CN111920822A - Medicine for effectively treating cerebral arterial thrombosis and application thereof - Google Patents
Medicine for effectively treating cerebral arterial thrombosis and application thereof Download PDFInfo
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- CN111920822A CN111920822A CN202010811768.2A CN202010811768A CN111920822A CN 111920822 A CN111920822 A CN 111920822A CN 202010811768 A CN202010811768 A CN 202010811768A CN 111920822 A CN111920822 A CN 111920822A
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Abstract
The invention belongs to the technical field of cerebral apoplexy medicines, and discloses a medicine for effectively treating cerebral arterial thrombosis and application thereof. The invention obtains the target of the triptolide for resisting cerebral ischemia reperfusion injury by combining network pharmacology with transcriptomics, performs enrichment analysis and metabolic pathway analysis on the obtained target by bioinformatics, and further performs cell experimental verification, thereby providing a basis for scientific research of cerebral ischemia reperfusion injury mechanisms and development and utilization of the triptolide, and providing a reference for research and development of multi-target new drugs. The invention screens out the pathway and protein of triptolide acting on CIRI; the valuable pathways and proteins which are not deeply involved in other researches are selected, the action mechanism and the target of triptolide to resist CIRI are revealed through cell model verification analysis, and new therapeutic drugs and therapeutic targets are searched for CIRI treatment.
Description
Technical Field
The invention belongs to the technical field of cerebral apoplexy medicines, and particularly relates to a medicine for effectively treating cerebral arterial thrombosis and application thereof.
Background
At present, pathological damage of brain tissues caused by cerebral ischemia-reperfusion relates to different cell components and multiple pathological effects, the current research result of drug therapy is not ideal, only a single therapeutic target is possibly regarded as important, and the protection of the overall function of the brain is neglected. Research shows that triptolide can resist oxidation and protect nerves through different approaches and different targets, but no report exists at present on the systemic pharmacology research aiming at the CIRI resistance multi-target and multi-path of triptolide.
Through the above analysis, the problems and defects of the prior art are as follows: the existing systematic pharmacology research aiming at the multiple target points and multiple channels of triptolide CIRI resistance is not reported at present.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a medicine for effectively treating cerebral arterial thrombosis and application thereof.
The invention is realized in such a way that the medicine for effectively treating the ischemic stroke is triptolide.
The invention also aims to provide application of the medicine for effectively treating cerebral arterial thrombosis in treating cerebral ischemia reperfusion injury in cerebral arterial thrombosis.
Another object of the present invention is to provide a method for screening a target of triptolide for resisting cerebral ischemia-reperfusion injury, which uses the drug for effectively treating ischemic stroke, wherein the method for screening the target of triptolide for resisting cerebral ischemia-reperfusion injury comprises:
(1) predicting a potential target point of triptolide through network pharmacology;
(2) constructing a CIRI model, and observing the protective effect of triptolide on the CIRI through TTC and neurological deficit score;
(3) obtaining triptolide CIRI resistant differential expression genes in animal samples through a transcriptomics technology;
(4) predicting a potential target point of triptolide by network pharmacology, and matching the potential target point with a triptolide CIRI resistant differential expression gene obtained by a transcriptomics technology to obtain the triptolide CIRI resistant target point;
(5) protein interaction PPI analysis is carried out on the targets by using a String database, GO enrichment analysis and KEGG metabolic pathway analysis are carried out on the targets by using a Clue GO plug-in of Cytoscape3.7.1 software, and key proteins, enrichment classification and action pathways of triptolide CIRI resistance are disclosed.
Further, in step (1), the method for predicting the target by cyber pharmacology is as follows:
drawing a triptolide structural formula through ChemDraw Professional 17.0 software, storing the triptolide structural formula as an sdf format, logging in a Pharmmapper server, obtaining a potential target point of the triptolide according to software operation instructions, and introducing the PDB ID of the target point and the triptolide structural formula into a systemsDock system for reverse molecular docking verification.
Further, in the step (4), the transcriptome sequencing method comprises:
after extracting total RNA of a sample and digesting DNA by using DNase, enriching mRNA by using magnetic beads with oligo (dT); adding an interrupting reagent to break mRNA into short segments, synthesizing first-strand cDNA by using a six-base random primer by using the broken mRNA as a template, preparing a second-strand synthesis reaction system to synthesize second-strand cDNA, and purifying the double-strand cDNA by using a kit; carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, then carrying out fragment size selection, and finally carrying out PCR amplification; the constructed library was qualified by Agilent2100Bioanalyzer quality testing and then sequenced using a sequencer such as Illumina HiSeq X Ten.
