering diabetes cognitive dysfunction, analyzes the differential expression genes of the hippocampal tissues of the mice based on transcriptome technology, constructing a differential gene protein interaction network, screening a basic drug and a prescription-reduced drug to regulate key differential genes of db/db mouse hippocampal tissues, and finally verifying key targets by an immune group. The results show that the average integrated optical density of the CA3 region and the DG region of the hippocampus of the mice is higher than that of Flt1, edn1, agt and Itga6 of the original drug group, and the expression of the CA3 region and the DG region Edn1 and the Spp1 of the hippocampus of the mice of the reduced seventy-ingredient pearl pill group is obviously increased compared with that of the model group, but the expression has no obvious regulation effect on the Flt1, agt and Itga6 targets. Thus, the zoteh realizes the regulation of cognitive dysfunction through regulating targets Flt1, agt and Itga 6.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of seventy-ingredient pearl pills in cognitive dysfunction regulation and a research method and application of molecular mechanisms thereof.
Background
Seventy-ingredient pearl pills (RANASAMPEL, RNSP) are rare Tibetan medicines with long history in China, and have the effects of calming and tranquillizing, promoting blood circulation and removing obstruction in collaterals, regulating qi and blood, inducing resuscitation and refreshing mind. Clinically, the traditional Chinese medicine composition is often used for treating diseases such as ischemic cerebral apoplexy, cerebral concussion, neurovascular headache, facial paralysis, alzheimer disease and the like, but is not reported in the research of diabetes cognitive dysfunction. In addition, zotai is one of the main components of seventy pearl pills and is an important raw material of many rare Tibetan medicines. Under the guidance of Tibetan pharmacology theory, mercury in zotai is processed, so that the acute toxicity is eliminated, and the Chinese medicinal composition has a magic curative effect. Researches show that the zoteur has the effect of enhancing the curative effects of various medicines in the compound medicine, but the effective form and mechanism of the synergistic effect are not clear.
Disclosure of Invention
Aiming at the problems, the invention aims to provide an application of seventy-ingredient pearl pills in cognitive dysfunction regulation and a research method and application of molecular mechanism thereof, and based on transcriptomics technology, the possible mechanism of main component zocine in seventy-ingredient pearl pills in treating diabetic cognitive dysfunction is researched and analyzed through technologies such as differential gene function enrichment analysis, key target spot screening immunohistochemical experiments and the like.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
The seventy-ingredient pearl pills are applied to the regulation of cognitive dysfunction, and particularly the seventy-ingredient pearl pills realize the regulation of cognitive dysfunction through regulating key targets Kdr, tek, flt, edn1, col18a1, agt and Itga 6.
More specifically, seventy-ingredient pearl pills improve cognitive dysfunction by increasing the expression of Kdr, tek, flt1, edn1, col18a1, agt and Itga 6.
Furthermore, the invention also comprises a molecular mechanism research method for the application of seventy-ingredient pearl pills in the regulation of cognitive dysfunction, which comprises the following steps,
S1: the experimental animals are respectively fed with seventy pearl pill raw medicines and the reduced seventy pearl pills without zotai, and a model group and a control group are arranged for control;
S2: differential gene screening by transcriptional sequencing;
s3: determining seventy pearl pills and key targets of the seventy pearl pills for regulating the hippocampal tissues of experimental animals by PPI network analysis and Hub gene identification;
s4: immunohistochemical detection of the expression of key target proteins.
Specifically, the PPI network analysis in step S3 includes Stress, radiality, closeness, EPC, degree, MCC, MNC parameter analyses.
Specifically, seventy pearl pills are used for regulating key targets of hippocampal tissues of experimental animals, including Kdr, tek, flt1, edn1, col18a1, agt and Itga6; the key targets of the seventy pearl pills for regulating the hippocampal tissues of experimental animals comprise Spp1, kdr and Edn1.
Specifically, in step S4, the seventy pearl pill group showed increased expression of Flt1, edn1, agt, itga6 in the CA3 and DG regions of the hippocampus of the experimental animals compared to the model group; the seventy pearl pill group had increased expression of Edn and Spp1 in the CA3 and DG regions of the hippocampus of the experimental animals compared to the model group.
