Application of tryptophan derived metabolite as marker in preparation of cerebral hemorrhage early diagnosis, treatment and prognosis evaluation products

Abstract

The invention provides an application of a tryptophan-derived metabolite as a marker in preparation of a product for early diagnosis, treatment and prognosis evaluation of cerebral hemorrhage, and the tryptophan-derived metabolite comprises kynurenine and / or kynurenine. According to the application, the tryptophan derived metabolite is used as the biomarker for early diagnosis, treatment and prognosis evaluation, the metabolite is used as the biomarker, the advantages of high detectability and high sensitivity are achieved, and the limitation of a traditional detection means is overcome.

Description

Technical Field

[0001] The present application relates to the field of medical testing technology, and in particular, to the use of a tryptophan-derived metabolite as a marker in the preparation of products for early diagnosis, treatment and prognosis assessment of cerebral hemorrhage. Background Art

[0002] Cerebral hemorrhage refers to the rupture of a cerebral blood vessel, with blood entering the brain parenchyma. Cerebral hemorrhage is the second most common cause of stroke after ischemic stroke. Among all strokes, cerebral hemorrhage has the highest mortality rate, with an early mortality rate of about 30%-40%. It also has the highest disability rate, with about 70% of patients experiencing long-term functional impairment.

[0003] Intracerebral hemorrhage is a medical emergency that requires rapid diagnosis and meticulous treatment. In clinical practice, focal neurological deficits often occur together with symptoms such as headache, vomiting, elevated blood pressure, and varying degrees of impaired consciousness, which may initially suggest a stroke diagnosis. However, neuroimaging (CT or MRI) should be performed promptly at the onset of the disease to clearly distinguish intracerebral hemorrhage from acute ischemic stroke. The National Institutes of Health Stroke Scale (NIHSS) and Glasgow Coma Scale (GCS) scores are commonly used standardized severity scores for stroke patients. However, patients with intracerebral hemorrhage often have impaired consciousness, which may limit the use of NIHSS. Existing literature reports that elevated troponin and blood glucose levels are associated with survival outcomes and mortality after intracerebral hemorrhage. Despite these advances, there is still a lack of readily available and fairly accurate metabolic biomarkers to predict prognosis and assess disease severity. Summary of the invention

[0004] In response to the shortcomings of existing methods, this application proposes the use of a tryptophan-derived metabolite as a marker in the configuration of early diagnosis, treatment and prognosis assessment products for cerebral hemorrhage, in order to solve the technical problems of unclear intervention targets and inaccurate prognosis diagnosis in related technologies.

[0005] The present application provides an application of a tryptophan-derived metabolite as a marker in the preparation of an early diagnosis, treatment and prognosis assessment product for cerebral hemorrhage, wherein the tryptophan-derived metabolite includes kynurenine and / or kynurenine.

[0006] Optionally, the product is used to detect the content of the tryptophan-derived metabolite in at least one of the cerebrospinal fluid, peripheral blood, and intestinal microbial metabolites of the recipient.

[0007] Further optionally, the product is used to intervene in the content of the tryptophan-derived metabolite in at least one of brain tissue, peripheral blood, and cerebrospinal fluid, and / or to intervene in the production or metabolism of the tryptophan derivative by intestinal microorganisms.

[0008] Further, the product is used to intervene in at least one of the following conditions by controlling the level of the tryptophan-derived metabolite: Levels of inflammatory factors in cerebrospinal fluid and / or brain tissue; The immune response level of glial cells in brain tissue; The level of neuroinflammation developed.

[0009] Specifically, the inflammatory factors include at least one of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, tumor necrosis factor-α, interferon-γ and interferon-α; the glial cells include microglia and / or astrocytes.

[0010] Optionally, the product is used to intervene in the enrichment of neutral amino acid transporters in brain tissue, so as to intervene in the content of the tryptophan-derived metabolite in at least one of brain tissue, peripheral blood and cerebrospinal fluid.

[0011] Furthermore, the neutral amino acid transporter comprises the transporter Slc7a5.

[0012] Optionally, the product is used to assess neurological function in a subject in the early to acute phase after an intracerebral hemorrhage.

[0013] The beneficial technical effects brought about by the technical solution provided by the embodiment of the present application include: (1) The application of this application utilizes tryptophan-derived metabolites as biomarkers for early diagnosis, treatment and prognosis evaluation. Metabolites as biomarkers have the advantages of strong detectability and high sensitivity, overcoming the limitations of traditional detection methods.

[0014] (2) The application of the present application can collect at least one of cerebrospinal fluid, peripheral blood and intestinal microbial metabolites as a test sample based on the in vivo metabolic pathway of tryptophan. The sampling process causes less trauma to the patient, and the number of sampling times is increased, which further increases the frequency of detection, improves the accuracy of detection, and extends the monitoring period.

