Biomarker, therapeutic drug and action target for traumatic brain injury

Abstract

The invention discloses a biological marker, a therapeutic drug and an action target spot for traumatic brain injury, and belongs to the technical field of biological medicine. The miRNA is selected from one or more of rno-miR-382-3p, rno-miR-21-5p, rno-miR-370-3p and the like, and the miRNA is one or more of the following components: the rno-miR-382-3p and the like. The invention also provides an application of the ginsenoside Rg1 in preparing a medicine for regulating and controlling the expression quantity of the miRNA in the claim 1 and an action target of the ginsenoside Rg1 for treating TBI by regulating and controlling the expression quantity of the miRNA in the claim 1, wherein the action target mainly comprises AKT1, EGFR, MMP9, SRC, STAT3 and CASP3. The method has the beneficial effects that the miRNAs expression change of exosomes in plasma after traumatic brain injury is evaluated through a high-throughput RNA sequencing technology, and 14 miRNAs are screened and proposed and can be used as biomarkers for diagnosing traumatic brain injury; and the influence of the Rg1 on the expression of the miRNAs in the plasma after trauma is further researched so as to determine the effectiveness and the targeting action way of treating the trauma by the Rg1.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to biomarkers, therapeutic drugs and action targets of traumatic brain injury. Background Art

[0002] Traumatic brain injury (TBI) is a traumatic structural injury or physiological destruction of the brain caused by external force. It is one of the most common brain diseases in neurosurgery and emergency departments. Due to the increasing incidence of traffic accidents and fall trauma, more and more people seek medical care for traumatic brain injury (TBI) every year. Among these individuals, 75% or more are considered to have mild or moderate TBI. Although severe TBI is one of the main causes of death or disability in patients, mild or moderate TBI is the main cause of long-term disability, including epileptic seizures and emotional and behavioral problems. TBI damage to the brain and intracranial contents can be divided into primary injury and delayed secondary injury. The latter is more serious, including axonal shearing, neuroinflammation, neurochemical changes, etc. Currently, there are limited diagnostic and treatment strategies for TBI.

[0003] Cerebrospinal fluid (CSF) or blood tests are useful in assessing the severity of injury and subsequent recovery of TBI patients by detecting protein or miRNA components in peripheral blood. However, these tests have detection sensitivity issues due to the low concentration of the substances.

[0004] miRNA is a single-stranded, endogenous, non-protein-coding small RNA molecule of about 22 nucleotides. miRNAs play a unique role in gene regulation at the post-transcriptional level. miRNAs can degrade the corresponding regions of specific target mRNAs and interact with them, resulting in degradation of mRNA or expression inhibition. To date, there are about 721 confirmed human miRNAs and about 579 mouse miRNAs, which regulate about 30% of human protein-coding genes. One miRNA can regulate multiple target genes, and at the same time, the same gene can be jointly regulated by multiple miRNAs. miRNAs are associated with the formation and development of a variety of diseases through upregulation or downregulation, such as atherosclerosis (AS), nasopharyngitis (nasopharyngitis), cerebrovascular disease (CNS), etc. After brain trauma, exosomes containing intracranial substances derived from brain cells cross the blood-brain barrier (BBB) ​​and enter the peripheral blood, and transfer brain substances to the peripheral blood through the BBB. Therefore, they may serve as biomarkers and regulators for the diagnosis and prognosis of TBI.

[0005] Ginseng is a classic medicine that has a wide range of effects on the central nervous system, cardiovascular system, digestive system, immune system, endocrine system and urogenital system. It can improve people's physical and intellectual activities and enhance the body's nonspecific resistance to harmful stimuli. Ginsenoside Rg1 is a sterol compound. It is mainly found in ginseng herbs. Ginsenoside Rg1 is the active ingredient in ginseng, which has the effects of improving neural plasticity, repairing function, anti-aging, and improving immunity. However, how ginsenoside Rg1 regulates miRNA and whether it has a therapeutic effect on traumatic brain injury has not been studied in detail. Summary of the invention

[0006] The technical problem to be solved by the present invention is how to propose a biomarker and a therapeutic drug for traumatic brain injury.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The first aspect of the present invention proposes the use of miRNA as a biomarker in the preparation of a reagent for diagnosing traumatic brain injury, wherein the miRNA is one or more of rno-miR-382-3p, rno-miR-21-5p, rno-miR-370-3p, rno-miR-122-5p, rno-miR-344b-1-3p, rno-miR-188-5p, rno-miR-3099, rno-miR-7a-2-3p, rno-miR-3576, rno-miR-7b, rno-miR-142-3p, rno-miR-7a-5p, rno-miR-137-3p, and rno-miR-410-3p (a total of 14 types);

[0009] The nucleotide sequences of rno-miR-382-3p to rno-miR-410-3p are shown in SEQ ID No.1 to 14.

[0010] Preferably, the reagent is used in a method for detecting miRNA as a biomarker.

[0011] Preferably, the method for detecting the miRNA content as a biomarker is southern blot, northern blot, polymerase chain reaction, reverse transcriptase PCR, quantitative real-time PCR, nanoarray, macroarray, autoradiography, Solexa sequencing, TLDA chip detection or in situ hybridization.