Further, the invention also includes the use of reagents for detecting Flt1, edn1, agt and Itga6 levels in the preparation of a product for diagnosing cognitive dysfunction.
Specifically, the products include kits and fluorescent probes.
The beneficial effects of the invention are as follows:
Based on transcriptome technology, the invention is verified by bioinformatics screening key targets and immunohistochemical experiments, and the possible mechanism of seventy pearl pills and the minus seventy pearl pills in treating diabetes cognitive dysfunction is discussed; as a result, it was found that key pathways shared by the seventy-ingredient drug group and the minus seventy-ingredient pearl pill group include neuroactive ligand-receptor interaction, renin secretion, calcium signaling pathway, and the like, and key target screening and immunohistochemical results show that the average integrated optical density of the CA3 and DG regions Flt1, edn1, agt, itga6 of the base drug group is higher (P <0.05 or P < 0.01) compared to the model group, and the expression of the CA3 and DG regions Edn and Spp1 of the minus seventy-ingredient pearl pill group is significantly increased (P < 0.01) compared to the model group, but no significant regulatory effect on the Flt1, agt, itga6 targets is achieved. Therefore, seventy-ingredient pearl pills can relieve the cognitive dysfunction of db/db mice by improving the expression of the hippocampus Flt1, edn1, agt and Itga6 of the db/db mice, and the pharmacodynamic basis of the reduced seventy-ingredient pearl pills and the original medicine is preliminarily compared, so that a certain difference exists between the two in key targets and regulating paths.
Drawings
FIG. 1 is a diagram of a pyrogram and KEGG enrichment pathway plot of differential analysis of hippocampal transcriptome sequencing data according to the present invention.
FIG. 2 is an enlarged view of the map of the transcription sequencing result db VS mm differential gene KEGG enrichment pathway of FIG. 1 according to the present invention.
FIG. 3 is an enlarged view of the amplification pathway diagram of the differential gene KEGG of the transcription sequencing result RP VSdb in FIG. 1 according to the present invention.
FIG. 4 is an enlarged view of the map of the RZ VSdb differential gene KEGG enrichment pathway of the sequencing result of FIG. 1 according to the present invention.
FIG. 5 is a PPI network topology diagram of the RP VSdb and RZ VSdb differential genes according to the present invention.
FIG. 6 shows a set map of a common gene key target obtained by 7 algorithms and PPI network GO and KEGG enrichment analysis.
FIG. 7 is an enlarged view of the enrichment analysis result of the RP VSdb differential gene PPI network GO of FIG. 6 according to the present invention.
FIG. 8 is an enlarged view of the result of the enrichment analysis of the PPI network KEGG of the RP VSdb differential gene of FIG. 6 according to the present invention.
FIG. 9 is an enlarged view of the result of the enrichment analysis of the PPI network GO of the RZ VSdb differential gene of FIG. 6 according to the present invention.
FIG. 10 is an enlarged view of the result of the PPI network KEGG enrichment analysis of the RZ VSdb differential gene of FIG. 6 according to the present invention.
FIG. 11 shows the expression of key indicators in the CA3 region of the hippocampus of mice in the present invention.
FIG. 12 shows the expression of key indicators in the hippocampal DG region of mice in the present invention.
Detailed Description
In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
The invention selects the seventy pearl pills with the adjuvant component removed, discusses the drug effect substance basis of the seventy pearl pills with the adjuvant component removed and the raw medicine (the seventy pearl pills contain the adjuvant component) in a mice model with cognitive dysfunction, and observes whether the seventy pearl pills with the adjuvant component removed have the change of action way compared with the raw medicine, thereby verifying the medicinal value of the adjuvant component in the seventy pearl pill formula, and further evaluating the pharmacological machine manufacture of the seventy pearl pill formula component.