[0015] (3) The application of this application experimentally confirms that controlling the content of tryptophan-derived metabolites in the body can affect the symptoms and prognosis of cerebral hemorrhage. Therefore, it is believed that tryptophan-derived metabolites can be used as intervention targets for the treatment of cerebral hemorrhage, providing a new idea for the intervention treatment of cerebral hemorrhage.

[0016] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which: Figure 1 A validation design and workflow diagram for the application of this application; Figure 2 This is the partial least squares discriminant analysis (PLS-DA) cluster analysis diagram of cerebrospinal fluid metabolites in patients with acute (Acute-phase) and recovery (Recovery-phase) cerebral hemorrhage; Figure 3 This is a heat map of the differences in cerebrospinal fluid metabolites between patients in the acute phase (Acute-phase) and the recovery phase (Recovery-phase) of cerebral hemorrhage; Figure 4 Variable projection importance plot (VIP plot) of cerebrospinal fluid metabolites in patients with acute-phase and recovery-phase ICH, showing the most discriminatory metabolites in the two groups; Figure 5 Variable projection importance plot (VIP plot) of cerebrospinal fluid metabolites in patients in the acute phase (Acute-phase) and recovery phase (Recovery-phase) of cerebral hemorrhage, showing the most important metabolites in the two groups; Figure 6 This is a comparison chart of the levels of inflammatory factors in the cerebrospinal fluid of healthy people (control group, Control), patients in the acute phase (Acute-phase) and the recovery phase (Recovery-phase) of cerebral hemorrhage; Figure 7 The results of heat map analysis of metabolites and inflammatory factors in cerebrospinal fluid of healthy people (control group), acute phase (Acute-phase) and recovery phase (Recovery-phase) of cerebral hemorrhage patients; Figure 8 The results of the correlation analysis between kynurenic acid and IL-6 and IL-4 in the cerebrospinal fluid of healthy people (control group), acute phase (Acute-phase) and recovery phase (Recovery-phase) of cerebral hemorrhage patients, respectively; Fig. 9 The results are the quantitative analysis of the kynurenic acid content in cerebrospinal fluid and plasma of healthy people (control group), acute phase (Acute-phase) and recovery phase (Recovery-phase) of cerebral hemorrhage patients; Fig.10 This is the flowchart of the operation scheme of the mouse cerebral hemorrhage modeling experiment and the quantitative analysis results of the kynurenic acid content in brain tissue; Fig.11Immunosections of brain tissues from the control group and model group, showing the localization of IBA1-positive microglia in the area surrounding the hemorrhage and related statistics; Fig.12 The first top significant bubble diagram of GO and KEGG pathway analysis in the brain tissues of the control group and the model group; Fig.13 The second top significant bubble diagram of GO and KEGG pathway analysis in the brain tissues of the control group and the model group; Fig.14 In vitro experiments verify the activation of microglia by kynurenic acid, where Figure A is a schematic diagram of cell culture treatment. Figure BE is the comprehensive results of kynurenic acid and vehicle (DMSO); Fig.15 The GO enrichment analysis diagram of the transporter proteins in the mice that received ICH modeling during the transport of neutral amino acids is shown; Fig.16 The heat map of the expression level of neutral amino acid transporters in the cerebral hemisphere tissues of ICH model mice is shown, and the relative gene expression of the transporter Slc7a5 is shown, as well as the quantitative analysis results of the kynurenine content in the brain of the ICH group and the CON group at this time; Fig.17 Schematic diagram showing peripheral kynurenine entering the brain via the transporter Slc7a5 and being converted to kynurenic acid; Fig.18 The figure shows a schematic diagram of the experimental process of applying kynurenine to the peripheral tissues of mice after ICH modeling, and the horizontal axis represents the time node; Fig.19 The graph shows the changes in the content of kynurenic acid in brain tissue after peripheral administration of kynurenic acid to mice after ICH modeling, as well as the changes in the mRNA expression levels of inflammatory factors (IL-4, IL-6, and TNF-α). DETAILED DESCRIPTION

[0018] The embodiments of the present application are described below in conjunction with the drawings in the present application. It should be understood that the implementation methods described below in conjunction with the drawings are exemplary descriptions for explaining the technical solutions of the embodiments of the present application and do not constitute a limitation on the technical solutions of the embodiments of the present application.

[0019] Those skilled in the art will appreciate that, unless expressly stated, the "said" and "the" used herein may also include plural forms. It should be further understood that the term "including" used in the specification of the present application refers to the presence of the features, integers, steps, operations, elements and / or components, but does not exclude the implementation of other features, information, data, steps, operations, elements, components and / or combinations thereof supported by the technical field. The term "and / or" used herein refers to at least one of the items defined by the term, for example, "A and / or B" may be implemented as "A", or as "B", or as "A and B".

[0020] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below with reference to the accompanying drawings.