[0012] The second aspect of the present invention proposes the use of ginsenoside Rg1 in the preparation of a drug for regulating the expression level of the above-mentioned miRNA.

[0013] The third aspect of the present invention proposes that ginsenoside Rg1 treats TBI targets by regulating the expression of the above-mentioned miRNAs, which are characterized in that they mainly include AKT1, EGFR, MMP9, SRC, STAT3, and CASP3.

[0014] The fourth aspect of the present invention provides a drug for regulating the expression level of the above-mentioned miRNA, wherein the active ingredient of the drug includes ginsenoside Rg1.

[0015] Preferably, it further contains a pharmaceutically acceptable carrier.

[0016] Preferably, the pharmaceutically acceptable carrier is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

[0017] Preferably, it is prepared into a pharmaceutically acceptable dosage form.

[0018] Preferably, the dosage form is tablet, pill, paste, capsule, oral solution or granule.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present invention uses high-throughput RNA sequencing technology to evaluate the changes in miRNAs expression in exosomes in plasma after traumatic brain injury, and screens and proposes 14 miRNAs that can be used as biomarkers for diagnosing traumatic brain injury; and further studies the effect of ginsenoside Rg1 on the expression of miRNAs in plasma after trauma, in order to determine the effectiveness and targeted action pathway of ginsenoside Rg1 in treating traumatic brain injury.

[0021] 2. The present invention reveals several new potential roles of these differentially expressed miRNAs in peripheral blood plasma exosomes after TBI, the most important of which is the prediction of the targeting pathway of Rg1 in treating TBI. The key targets of Rg1 in treating traumatic brain injury mainly include AKT1, EGFR, MMP9, SRC, STAT3, and CASP3. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a comparison of HE staining of rat brain tissue sections in the control group, TBI model group, Rg1 administration group and blebbistatin group in Example 1 of the present invention;

[0023] Figure 2 This is a diagram of the detection and analysis of exosomes in Example 1 of the present invention, wherein Figure 2 a is the TEM image of exosome identification, Figure 2 b is the nanoparticle tracking analysis (NTA) diagram, Figure 2 c is a video capture of exosome movement, Figure 2 d is the marker detection image by western blot;

[0024] Figure 3 is a flowchart of sequencing of miRNA in Example 1 of the present invention;

[0025] Figure 4 This is a cluster analysis diagram of differential miRNAs in Example 1 of the present invention;

[0026] Figure 5 It is a bar chart of GO classification of differential miRNAs target genes in Example 1 of the present invention;

[0027] Figure 6 This is a bubble chart of differential miRNAs target gene enrichment hierarchical analysis in Example 1 of the present invention, wherein Figure 6 A is the differentially expressed miRNAs associated with the following terms: molecular function, kinase activity, ATP binding, nucleotide binding, and zinc ion binding, Figure 6 B is the significant GO CC terms of differentially expressed mirna, including cytoplasm, microtubules, cell components and endosomal membranes. Figure 6 C is the significant GO BP terms of differentially expressed miRNAs including: biological process, phosphorylation, and protein N-linked glycosylation;

[0028] Figure 7 It is a bubble diagram of KEGG pathway enrichment analysis of differential miRNAs in Example 1 of the present invention;

[0029] Figure 8 This is a diagram showing the expression verification results of miRNAs in Example 1 of the present invention;

[0030] Fig. 9 The venny diagram of the Rg1 and TBI targets in Example 1 of the present invention;

[0031] Fig.10 This is a diagram of the protein interaction network between Rg1 and TBI in Example 1 of the present invention;

[0032] Fig.11 It is a bar chart of GO molecular function (MF), biological process (BP), and cellular component (CC) analysis between Rg1 and TBI in Example 1 of the present invention;

[0033] Fig.12 This is a bubble diagram of KEGG analysis between Rg1 and TBI in Example 1 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0036] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0037] Embodiment 1:

[0038] Materials and methods

[0039] SPF-grade male adult SD rats (12-16 weeks old, 300-400 g, purchased from the Animal Center of Wuhan University) were used in the experiment and acclimated for at least 1 week before surgery. All animal experimental protocols were approved by the Animal Experiment Center and Ethics Committee of Zhongnan Hospital of Wuhan University and followed the Guide for the Care and Use of Laboratory Animals.

[0040] Traumatic brain injury (TBI) model and experimental groups

[0041] In the present study, we induced moderate TBI in rats using the free-fall impact (weight-drop, WD) method described in previous studies. Rats were briefly anesthetized with 1% pentobarbital (30 mg / kg) intraperitoneally. After anesthesia, the rats were fixed, shaved, and the skin was disinfected and incised. The fracture zone was exposed, and a 5-mm diameter hole was drilled in the skull (2.0 mm posterior to the fracture zone and 2.0 mm right of the sagittal suture); then, the dura mater was exposed. A 40-g weight was used to perform a vertical impact at a distance of 25 cm to induce moderate TBI. The control group received surgery but no WD. The treatment groups were surgically treated after moderate TBI, and the Rg1-administered group was injected intraperitoneally with a 5-ml syringe and a limit of 7 ml / kg. The inhibitor blebbistatin group was injected intraperitoneally (10 mg / kg). After 24 hours, all rats were anesthetized, and peripheral blood was collected in tubes containing EDTA as an anticoagulant.