Another object of the present invention is to provide a method for verifying a target of triptolide for resisting cerebral ischemia-reperfusion injury, which uses the drug for effectively treating ischemic stroke, the method comprising:
(I) establishing a CIRI model in vitro by HT22 cells, and observing the influence of triptolide with different concentrations on the viability and toxicity of the neuron cells in the OGD environment by an MTT method and an LDH method;
(II) verifying the screened key target by a Westernblot, RT-PCR and immunofluorescence method.
Further, in step (I), the HT22 cell culture method comprises the following steps:
cell culture HT22 cell is an immortalized mouse hippocampal neuron, and is cultured in F12 medium containing 10% fetal bovine serum and 1% double antibody under 5% CO2And an incubator at 37 ℃, HT22 cells in the logarithmic growth phase are taken, 0.25% of pancreatin is digested, and the cells are inoculated on a 96-well plate according to the density of about 5000 cells per well for subsequent experimental study.
The method for establishing the OGD/R model of the HT22 cells comprises the following steps:
taking HT22 cells in a logarithmic phase of culture, carrying out trypsinization, preparing a single cell suspension, inoculating the single cell suspension on a 96-well plate according to the density of 5000 cells per well, after the cells are attached to the wall, normally culturing for 24h, then removing the original culture medium, washing the cells for 3 times by PBS, replacing the cells with a DMEM sugar-free culture medium, placing the cells in an anaerobic incubator with preset parameters for culturing, starting timing when the oxygen concentration is reduced to 1%, after the oxygen sugar is deprived for 2h, replacing the DMEM sugar-free culture medium with a complete culture medium, and placing the DMEM sugar-free culture medium in a constant oxygen incubator again for 24h with reoxygenation.
Further, in step (I), the method for identifying the activity of HT22 cells and determining the injury rate comprises the following steps:
HT22 cell activity determination cells were inoculated into a 96-well plate, each group had 6 duplicate wells, each well had about 5000 cells, each group was repeated three times, each group was treated, the original medium was aspirated and discarded, PBS was washed 3 times, 100. mu.L DMEM and 10. mu.L MTT solution were added to each well, incubation was carried out at 37 ℃ for 1h, absorbance value (OD value) was determined at 450nm in a microplate reader, and cell activity was calculated by the ratio of OD value of each group to OD value of a normal control group.
HT22 cell injury rate determination cells were inoculated into 96-well plates, each group was provided with 6 multiple wells, each well was 5000 cells, each group was repeated three times, each group cell treatment was completed, 150. mu.L of each group cell culture supernatant was placed in a new 96-well plate, LDH reaction solution in the kit was added, incubation at 37 ℃ for 30min, and then the absorbance value (OD value) of the sample was measured at 490nm with a microplate reader. Cell LDH release rate (%). unit of enzyme activity measured in cell culture medium/(unit of enzyme activity measured in cell lysate + unit of enzyme activity measured in cell culture medium) × 100%. And averaging the results for statistical analysis.
Further, in the step (II), the method for measuring the expression of the screening protein by immunofluorescence comprises:
expression of the screening protein by immunofluorescence 4% paraformaldehyde fixation for 10min was performed on HT22 cells after each treatment. The serum was blocked and incubated with the selected protein antibody overnight at 4 ℃. The cells were washed three times with PBS and incubated for 30min at 37 ℃ with anti-rabbit secondary antibody. Staining the cell nucleus with a staining solution. Imaging was observed with a fluorescence microscope (Eclipse C1, nikon, japan).
Further, in the step (II), the RT-PCR method comprises the following steps:
extracting total RNA by using a Trizol method, designing primers and experimental conditions of target protein, and completing real-time quantitative PCR analysis strictly according to the operation flows of a reverse transcription kit and a real-time quantitative PCR kit instruction.
The method for measuring and screening the expression quantity of the related protein by using Western-Blot comprises the following steps:
western-blot method for determining expression level of screening-related protein Each group of cells was lysed with lysis buffer containing protease inhibitor, and adherently grown HT22 cells were carefully scraped off with a spatula and carefully collected in a 2ml centrifuge tube, and then placed in a refrigerated centrifuge for centrifugation at 12000r/min at 4 ℃ for 10min, and the supernatant was extracted. The BCA method determines the protein concentration. Carrying out Western blotting experiment on 50 mu g of protein, carrying out electrophoresis, carrying out membrane transfer, blocking 5% fetal Bovine Serum (BSA) at room temperature for 2h, adding a primary antibody, then incubating overnight at 4 ℃, washing the membrane for 3 times and 5 min/time by TBST, adding an anti-rabbit secondary antibody, incubating at room temperature for 1h, and washing the membrane for 3 times and 5 min/time by TBST. ECL chemiluminescence was then repeated three times for each experiment.