1. Materials and methods
1.1 Experimental animal
SPF class 8 week old male db/db mice 36, d/m mice 12 were bred in SPF class animal houses at Beijing university of traditional Chinese medicine. The room temperature is maintained at 22-24 ℃, the humidity is maintained at 40-50%, 12 h/12 h are alternately illuminated day and night, and conventional pellet feed and drinking water are fed. The experimental scheme is approved by the ethical committee of the biomedical science of Beijing university, and the approval number is LA2017282.
1.2 Experimental medicine
Seventy pearl pills are purchased from Tibetan manna Tibetan medicine Co., ltd, and each pill weighs 1g. The medicine of the seventy-ingredient pearl pill (the adjuvant ingredient in the seventy-ingredient pearl pill is removed, and other ingredients are completely the same as the seventy-ingredient pearl pill) is provided by pharmaceutical factories of Tibetan medicine universities.
1.3 Group administration
After the mice were adaptively fed for one week, 36 db/db mice were randomly divided into a model group (db), seventy pearl pill administration groups (RP), and a reduced seventy pearl pill administration group (RZ), each group being 12; 12 d/m mice were control group (mm). Seventy pearl pill group mice were filled with seventy pearl pill solution (1.25 g/kg/d) daily, and the seventy pearl pill group mice were reduced with the same amount of drug solution as the seventy pearl pill group, and the control group and model group mice were filled with equal volumes of deionized water daily, and the filling was continued for 4 weeks.
1.4 Transcriptional sequencing and differential gene screening
Tissue sampling is carried out after the behavioural experiment is finished, 6 mouse hippocampal tissues are randomly taken from each group of mice respectively, total RNA is extracted, library building and sequencing are carried out on the mice, log2 conversion is carried out by using R language, and data pretreatment is carried out. And performing data processing such as background correction, quantile standardization and the like by using an automatic multi-chip averaging method of Affy sub-packets in a Bioconductor library. Then, using Limma sub-package in R language to screen difference gene (DEGs), using Fold Change not less than 1.5 and P-value <0.05 as standard to screen difference gene between mm VS db, RP VSdb and RZ VSdb. The volcanic mapping pair DEGs was visually analyzed by R software package (ggplot 2), the KEGG pathway analysis was performed on DEGs using R package (clusterProfiler), and adjusted P-value <0.05 was considered to have significant differences.
1.5 PPI network analysis and Hub gene identification
PPI networks of RP VSdb and RZ VSdb differential genes are respectively constructed by using STRING database (https:// STRING-db. Org /), and interaction score (interaction score) is more than or equal to 0.4, which is considered to have statistical significance. The PPI network was then analyzed by means of the cytoHubba 7 parameters (Stress, radiality, closeness, EPC, degree, MCC, MNC) in Cytoscape.7.2 software to screen for key targets. And performing GO and KEGG enrichment analysis on the PPI network respectively, and finally synthesizing key genes and enrichment analysis results, and determining key differential genes of seventy pearl pills and the hippocampal tissues of the minus seventy pearl pills for regulating db/db mice by combining literature researches.
1.6 Immunohistochemical detection of the expression of key differential Gene
Brain tissue of each group of 5 mice was fixed in 4% paraformaldehyde solution, brain sections including hippocampal CA1, CA3 and Dentate Gyrus (DG) regions were prepared according to the stereotactic map of the mice brain, and then dehydrated, transparent, embedded, and coagulated. Paraffin sections were dewaxed with gradient ethanol, subjected to antigen retrieval, blocked with 0.3% H 2O2 for endogenous peroxidase, blocked with 5% BSA solution for 1H at room temperature, then blocked with Flt1, edn1, agt, itga6 and Spp1 antibodies overnight at 4 ℃, incubated with secondary antibody in an oven at 37 ℃ for 0.5H, developed with DBA chromogenic solution, observed under a microscope and controlled in reaction extent, and the reaction was terminated in time with purified water.