[0042] The samples were briefly stored at 4°C and immediately centrifuged at 4°C and 3000 rpm for 10 min. The supernatant was carefully collected and transferred to a new tube without disturbing the intermediate gray-white coating, and then passed through a 0.8 μm filter (SLAA033SB, Millex, Nanopore) to remove additional cell debris and cell debris. The samples were then collected for further steps. A total of 21 samples (7 from the TBI group, 7 from the control group, and 7 from the treatment group) were used to isolate exosomes and extract miRNA. Among them, 9 samples (3 in the control group, 3 in the TBI group, and 3 in the treatment group) were subjected to miRNA sequencing, and 12 samples (4 in the control group, 4 in the TBI group, and 4 in the treatment group) were selected for qRT-PCR validation.

[0043] Hematoxylin and eosin (HE) staining

[0044] The brain tissue was taken, rinsed with PBS buffer, part of the tissue was cut, fixed with OCT embedding glue, and frozen. The brain tissue was processed into thin slices with a thickness of 4 μm and attached to a slide, thus completing the slice preparation process and performing HE staining.

[0045] HE staining steps: fix the frozen sections with 4% paraformaldehyde for 20 minutes, rinse with distilled water; stain with hematoxylin for 3 minutes, rinse with distilled water to wash away the residual hematoxylin staining solution, and then stain with eosin for 1 minute; dehydrate with ethanol gradient, and finally cover with a coverslip. Observe the histopathological changes under a light microscope.

[0046] Isolation and identification of plasma exosomes

[0047] Plasma exosomes were isolated from rat plasma using the exoEasy Maxi kit (Qiagen, 76064) according to the manufacturer's instructions. Exosomes were observed under a transmission electron microscope (Tecnai G2 Spirit TEM, Zeiss, Oberkochen, Germany), stained with phosphotungstic acid, and detected at 80 kV. The size distribution of plasma-derived exosomes was determined using a nanoparticle tracking analyzer (NTA; Particle Metrix, Germany) ZetaView). Proteins were extracted from three groups of plasma exosomes. Typical exosome markers were detected by Western blot, among which common exosome markers were identified: CD63 / CD9 / TSG101.

[0048] MicroRNA Sequencing

[0049] miRNA was isolated from exosome samples using the exoRNeasy serum / plasma Maxi kit (Qiagen, 76064) according to the manufacturer's instructions. The sequence library was determined by Agilent 2100 Bioanalyzer. After we extracted miRNA from exosomes, we performed high-throughput RNA sequencing on these miRNAs.

[0050] First, 1830nt RNA was cut by PAGE electrophoresis to isolate total RNA. 5-adenylated, 3-blocked single-stranded DNA connectors were attached to the 3' end of RNA in these miRNAs. RT primers with UMI were added to the system to hybridize with the 3' adapter attached to the RNA and the excess free 3' adapter. The 3' adapter was then removed by digestion enzyme, and the 5' ends of these miRNAs were connected to 5' adapters. The cDNA chain was then reverse transcribed and synthesized using uni-belt RT primers. The cDNA was amplified using ultrasensitive polymerase, and after enrichment, the cDNA was connected with 3' and 5' connectors to amplify the library yield. PAGE electrophoresis was then used to separate PCR products in the range of 100-120bp to remove primer dimers and other byproducts. The constructed library was quantified, summarized, and quality checked. The qualified library was sent to the machine for sequencing. Clean tags were filtered from the raw data of the above operation, and then the sequences were compared.

[0051] High-throughput sequencing process

[0052] 1) RNA fragment selection: Total RNA was subjected to PAGE electrophoresis to separate RNA fragments of 18-30 nt.

[0053] 2) 3' adapter ligation: Use a 5-adenylated, 3-blocked single-stranded DNA adapter to ligate to the 3' end of the RNA in (1).

[0054] 3) Add the RT primer with the UMI to the system, hybridize with the 3' adapter attached to the RNA, and hybridize with the excess free 3' adapter.

[0055] 4) Add digestion enzyme to remove the 3' linker.

[0056] 5) 5' adapter ligation: The 5' adapter is ligated to the 5' end of the product in (3). Since the adapter preferentially ligates to the single-stranded molecule rather than to the hybridized strand of the 3' adapter and RT primer, adapter self-ligation is greatly reduced.

[0057] 6) Single-strand cDNA synthesis: Reverse transcription extension is performed using RT primers with UMI to synthesize single-strand cDNA.

[0058] 7) PCR amplification: Use a highly sensitive polymerase to amplify the cDNA, enrich the cDNA connected to both the 3' adapter and the 5' adapter, and amplify the library yield.

[0059] 8) Library fragment selection: PAGE electrophoresis was used to separate PCR products in the range of 100-120 bp, effectively removing byproducts such as primer dimers.

[0060] 9) Library quantification and pooling.

[0061] 10) Perform quality check on the constructed library.

[0062] 11) The libraries that have passed the quality inspection are sequenced.