Statistical processing was analyzed using GraphpadPrism 6.0 software. Data are expressed as means ± standard deviation (x ± s), One-way analysis of variance (One-way anova) is used for the comparison between groups, and P < 0.05 indicates that the difference is statistically significant.
By combining all the technical schemes, the invention has the advantages and positive effects that: the medicine for effectively treating ischemic stroke provided by the invention obtains the target of the triptolide for resisting cerebral ischemia reperfusion injury by combining network pharmacology with transcriptomics, and then carries out enrichment analysis and metabolic pathway analysis on the obtained target by bioinformatics, and further carries out cell experimental verification, thereby providing a basis for scientific research of cerebral ischemia reperfusion injury mechanism and development and utilization of the triptolide, and providing a reference for research and development of multi-target new medicines.
The invention screens out the pathway and protein of triptolide acting on CIRI; the valuable pathways and proteins which are not deeply involved in other researches are selected, the action mechanism and the target of triptolide to resist CIRI are revealed through cell model verification analysis, and new therapeutic drugs and therapeutic targets are searched for CIRI treatment.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
FIG. 1 is a flow chart of a method for screening a target of triptolide for resisting cerebral ischemia-reperfusion injury provided by an embodiment of the invention.
Fig. 2 is a flowchart of a method for verifying a target point of triptolide for resisting cerebral ischemia-reperfusion injury provided by an embodiment of the invention.
Fig. 3 is a technical route diagram of a triptolide CIRI-resistance mechanism combining network pharmacology and transcriptomics provided in an embodiment of the present invention.
Fig. 4 is a cell experimental verification roadmap of the triptolide anti-CIRI mechanism of action provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a medicament for effectively treating cerebral arterial thrombosis and application thereof, and the invention is described in detail below with reference to the accompanying drawings.
The medicine for effectively treating the cerebral arterial thrombosis provided by the embodiment of the invention is triptolide.
As shown in fig. 1, the method for screening a target of triptolide for resisting cerebral ischemia-reperfusion injury provided by the embodiment of the invention comprises:
s101, predicting the potential target of triptolide through network pharmacology.
S102, constructing a CIRI model, and observing the protective effect of triptolide on the CIRI through TTC and neurological deficit score.
S103, obtaining the triptolide CIRI resistant differential expression gene in the animal specimen through a transcriptomics technology.
S104, predicting the potential target of triptolide through network pharmacology, and matching the potential target with the triptolide CIRI resistant differential expression gene obtained through the transcriptomics technology to obtain the triptolide CIRI resistant target.
S105, carrying out protein interaction PPI analysis on the target by using a String database, and carrying out GO enrichment analysis and KEGG metabolic pathway analysis on the target by using a Cytoscape3.7.1 software Clue GO plug-in, thereby revealing key proteins, enrichment classification and action pathways of triptolide CIRI resistance.
As shown in fig. 2, the method for verifying the target point of triptolide for resisting cerebral ischemia-reperfusion injury provided by the embodiment of the invention comprises:
s201, establishing a CIRI model in vitro through HT22 cells, and observing the influence of triptolide with different concentrations on the viability and toxicity of the neuron cells in the OGD environment through an MTT method and an LDH method.
S202, verifying the screened key target by a Westernblot, RT-PCR and immunofluorescence method.
The technical solution of the present invention is further described with reference to the following examples.
Example 1: research on molecular mechanism of triptolide for resisting cerebral ischemia-reperfusion injury based on transcriptomics and cybepharmacology, namely the triptolide resists the cerebral ischemia-reperfusion injury through a PI3K signal channel.
Brief introduction of items: researches find that the stroke incidence rate of people in China is higher than that of people in Europe and America, and the research is carried out deeply on the pathophysiological mechanism of reperfusion injury after cerebral ischemia, so that the research has important research value and clinical significance for finding a medicament for effectively treating ischemic stroke. Triptolide is an epoxy diterpene lactone compound extracted from roots, leaves, flowers and fruits of tripterygium wilfordii which is a traditional Chinese medicine Celastraceae plant in China, is a main active ingredient of tripterygium wilfordii extract, and early researches show that the triptolide has pharmacological activities of resisting oxidation, resisting rheumatoid diseases, resisting senile dementia, resisting cancers, resisting acute cerebral ischemia injury and the like. However, the triptolide target is comprehensively predicted in an ischemia-reperfusion injury model through a network pharmacology and transcriptomics research method, and the mechanism for revealing the anti-cerebral ischemia-reperfusion injury effect of the triptolide target is not reported. The subject aims to obtain a differential expression gene of triptolide for resisting cerebral ischemia reperfusion injury in an animal model through a transcriptomics technology, match the differential expression gene with a triptolide potential target obtained through network pharmacology, and perform PPI, GO enrichment, metabolic pathway and other bioinformatics analysis on the matched target to obtain valuable pathways and proteins which are not deeply involved in other researches. And finally, in an in vitro cerebral ischemia reperfusion injury model, verifying the authenticity of the pathway and the protein by Western blot, RT-PCR, immunofluorescence and other methods. Further reveals the action mechanism and target of triptolide for resisting cerebral ischemia reperfusion injury.