2. Results
2.1 Screening and analysis of Key differential genes
By analyzing the hippocampal transcriptome sequencing data, the obtained db VSmm, RP VSdb and RZ VSdb differential genes are shown in the accompanying figure 1, and the accompanying figures 2-4 are respectively enlarged diagrams of the enrichment pathway point diagrams of the sequencing db VSmm, RP VSdb and RZ VSdb differential genes KEGG in the figure 1. Wherein 1060 differential genes of db VS mm are added; 321 different genes of RP VSdb; there are 382 different genes of RZ VSdb. KEGG analysis of the differential gene shows that the key pathways enriched in db VS mm differential gene have neuroactive ligand-receptor interactions (Neuroactive ligand-receptor interaction), axon guidance (Axon guidance), morphine addiction (Morphine addiction), nicotine addiction (Nicotine addiction), gabaergic synapses (GABAergic synapse); key pathways enriched for RP VS db differential gene are renin secretion (Renin secretion), melanogenesis (Melanogenesis), cancer pathway (PATHWAYS IN CANCER), neuroactive ligand-receptor interactions (Neuroactive ligand-receptor interaction), vascular smooth muscle contraction (Vascular smooth muscle contraction); the key pathways enriched for RZ VSdb differential gene are neuroactive ligand-receptor interactions (Neuroactive ligand-receptor interaction), circadian traction (CIRCADIAN ENTRAINMENT), renin secretion (Renin secretion), african trypanosomiasis (African trypanosomiasis), hypertrophic cardiomyopathy (Hypertrophic cardiomyopathy). This result suggests that seventy-ingredient pearl pills and subtractive seventy-ingredient pearl pills have the same and different mechanisms for their modulating action on the hippocampus of db/db mice.
2.2 PPI protein network analysis and Hub gene screening
To further screen out the key differential genes of seventy-ingredient pearl pills and subtractive seventy-ingredient pearl pills, PPI network topology analysis of RP VSdb and RZ VSdb differential genes was performed using STRING database, and PPI networks comprising 216 nodes (RP VSdb) and 265 nodes (RZ VSdb) were obtained, respectively, as shown in FIG. 5. The key PPI network targets (Kdr, pecam1, cdh5, tek, flt1, cav1, MKi67, edn1, col18a1, itga6, gata2, agt) of 12 RP VSdb and the key PPI network targets (Pecam 1, spp1, kdr, ctgf, edn1, tac 1) of 6 RZ VSdb were obtained by the cytoHubba algorithms, upSet charts were used to display common genes obtained by the 7 algorithms, as shown in FIG. 6, FIG. 7 is an enlarged graph of the PPI network GO enrichment analysis result of the RP VSdb difference gene in FIG. 6, FIG. 8 is an enlarged graph of the PPI network KEGG enrichment analysis result of the RP VSdb difference gene in FIG. 6, FIG. 9 is an enlarged graph of the PPI enrichment analysis result of the RZ VSdb difference gene in FIG. 6, and FIG. 10 is an enlarged graph of the PPI network KEGG enrichment analysis result of the RZ db difference gene in FIG. 6.
As can be seen from fig. 6-10, through GO functional analysis, PPI networks of RP VS db and RZ VS db retrieve 1088 and 1025 entries (Padjust < 0.05), respectively. Wherein the GO entries of the RP VSdb PPI network are mainly related to collagen-containing extracellular matrix (collagen-containing extracellular matrix), vascular processes in the circulatory system (vascular process in circulatory system), amoeba cell migration (ameboidal-type cell migration), neurotransmitter transport (neurotransmitter transport), Regulation of blood circulation (regulation of blood circulation), hormone transport (hormone transport), regulation of neurotransmitter levels (regulation of neurotransmitter levels), regulation of cellular responses to growth factor stimulation (regulation of cellular response to growth factor stimulus), neurotransmitter secretion (neurotransmitter secretion), synaptic signal release (SIGNAL RELEASE from synapse) and the like; The GO entries of the RZ VS db PPI network are mainly related to presynaptic (presynapse), ion channel complex (ion channel complex), distal axon (distal axon), receptor complex (receptor complex), synaptic membrane (synaptic membrane), collagen-containing extracellular matrix (collagen-containing extracellular matrix), gated channel activity (GATED CHANNEL ACTIVITY), and, Modulation of membrane potential (regulation of membrane potential), amoeba cell migration (ameboidal-type cell migration), gland development (gland development). Through KEGG analysis, PPI networks of RP VS db and RZ VS db were found to retrieve 36, 47 pathways (Padjust < 0.05), respectively. Wherein the