[0063] The raw data obtained through the above operations are filtered to obtain clean tags, and then sequence alignment is performed.

[0064] Analysis of differentially expressed miRNAs

[0065] Base reads were converted to raw data by base calling and obtained from Illumine Hiseq4000 sequencer. Low-quality reads were filtered and reads containing 5' primer contaminants or poly(A) sequences were removed. Reads without 3' adapters and inserted tags, as well as reads shorter than 15nt or longer than 41nt were filtered out from the raw data to obtain clean reads. The length distribution of clean sequences in the reference genome was determined by preliminary analysis. Noncoding RNAs were annotated as RNAs, tRNAs, small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs). Known miRNAs were identified by alignment with the miRBase v.21 database, and the expression patterns of known miRNAs in different samples were analyzed. Unannotated small RNAs were analyzed by miRDeep2 to predict novel miRNAs. The corresponding miRNA star sequences were identified based on the hairpin structure of premiRNA and the miRbase database. Differentially expressed miRNAs were identified with a threshold of p < 0.05. The p-value was calculated using the DEG algorithm in the R package.

[0066] Bioinformatics analysis

[0067] Targets of differentially expressed miRNAs were predicted using Targetscan, PITA, Miranda, and RNA hybridization software, and interactions were selected as the final decision. GO enrichment (http: / / www.geneontology.org) and KEGG (http: / / www.genome.jp / kegg) pathway enrichment analysis was performed on differentially expressed miRNA target genes using R based on hypergeometric distribution.

[0068] qRT-PCR validation of miRNA-seq data

[0069] In order to verify the accuracy of miRNA sequencing data, qRT-PCR was used for verification. RNA was reverse transcribed and cDNA was synthesized using a one-step cDNA first-strand rapid synthesis kit (Nanjing Yifeixue Biotechnology Co., Ltd., catalog number: YFXM009): PCR and preliminary design were performed according to the manufacturer's instructions.

[0070] The qRT-PCR method is as follows: after obtaining miRNAs, U6snRNA is used as the internal reference gene, and the differentially expressed miRNAs are reverse transcribed using a cDNA synthesis kit, so that all samples are normalized to the internal control, and the difference fold is calculated by relative quantification.

[0071] The specific steps include:

[0072] (1) Synthesize cDNA

[0073] ① Prepare the following system in an RNase-Free centrifuge tube in an ice bath:

[0074] Table 4 Reaction solution components

[0075]

[0076] ② Mix gently and centrifuge briefly (so that all the liquid flows to the bottom of the centrifuge tube).

[0077] ③According to the conditions in Table 5, carry out the reaction in a reverse transcription PCR instrument.

[0078] Table 5 PCR reaction conditions

[0079]

[0080] ④ After reverse transcription, take 2 μL of the cDNA obtained and use it for detection of significantly different miRNA quantitative PCR kit.

[0081] (2) miRNA qPCR reaction

[0082] ① Prepare the reaction system: Prepare 20 μL of PCR reaction system on ice according to the components in Table 8, and set up duplicate wells for all samples.

[0083] Table 8 PCR reaction solution system

[0084]

[0085] After the above PCR reaction solution is fully mixed, it is slowly added to the 96-well plate required for the PCR reaction and centrifuged briefly.

[0086] ② Perform Real-Time PCR reaction: Place the 96-well PCR reaction plate with the reaction solution in the PCR instrument and set the following program according to Table 9:

[0087]

[0088] After the reaction is completed, the CT value of cDNA amplified in the PCR instrument can be obtained, and the final CT value is the average value of three replicate wells.

[0089] ③Experimental result processing: adopt △△ The CT method was used to perform relative quantitative analysis of the gene expression of three up-regulated and three down-regulated miRNAs in each sample using U6 as the internal reference. The calculation method was:

[0090] Treatment group △CT = CT (target gene, sample to be tested) - △CT (reference gene, sample to be tested)

[0091] Untreated group △CT = CT (target gene, control sample) - CT (reference gene, control sample)

[0092] △△CT=△CT of treatment group-△CT of untreated group

[0093] Expression multiple = 2 -△△CT .

[0094] ④According to the quantitative analysis in step ③, the verification results of qRT-PCR were obtained.

[0095] Network pharmacology study of ginsenoside RG1 against traumatic brain injury

[0096] 1. Screening of active ingredient targets and disease targets

[0097] The SMILES number of ginsenoside Rg1 was found in the Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov), and the SMILES of ginsenoside Rg1 was imported into the SwissTargetPrediction database (http: / / www.swisstargetprediction.ch / ), and the species was selected as "Homo sapiens" to obtain its corresponding target data. The GeneCards database (https: / / www.genecards.org / ) was used to search for relevant targets of traumatic brain injury with the keyword "Traumatic brain injury". All active ingredient targets were pooled together for screening, and the targets of traumatic brain injury were pooled together for screening. Ginsenoside Rg1 and targets related to traumatic brain injury were imported into the Venny drawing software (http: / / bioinformatics.psb.ugent.be / webtools / Venn / ) to obtain the intersection, so as to obtain the common targets of ginsenoside Rg1 against traumatic brain injury.