[0021] The pathophysiology of intracerebral hemorrhage is very complex and includes mechanical expansion of hematoma, mass effect, and secondary injury. There is increasing evidence that neurological dysfunction is closely related to secondary brain injury, and survivors suffer from severe neurological dysfunction for a long time. Early active bleeding and hematoma expansion usually lead to neurological deterioration and poor functional prognosis. Advances have been made in the acute treatment of patients, such as blood pressure control, neurosurgery, and hemostatic strategies. However, less than half of the patients survive for 1 year, and less than one-third of the patients survive for 5 years. There is currently a lack of clear primary treatments as well as treatments for peripheral hematomas and related secondary injuries.

[0022] However, the chronic nature of neuroinflammation in ICH provides a therapeutic window to mitigate its adverse consequences. Local microglia and astrocytes are the first responders, and their proinflammatory activation promotes the influx of circulating immune cells (mainly macrophages). The release of inflammatory factors (such as interleukin-1β, tumor necrosis factor), free radicals, and chemokines increases. This attracts and activates lymphocytes. Importantly, these processes also impair the integrity of the blood-brain barrier (BBB), leading to the formation of peritumoral edema and the potential growth of ICH. Proinflammatory microglia are considered promising therapeutic targets for early ICH because they induce neuroinflammation, which is mainly associated with poor prognosis. Based on the above studies, microglia may be a potential therapeutic target. There is an urgent need for a metabolic indicator that can serve as both a target for neuroinflammatory intervention and a prognostic marker for ICH.

[0023] Therefore, the technical problem to be solved in this application is to identify new metabolites as neuroinflammatory intervention targets in the early stages of cerebral hemorrhage, discover new pathophysiological mechanisms, and provide a basis for the development of therapeutic intervention measures and prognostic biomarkers.

[0024] refer to Figure 1, this application adopted a multi-omics approach, using human samples and mouse models to discover a metabolite associated with neuroinflammation after intracerebral hemorrhage and elucidate its potential mechanism.

[0025] To identify specific metabolites that cause activation or aggravation of neuroinflammation in ICH, cerebrospinal fluid (CSF) samples from patients with ICH were analyzed, including cerebrospinal fluid (CSF) and plasma samples from 50 subjects in the study cohort (38 patients with ICH and 12 controls without ICH). The inclusion criteria for patients with ICH were hemorrhage on computed tomography of the head and lumbar puncture as part of the diagnostic procedure for screening ICH. Exclusion criteria for both groups were dementia or severe physical illness. Participants underwent Glasgow Coma Scale, plasma, and cerebrospinal fluid sample collection according to the study protocol at admission and discharge within the specified time period. The cerebrospinal fluid samples of the subjects were analyzed for metabolite differences between sample groups based on the UPLC-MS / MS detection platform, standards, and statistical analysis. Kynuric acid, a metabolic marker that can be used for the prognosis assessment of ICH, was screened out. The screening results showed that kynurenic acid was significantly associated with neuroinflammation.

[0026] Specifically, the experimental method is as follows: (1) Research design of population studies The overall design of the study is as follows Figure 1 Shown: A total of 38 ICH patients and 12 control participants (aged between 12 and 81 years) were screened for this study. The inclusion criteria for ICH patients were hemorrhage on head computed tomography and lumbar puncture as part of the ICH screening diagnostic procedure. Exclusion criteria for both groups were dementia or severe physical illness. Participants underwent Glasgow Coma Scale, plasma, and cerebrospinal fluid samples at admission and discharge according to the study protocol.

[0027] (2) Human cytokine detection The levels of 12 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interferon-α (IFN-α)) were quantified using a human multi-cytokine assay kit. The minimum and maximum limits of detection for all cytokines were 0 and 10,000 pg / ml, respectively. Samples were analyzed using a Beckman Dxflex flow cytometer. Data were generated in graphical form using Kaluza Array software developed by Beckman Instruments (Kramer Avenue Brea, California, USA).

[0028] (3) LC-MS / MS analysis ①Sample preparation Brain tissue samples were homogenized with 0.1 mL of water for 30 s using a fully automatic sample grinder (JXFSTPRP-32). 50 μL of brain homogenate was mixed with 10 μL of trans fatty acids and proteins were precipitated under a fume hood. After 2 h, the mixture was vortexed and centrifuged at 20,000 g for 15 min at 4 °C. The supernatant was then collected into a mass spectrometer vial and prepared for analysis. The preparation methods of serum samples, calibration standards, and quality control samples were the same as those of brain homogenate samples.

[0029] ②LC / MS instrument and chromatographic conditions Chromatographic separation was performed using a Water Acquity UPLC HSS T3 (2.1x100mm 1.8um) column with the column oven set at 40°C. The sample was placed in an autosampler at 5°C and 1 µL was injected into the system with the following mobile phases: (A) 0.5% formic acid in water (v / v) and (B) acetonitrile. The gradient elution was as follows: organic phase (B) was maintained at 8% from 0 to 1.5 min, increased to 95% from 1.5 to 7 min (linear gradient), and decreased to 5% from 7 to 10 min (linear gradient). The total run time of the method was 10 min with a flow rate of 0.28 mL / min. The mass spectrometer monitored the transitions of m / z 189.9 → 143.9 (Kyna, kynurenic acid) and 209.4 → 192.0 (Kyn, kynurenine) at appropriate collision energies.