pathways of the RP VSdb PPI network are mainly focused on renin secretion (Renin secretion), neuroactive ligand-receptor interactions (Neuroactive ligand-receptor interaction), calcium signaling pathway (Calcium SIGNALING PATHWAY), inflammatory mediator modulation of TRP channels (Inflammatory mediator regulation of TRP CHANNELS), circadian traction (CIRCADIAN ENTRAINMENT), and, wnt signaling pathway (WNT SIGNALING PATHWAY), cAMP signaling pathway (CAMP SIGNALING PATHWAY), glutamatergic synapse (Glutamatergic synapse), gnRP signaling pathway (GNRP SIGNALING PATHWAY), PI3K-Akt signaling pathway (PI 3K-AKT SIGNALING PATHWAY), and the like; The pathways of the RZ VSdb PPI network are focused primarily on the neuroactive ligand-receptor interactions (Neuroactive ligand-receptor interaction), circadian traction (CIRCADIAN ENTRAINMENT), renin secretion (Renin secretion), glutamatergic synapses (Glutamatergic synapse), GABAergic synapses (GABAergic synapse), cAMP signaling pathway (CAMP SIGNALING PATHWAY), and, Serotonergic synapses (Serotonergic synapse), PI3K-Akt signaling pathway (PI 3K-AKT SIGNALING PATHWAY), calcium signaling pathway (Calcium SIGNALING PATHWAY), wnt signaling pathway (WNT SIGNALING PATHWAY), and the like.
From this, seventy-ingredient pearl pills and subtractive seventy-ingredient pearl pills have certain differences in key targets and regulatory pathways, and by further analysis, kdr, tek, flt, edn1, col18a1, agt, itga6 related to key pathways in the key targets of the RP VS db PPI network are found; whereas the key target points of the RZ VSdb PPI network involve Spp1, kdr and Edn1 of key paths.
2.3 Expression and distribution of key differential genes in hippocampus
The binding literature studies found that Flt1, edn key targets, agt, itga6, spp1 were highly correlated with cognitive dysfunction, so their distribution in the hippocampus was observed by immunohistochemistry, further validating their expression. FIGS. 11 and 12 show the expression of Flt1, edn1, agt, itga6, spp1 in the CA3 and DG regions of the hippocampus of mice in the control, model, seventy pearl pill and subtractive seventy pearl pill groups. The mean integrated optical densities of the seventy pearl pill group mice hippocampal CA3 region and DG region Flt1, edn1, agt, itga6 were higher (P <0.05 or P < 0.01) compared to the model group, indicating that seventy pearl pill drugs were able to modulate the expression of these key targets, which is also consistent with sequencing results. Meanwhile, the expression of the CA3 region and the DG regions Edn and the Spp1 of the hippocampus of the mice in the reduced seventy-ingredient pearl pill group is obviously increased (P is less than 0.01) compared with that in the model group, but the key targets Flt1, agt and Itga6 of the seventy-ingredient pearl pill group have no obvious regulation effect, so that a certain difference exists between the efficacy of the seventy-ingredient pearl pill after removing the adjuvant ingredient and the efficacy of the seventy-ingredient pearl pill group.
In conclusion, according to the invention, through KEGG analysis of the sequencing differential genes, the neural activity ligand-receptor interaction is a key way for enriching db/db mouse hippocampal differential genes, and is also one of key ways for enriching seventy pearl pills and minus seventy pearl pill group differential genes. The interaction of neuroactive ligands and receptors plays an important role in regulating nervous system function. Different types of neurotransmitters and neuromodulatory substances regulate the activity and information transmission of neurons in the brain by binding to their corresponding receptors. Dysfunction of some neuroactive ligands and receptors is associated with cognitive dysfunction, such as neurological diseases like alzheimer's disease, parkinson's disease, depression, etc. In the treatment of cognitive dysfunction, modulation of neuroactive levels and receptor function is one of the basic strategies to improve cognitive and emotional function. The seventy-ingredient pearl pill can regulate the interaction path of the ligand-receptor of the hippocampal nerve activity, which also indicates that the pearl pill has a pharmacological mechanism for relieving the cognitive dysfunction of db/db mice, and the analysis result of the differential gene KEGG of the pharmaceutical intervention group indicates that the medicinal efficacy of the seventy-ingredient pearl pill and the medicinal efficacy of the minus seventy-ingredient pearl pill also have different pharmacological mechanisms.