First, objective and task demand analysis
The pathological damage of brain tissue caused by cerebral ischemia-reperfusion relates to different cell components and multiple pathological effects, the current research result of the drug therapy is not ideal, only a single therapeutic target is possibly regarded as important, and the protection of the whole function of the brain is neglected. Research shows that triptolide can resist oxidation and protect nerves through different approaches and different targets, but no report exists at present on the systemic pharmacology research aiming at the CIRI resistance multi-target and multi-path of triptolide. In the early prediction research of the invention, the triptolide can act on 16 CIRI targets, and the KEGG metabolic pathway result shows that the targets participate in pathways such as a neurotrophic factor signal pathway, an IL-17 signal pathway, apoptosis and the like. Through transcriptomics analysis, the related information of the RNA level of gene expression is obtained, the internal relation between the gene expression and the life phenomenon of the organism in physiological and pathological states can be explored, and the research on the pathogenesis of CIRI and the development of multi-target new drugs are facilitated. The subject aims to obtain triptolide CIRI-resistant male C57BL/6 mouse differential expression genes through a transcriptomics technology, match the triptolide potential targets obtained through network pharmacology, perform bioinformatics analysis on the matched targets, comprehensively screen out triptolide CIRI-resistant mechanisms, select valuable pathways and proteins which are not deeply involved in other researches, and finally verify the authenticity of the pathways and proteins in an in-vitro CIRI model through Western blot, RT-PCR, immunofluorescence and other methods. Further reveals the action mechanism and target of triptolide for resisting cerebral ischemia reperfusion injury.
Second, feasibility analysis
The invention combines bioinformatics with a transcriptome sequencing method in an animal model to find a new path and a target point of triptolide acting on CIRI, and the method is feasible. The early prediction result of the invention shows that multiple pathways and targets of triptolide action are related to CIRI, and the theory is feasible. The project declarers have a solid bioinformatics theoretical basis and practice, and the subject groups are engaged in CIRI related research for a long time, so that the smooth experiment can be better ensured.
Second, feasibility analysis
The invention combines bioinformatics with a transcriptome sequencing method in an animal model to find a new path and a target point of triptolide acting on CIRI, and the method is feasible. The early prediction result of the invention shows that multiple pathways and targets of triptolide action are related to CIRI, and the theory is feasible. The project declarers have a solid bioinformatics theoretical basis and practice, and the subject groups are engaged in CIRI related research for a long time, so that the smooth experiment can be better ensured.
Third, research content
1. Screening of target point of triptolide for resisting cerebral ischemia reperfusion injury
(1) Predicting the potential target of triptolide through network pharmacology.
(2) And (3) constructing a CIRI model, and observing the protective effect of triptolide on the CIRI through TTC and neurological deficit score.
(3) Obtaining the triptolide CIRI resistant differential expression gene in the animal specimen by transcriptomics technology.
(4) And (3) predicting the potential target of triptolide by network pharmacology, and matching the potential target with the triptolide CIRI resistant differential expression gene obtained by the transcriptomics technology to obtain the triptolide CIRI resistant target.
(5) And (3) carrying out protein interaction (PPI) analysis on the target by using a String database, and carrying out GO enrichment analysis and KEGG metabolic pathway analysis on the target by using a Clue GO plug-in of Cytoscape3.7.1 software to reveal key proteins, enrichment classification, action pathways and the like of triptolide CIRI resistance.
2. Verification of target point of triptolide for resisting cerebral ischemia reperfusion injury
(1) A CIRI model is constructed in vitro by HT22 cells, and the influence of triptolide with different concentrations on the viability and toxicity of the neuron cells in the OGD environment is observed by an MTT method and an LDH method.
(2) The screened key target spot is verified by a Westernblot, RT-PCR and immunofluorescence method.