[0098] 2. Construct a protein-protein interaction network diagram

[0099] The common targets of ginsenoside Rg1 against traumatic brain injury were imported into the String database (https: / / cn.string-db.org / ), and the PPI network model with biological species set to "Human" was constructed; the confidence>0.7 and the disconnected nodes in the network were hidden, and the other parameters were set to the default values. The data graph of protein-protein interaction was obtained and saved in TSV format, which was then imported into Cytoscape 3.8.2 software for visualization of protein-protein interaction (PPI).

[0100] 3. GO function and KEGG pathway enrichment analysis of potential targets

[0101] The common targets of ginsenoside Rg1 against traumatic brain injury were imported into the DAVID database (https: / / david.ncifcrf.gov / ), and the top 10 entries in GO analysis and the top 20 entries in KEGG analysis were selected for drawing graphs with P < 0.05 as the screening condition. The results were visualized and analyzed, and the GO enrichment analysis results were obtained, including biological process (BP), molecular function (MF) and cellular component (CC). The pathway enrichment results were obtained through KEGG analysis, and then the microbial information database (https: / / www.bioinformatics.com.cn / ) was used to draw a graph for visualization analysis.

[0102] 4. Animal in vivo validation

[0103] (1) Protein extraction

[0104] Add lysis buffer (RIDA lysis buffer (A): protein phosphatase inhibitor: RIDA lysis buffer (B) = 100:1:1) to rat brain tissue, shake gently, freeze and lyse, then place in a 4°C refrigerator for 40 min, estimate the volume of each sample, take out 4 μL of each sample for later use, add Loading to the remaining sample and centrifuge briefly; incubate at 100°C for 7 min, and store in a -20°C refrigerator.

[0105] (2) Protein quantification

[0106] Take a 96-well plate and first add BCA (A solution: B solution = 50:1) standard curve, then add 2 μL of the sample reserved in the previous step to each well, and finally add BCA. Incubate in a 37°C incubator for 30 minutes; place it in an enzyme reader to measure the concentration at a wavelength of 562nm, and calculate the concentration of the protein sample according to the standard curve.

[0107] (3) SDS-PAGE electrophoresis

[0108] Gel preparation: slowly add the prepared 10% separation gel into the assembled plate, and inject distilled water to seal the gel surface to prevent air from entering the gel. Let it stand for 40 minutes. Add the prepared stacking gel into the glass plate until it overflows, insert a comb, let it stand for 40 minutes, place the plate in the electrophoresis buffer, and quickly pull out the comb vertically upwards.

[0109] Loading: Add the marker and protein sample to the comb teeth of the gel plate in sequence.

[0110] Gel running: Place the electrophoresis box in a 4°C refrigerator and select 80V constant voltage for electrophoresis.

[0111] (4) Transfer

[0112] Cut the target protein according to the marker. Place it on the methanol-activated PVDF membrane and immerse it in the transfer solution. Fill the transfer tank with transfer solution, place it in a 4°C refrigerator, select 200mA constant current for transfer, and determine the transfer time according to the relative molecular mass of the target protein.

[0113] (5) Closed

[0114] After the transfer, the membrane was placed in a TBST solution containing 5% BSA and shaken at room temperature for 2 h. The membrane was washed again with TBST for 3 times, each time for 8 min.

[0115] (6) Primary antibody incubation

[0116] Dilute the primary antibody with 5% BSA, add the diluted primary antibody to the sealed PVDF membrane, and incubate at 4°C overnight. Remove and wash with TBST 3 times, 8 minutes each time.

[0117] (7) Secondary antibody incubation

[0118] Recover the primary antibody, soak the PVDF membrane in the secondary antibody diluted with 5% BSA, shake slowly on a shaker at room temperature for 2 hours, wash with TBST three times, each time for 8 minutes, add HRP-labeled goat anti-rabbit secondary antibody after washing, shake slowly on a shaker at room temperature for 2 hours, recover the secondary antibody, and wash with TBST three times, each time for 8 minutes.

[0119] (8) ECL color development

[0120] A color developing solution was prepared by mixing solution A and solution B in a ratio of 1:1, and the solution was evenly added onto the PVDF membrane. The membrane was then placed in a chemiluminescent gel imaging system for observation and photographing.

[0121] (9) Results Analysis

[0122] Image J analysis software was used to digitize the grayscale of the target protein band and the internal reference protein band to compare the protein expression.

[0123] result

[0124] HE

[0125] Comparison of HE staining (×200) of rat brain tissue sections in the control group, TBI model group, Rg1 administration group and blebbistatin group Figure 1 As shown, scale bar is 100 μm.

[0126] from Figure 1 It can be seen that the brain tissue structure of the control group was intact, the staining was uniform, there was no obvious pathological change, no bleeding, obvious inflammatory cell infiltration and other phenomena; the brain tissue of the TBI model group showed obvious brain tissue structural damage, small and ruptured cell nuclei, and a large number of inflammatory cells infiltrated, and the surrounding tissue was obviously edematous; compared with the TBI group, the brain tissue structural damage of the Rg1-treated group and the blebbistatin group was significantly improved, the inflammatory cell infiltration phenomenon was reduced, and the cell edema was alleviated.

[0127] Exosome detection

[0128] Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis were used to identify the obtained exosomes. Figure 2 In a), the exosomes are irregular spheres with a diameter of 30-150nm, with a clear and relatively intact membrane. The NTA results show that the range of exosomes is continuous, with a peak of 137.6nm, indicating that the range of exosomes is large ( Figure 2 bc). Western blot analysis showed ( Figure 2 d), All three groups of exosomes expressed typical exosomes.

[0129] Classification of miRNA

[0130] The microRNA annotation statistics were determined by comparison with the known sRNA database. In order to make each unique miRNA have a unique annotation, the miRNAs were traversed and annotated in the priority order of miRNA>pi-RNA>snoRNA>R-fam>othersRNA. The mature and precursor information annotation results of the miRNAs were selected by comparison and classification with the known mature miRNAs in the miRNA database. The known miRNAs were identified by comparison with the known miRNA database (Table 1). The remaining unknown miRNAs were used to predict new miRNAs. We predicted some new miRNAs during the sequencing process.

[0131] Table 1. Results of miRNAs classification and annotation

[0132]

[0133] Differential expression of miRNAs in plasma exosomes after TBI and Rg1

[0134] High-throughput RNA sequencing technology screens out miRNAs with significant differences. The entire sequencing process is as follows Figure 3 As shown. We performed multiple hypothesis test corrections on the p-value of the difference test and determined the domain value of the p-value by controlling the FDR (False Discovery Rate). The smaller the FDR value, the greater the multiple of the difference between the three groups, indicating that the gene expression difference between the three groups is more obvious. At the same time, the FDR value of the difference test was obtained, and the multiple of the gene expression difference between different samples was calculated according to the expression level (FPKM value). We set the difference multiple to greater than 2 times (|log2)(FPKM of the treatment group / FPKM of the control group)|≥1) and FDR<0.001 to screen the miRNAs with extremely significant expression differences between TBI rats and normal rats. A total of 92 miRNAs were obtained, including 7 upregulated miRNAs and 85 downregulated miRNAs.

[0135] Since the processing of these 92 miRNAs was difficult, we set the difference multiple to more than 32 times (|log2(FPKM of treatment group / FPKM of control group)|≥5) and FDR<0.001 to screen out miRNAs with extremely significant expression differences between TBI rats and normal rats. Three up-regulated miRNAs (rno-miR-382-3p, rno-miR-21-5p, and rno-miR-370-3p) and 11 down-regulated miRNAs (rno-miR-122-5p, rno-miR-344b-1-3p, rno-miR-188-5p, rno-miR-3099, rno-miR-7a-2-3p, rno-miR-3576, rno-miR-7b, rno-miR-142-3p, rno-miR-7a-5p, rno-miR-137-3p, and rno-miR-410-3p) were screened (Table 2 ), and the specific nucleotide sequences are shown in Table 3 . In addition, up-regulated miRNAs with significant expression differences between TBI rats and normal rats were down-regulated between Rg1 rats and TBI rats, while down-regulated miRNAs between TBI rats and normal rats were up-regulated between Rg1 rats and TBI rats. This means that these 14 miRNAs, especially rno-miR-7a-5p and rno-miR-7b, changed significantly before and after the lesion. Ginsenoside Rg1 has a therapeutic effect on traumatic brain injury by regulating the expression of the above miRNAs, and the effect after treatment is significant.

[0136] Table 2. Differentially expressed miRNAs among different groups

[0137]

[0138]

[0139] Table 3. Differential miRNA nucleotide sequences

[0140]

[0141]

[0142] Differential miRNA clustering analysis

[0143] The Heatmap function in R software was applied to hierarchical cluster analysis. The cluster analysis results (such as Figure 4 The results show that 14 differentially expressed miRNAs can be correctly isolated between the model group and the control group. Therefore, these 14 miRNAs may become biomarkers for detecting TBI lesions, and Rg1 may have a therapeutic effect on TBI by intervening in the markers.

[0144] Rg1 regulates the expression of TBI signature miRNAs

[0145] By comparing the 92 significantly differentially expressed miRNAs in the three groups, the model group had 7 identical upregulated miRNAs and 85 identical downregulated miRNAs relative to the control group and the Rg1 group. At the same time, 3 extremely significantly upregulated miRNAs and 11 extremely downregulated miRNAs were confirmed between the TBI group and the control group or the Rg1 group, suggesting that these miRNAs may regulate TBI through the action of Rg1.

[0146] Prediction and functional analysis of miRNA target genes

[0147] Possible target genes of differentially expressed miRNAs are necessary for subsequent analysis. In order to find more accurate results, we used multiple software for prediction. The prediction was performed in combination with corresponding filtering conditions such as free energy, score value, etc. (Additional file 2). GO and KEGG analysis were performed in the common areas of the prediction results of the three software. GO analysis was divided into three parts: biological process (BP), cellular component (CC), and molecular function (MF). GO analysis showed that the differentially expressed miRNAs were significantly associated with the following terms: cellular process, biological regulation, metabolic process, biological process regulation, cell part, organelle, and binding (such as Figure 5 For MF, differentially expressed miRNAs were associated with the following terms: molecular function, kinase activity, ATP binding, nucleotide binding, and zinc ion binding (as shown in Figure 6 The significant GO CC terms of differentially expressed miRNAs include: cytoplasm, microtubules, cellular components and endosomal membrane (as shown in Figure 2A). Figure 6 B). The significant GO BP terms of differentially expressed miRNAs include: biological process, phosphorylation, and protein N-linked glycosylation (e.g. Figure 6 C). Secondly, the significance analysis of metabolic pathways was performed through the KEGG pathway database, and it was found that the main metabolic pathways involved were phosphatidylinositol signaling system, ubiquitin-mediated proteolysis, metabolic pathways, Autophagy-other, Autophagy-yeast and other metabolic pathways (such as Figure 7 shown).

[0148] qRT-PCR validation of miRNA-seq data

[0149] The qRT-PCR method was used to verify the three up-regulated miRNAs (i.e., rno-miR-6382-3p, rno-miR-21-5p, and rno-miR-370-3p) and three down-regulated miRNAs (i.e., rno-miR-122-5p, rno-miR-344b-1-3p, and rno-miR-188-5p) among the 14 differentially expressed miRNAs screened (the results are shown in Figure 8 shown).

[0150] from Figure 8 The relative level of each miRNA can be obtained from the results. The results showed that rno-miR-6382-3p, rno-miR-21-5p and rno-miR-370-3p were all lowly expressed in the control group, and were highly expressed in the model group, and the expression of the three miRNAs was significantly downregulated after drug treatment. rno-miR-122-5p, rno-miR-344b-1-3p and rno-miR-188-5p were all highly expressed in the control group, and were lowly expressed in the model group, and the expression of the three miRNAs was significantly upregulated after drug treatment, which was consistent with the deep sequencing results, indicating that the sequencing results are reliable and can be used as biomarkers for diagnosing TBI, and the drug Rg1 is effective for the treatment of TBI.

[0151] Prediction of targets of ginsenoside Rg1 against traumatic brain injury

[0152] The SwissTargetPrediction database was used to predict the targets of ginsenoside RG1, and 100 targets corresponding to ginsenoside RG1 were finally screened out after removing duplicates. 2570 targets related to traumatic brain injury were obtained by screening the GeneCards database. Finally, the Venny drawing software was used to intersect the targets of ginsenoside RG1 and the targets related to traumatic brain injury, and 57 targets were obtained. Fig. 9 shown.

[0153] PPI network construction and analysis

[0154] The common targets of ginsenoside Rg1 against traumatic brain injury were imported into the STRING database, and the obtained data information was then imported into the Cytoscape 3.8.2 software for analysis. After removing the free nodes, a total of 57 nodes were obtained, resulting in 427 edges. The larger the node and the darker the color, the greater the degree of the target, and the more important the node is in the network. Fig.10 As shown, the key targets of ginsenoside Rg1 in anti-traumatic brain injury mainly include AKT1, EGFR, MMP9, SRC, STAT3, and CASP3.

[0155] Gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of ginsenoside Rg1 against traumatic brain injury

[0156] The 57 potential targets of ginsenoside Rg1 in the treatment of traumatic brain injury were imported into the DAVID database for GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis. The results of GO (Gene Ontology) analysis showed (see Fig.11 As shown in the figure, the biological process (BP) mainly includes proteolysis, negative regulation of apoptosis, positive regulation of protein kinase B signaling, positive regulation of protein phosphorylation, extracellular matrix decomposition, response to external stimuli, response to hypoxia, phosphorylation, positive regulation of peptide serine phosphorylation, negative regulation of gene expression, etc.; from the cell components (CC), it can be inferred that these targets are mainly concentrated in the plasma membrane, extracellular space, extracellular region, extracellular matrix, membrane rafts, lysosomes, ficolin-1-rich granule cavity, neuronal cell body, macromolecular complex, dendrites and other regions; molecular functions (MF) are mainly concentrated in endopeptidase activity, serine endopeptidase activity, peptidase activity, protein phosphatase binding, enzyme binding, heparin binding, kinase activity, sphingolipid binding, integrin binding, same protein binding, etc. The results of KEGG pathway enrichment analysis show (see Fig.12 The main pathways focus on cancer pathways, lipids and atherosclerosis, EGFR tyrosine kinase inhibitor resistance, proteoglycans in cancer, chemical carcinogenesis-receptor activation, relaxin signaling pathway, apoptosis, pancreatic cancer, coronavirus disease-COVID-19, PI3K-Akt signaling pathway, endocrine resistance, the role of AGE-RAGE signaling pathway in diabetic complications, Kaposi's sarcoma-associated herpes virus infection, diabetic cardiomyopathy, insulin resistance, bladder cancer, Ras signaling pathway, estrogen signaling pathway, fluid shear stress and atherosclerosis, calcium signaling pathway, etc.

[0157] Summarize

[0158] By high-throughput whole transcriptome sequencing and bioinformatics analysis, we first investigated the changes in the expression profiles of miRNAs in peripheral blood exosomes and Rg1 after TBI in rats. We made some new observations that improved the understanding of the molecular and intermolecular interactions after TBI and the key signaling pathways in which they participate. First, we demonstrated that the composition of plasma exosomes changed after TBI. Exosomes were isolated and identified by TEM, NTA, and Western blot. The changes in biological substances in plasma exosomes can reflect the physiological and pathological processes after TBI, and Rg1 may be a potential therapeutic target for the treatment of traumatic brain injury, which also suggests that Rg1 can be used to treat TBI.

[0159] In this study, we found that a total of 92 miRNAs were differentially expressed in exosomes after TBI, including 7 upregulated miRNAs and 85 downregulated miRNAs. Meanwhile, we found 14 extremely differentially expressed miRNAs, of which 3 were upregulated and 11 were downregulated. In addition, the differential expression of miRNAs between TBI rats and Rg1 rats was the same as that between TBI rats and normal rats. These results not only suggest that the alteration of miRNAs may be involved in the progression of TBI, but also may reveal that Rg1 may have an effective role in the treatment of TBI.

[0160] Several previous studies have demonstrated miRNAs from different sources during TBI, including from cerebrospinal fluid (CSF), serum, brain tissue, and saliva. Compared with these samples, it is difficult to obtain CSF or brain tissue in the emergency room (ER), and the preparation of serum usually takes hours, while in the ER, plasma can be obtained relatively easily along with blood tests. In this study, miRNA samples were obtained from plasma exosomes. We found some miRNAs with extremely different expressions, such as rno-miR-7b, rno-miR-7a-5p. Some miRNAs have been discovered in previous studies, and they have different roles. These miRNAs are most likely to be regulators of TBI.

[0161] In our previous studies, we found that Rg1 has multiple effects such as improving neuroplasticity, repairing function, anti-aging, and improving immunity, suggesting that Rg1 may be a therapeutic drug for TBI. In this study, we found that the differential expression levels of miRNAs between TBI rats and normal rats in the exosomes of Rg1-treated rats were the same as the differential expression levels of miRNAs in the exosomes of normal rats. Therefore, we can speculate that Rg1 can be used for the treatment of TBI.

[0162] Although we have discovered the possible role of Rg1 in TBI treatment, we still need further research. In previous studies, the target pathways of Rg1 in TBI treatment were very unclear, so we need to further study the differentially expressed miRNAs. We predicted the target genes of these differentially expressed miRNAs by prediction software, and performed GO and KEGG analysis on the overlapping parts of different prediction software. We found that cellular process, biological regulation, metabolic process, biological process regulation, cell part, organelle and binding were significantly associated with target genes, and target genes were related to differentially expressed miRNAs. In addition, the results of KEGG analysis showed that the main metabolic pathways included phosphatidylinositol signaling system, ubiquitin-mediated proteolysis, metabolic pathways, autophagy-other, autophagy-yeast and other metabolic pathways. When we analyzed by bioinformatics, we obtained the target pathways of Rg1 in TBI treatment. These pathways we found can provide some information for further research on TBI and Rg1, which may help us make new progress in the field of TBI treatment.

[0163] In the field of traditional medicine, the effect of drugs mainly depends on the regulation of protein or other compound levels. However, in our research, we found that drugs may work by regulating the miRNAs network and the corresponding biological metabolic pathways, which suggests that the research direction of medicine and diseases can pay more attention to the regulation of miRNAs.

[0164] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of miRNA as a biomarker in the preparation of a reagent for diagnosing traumatic brain injury, characterized in that: The miRNA is one or more of rno-miR-382-3p, rno-miR-21-5p, rno-miR-370-3p, rno-miR-122-5p, rno-miR-344b-1-3p, rno-miR-188-5p, rno-miR-3099, rno-miR-7a-2-3p, rno-miR-3576, rno-miR-7b, rno-miR-142-3p, rno-miR-7a-5p, rno-miR-137-3p, and rno-miR-410-3p; the nucleotide sequences of rno-miR-382-3p to rno-miR-410-3p are shown in SEQ ID No.1 to 14.

2. The use according to claim 1, characterized in that: The reagent is used in a method for detecting miRNA as a biomarker.

3. The use according to claim 1, characterized in that: The method for detecting the content of miRNA as a biomarker is southern blot, northern blot, polymerase chain reaction, reverse transcriptase PCR, quantitative real-time PCR, nanoarray, macroarray, autoradiography, Solexa sequencing, TLDA chip detection or in situ hybridization.

4. Use of ginsenoside Rg1 in the preparation of a drug for regulating the expression of the miRNA according to claim 1.

5. Ginsenoside Rg1 treats TBI targets by regulating the expression of miRNA according to claim 1, characterized in that: Mainly including AKT1, EGFR, MMP9, SRC, STAT3, and CASP3.

6. A drug for regulating the expression of the miRNA according to claim 1, characterized in that: Its active ingredients include ginsenoside Rg1.

7. The drug according to claim 6, characterized in that It also contains a pharmaceutically acceptable carrier.

8. The drug according to claim 7, characterized in that The pharmaceutically acceptable carrier is selected from one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

9. The drug according to claim 6, characterized in that Prepared into pharmaceutically acceptable dosage forms.

10. The drug according to claim 9, characterized in that The dosage form is tablet, pill, paste, capsule, oral liquid or granule.

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