In order to deeply explore the pharmacological mechanism of the presence or absence of zotai components of seventy pearl pills, the differential genes are further constructed into a PPI network, key differential genes are screened, and enrichment analysis is carried out. The results show that besides the neuroactive ligand-receptor interaction, the common pharmacological mechanism of seventy-ingredient pearl pills and subtractive seventy-ingredient pearl pills (without zotai) also relates to renin secretion, calcium signal pathway, circadian rhythm traction, wnt signal pathway, cAMP signal pathway and the like, and through the comprehensive analysis of screened differential genes and KEGG pathway, 6 (Kdr, tek, flt, edn1, col18a1, agt, itga 6) seventy-ingredient pearl pills and 3 (Spp 1, kdr, edn 1) seventy-ingredient pearl pills (without zotai) key targets are respectively obtained.
The invention also observes the expression condition of the key target points in the hippocampal tissues through immunohistochemistry, wherein seventy pearl pills improve the expression of the hippocampus Flt1, edn1, agt and Itga6 of the db/db mouse, and the seventy pearl pills (without zotai) reduce the expression of the hippocampus Edn1 and Spp1 of the db/db mouse, so that the invention further explains that the two have a certain difference in the drug effect for treating the cognitive dysfunction.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. The application of seventy-ingredient pearl pills in the regulation of cognitive dysfunction is characterized in that: seventy-ingredient pearl pills achieve the regulation of cognitive dysfunction by modulating key targets Kdr, tek, flt, edn1, col18a1, agt and Itga 6.
2. The use according to claim 1, characterized in that: seventy-ingredient pearl pills improve cognitive dysfunction by increasing the expression of Kdr, tek, flt1, edn1, col18a1, agt and Itga 6.
3. A molecular mechanism investigation method for seventy ingredient pearl pills for use in the modulation of cognitive dysfunction according to claim 1, comprising the steps of,
S1: the experimental animals are respectively fed with seventy pearl pill raw medicines and the reduced seventy pearl pills without zotai, and a model group and a control group are arranged for control;
S2: differential gene screening by transcriptional sequencing;
s3: determining seventy pearl pills and key targets of the seventy pearl pills for regulating the hippocampal tissues of experimental animals by PPI network analysis and Hub gene identification;
s4: immunohistochemical detection of the expression of key target proteins.
4. A method of investigation according to claim 3, characterized in that: the PPI network analysis in step S3 includes Stress, radiality, closeness, EPC, degree, MCC, MNC seven parameter analyses.
5. The method of investigation of claim 4, wherein: seventy pearl pills are used for regulating key targets of hippocampal tissues of experimental animals, including Kdr, tek, flt1, edn1, col18a1, agt and Itga6; the key targets of the seventy pearl pills for regulating the hippocampal tissues of experimental animals comprise Spp1, kdr and Edn1.
6. The method of investigation of claim 5, wherein: in step S4, the seventy pearl pill group has increased Flt1, edn1, agt and Itga6 expression in the CA3 region and DG region of the hippocampus of the experimental animal compared with the model group; the seventy pearl pill group had increased expression of Edn and Spp1 in the CA3 and DG regions of the hippocampus of the experimental animals compared to the model group.
7. Use of an agent that detects Flt1, edn1, agt, and Itga6 levels in the manufacture of a product for diagnosing cognitive dysfunction.