Fourth, research method
The invention relates to a research project of interdisciplinary and interpenetration of multiple subjects such as transcriptomics, cybepharmacology, bioinformatics and the like, which starts from the overall view, uses triptolide as an object, uses male C57BL/6 mice and HT22 Cell as carriers, and discloses an action mechanism and a target of triptolide for resisting CIRI. The invention discloses the action target and metabolic pathway of triptolide for resisting CIRI by using a mode of multidisciplinary crossing and mutual complementation verification such as bioinformatics, transcriptomics and the like, establishes further verification in cells through a construct external model, and is expected to provide a basis for scientific research of CIRI mechanism and development and utilization of triptolide and provide a reference for research and development of multi-target new drugs.
Fifth, technical route
Animal experiments: the technical route diagram of triptolide anti-CIRI mechanism combining network pharmacology and transcriptomics is shown in fig. 3.
Cell validation experiment: a cell experiment verification route chart of the action mechanism of triptolide against CIRI is shown in FIG. 4.
Sixth, concrete experimental scheme
1. Network pharmacology prediction target
Drawing a triptolide structural formula through ChemDraw Professional 17.0 software, storing the triptolide structural formula as an sdf format, logging in a Pharmmapper server, obtaining a potential target point of the triptolide according to software operation instructions, and introducing the PDB ID of the target point and the triptolide structural formula into a systemsDock system for reverse molecular docking verification.
2. Grouping and administration of C57B/L mice
30 male mice of C57/BL were randomly assigned to a Sham group (Sham), a model group (CIRI) and a Dendrobine administration group (5mg/kg), and Dendrobine was mixed with DMSO1:1 and dissolved in 0.9% physiological saline to prepare a Dendrobine solution at a concentration of 5 mg/kg. At 72 hours before operation, mice in the Dendrobine group were intraperitoneally injected with Dendrobine (5mg/kg), and mice in the Sham group and CIR group were injected with the same amount of solvent.
3. Establishment of C57B/L mouse CIRI model
A mouse model of cerebral central artery ischemia was established with reference to the modified line-embolization method of Longa et al. Using 1% sodium isoamyl barbital, 80mg/Kg, anaesthetizing by intraperitoneal injection, fixing in supine position, preparing skin, and cutting an incision of about 2cm along the median line of the neck at 1cm of the lower jaw. The right common carotid artery and the internal and external carotid arteries were separated and exposed with a glass needle. Ligating the root of right common carotid artery and external carotid artery, clamping the internal carotid artery with a arteriole clamp, cutting a small opening at the proximal bifurcation of the common carotid artery, inserting MCAO embolus to the internal carotid artery, stopping when slight resistance is felt, and drawing out the thread to realize reperfusion after 2h of ischemia. The electric blanket is used for heating in the operation process, so that the body temperature of the mouse is maintained at about 37 ℃, and the skin is sutured. Sham group (sham group) treatment procedure was the same as the surgical group, but no plug wire was inserted.
4. Neuroethological scoring
All mice were evaluated for neurological deficits using Longa5 grade 4 scoring. The scoring criteria were as follows: zero order: no neurological deficit; first-stage: the contralateral forelimb cannot be fully extended when lifted by the tail; and (2) second stage: turning to the paralytic side spontaneously when walking; third-stage: the opposite side is not consciously fallen down when walking; and (4) fourth stage: the user can not walk independently and lose consciousness.
5. Cerebral infarction volume determination
Brain sections were stained using TTC to assess brain infarct volume. After 24 hours of reperfusion, 4% pentobarbital sodium (40mg/kg) was intraperitoneally injected to anesthetize the mice, followed immediately by decapitation, and the mouse brains were completely and carefully removed, and then each coronal section was cut into 5 pieces, each about 2 mm thick. Incubating the sample in 2% TTC dye solution for 30, heating in water bath to ensure that the temperature is 37 ℃ and keeping away from light, separating for 15min midway, slicing the sample, turning over, and finally fixing 4% paraformaldehyde overnight and then taking a picture. The unstained area (white area) was considered infarct and the percentage of infarct volume was then quantitatively assessed using Image-pro Plus software.
6. Transcriptome sequencing
After extracting total RNA of a sample and digesting DNA by using DNase, enriching mRNA by using magnetic beads with oligo (dT); adding an interrupting reagent to break mRNA into short segments, synthesizing first-strand cDNA by using a six-base random primer by using the broken mRNA as a template, preparing a second-strand synthesis reaction system to synthesize second-strand cDNA, and purifying the double-strand cDNA by using a kit; carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, then carrying out fragment size selection, and finally carrying out PCR amplification; the constructed library was qualified by Agilent2100Bioanalyzer quality testing and then sequenced using a sequencer such as Illumina HiSeq X Ten.
7. Bioinformatics analysis
Introducing the triptolide CIRI-resistant target spot into a String database for PPI analysis, then introducing the triptolide CIRI-resistant target spot into cytoscape3.7.1 software for visualization, and analyzing key target protein by using a cytoshubba plug-in; in addition, the Clue GO plug-in is used for carrying out GO enrichment analysis and KEGG metabolic pathway analysis on the target spots.
8. HT22 cell culture
Cell culture HT22 cell is an immortalized mouse hippocampal neuron, and is cultured in F12 medium containing 10% fetal bovine serum and 1% double antibody under 5% CO2And an incubator at 37 ℃, HT22 cells in the logarithmic growth phase are taken, 0.25% of pancreatin is digested, and the cells are inoculated on a 96-well plate according to the density of about 5000 cells per well for subsequent experimental study.
9. Establishment of OGD/R model of HT22 cells
Taking HT22 cells in a logarithmic phase of culture, carrying out trypsinization, preparing a single cell suspension, inoculating the single cell suspension on a 96-well plate according to the density of 5000 cells per well, after the cells are attached to the wall, normally culturing for 24h, then removing the original culture medium, washing the cells for 3 times by PBS, replacing the cells with a DMEM sugar-free culture medium, placing the cells in an anaerobic incubator with preset parameters for culturing, starting timing when the oxygen concentration is reduced to 1%, after the oxygen sugar is deprived for 2h, replacing the DMEM sugar-free culture medium with a complete culture medium, and placing the DMEM sugar-free culture medium in a constant oxygen incubator again for 24h with reoxygenation.
10. HT22 cell activity identification and injury rate determination
HT22 cell activity determination cells were inoculated into a 96-well plate, each group had 6 duplicate wells, each well had about 5000 cells, each group was repeated three times, each group was treated, the original medium was aspirated and discarded, PBS was washed 3 times, 100. mu.L DMEM and 10. mu.L MTT solution were added to each well, incubation was carried out at 37 ℃ for 1h, absorbance value (OD value) was determined at 450nm in a microplate reader, and cell activity was calculated by the ratio of OD value of each group to OD value of a normal control group.
HT22 cell injury rate determination cells were inoculated into 96-well plates, each group was provided with 6 multiple wells, each well was 5000 cells, each group was repeated three times, each group cell treatment was completed, 150. mu.L of each group cell culture supernatant was placed in a new 96-well plate, LDH reaction solution in the kit was added, incubation at 37 ℃ for 30min, and then the absorbance value (OD value) of the sample was measured at 490nm with a microplate reader. Cell LDH release rate (%). unit of enzyme activity measured in cell culture medium/(unit of enzyme activity measured in cell lysate + unit of enzyme activity measured in cell culture medium) × 100%. And averaging the results for statistical analysis.
11. Grouping of HT22 cells
Cells were randomly divided into 3 groups: normal Control group without any treatment (Control group), OGD/R group, OGD/R + triptolide treatment group (3 mg/ml).
12. Immunofluorescence assay screening for protein expression
Expression of the screening protein by immunofluorescence 4% paraformaldehyde fixation for 10min was performed on HT22 cells after each treatment. The serum was blocked and incubated with the selected protein antibody overnight at 4 ℃. The cells were washed three times with PBS and incubated for 30min at 37 ℃ with anti-rabbit secondary antibody. Staining the cell nucleus with a staining solution. Imaging was observed with a fluorescence microscope (Eclipse C1, nikon, japan).
13、RT-PCR
Extracting total RNA by using a Trizol method, designing primers and experimental conditions of target protein, and completing real-time quantitative PCR analysis strictly according to the operation flows of a reverse transcription kit and a real-time quantitative PCR kit instruction.
14. Protein expression quantity related to screening by Western blotting (Western-Blot) assay
Western-blot method for determining expression level of screening-related protein Each group of cells was lysed with lysis buffer containing protease inhibitor, and adherently grown HT22 cells were carefully scraped off with a spatula and carefully collected in a 2ml centrifuge tube, and then placed in a refrigerated centrifuge for centrifugation at 12000r/min at 4 ℃ for 10min, and the supernatant was extracted. The BCA method determines the protein concentration. Performing Western blotting experiment on 50 mu g of protein, performing electrophoresis, transferring a membrane, blocking 5% fetal Bovine Serum (BSA) at room temperature for 2h, adding a primary antibody, incubating at 4 ℃ overnight, washing the membrane for 3 times (5 min/time) with TBST, adding an anti-rabbit secondary antibody, incubating at room temperature for 1h, and washing the membrane for 3 times (5 min/time) with TBST. ECL chemiluminescence was then repeated three times for each experiment.
15. Statistical treatment
Statistical processing was analyzed using GraphpadPrism 6.0 software. Data are expressed as means ± standard deviation (x ± s), One-way analysis of variance (One-way anova) is used for the comparison between groups, and P < 0.05 indicates that the difference is statistically significant.
Seventh, innovation point and technical difficulty
The invention obtains the target of the triptolide for resisting cerebral ischemia reperfusion injury by combining network pharmacology with transcriptomics, performs enrichment analysis and metabolic pathway analysis on the obtained target by bioinformatics, and further performs cell experimental verification, thereby providing a basis for scientific research of cerebral ischemia reperfusion injury mechanisms and development and utilization of the triptolide, and providing a reference for research and development of multi-target new drugs.
Screening out the pathway and protein of triptolide acting on CIRI; the valuable pathways and proteins which are not deeply involved in other researches are selected, the action mechanism and the target of triptolide to resist CIRI are revealed through cell model verification analysis, and new therapeutic drugs and therapeutic targets are searched for CIRI treatment.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The medicine for effectively treating the ischemic stroke is characterized in that the medicine for effectively treating the ischemic stroke is triptolide.
2. The use of the agent of claim 1 for treating ischemic stroke in the treatment of cerebral ischemia reperfusion injury in stroke.
3. A method for screening the target of triptolide for resisting cerebral ischemia-reperfusion injury, which is applied to the medicine for effectively treating ischemic stroke according to claim 1, wherein the method for screening the target of triptolide for resisting cerebral ischemia-reperfusion injury comprises:
(1) predicting a potential target point of triptolide through network pharmacology;
(2) constructing a CIRI model, and observing the protective effect of triptolide on the CIRI through TTC and neurological deficit score;
(3) obtaining triptolide CIRI resistant differential expression genes in animal samples through a transcriptomics technology;
(4) predicting a potential target point of triptolide by network pharmacology, and matching the potential target point with a triptolide CIRI resistant differential expression gene obtained by a transcriptomics technology to obtain the triptolide CIRI resistant target point;
(5) protein interaction PPI analysis is carried out on the targets by using a String database, GO enrichment analysis and KEGG metabolic pathway analysis are carried out on the targets by using a Clue GO plug-in of Cytoscape3.7.1 software, and key proteins, enrichment classification and action pathways of triptolide CIRI resistance are disclosed.
4. The method for screening the target point of triptolide for resisting cerebral ischemia-reperfusion injury according to claim 3, wherein in the step (1), the method for predicting the target by network pharmacology comprises the following steps:
drawing a triptolide structural formula through ChemDraw Professional 17.0 software, storing the triptolide structural formula as an sdf format, logging in a Pharmmapper server, obtaining a potential target point of the triptolide according to software operation instructions, and introducing the PDB ID of the target point and the triptolide structural formula into a systemsDock system for reverse molecular docking verification.
5. The method for screening the target point of triptolide for resisting cerebral ischemia-reperfusion injury according to claim 3, wherein in the step (4), the transcriptome sequencing method comprises:
after extracting total RNA of a sample and digesting DNA by using DNase, enriching mRNA by using magnetic beads with oligo (dT); adding an interrupting reagent to break mRNA into short segments, synthesizing first-strand cDNA by using a six-base random primer by using the broken mRNA as a template, preparing a second-strand synthesis reaction system to synthesize second-strand cDNA, and purifying the double-strand cDNA by using a kit; carrying out end repair on the purified double-stranded cDNA, adding A tail and connecting a sequencing joint, then carrying out fragment size selection, and finally carrying out PCR amplification; the constructed library was qualified by Agilent2100Bioanalyzer quality testing and then sequenced using a sequencer such as Illumina HiSeq X Ten.
6. The method for verifying the target of triptolide for resisting cerebral ischemia-reperfusion injury by using the medicine for effectively treating ischemic stroke according to claim 1, wherein the method for verifying the target of triptolide for resisting cerebral ischemia-reperfusion injury comprises:
(I) establishing a CIRI model in vitro by HT22 cells, and observing the influence of triptolide with different concentrations on the viability and toxicity of the neuron cells in the OGD environment by an MTT method and an LDH method;
(II) verifying the screened key target by Western blot, RT-PCR and immunofluorescence method.
7. The method for verifying the target of triptolide for resisting cerebral ischemia-reperfusion injury according to claim 6, wherein in the step (I), the HT22 cell culture method comprises the following steps:
cell culture HT22 cell is an immortalized mouse hippocampal neuron, and is cultured in F12 medium containing 10% fetal bovine serum and 1% double antibody under 5% CO2Incubating at 37 deg.C, collecting HT22 cells in logarithmic growth phase, and pancreatin 0.25%Inoculating the cells on a 96-well plate according to the density of 5000 cells per well for subsequent experimental study;
the method for establishing the OGD/R model of the HT22 cells comprises the following steps:
taking HT22 cells in a logarithmic phase of culture, carrying out trypsinization, preparing a single cell suspension, inoculating the single cell suspension on a 96-well plate according to the density of 5000 cells per well, after the cells are attached to the wall, normally culturing for 24h, then removing the original culture medium, washing the cells for 3 times by PBS, replacing the cells with a DMEM sugar-free culture medium, placing the cells in an anaerobic incubator with preset parameters for culturing, starting timing when the oxygen concentration is reduced to 1%, after the oxygen sugar is deprived for 2h, replacing the DMEM sugar-free culture medium with a complete culture medium, and placing the DMEM sugar-free culture medium in a constant oxygen incubator again for 24h with reoxygenation.
8. The method for verifying the target of triptolide for resisting cerebral ischemia-reperfusion injury according to claim 6, wherein in the step (I), the HT22 cell activity identification and injury rate determination method comprises the following steps:
HT22 cell activity determination is carried out on inoculated cells in a 96-well plate, each group is provided with 6 multiple wells, each well is provided with 5000 cells, each group is repeated for three times, the cell treatment of each group is finished, the original culture medium is removed, PBS is washed for 3 times, 100 mu L DMEM and 10 mu L MTT solution are added into each well, the incubation is carried out for 1h at 37 ℃, the absorbance value (OD value) is determined at 450nm of an enzyme labeling instrument, and the cell activity is calculated by the ratio of the OD value of each group to the OD value of a normal control group;
HT22 cell damage rate determination is carried out on inoculated cells in a 96-well plate, each group is provided with 6 multiple wells, each well is provided with 5000 cells, each group is repeated for three times, the cell treatment of each group is finished, 150 mu L of cell culture solution supernatant of each group is taken and placed in a new 96-well plate, LDH reaction solution in the kit is added, incubation is carried out for 30min at 37 ℃, and then the absorbance value (OD value) of a sample is measured at 490nm of an enzyme-labeling instrument; cell LDH release rate (%). enzymatic activity units measured in cell culture medium/(enzymatic activity units measured in cell lysate + enzymatic activity units measured in cell culture medium) × 100%; and averaging the results for statistical analysis.
9. The method for verifying the target point of triptolide for resisting cerebral ischemia-reperfusion injury according to claim 6, wherein in the step (II), the method for detecting the expression of the screening protein by immunofluorescence comprises the following steps:
detecting the expression of the screening protein by an immunofluorescence method, and fixing HT22 cells subjected to treatment in each group for 10min by 4% paraformaldehyde; sealing the mountain blood serum, and then incubating overnight at 4 ℃ by using a screening protein antibody; washing the cells with PBS for three times, and incubating the anti-rabbit secondary antibody at the constant temperature of 37 ℃ for 30 min; adopting a dye solution to dye cell nucleuses; the image was observed with a fluorescence microscope.
10. The method for verifying the target point of triptolide for resisting cerebral ischemia-reperfusion injury according to claim 6, wherein in the step (II), the RT-PCR method comprises:
extracting total RNA by using a Trizol method, designing primers and experimental conditions of target protein, and completing real-time quantitative PCR analysis strictly according to the operation flows of a reverse transcription kit and a real-time quantitative PCR kit instruction;
the method for measuring and screening the expression quantity of the related protein by using Western-Blot comprises the following steps:
the expression quantity of the relevant proteins for screening is measured by a Western-blot method, each group of cells is cracked by using a cracking buffer solution containing a protease inhibitor, the HT22 cells which grow by adherence are carefully scraped by a scraper and carefully collected in a 2ml centrifugal tube, and then the cell is placed in a refrigerated centrifuge for centrifugation at 12000r/min at 4 ℃ for 10min to extract supernatant; determining the protein concentration by using a BCA method; carrying out Western blotting experiment on 50 mu g of protein, carrying out electrophoresis, carrying out membrane transfer, sealing 5% fetal bovine serum BSA at room temperature for 2h, adding a primary antibody, then incubating overnight at 4 ℃, washing the membrane for 3 times and 5 min/time by TBST, adding an anti-rabbit secondary antibody, incubating at room temperature for 1h, and washing the membrane for 3 times and 5 min/time by TBST; ECL chemiluminescence was then repeated three times per experiment;
statistical processing was analyzed using GraphpadPrism 6.0 software; data are expressed as means ± standard deviation (x ± s), One-way analysis of variance (One-way anova) is used for the comparison between groups, and P < 0.05 indicates that the difference is statistically significant.
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