[0030] Furthermore, the experimental results of the cohort study are as follows Figures 2~9 shown.

[0031] Figures 2~5 showed that non-targeted metabolome analysis revealed changes in cerebrospinal fluid metabolites in patients after the acute phase.

[0032] Figure 2 The difference in cerebrospinal fluid metabolites between patients in the acute phase (Acute-phase) and the recovery phase (Recovery-phase) of cerebral hemorrhage clustering by partial least squares discriminant analysis (PLS-DA) is shown. Partial least squares discriminant analysis (PLS-DA) is a statistical method used to process data with multiple predictor and response variables. The principle is to rotate the data by partial least squares method to distinguish samples between different groups. PLS-DA analysis is based on two data sets X and Y, where X is the predictor variable (independent variable) and Y is the response variable (dependent variable, classification information). Through analysis, the principal component axes of the X and Y spaces and the scores of the sample points can be obtained, thereby achieving classification and discrimination of the data. Figure 2Two obvious clusters were shown, namely the clusters of metabolites that fluctuated significantly in the acute phase and the recovery phase.

[0033] Figure 3 The heat map of the difference in cerebrospinal fluid metabolites between patients in the acute phase and recovery phase of cerebral hemorrhage was displayed. The metabolites with significant differences between the two groups included gluconic acid, kynurenic acid, porphobilinogen, and 5-aminopentanoic acid, suggesting that these metabolites have the potential to be used as biomarkers, but further verification is needed. Among them, gluconic acid has multiple biological activities, including providing energy and nutritional supplements in the body, promoting metabolism and chelation, etc. Kynurenic acid is a derivative metabolite of the kynurenine pathway of tryptophan; porphobilinogen has a wide range of physiological functions and metabolic pathways in the body, such as participating in heme biosynthesis; 5-aminopentanoic acid has amino and carboxyl reaction activity, and 5-aminopentanoic acid is considered to be a methylene homologue of γ-aminobutyric acid (GABA) and acts as a weak GABA agonist. In humans, it is a normal metabolite present in saliva, and its concentration tends to be elevated in the saliva of patients with chronic periodontitis. In addition, it is also believed to have antifibrinolytic effects, and thus can act as a weak inhibitor of the blood coagulation pathway.

[0034] Figure 4 The VIP diagram shows the most discriminatory metabolites between the acute phase (Acute-phase) and the recovery phase (Recovery-phase) of cerebral hemorrhage. In statistics, especially in the field of omics data analysis, the VIP diagram is an important visualization tool for evaluating the importance of individual variables (such as metabolites, genes, etc.) in the projection or classification process. VIP is the abbreviation of Variable Importance in Projection, which reflects the contribution of each variable to the model classification or projection. The VIP value is calculated by comparing the weight of each variable with the standard deviation of the weights of all variables in the model. The higher the VIP value, the stronger the discriminatory ability of the variable in the model and the greater its contribution to the classification or projection. The VIP diagram is usually presented in the form of a combination of a scatter plot and a heat map, showing the VIP values ​​of each variable and the content change trends of these variables among the groups. This combination enables the VIP diagram to intuitively show which variables contribute most to distinguishing different groups, which helps to quickly identify important variables. Therefore, combined with Figure 3 and Figure 4, we were able to further identify the most discriminative metabolites involved in the following metabolic pathways: Central carbon metabolism cancer, Protein digestion and absorption, Phenylalanine tyrosine and tryptophan biosynthesis, Phenyialanine metabolsm, Aminoacyl-tRNA biosynthesis.

[0035] Figure 5 The VIP diagram shows the most important metabolites. The metabolic pathways involved include phenylalanine metabolism, aminoacyl-tRNA biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis. The diagram shows that the following metabolites were significantly increased: Cytidine Monophosphate (CMP), Ribose 1,5-bisphosphate, Gluconic acid, Kynurenic acid, N6-(δ2-Isopentenyl)-sdenine; the following metabolites were significantly decreased: 1-Hydroxy-2-naphthoate, 5-Aminopentanoic acid, Porphobilinogen, beta-Alanyl-L-lysine, Glucicacid.

[0036] from Figures 2 to 5 It is a step-by-step focused analytical process that provides clues for confirming target metabolites.

[0037] Further, the correlation between the content of inflammatory factors and metabolites in cerebrospinal fluid was analyzed. Inflammatory factors are a class of small molecule proteins or polypeptides with multiple biological activities produced by inflammatory cells (such as macrophages, monocytes, lymphocytes, endothelial cells, etc.) or other cells (such as fibroblasts, keratinocytes, etc.) when the body is infected, tissue damaged or otherwise stimulated. These factors play an important role in the body's immune response and inflammatory process, and can regulate immune response, promote leukocyte migration, adhesion and activation, and increase vascular permeability, thereby inducing and maintaining inflammatory response. According to the specific effects, inflammatory factors can also be classified into cytokines, chemokines, leukotrienes and prostaglandins. This example focuses on the detection of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and interferon-α (IFN-a).

[0038] Figure 6 The results show the changes in inflammatory factors in the cerebrospinal fluid of the control group, acute phase of cerebral hemorrhage, and recovery phase. The control group is samples from healthy people. The results show that inflammatory factors accumulate in the cerebrospinal fluid of patients with cerebral hemorrhage, and the levels of IL-10, IL-1β, TNF-α, and IL-6 are significantly reduced after conventional treatment.

[0039] Furthermore, the correlation between the metabolites and inflammatory factor levels in cerebrospinal fluid was studied. Figure 7As shown in the heat map, the metabolites most significantly correlated with the levels of various inflammatory factors are kynurenic acid, pyrimidodiazepine and isocitric acid. Other metabolites positively correlated with the levels of inflammatory factors include quinaldic acid, (R)-4-hydroxymandelate, L-Histidine trimethylbetaine, 4-Methylcatechol, Caffeine, 5-Keto-D-gluconate, Ursodeoxycholic acid, alpha-Ketaisovaleric acid, Succinic acid semialdehyde, and 2-Ketobutyric acid. , Na-Acetyicitrulline, 7,8-Dihydromonapterin, Mefloquine, Rosmarinic acid, N-Acetylaspartyiglutamic acid, Isovitexin, etc.

[0040] Linear regression and Spearman correlation analysis showed the correlation between kynurenic acid and inflammatory factors. Figure 8 As shown, kynurenic acid showed a significant linear positive correlation trend with IL-6 and IL-4, respectively. Kynurenic acid had a significant correlation with IL-6 and IL-4, respectively.

[0041] Furthermore, the quantitative analysis of the kynurenic acid content in the cerebrospinal fluid of the three groups showed that Fig. 9 shown. Fig. 9 In the figure, the left picture shows that the kynuric acid content in the cerebrospinal fluid of patients with cerebral hemorrhage is significantly increased, but the content decreases after conventional treatment; the middle picture shows the Spearman correlation analysis between the kynurenic acid content and the GCS score after cerebral hemorrhage, and the kynurenic acid content is linearly negatively correlated with the GCS score; the right picture shows the quantitative analysis of the kynurenic acid content in plasma, and the kynurenic acid content in the plasma of patients with cerebral hemorrhage is significantly reduced, and the kynurenic acid content in the plasma has appropriately recovered after conventional treatment.

[0042] The above results indicate that changes in the content of kynurenic acid in cerebrospinal fluid are related to the development of cerebral hemorrhage symptoms, and that kynurenic acid in plasma also changes concomitantly.

[0043] In order to further clarify the correlation between kynurenic acid and cerebral hemorrhage, in vivo verification was performed using model animals, specifically ICH (intracerebral hemorrhage) model mice.

[0044] The relevant experimental methods are as follows: (4) ICH model refer to Fig.10 , 1.5% Avertin was used to induce anesthesia, and mice (strain C57BL / 6J) were positioned using a stereotaxic apparatus (Shenzhen Rongweida Life Science Co., Ltd.). Then, 0.05 U collagenase VII (#C0773, Sigma-Aldrich, St. Louis, MO, USA) was prepared with 0.2 μL of saline and stereotaxically injected into the left striatum (2.0 mm lateral, 0.4 mm anterior to bregma, 3.2 mm deep). During the operation, the body temperature of the mice was maintained at 37.0±0.5 ℃. Sham-operated mice (CON group) were injected with saline at the same location and injection volume.

[0045] (5) Transcriptome sequencing RNA quantification of brain tissue samples was performed on a Bioanalyzer2100 system (Agilent Technologies, CA, USA). Subsequently, RNA was reverse transcribed to obtain cDNA for library construction, and the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Index-encoded samples were clustered on the cBot Cluster Generation System using the TruSeq PECluster Kit v3-cBot-HS (llumia) according to the manufacturer's instructions. After cluster generation, library preparations were sequenced on the llluminaNovaseq platform to obtain sequencing data for all samples with a read length of 150 bp (paired end).

[0046] (6) Real-time quantitative PCR Total RNA was extracted with TRIzol reagent, and cDNA was synthesized using a reverse transcription kit (R333-01, Vazyme). Quantitative PCR was performed using QuantStudio 7 Flex (Thermo Fisher Scientific) and Universal SYBR qPCR Master Mix (Q712-02, Vazyme). Relative RNA levels were compared with Hprt1 or Gapdh as standards.

[0047] The experimental results verified in mice are as follows Figures 10 to 13 As shown, specifically: Fig.10 The results of quantitative analysis of the kynurenic acid content in brain tissue are shown, and the results show that the kynurenic acid content in the brain tissue of ICH model mice is significantly increased.

[0048] Fig.11 It was shown that the localization of IBA1-positive microglia in the peri-bleeding areas of the two groups in the immune sections of brain tissue was different, and the IBA1-positive areas of the two groups were quantified and statistically analyzed, and the relative abundance of the AIF gene encoding the IBA1 protein in the brains of the two groups of mice was statistically analyzed. IBA1 protein, also known as ionized calcium-binding adapter molecule 1 or allogeneic inflammatory factor 1 (AIF1), has one of its physiological functions of activating macrophages, that is, IBA1 plays an important role in macrophage activation and function, and is involved in morphological changes related to the state of activated microglia / macrophages. Therefore, by detecting the expression level of IBA1, we can indirectly understand the immune response level of microglia. The results showed that the expression level of IBA1 in the ICH model group was significantly increased, reaching a statistically extremely significant level ( ).

[0049] Fig.12 and 13The top significant bubble charts of GO and KEGG pathway analysis of two groups are shown. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis are often used in the fields of bioinformatics and systems biology. GO is a standardized language used to describe the functions of genes and proteins. It mainly annotates gene products from three aspects: molecular function (MF), biological process (BP) and cellular component (CC). GO pathway analysis uses GO terms and known biological pathway information to parse gene expression data, aiming to reveal how genes work together in specific biological processes and how these processes are affected by experimental conditions (such as disease states, drug treatments, etc.); KEGG is a comprehensive database that integrates genomic, chemical and system functional information. KEGG pathway analysis is a functional annotation and enrichment analysis method based on the KEGG database, aiming to find genes related to certain metabolic pathways in a gene set, thereby revealing the biological significance of the gene set.The results showed that the signaling pathways with a high correlation with cerebral hemorrhage included at least: external side of defense response, positive regulation of defense response, T cell activation, adaptive immune response, degense response to other organisms, leukocyte proliferation, lymphocyte proliferation, mononuclear cell proliferation, positive regulation of immune response, leukocyte-mediated immunity, Epstein-Barr virus infection, proteoglycans in cancer, chemokine signaling pathway, phagosome, lipid and atherosclerosis, tuberculosis, cytokine-cytokine receptor interaction. ) (including cytokine receptor binding and cytokine binding), NOD-like receptor signaling pathway.

[0050] In summary, through in vivo experiments in ICH model mice, some signal pathways related to cerebral hemorrhage can be screened and the immune response status of microglia can be detected.

[0051] Furthermore, this example also conducts in vivo verification, specifically using mouse microglia (BV2 cells) cultured in vitro to examine the effects of kynurenic acid (Kyna). The experimental method and results are as follows: (7) Culture and treatment of mouse microglia Microglial cells were cultured in DMEM (C11995500BT, Gibco) containing 100 U / mL penicillin-streptomycin (15140122, Gibco) and 10% fetal bovine serum (16140071, Gibco) at 37°C in an incubator with 5% CO2 humidity.

[0052] After 24 h of culture, microglia were washed twice with PBS and then stimulated with 1000 ng / ml LPS (L2880, Sigma) and 10 uM Kyna (CAS 492-27-3). After 24 h of treatment, cells were collected for quantitative PCR detection.

[0053] (8) CCK8 experiment Microglia were seeded in 96-well plates, 5000 cells per well, and cultured for 24 h. Then, 1000 ng / mL LPS and different concentrations of Kyna (10μM, 30μM, and 300μM) were added to each well. After 24 h of culture, 10μL of CCK8 reagent was added to each well. Cultured for 1-4 h to allow the reagent to react with NADPH in the cells, and then the absorbance was measured at a wavelength of 450 nm. The proliferation rate was calculated.

[0054] like Fig.14 As shown in Figure 2, BV2 cells were treated with vehicle (DMSO) and kynurenic acid for 24 h, respectively. The treatment gradients of the kynurenic acid group were set to 10 μM, 30 μM, and 100 μM, respectively. Cell counting kit-8 was used to detect cell viability. The results showed that kynurenic acid promoted the proliferation of BV2 cells, but did not show concentration dependence. The proliferation rate of BV2 cells was the highest under the treatment of 30 μM concentration (as shown in Figure B, ); CE graphs show the expression levels of IL-4, IL-6 and TNF-α mRNA genes in the control group and BV2 cells. The expression of IL-4 and TNF-α in the treated BV2 cells increased significantly ( ).

[0055] Kynurenine has a significant effect on BV2 cells cultured in vitro, and the effect of kynurenic acid on BV2 cells was verified in vivo. Exogenous kynurenine (Kyn) was applied to ICH model mice and the state of mouse brain tissue was further analyzed. Kynurenine is a metabolic intermediate of tryptophan. In the animal body, kynurenine can be further metabolized into kynurenic acid, which plays a role in neuroregulation, immunoregulation or vasodilation. The corresponding experimental results are as follows Figures 15 to 19 As shown: Fig.15 The GO enrichment analysis of transport proteins in ICH-induced mice during neutral amino acid transport is shown. GO enrichment analysis (Gene Ontology Enrichment Analysis) is a bioinformatics method that is mainly used to analyze whether a specific gene set or protein set is significantly clustered in certain functional categories or pathways in Gene Ontology (GO). According to the specified classification criteria, the genes that were significantly detected were divided into three categories: Molecular Function (MF), Biological Process (BP), and Cellular Component (CC). The results showed that the genes with the highest enrichment in BP were involved in the process of neutral amino acid transport, and the genes with the highest enrichment in MF were involved in the function of neutral amino acid transmembrane transporter activity.

[0056] Fig.16 The heat map in the figure shows the expression levels of proteins related to neutral amino acid transport (GO:0015804) in the cerebral hemisphere tissues, among which the transport protein Slc7a5 increased most significantly in the ICH group. The relative gene expression of the transport protein Slc7a5 was further statistically analyzed (lower left figure, The lower right figure is the quantitative analysis of kynurenine content in the brain of the ICH group and the CON group (n=8-10, ), it can be seen that exogenous kynurenine enters the brain tissue through the blood-brain barrier and activates microglia, which may increase neuroinflammation in the brain.

[0057] Based on the above inference, a schematic diagram of peripheral kynurenine entering the brain through the transporter Slc7a5 and being converted into kynurenic acid is given ( Fig.17 ): There is a blood-brain barrier between plasma and cerebrospinal fluid in brain tissue. Kynuric acid (Kyna) cannot pass through the blood-brain barrier to enter brain cells, but kynurenine (Kyn) can use the transporter Slc7a5 on the blood vessel wall (endothelial cells) to cross the blood-brain barrier and use kynurenine aminotransferase (KAT) to convert into kynurenic acid in the cerebrospinal fluid. This process is an important link in the kynurenine pathway of tryptophan metabolism and has an important impact on the production of neurotransmitters and neuromodulators. Kynuric acid in the cerebrospinal fluid induces neuroinflammation. ICH model mice with aggravated neuroinflammation eventually died ( Fig.18 as shown).

[0058] Fig.19 From left to right, the changes in the content of kynurenic acid in brain tissue after peripheral administration of kynurenic acid to mice after ICH modeling, as well as the changes in the mRNA expression levels of inflammatory factors (IL-4, IL-6 and TNF-α), are shown. All monitoring items reached significant or extremely significant levels ( ). This further confirmed that exogenous kynurenine increased neuroinflammation in the brain of mice with an ICH model.

[0059] In summary, in vivo and in vitro experiments have verified that the inflammatory response in the brain after cerebral hemorrhage involves significant activation of microglia, and the significant increase in kynurenic acid in brain tissue promotes the inflammatory response in the brain after cerebral hemorrhage. Tracing the source of kynurenic acid, its precursor kynurenine was found to be elevated, and exogenous kynurenine promoted the rise of kynurenic acid in brain tissue and the aggravation of neuroinflammation.

[0060] In this study, mice were modeled with intracerebral hemorrhage, and collagenase was stereotactically injected into the striatum of mice. The content of kynurenic acid and kynurenine and inflammatory changes in the brain tissue of mice with intracerebral hemorrhage were detected. The content of kynurenic acid and kynurenine in the brain tissue of mice with intracerebral hemorrhage was increased, which is consistent with the results of previous human studies. Microglia in the hemorrhage area release proinflammatory cytokines and initiate early inflammatory responses. The data showed that the staining intensity of IBA1 in the perihematomal area of ​​mice with intracerebral hemorrhage was significantly higher than that in the control group. In addition, the mRNA expression of AIF1 in the brain tissue of mice with intracerebral hemorrhage was higher. The immune response and cytokine binding of mice with intracerebral hemorrhage were significantly activated. Combined with transcriptome data analysis, kynurenic acid in the brain is derived from the precursor substance kynurenine, and most of its increase comes from kynurenine in peripheral blood. Exogenous kynurenine increased the expression of inflammatory factors in the brain in mice with intracerebral hemorrhage model. These data indicate that the significant increase of kynurenic acid and kynurenine in the brain after intracerebral hemorrhage is related to neuroinflammation. In vivo and in vitro experiments also further proved that kynurenic acid and kynurenine can promote neuroinflammation and affect the occurrence and development of the disease.

[0061] Elevated levels of kynurenic acid are significantly correlated with neuroinflammation and neurological prognosis, indicating its potential as a biomarker. In vivo and in vitro experiments have shown that increased kynurenic acid in brain tissue regulates cytokine interactions and promotes the increase of inflammatory cytokines (such as TNF-α, IL6, and IL4). However, the concentration of kynurenic acid in the blood is low and has limited permeability through the blood-brain barrier (BBB). In brain tissue, kynurenic acid is derived from the metabolism of its precursor kynurenine mediated by the kynurenine aminotransferase (KAT) enzyme, because kynurenine can cross the BBB through the transporter Slc7a5. It is speculated that the increase in brain kynurenic acid is derived from the increase in kynurenine in the brain. The increase in the activity of the transporter Slc7a5 indicates that part, if not all, of the surge in kynurenine in the brain is absorbed from the blood. Experiments have shown that exogenous kynurenine upregulates the total level of kynurenic acid in brain tissue and exacerbates the expression of inflammatory factors in the brain. The production of peripheral kynurenine is closely related to intestinal microorganisms, which provides a potential new target for intestinal microbial intervention. Based on this, it is believed that tryptophan-related biomarkers - kynurenine and / or kynurenine - can be used as intervention targets and prognosis assessment for cerebral hemorrhage.

[0062] Furthermore, the use of tryptophan-derived metabolites as markers in the preparation of products for early diagnosis, treatment and prognosis assessment of cerebral hemorrhage can be specifically carried out in at least one of the following ways: The product is used to detect the content of tryptophan-derived metabolites derived from at least one of the cerebrospinal fluid, peripheral blood, and intestinal microbial metabolites of the recipient; The product is used to intervene in the content of at least one of the tryptophan-derived metabolites in brain tissue, peripheral blood, and cerebrospinal fluid; The product is used to intervene in the production or metabolism of tryptophan-derived metabolites by intestinal microorganisms; This product is used to intervene in the enrichment of neutral amino acid transporters (such as transporter Slc7a5); This product is used to assess the neurological function of subjects in the early to after acute phase of cerebral hemorrhage.

[0063] In one possible embodiment, the product is used to intervene in at least one of the following conditions by controlling the level of tryptophan-derived metabolites: Levels of inflammatory factors in cerebrospinal fluid and / or brain tissue; The immune response level of glial cells (e.g., microglia and / or astrocytes) in brain tissue; The level of neuroinflammation developed.

[0064] The application based on this application is expected to achieve early intervention and early treatment, and reduce the huge risks that cerebral hemorrhage brings to patients.

[0065] The experimental methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources. Statistical analysis involved in the examples: All statistical processes were performed using R software version 4.0.3, SPSS version 22.0, and GraphPad Prism version 9.0. The differences in normally distributed quantitative data were evaluated using a two-tailed independent sample Student's t test. For non-normally distributed variables, the Wilcoxon rank sum test was used. Categorical variables were evaluated by Pearson's χ^2 test or Fisher's exact test. For the relationship between two groups of non-normally distributed variables, a Spearman correlation analysis was performed.

[0066] Those skilled in the art will appreciate that the various operations, methods, steps, measures, and schemes in the processes discussed in this application may be alternated, changed, combined, or deleted. Furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and schemes in the related art that are similar to those disclosed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted.

[0067] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0068] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

[0069] The above is only a partial implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the technical concept of the scheme of the present application, other similar implementation methods based on the technical ideas of the present application are also within the protection scope of the embodiments of the present application.

Claims

1. The use of tryptophan-derived metabolites as markers in the preparation of products for early diagnosis, treatment and prognosis assessment of cerebral hemorrhage, characterized in that: The tryptophan-derived metabolites include kynurenine and / or kynurenine.

2. The use according to claim 1, characterized in that The product is used to detect the content of the tryptophan-derived metabolite in at least one of the cerebrospinal fluid, peripheral blood, and intestinal microbial metabolites of a recipient.

3. The use according to claim 1, characterized in that The product is used to intervene in the content of the tryptophan-derived metabolite in at least one of brain tissue, peripheral blood, and cerebrospinal fluid, and / or to intervene in the production or metabolism of the tryptophan derivative by intestinal microorganisms.

4. The use according to claim 3, characterized in that The product is used to intervene in at least one of the following conditions by controlling the level of the tryptophan-derived metabolite: Levels of inflammatory factors in cerebrospinal fluid and / or brain tissue; The immune response level of glial cells in brain tissue; The level of neuroinflammation developed.

5. The use according to claim 4, characterized in that The inflammatory factors include at least one of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, tumor necrosis factor-α, interferon-γ and interferon-α; the glial cells include microglia and / or astrocytes.

6. The use according to claim 3, characterized in that The product is used to intervene in the enrichment of neutral amino acid transporters in brain tissue, so as to intervene in the content of the tryptophan-derived metabolites in at least one of brain tissue, peripheral blood and cerebrospinal fluid.

7. The use according to claim 6, characterized in that The neutral amino acid transporters include the transporter Slc7a5.

8. The use according to claim 1, characterized in that The product is used to assess the neurological function of a subject in the early to acute phase of cerebral hemorrhage.

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