8. The use of claim 7, wherein the product comprises a kit and a fluorescent probe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410435243.1A CN118389663A (en) | 2024-04-11 | 2024-04-11 | Application of seventy-ingredient pearl pills in regulation of cognitive dysfunction and research method and application of molecular mechanism of seventy-ingredient pearl pills |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410435243.1A CN118389663A (en) | 2024-04-11 | 2024-04-11 | Application of seventy-ingredient pearl pills in regulation of cognitive dysfunction and research method and application of molecular mechanism of seventy-ingredient pearl pills |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118389663A true CN118389663A (en) | 2024-07-26 |
Family
ID=92006661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410435243.1A Pending CN118389663A (en) | 2024-04-11 | 2024-04-11 | Application of seventy-ingredient pearl pills in regulation of cognitive dysfunction and research method and application of molecular mechanism of seventy-ingredient pearl pills |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118389663A (en) |
-
2024
- 2024-04-11 CN CN202410435243.1A patent/CN118389663A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | MiR‐92b‐3p promotes neurite growth and functional recovery via the PTEN/AKT pathway in acute spinal cord injury | |
| WO2017157131A1 (en) | Use of chlorogenic acid in preparing pharmaceuticals for treatment of lag-3-mediated disease | |
| Kang et al. | Downregulation of microRNA‐124‐3p promotes subventricular zone neural stem cell activation by enhancing the function of BDNF downstream pathways after traumatic brain injury in adult rats | |
| Zhang et al. | Autophagy collaborates with apoptosis pathways to control oligodendrocyte number | |
| Rajagopalan et al. | Understanding the multi-functional role of TCTP in the regeneration process of Earthworm, Perionyx excavatus | |
| Lv et al. | An evidence update on the protective mechanism of tangeretin against neuroinflammation based on network pharmacology prediction and transcriptomic analysis | |
| CN118389663A (en) | Application of seventy-ingredient pearl pills in regulation of cognitive dysfunction and research method and application of molecular mechanism of seventy-ingredient pearl pills | |
| CN113440600A (en) | Application of lycium barbarum glycopeptide in preparation of medicine for relieving anxiety | |
| Qu et al. | Proteomic analysis of the sphincter in a neurogenic bladder caused by T10 spinal cord injury | |
| Gao et al. | Optogenetics stimulates nerve reorganization in the contralesional anterolateral primary motor cortex in a mouse model of ischemic stroke | |
| Clark et al. | Age‐related changes to macrophage subpopulations and TREM2 dysregulation characterize attenuated fracture healing in old mice | |
| Sun et al. | Unveiling proteomic targets in the hypothalamus of ovariectomized and estradiol-treated rats: Insights into menopausal syndrome mechanisms | |
| CN115105508B (en) | Application of Fei Dela tenib in preparation of medicine for treating head and neck squamous cell carcinoma with KRT18 low expression | |
| CN109890403A (en) | S100A8/S100A9-induced immune tolerance in neonatal subjects | |
| Khoder | Role of the prefrontal-brainstem pathway in mediating avoidance behavior | |
| Emmerich | DIVERGENT MECHANISMS UNDERLYING CELLULAR REGENERATION IN THE ZEBRAFISH RETINA | |
| CN108434175A (en) | Application of the intestinal flora in Dcf1 correlation Depressive behaviors | |
| CN117652457A (en) | Preparation method and application of rodent model of neurodegenerative disease | |
| Fei et al. | Timing dependent neuronal migration is regulated by Cdk5-mediated phosphorylation of JIP1 | |
| EP1404870A2 (en) | Function and application of tob gene in central nervous system of mammal | |
| CN117122705A (en) | Method for restoring adult neurogenesis of old hippocampus | |
| CN119488538A (en) | Application of lactobacillus plantarum in preparation of drug for preventing and/or treating Alzheimer's disease and drug containing lactobacillus plantarum | |
| Quansah et al. | Tet2 loss suppress α-synuclein pathology by stimulating ciliogenesis | |
| CN119280406A (en) | Use of ventral hippocampal transferrin receptor 1 inhibitor in the preparation of drugs for preventing and/or treating anxiety disorders | |
| Quintanilla | Septohippocampal Cholinergic Regulation of Distinct Hippocampal Functions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |