Use of reagent in preparing drugs for treating or improving heart failure and drug screening method

Abstract

The present invention discloses the use of reagents in the preparation of drugs for treating or improving heart failure and a drug screening method. The present invention discovers for the first time the connection between kynurenine pathway dysregulation and depletion of Parabacteroides dieldrinii strains in the progression of heart failure. Validation analysis not only confirms the bidirectional relationship between the kynurenine pathway and Parabacteroides dieldrinii, but also determines that these pathways can be targeted by inhibiting the kynurenine pathway or supplementing Parabacteroides dieldrinii, thereby treating heart failure through a bidirectional loop, which provides a promising therapeutic target and method for the treatment of heart failure.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of a reagent in preparing a drug for treating or improving heart failure and a drug screening method. Background Art

[0002] Heart failure (HF) is a multifaceted, life-threatening syndrome characterized by high morbidity and mortality, poor functional capacity and quality of life, and high treatment costs. Despite major advances in the medical management of HF, the prognosis remains poor. The pathogenesis and progression of HF are complex and include cardiac impairment, systemic inflammation, hemodynamic deterioration, and neurohumoral changes. Recently, researchers have focused on the role of intestinal microbiota in the progression of HF and have conducted a series of studies on the heart-gut axis in HF. Elucidating the mechanisms of HF is of great significance for the diagnosis and treatment of HF.

[0003] Metabolic impairment is an intrinsic component of the pathophysiology of heart failure. Most studies have shown that HF ​​triggers widespread changes in systemic metabolism, including lipid metabolism disorders, energy metabolism dysregulation, and amino acid metabolism imbalance. Notably, amino acid disturbances play an important role in the development of heart failure. A range of amino acids are significantly altered after heart failure. Metabolomics is a key approach to deepen the understanding of the pathophysiology of cardiovascular diseases and clinicians' insights into their pathogenesis. Using metabolomics tools to study amino acid perturbations after HF has great potential to identify new biomarkers and elucidate the underlying mechanisms of HF, thereby facilitating targeted therapeutic interventions.

[0004] Increasing evidence suggests that the gut microbiota may be a novel target for the treatment of heart failure. However, the impact of the gut microbiome on HF remains elusive. A deeper investigation of HF-associated microbiota characteristics may provide new insights into the potential role of the heart-gut axis in HF.

[0005] Studies have shown that tryptophan (TRP) is an effective marker for predicting HF recurrence in patients with acute decompensated heart failure (ADHF), and TRP metabolism changes significantly after HF recurrence. In addition, many reports have shown that the intestinal flora is involved in TRP metabolism. For example, abnormal components of the intestinal microbiota lead to the onset of schizophrenia through TRP-KYN metabolism. However, how the TRP metabolic process associated with the intestinal flora affects HF through the heart-gut axis is still unclear. Summary of the invention

[0006] In order to further explore the pathological mechanism of HF, the present invention combined intestinal microbiota and amino acid metabolomics to study the pathogenesis of HF. Amino acid metabolomics analysis, mass spectrometry imaging (MSI) and 16S ribosomal DNA (16S rDNA) sequencing were performed using the established HF rat model. By combining various detection and verification techniques, the potential role of the KYN pathway in the pathogenesis of HF and changes in intestinal microbial composition was explored. In addition, indoleamine 2,3-dioxygenase (IDO) inhibitors and Parabacteroides distilogenes were used to detect and validate the KYN pathway. Parabacteroides_distasonis(PD) It was used to treat HF ​​rats and verified that the KYN pathway is associated with the progression of HF. PD Specifically, the present invention includes the following contents.

[0007] In a first aspect of the present invention, a method for preparing a drug for treating or improving heart failure is provided, wherein the reagent comprises Parabacteroides dieldrin or its culture, lysate, extract, content, metabolite, or a Parabacteroides dieldrin promoter.

[0008] In certain embodiments, according to the use of the present invention, the promoter comprises:

[0009] (1) agents that activate or promote the growth or proliferation of Parabacteroides distichous; and / or

[0010] (2) Agents that enhance the activity of Parabacteroides distichous.

[0011] In certain embodiments, according to the use of the present invention, the promoter comprises a kynurenine pathway inhibitor.

[0012] In certain embodiments, according to the use of the present invention, the kynurenine pathway inhibitor comprises an indoleamine 2,3-dioxygenase inhibitor.

[0013] In certain embodiments, according to the use of the present invention, the kynurenine pathway inhibitor is 1-methyltryptophan (also referred to herein as "1-MT") or a derivative thereof.

[0014] In certain embodiments, according to the use of the present invention, the treatment or improvement includes curing, alleviating, alleviating, preventing the occurrence or development of heart failure.

[0015] A second aspect of the present invention provides a method for screening a drug for treating heart failure, comprising:

[0016] a. using a reagent to measure the amount of Parabacteroides distichous in a sample collected from a subject suffering from heart failure to obtain a first measurement value;

[0017] b. a step of administering the test drug to a subject suffering from heart failure;

[0018] c. measuring the amount of Parabacteroides distichous in a sample collected from a subject with heart failure after administration of the test drug to obtain a second measurement value;

[0019] d. comparing the first measurement value and the second measurement value;

[0020] e. When the second measurement value is less than or equal to the first measurement value, the drug to be tested is screened as a drug that is useless for treating or alleviating heart failure; when the second measurement value is greater than the first measurement value, the drug to be tested is screened as a drug that is useful for treating or alleviating heart failure.

[0021] In certain embodiments, according to the method of the present invention, the drug to be tested includes at least one of a small molecule compound, an antibody, a protein, an enzyme, and a nucleic acid.

[0022] According to a third aspect of the present invention, there is provided use of a reagent for displaying Parabacteroides distichous in the preparation of a product for predicting, detecting or diagnosing heart failure.

[0023] In certain embodiments, according to the use of the present invention, the product comprises a kit, a diagnostic device and / or a computer system.

[0024] In certain embodiments, according to the use of the present invention, the product further comprises a reagent for detecting kynurenine.

[0025] In certain embodiments, according to the use of the present invention, wherein the prediction, detection or diagnosis comprises the following steps:

[0026] (1) measuring the amount of Parabacteroides distichoui in a sample collected from a subject using a detection reagent to obtain a measurement value;

[0027] (2) comparing the measured value with a standard value; and

[0028] (3) When the measured value is lower than the standard value, the subject is predicted to suffer from heart failure, or the subject is predicted to be at risk of suffering from heart failure; when the measured value is higher than the standard value, the subject is predicted to not suffer from heart failure, or the subject is predicted to not be at risk of suffering from heart failure.

[0029] In certain embodiments, according to the use of the present invention, the standard value is a value obtained from a biological sample of a normal subject of comparable age to the subject.

[0030] Although there is evidence that the gut microbiota may play a key role in the pathogenesis of cardiovascular diseases. However, the mechanisms of heart-gut axis interactions in heart failure are still unclear. In this study, amino acid metabolomics and gut microbiota diversity analysis were performed to explore metabolic disorders and gut microbial community dysbiosis in HF. The results showed that the kynurenine pathway was strongly activated in the circulation and cardiac tissues of HF rats. In addition, 16S rDNA sequencing revealed changes in the microbial profile after HF. In particular, the bacteria Parabacteroides distichum associated with the KYN pathway Parabacteroides_distasonis(PD) In order to verify the KYN pathway and PD The relationship between KYN pathway inhibitors and HF rats was found, which improved the cardiac function damage and increased PD Level. Application PD The results showed that the KYN pathway disorder was reversed in HF rats after HF and HF was relieved, which can be used as a drug to treat or improve HF. These results innovatively demonstrated that the KYN pathway and PD The bidirectional circuit response on the heart-gut axis in HF treatment provides new insights into the heart-gut axis in the development of HF and suggests new potential targets for the treatment of HF. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The ejection fraction (EF) and fractional shortening (FS) of heart failure model rats were detected using an ultrasound system.

[0032] Figure 2 The maximum rise and fall rates of left ventricular pressure (dp / dt max and dp / dt min ), left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP).

[0033] Figure 3 B-type natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) in rats with heart failure model were detected by enzyme-linked immunosorbent assay (ELISA).

[0034] Figure 4 Changes of kynurenic acid (KYNA), tryptophan (TRP) and KYNA / TRP ratio in sham-operated and model groups of rats with heart failure model.

[0035] Figure 5 Heat map of the results of Spearman correlation analysis examining the associations between serum KYNA / TRP levels, cardiac function, and inflammation-related parameters.

[0036] Figure 6The spatial distribution of TRP, KYNA and KYN in the sham operation group and the model group was obtained by mass spectrometry imaging (MSI).

[0037] Figure 7 Chao index analysis was used to evaluate the alpha diversity of the intestinal microbiota in the sham and model groups.

[0038] Figure 8 Principal coordinate analysis (PCoA) plot of intestinal flora of rats in each group at the genus level.

[0039] Fig. 9 Relative abundance distribution of intestinal flora at the genus level in each group of rats.

[0040] Fig.10 Wilcoxon rank sum test bar chart of intestinal flora of rats in each group at the genus level.

[0041] Fig.11 Intestinal flora of rats in each group Parahacteroides_distasonis Bacterial strain comparison.

[0042] Fig.12 Linear discriminant analysis effect size (LEfSe) analysis ( Fig.12 Parabacteroides distiliensis (PD) The correlation between the relative abundance of KYNA and TRP ( Fig.12 ).

[0043] Fig.13 Concentrations of 2,3-dioxygenase (IDO) and kynurenine aminotransferase (KAT) after IDO inhibitor treatment.

[0044] Figure 14-16 Echocardiography, hemodynamic measurements, and HF-related biochemical parameters after 2,3-dioxygenase (IDO) inhibitor therapy.

[0045] Fig.17 Kynurenine, tryptophan, kynurenic acid, and kynurenic acid / tryptophan levels after 2,3-dioxygenase (IDO) inhibitor treatment.

[0046] Fig.18 Heat map of the results of Spearman correlation analysis examining the correlation between IDO and KAT and cardiac function.

[0047] Fig.19 Parabacteroides dissimilar (PD) Relative quantitative polymerase chain reaction (qPCR) analysis.

[0048] Fig. 20 Parabacteriodes_distasonis(PD)After treatment, the ejection fraction (EF) and fractional shortening (FS) were detected using an ultrasound system in the sham group, model group, low-dose group, and high-dose group.

[0049] Figure 21-22 Parabacteriodes_distasonis(PD) Echocardiography, hemodynamic measurements, and biochemical tests related to heart failure were performed after treatment.

[0050] Fig.23 Parabacteriodes_distasonis(PD) KYN, KYNA, and TRP levels in rat serum after treatment.

[0051] Fig.24 Parabacteriodes_distasonis(PD) Pathological evaluation results of rats after treatment.

[0052] Fig.25 Pathological staining images of the model group and the drug-treated group. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0055] Unless otherwise stated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. Unless otherwise stated, "%" is a percentage based on weight.

[0056] The present invention is described in detail below.

[0057] In the present invention, the term "amount" includes content, level, absolute amount, relative amount and the like.

[0058] The terms "heart failure", "HF" and "heart failure" used in this article are used interchangeably. They refer to the inability of the heart's contractile and / or diastolic function to adequately discharge the venous return blood from the heart, resulting in blood congestion in the venous system and insufficient blood perfusion in the arterial system, thereby causing a syndrome of cardiac circulatory disorders.

[0059] Use of reagent in preparing medicine for treating or improving heart failure

[0060] In one aspect of the present invention, a reagent is provided for preparing a drug for treating or improving heart failure, wherein the reagent comprises Parabacteroides districhiae or its culture, lysate, extract, content, metabolite, or a promoter of Parabacteroides districhiae. Promoters refer to any small or macromolecular substance that can (1) activate or promote the growth or proliferation of Parabacteroides districhiae; and / or (2) enhance the activity of Parabacteroides districhiae.

[0061] It is understood that as long as the complete bacterial body of Parabacteroides distichous or the material derived from the bacteria (including but not limited to the lysate, extract, content, metabolite of the bacteria) is contained, it is within the scope of the present invention. In a specific embodiment, the agent used for treatment is a liquid preparation containing Parabacteroides distichous bacteria, or a culture solution with a suitable number of viable bacteria obtained by Parabacteroides distichous under conditions suitable for its growth and amplification. Suitable culture conditions for Parabacteroides distichous are well known in the art and are not particularly limited thereto.

[0062] The present invention verifies the bidirectional circular pathway of Parabacteroides dieldii-kynurenine. Therefore, the promoter of the present invention includes but is not limited to a kynurenine pathway inhibitor, which may also be an inhibitor of an enzyme involved in the kynurenine pathway, for example, an inhibitor of indoleamine 2,3-dioxygenase.

[0063] In the present invention, the levels of the precursors or metabolites corresponding to the upstream and downstream of kynurenine and Parabacteroides distichous can be detected and quantified by any method known in the art. For example, mass spectrometry, such as UPLC-ESI-MS / MS or NMR spectroscopy, can be used. 1 H-NMR spectroscopy, or various appropriate technical means such as fluorescence quantitative PCR for detection.

[0064] The treatment or improvement of heart failure can be achieved by administering a therapeutically effective amount of the agent to a subject in need thereof. The term "therapeutically effective amount" used in the present invention refers to an effective dosage considered pharmaceutically, that is, the amount of the drug administered is sufficient to significantly improve the condition without causing serious side effects. The dosage depends on many factors, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the number of administrations, and the purpose of treatment. Therefore, the therapeutic dose of the present invention can vary widely.

[0065] Use of reagents in the preparation of products for predicting, detecting or diagnosing heart failure

[0066] In one aspect of the present invention, a reagent is provided for use in preparing a product for predicting, detecting or diagnosing heart failure, wherein the product is selected from a kit, a diagnostic device and / or a computer system, and the reagent used for detection includes any reagent capable of displaying Parabacteroides distichous, for example, it can be a primer and / or probe used for detecting Parabacteroides distichous, etc. The specific sequence can be found in CN117683849A, the content of which is incorporated herein by reference in its entirety.

[0067] In the present invention, the diagnosis or prediction at least includes:

[0068] (1) measuring the amount of Parabacteroides distichoui in a sample collected from a subject using a detection reagent to obtain a measurement value;

[0069] (2) comparing the measured value with a standard value; and

[0070] (3) When the measured value is lower than the standard value, the subject is predicted to suffer from heart failure, or the subject is predicted to be at risk of suffering from heart failure; when the measured value is higher than the standard value, the subject is predicted to not suffer from heart failure, or the subject is predicted to not be at risk of suffering from heart failure.

[0071] In a preferred embodiment, the measured values ​​also include additional measured values ​​for combined detection with the amount of Parabacteroides distichous, including but not limited to KYN, KYNA, TRP levels and / or kynurenic acid / tryptophan ratio (KYNA / TRP). Similarly, the standard values ​​also include KYN, KYNA, TRP levels and / or kynurenic acid / tryptophan ratios obtained from biological samples of normal subjects of comparable age to the subject.

[0072] Step (1)

[0073] Step (1) of the present invention is a step for obtaining the amount of Parabacteroides distichous in a sample from a subject, and / or the amount of KYN, KYNA, TRP and / or the kynurenic acid / tryptophan ratio. The type of sample is not limited, and examples of biological sample types that can be used in the present invention include, but are not limited to, one or more of the following: urine, feces, tears, whole blood, serum, plasma, blood components, bone marrow, cells, tissues, organs, body fluids, saliva, cheek swabs, lymph, cerebrospinal fluid, lesion exudates, and other fluids produced by the body. The biological sample can also be a frozen, fixed, paraffin-embedded or fresh biopsy sample.

[0074] In step (1) of the present invention, the detection reagent may further include other components, examples of which include but are not limited to LC-MS / MS, amplicon sequencing, and flow cytometry-related assay reagents.

[0075] The detection reagent of the present invention can be further prepared as a diagnostic agent for detecting heart failure in a subject, which can be in the form of a diagnostic composition, a diagnostic kit, or any other form of a combination of multiple reagents existing alone.

[0076] Step (2)

[0077] Step (2) of the present invention is a step of comparing the measured value with a standard value, wherein the standard value can be a specific value or a range of values.

[0078] In certain embodiments, the standard value can be a sample test value from a normal subject. Preferably, the sample value from a normal subject of the same age as the subject to be tested is used, and preferably, the standard value and the measured value are obtained by the same method.

[0079] In certain embodiments, the standard value may also be derived from different time periods of the same subject. For example, the amount in the sample T1 collected from the subject at the first time point is measured as the standard value, and the amount in the sample T2 collected from the same subject at the second time point is measured as the measured value. The first time point T1 is preferably a time point before any sign of heart failure is shown or does not appear, and the time point when the second time point T2 is detected is preferably a time point after any sign of heart failure appears.

[0080] Step (3)

[0081] Step (3) of the present invention is a result determination step. Specifically, when the measured value is lower than the standard value, the subject is predicted to suffer from heart failure, or is predicted to be at risk of suffering from heart failure; when the measured value is higher than the standard value, the subject is predicted to not suffer from heart failure, or is predicted to not be at risk of suffering from heart failure.

[0082] When the test is performed in combination with other indicators, for example, when the kynurenic acid / tryptophan ratio KYNA / TRP is higher than the reference value and the amount of Parabacteroides distilogenes is lower than the reference value, the subject is predicted to suffer from heart failure, or is predicted to be at risk of suffering from heart failure; otherwise, the subject is predicted to not suffer from heart failure, or is predicted to not be at risk of suffering from heart failure.

[0083] Drug Screening Methods

[0084] One aspect of the present invention provides a method for screening a drug for treating heart failure, the method comprising:

[0085] a. using a reagent to measure the amount of Parabacteroides distichous in a sample collected from a subject suffering from heart failure to obtain a first measurement value;

[0086] b. a step of administering the test drug to a subject suffering from heart failure;

[0087] c. measuring the amount of Parabacteroides distichous in a sample collected from a subject with heart failure after administration of the test drug to obtain a second measurement value;

[0088] d. comparing the first measurement value and the second measurement value;

[0089] e. When the second measurement value is less than or equal to the first measurement value, the drug to be tested is screened as a drug that is useless for treating or alleviating heart failure; when the second measurement value is greater than the first measurement value, the drug to be tested is screened as a drug that can treat or alleviating heart failure.

[0090] In the screening method of the present invention, the subject is preferably an animal model suffering from heart failure, for example, rats, mice, dogs, pigs, monkeys, orangutans, etc. Such animals can be artificially induced to suffer from heart failure or show typical clinical features of heart failure (including detectable biochemical indicators).

[0091] In a preferred embodiment of combining other indicators for drug screening, the drug screening method of the present invention comprises:

[0092] (1) using reagents to measure the kynurenic acid / tryptophan ratio KYNA / TRP and the amount of Parabacteroides distichous in a sample collected from a subject with heart failure, respectively, to obtain a first measurement value;

[0093] (2) administering the drug to a subject with heart failure;

[0094] (3) respectively measuring the kynurenic acid / tryptophan ratio KYNA / TRP and the amount of Parabacteroides distichous in the sample collected from the subject after the administration in step (2) to obtain a second measurement value;

[0095] (4) comparing the first measurement value of step (1) and the second measurement value of step (3), wherein when the KYNA / TRP in the second measurement value is less than the KYNA / TRP in the first measurement value, and the amount of Parabacteroides distichous in the second measurement value is greater than the amount of Parabacteroides distichous in the first measurement value, the drug is screened as a drug useful for treating or alleviating heart failure; otherwise, the drug is screened as a drug that is not useful for treating or alleviating heart failure. Example

[0096] 1. Materials and Methods

[0097] 1. Materials and Methods

[0098] 1.1 Preparation of the rat left anterior descending coronary artery ligation (LAD) model

[0099] SPF male SD rats (purchased from Beijing Huafukang Biotechnology Co., Ltd.) weighing 240±10g were used. After acclimating to the environment for 3 days, the rats were fasted for 12 hours before modeling. They were randomly divided into a sham operation group and a model group. The model group was anesthetized intraperitoneally with 3% sodium pentobarbital solution prepared with sterile saline at a dose of 0.2 ml / 100 g. The rats were depilated and skinned, disinfected, and the skin was cut longitudinally about 2 cm along the left edge of the left sternum under sterile conditions. The muscle tissue was bluntly separated between the fourth or fifth ribs, the chest cavity was opened and the pericardium was cut open, and the heart was hooked out with a blunt retractor. The left atrial appendage and the pulmonary artery cone were threaded, and the left anterior descending branch of the coronary artery were ligated. Then the heart was quickly returned to the chest cavity, and the chest cavity and skin were sutured. After the ligation of the left anterior descending branch of the coronary artery, the electrocardiogram of each rat was observed, and the ST segment arch elevation ≥0.1mV was retained, that is, the rat had symptoms of myocardial ischemia. The sham operation group was treated in the same manner as the model animals except that the coronary artery was not ligated.

[0100] Blood samples were collected 28 days after modeling, serum was placed in an ice bath for 30 minutes, centrifuged at 4°C and 3000 rpm for 10 minutes, and the supernatant was aliquoted and frozen in a -80°C refrigerator for later use. Fresh feces of rats were collected by abdominal massage for intestinal flora analysis and stored in a -80°C ultra-low temperature refrigerator for later use.

[0101] 1.2 Confirmation of rat heart failure model

[0102] Cardiac function, hemodynamics and biochemical indexes were tested 28 days after modeling.

[0103] The small animal ultrasound system was used for measurement, and the results of cardiac function test were as follows Figure 1 The results showed that compared with the sham operation group, the left ventricular ejection fraction and left ventricular fractional shortening of the rats in the model group were significantly reduced on the 28th day after acute myocardial infarction induced by coronary artery ligation, indicating that heart failure may occur in the rats.

[0104] The carotid artery intraventricular catheterization method was used to evaluate the cardiac hemodynamic parameters of rats in each group. Figure 2 The hemodynamic evaluation showed that the left ventricular end-diastolic pressure in the model group was higher than that in the sham operation group. On the contrary, compared with the sham operation group, the left ventricular systolic pressure and the maximum rate of decrease of left ventricular pressure (±dp / dt min ) and the maximum rate of rise of left ventricular pressure (±dp / dt max ) was significantly reduced.

[0105] The serum of rats was collected 28 days after modeling and the levels of rat B-type natriuretic peptide and N-terminal pro-brain natriuretic peptide were detected using the THERMO fully automatic microplate reader MK3 according to the ELISA kit. Figure 3 Biochemical indexes showed that the concentrations of B-type natriuretic peptide and N-terminal pro-brain natriuretic peptide in the model group were significantly higher than those in the sham-operated rats. The above results showed that the rat heart failure model was successfully established.

[0106] 1.3 Sample collection and processing

[0107] After the hemodynamic analysis, the rats were euthanized, and the heart tissue samples were collected, rinsed with chilled saline, and divided into three groups; the samples were frozen at −80°C, fixed with 4% paraformaldehyde and embedded in paraffin, or embedded in optimal cutting temperature (OCT) complex medium for histopathological analysis.

[0108] 1.4 Histopathological analysis

[0109] Frozen heart tissue and 4% paraformaldehyde-fixed heart tissue were embedded in OCT and paraffin, respectively, and cut into 6-µm sections. Samples were stained with hematoxylin and eosin and Masson's trichrome stain (B1011) according to the manufacturer's instructions and imaged using an appropriate microscope (Nikon ECLIPSCI). Histopathological parameters of H&E and Masson's staining were quantified by pathological evaluation and identification of fibrosis-positive areas.

[0110] 1.5 Analysis of serum inflammatory cytokine levels

[0111] Serum samples collected as described above were analyzed for inflammatory cytokine content using rat LegendPlex interleukin-6 (IL-6) and interleukin-17A (IL-17A) capture beads and a BD FACSMelody™ flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA). FCS data were analyzed using the provided tool (https: / / www.biolegend.com / en-us / legendplex).

[0112] 1.6 Serum metabolomics analysis

[0113] 1.6.1 Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS / MS) conditions for targeted metabolomics analysis

[0114] Targeted metabolomics analysis was performed using UPLC-MS / MS using a Waters ACQUITY UPLC system coupled with a triple quadrupole mass spectrometer and a Xevo TQ-S (Waters Corp., Milford, MA) equipped with an electrospray ionization (ESI) source. The mass spectrometer (MS) was operated in positive and negative ion modes with multiple reaction monitoring (MRM). Data acquisition and peak processing were performed using Waters TargetLynx software.

[0115] 1.6.2 UPLC-MS / MS for quantitative analysis of TRP, kynurenic acid (KYNA), and KYN

[0116] Serum TRP, KYNA, and KYN concentrations were quantified using a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) (AB Sciex Corp., Boston, USA) system in ESI positive ion mode.

[0117] Peak area integration was performed using MultiQuant 3.0.3 (AB Sciex Corp, Boston, USA), and sample concentration was calculated using the calibration curve according to the following formula: sample concentration = sample area × internal standard (IS) concentration / IS area.

[0118] Linear regression analysis was used to evaluate the slope, intercept and correlation coefficient values ​​of the individual calibration curves using a weighting factor of 1 / x. The calibration curves showed good linearity (r>0.99) over a wide range.

[0119] 1.6.3 Quantitative analysis of TRP, KYNA, and KYN in serum of rats treated with 1-MT by UPLC-MS / MS

[0120] A Waters ACQUITY UPLC system coupled with a triple quadrupole mass spectrometer, a Xevo TQ-S (Waters Corp., Milford, MA) system and an ESI source was used to quantify serum TRP, KYNA, and KYN concentrations in the ESI positive ion mode.

[0121] Waters TargetLynx software was used for data acquisition and peak processing. The calibration curve showed good linearity (r>0.99) over a wide range.

[0122] 1.6.4 UPLC-MS / MS quantitative analysis PD TRP, KYNA and KYN in serum of treated rats

[0123] The detection was performed using the same method as described above. The calibration curve showed good linearity (r>0.99) over a wide range.

[0124] 1.7 Mass spectrometry imaging

[0125] Heart tissue samples frozen in liquid nitrogen and randomly selected from each group were cut into 15-µm-thick sections. These sections were then analyzed using an airflow-assisted desorption electrospray ionization mass spectrometry platform (AFADESI-MSI) following the methods outlined in previous studies.

[0126] 1.8 16S rDNA sequencing

[0127] On day 28 after the establishment of the HF model, fecal samples were collected from each rat, quickly frozen in liquid nitrogen, and stored at −80 °C until 16S rDNA sequencing.

[0128] 1.9 Verification of 1-MT administration

[0129] After 7 days of acclimatization, the HF model was established in SD rats, which were then randomly divided into sham operation, HF model, low-dose (250 mg / kg / day, MT-L), and high-dose (500 mg / kg / day, MT-H) groups. Rats in the low-dose and high-dose groups were treated with 1-MT (L-1-MT in drinking water) by oral gavage for 7 days before ligation, and then until euthanasia 28 days after LAD ligation.

[0130] 1.10 PD Verification of drug administration

[0131] After 3 days of adaptation, the HF model was established in SD rats and then randomly divided into the following groups: sham operation group, HF model group, low dose (1×10 8 CFU / rat) and high dose (1×10 9 CFU / rat) group. After ligation, rats in the low-dose group and the high-dose group were gavaged PD 28 days until euthanasia.

[0132] 2. Results

[0133] 2.1 Animal model establishment

[0134] 2.1.1 Cardiac function and hemodynamic parameters of the heart failure rat model

[0135] In order to elucidate the potential pathological mechanism of heart failure, this example designed a heart failure rat model established by ligating the LAD coronary artery branches. On the 28th day after ligation, echocardiographic evaluation showed that the left ventricular ejection fraction (LVEF) and shortening fraction (FS) in the HF model group were significantly reduced compared with the sham control group ( Figure 1 These findings are consistent with the characteristics of the CHF model. In addition, hemodynamic evaluation performed on the 33rd day after ligation showed that the left ventricular end-diastolic pressure (LVEDP) of the HF model rats was significantly increased, and the left ventricular systolic pressure (LVSP) was significantly decreased. The maximum rate of increase and decrease of left ventricular intraventricular pressure (dp / dt, respectively) was calculated. max and dp / dt min )( Figure 2 ). Together, these changes indicate that a rat model that recapitulates key features of HF has been successfully established.

[0136] 2.1.2 Serological indices of heart failure rat model

[0137] Since serum levels of brain natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) are gold standard biomarkers for diagnosing HF and assessing the prognosis of affected patients, the levels of these two proteins were determined in both groups of rats. Compared with the sham group, serum BNP and NT-proBNP levels in the HF model group were significantly increased ( Figure 3 ).

[0138] 2.1.3 Histopathological examination of the heart failure rat model

[0139] In addition, to more clearly characterize the characteristics of HF rats, the histomorphological changes of the myocardium were analyzed using H&E and Masson's trichrome staining. H&E and Masson's trichrome staining showed that the pathological evaluation results and fibrosis-positive areas were significantly increased after HF. The myocardial lesions in the HF model group were mainly confined to the middle part of the outer wall of the left ventricle, characterized by myocardial fiber fragmentation, disorganized cell structure, severe nuclear staining, and interstitial edema with inflammatory cell infiltration. Notably, the left ventricular wall of HF rats showed considerable collagen fiber deposition, indicating the presence of fibrosis. In addition, these animals showed compensatory right ventricular (RV) hypertrophy and ventricular cavity dilation, further emphasizing the structural remodeling associated with HF.

[0140] 2.2 Metabolomics analysis of serum and myocardial tissue in rats with heart failure model

[0141] 2.2.1 Analysis of serum amino acid metabolomics in HF model rats

[0142] Amino acid metabolomics analysis was also performed to investigate metabolic disturbances after HF. Two metabolites with different abundances, tryptophan TRP and kynurenic acid KYNA, were identified. Compared with the sham group, serum KYNA and TRP levels were increased in the HF model group, as was the KYNA / TRP ratio ( Figure 4 ). There are three metabolic pathways for TRP metabolism, namely the KYN pathway, the indole pathway, and the serotonin pathway, among which the KYN pathway is dominant. The rate-limiting enzyme of the KYN pathway is IDO, and the KYNA / TRP ratio reflects the activity of the KYN pathway.

[0143] 2.2.2 Spearman correlation analysis between KYNA / TRP and cardiac function

[0144] In order to more clearly determine the relationship between KYNA / TRP and cardiac function, Spearman correlation analysis was used. The results showed that KYNA / TRP and dp / dt min There was a significant positive correlation between BNP, NT-proBNP and LVSP, and a significant negative correlation between EF%, FS% and LVEDP ( Figure 5 These data suggest that activation of the KYN pathway of TRP metabolism is involved in the development and progression of HF.

[0145] 2.2.3 Myocardial MSI in rat heart failure model

[0146] To confirm the above hypothesis, metabolomics analysis was performed using MSI in the two groups of rats. MSI data showed that the levels of KYNA, TRP, and KYN in myocardial tissue of the HF model group were significantly increased compared with the sham group ( Figure 6). Collectively, these findings suggest that the KYN pathway of TRP metabolism exacerbates HF development and progression.

[0147] 2.3 Microbial characteristics of HF model rats

[0148] The gut microbiota has been identified as a core factor affecting human health and disease, and the gut microbiota has a certain influence on the pathogenesis of cardiovascular disease. In this example, 16S rDNA sequencing was performed on the fecal samples of these HF model rats to investigate the dysbiosis of the gut microbiota and to elucidate the relationship between the gut microbiota and TRP metabolism after HF. First, the differences in the composition of the gut microbiota between the two groups were analyzed by evaluating α diversity. The Chao index of the HF model group was significantly higher than that of the sham operation group ( Figure 7 ), indicating changes in the gut microbiota.

[0149] To further explore the changes in the composition of the rat gut microbiota, principal coordinate analysis (PCoA) was used to measure β diversity. In PCoA, the HF model group and the sham operation group could be clearly separated. That is, at the genus level, there were significant differences in the distribution of bacteria with higher relative abundance between the HF model group and the sham operation group ( Figure 8 ).

[0150] In order to understand the changes in the intestinal microbiome more precisely, differential analysis was performed at the genus and species levels. Fig. 9 The results showed that there were four genera between the HF model group and the sham operation group. (Erysipelatocostridium, Parabacteroides, Turicibacter and Dielma There are significant differences ( Fig.10 ).

[0151] The species-level community histogram analysis is as follows Fig.11 Screening of differential bacterial species showed that compared with the sham operation group, the number of Parabacteroides distilogenes in the HF model group was significantly higher than that in the sham operation group. PD To verify the accuracy of the results, a linear discriminant analysis effect size (LEfSe) analysis was further performed. At the genus level, the abundance of Parabacteroides distiliensis in the sham operation group was significantly higher than that in the model group. PD The abundance was significantly higher than that in the model group ( Fig.12 To explore PD The relationship between KYN pathway and PD The relative abundance and KYNA / TRP ratio of the two indicators were linearly analyzed. The results showed that there was a negative correlation between the two indicators ( Fig.12 ).

[0152] 2.4 Inhibition of the KYN pathway of TRP metabolism improves cardiac function in HF model rats

[0153] Although the concentrations of TRP and KYNA in serum and myocardial tissue of HF model rats were significantly increased, the causal relationship between these levels and HF remains to be verified. The rate-limiting enzyme in the TRP metabolism KYN pathway is IDO, therefore, the IDO inhibitor 1-MT (250 mg / kg / day and 500 mg / kg / day) was administered to HF model rats by gavage. The concentrations of IDO and kynurenine aminotransferase (KAT) in the serum of rats in the MT-L and MT-H groups were decreased ( Fig.13 In addition, echocardiography, hemodynamic measurements, and HF-related biochemical parameters were performed on day 28 after the start of 1-MT treatment ( Figure 14-16 ). Compared with the model group, all parameters related to cardiac function in the MT-L group showed a downward trend. In particular, EF%, FS% and dp / dt max Significant improvement. Short duration of HF (≤4 years) is one of the determining factors for the improvement of EF% and FS% in HF patients. Therefore, the results indicate that 250 mg / kg / day of 1-MT can partially improve HF. All parameters related to cardiac function were significantly improved in the MT-H group compared with the model group. These findings further suggest that inhibition of the KYN pathway can alleviate HF. In addition, serum targeted metabolomics analysis of rats taking 1-MT showed that kynurenine, tryptophan, kynurenic acid, and kynurenic acid / tryptophan levels were significantly reduced after 1-MT treatment ( Fig.17 ). Spearman correlation analysis was further performed on IDO and KAT and cardiac function indicators. The results showed that IDO and KAT were significantly positively correlated with BNP, NT-proBNP and LVEDP, but negatively correlated with EF% and FS% ( Fig.18 ). These data suggest that inhibition of the KYN pathway of TRP metabolism could alleviate HF.

[0154] To investigate the mechanism by which the KYN pathway affects HF and its relationship with the intestinal microbiota, relative quantitative PCR was used to measure the expression of KYN in the gut microbiota after 1-MT treatment. PD In HF rats, PD However, inhibition of the KYN pathway by 1-MT treatment resulted in PD The content gradually increased ( Fig.19 These findings suggest that the KYN pathway has an impact on HF progression. PD Level related.

[0155] To further verify the above hypothesis that inhibition of the KYN pathway of TRP metabolism can alleviate HF, H&E and Masson pathological staining of cardiac tissues were performed. The staining results showed that the pathological evaluation results and fibrosis-positive area increased significantly after HF and decreased significantly after 1-MT treatment. Rats in the HF model group showed myocardial fiber damage, disordered myocardial cell arrangement, and extensive myocardial infarction. In contrast, the 1-MT-treated group showed a significant reduction in myocardial infarction volume, reduced myocardial fiber damage, normal myocardial cell arrangement, and overall improvement in HF-related pathological morphology. In addition, the degree of improvement was positively correlated with the 1-MT dose.

[0156] 2.5 Verification through KYN PD Protective effect against HF

[0157] Based on previous experimental studies, it can be concluded that during HF PD The level was significantly reduced and negatively correlated with the degree of activation of the KYN pathway. In addition, inhibition of the KYN pathway with 1-MT significantly restored PD levels and reduce HF damage. PD In order to investigate the relationship between the KYN pathway and HF, a set of additional validation experiments were conducted. PD , the dose is 1×10 8 CFU / cell and 1×10 9 CFU / mouse. On the 28th day after administration, echocardiography, hemodynamic measurements, and biochemical tests related to heart failure were performed. Compared with the model group, the low-dose (L) group and the high-dose (H) group showed significant improvements in various cardiac function parameters ( Figure 20-22 ). In addition, serum targeted metabolomics analysis showed that PD The levels of KYN, KYNA and TRP in the serum of treated rats were significantly decreased ( Fig.23 ).

[0158] To prove PD To investigate the protective effect of HF, H&E and Masson pathological staining were performed on the heart tissues of each group of rats. H&E and Masson staining showed that the pathological evaluation results and the positive area of ​​fibrosis increased significantly after HF. PD After treatment, the Fig.24 ). The heart tissue of rats in the HF model group showed myocardial fiber damage, disordered myocardial cell arrangement and large-area myocardial infarction. Compared with the model group, PD The volume of myocardial infarction in the group was significantly reduced, myocardial fiber damage was alleviated, myocardial cell arrangement tended to be normal, and the overall pathological morphology related to HF was alleviated. PD There is a positive correlation between the dose ( Fig.25 ).

[0159] 3. Discussion

[0160] The intestinal microbiota plays a key role in the occurrence and development of HF and may be a new approach to explore the mechanism of HF and alleviate its symptoms. However, the mechanism of the heart-gut axis interaction in heart failure is still unclear. In this example, amino acid metabolomics and MSI were performed to explore metabolic disorders after HF. Notably, the KYN pathway was strongly activated in the circulation and cardiac tissues of HF rats. 16S sequencing revealed the disturbance of intestinal flora after HF, indicating that the KYN pathway is involved in the PD This relationship was confirmed by 1-MT administration and PD These findings suggest that KYN pathway inhibition by administration of 1-MT in HF rats increases PD levels and treat HF. In addition, HF rats were PD Therapeutic methods that affect the KYN pathway through the heart-gut axis to treat HF ​​have been demonstrated to be valuable. Therefore, the present invention provides new insights into the heart-gut axis in the development of HF and suggests new potential targets for the treatment of HF.

[0161] Metabolic disturbances are intrinsic factors in the pathophysiology of heart failure. In particular, imbalances in amino acid metabolism have been widely reported after HF. In this example, a rat model of heart failure characterized by cardiac dysfunction and hemodynamic disturbances was successfully established. Subsequently, amino acid metabolism was studied, with a particular focus on the KYN pathway. The results showed that this pathway was significantly upregulated compared with sham-operated rats, characterized by significantly increased serum concentrations of TRP and KYNA and an increased KYNA / TRP ratio. In addition, meticulous analysis of myocardial samples by MSI technology confirmed the parallel increase in TRP, KYN and KYNA levels in cardiac tissue. These cumulative observations highlight the systemic and local upregulation of the KYN pathway in the context of HF, indicating its involvement in the pathophysiological processes underlying this complex disease state.

[0162] TRP is one of the nine essential amino acids that must be obtained through dietary sources such as legumes and meat. TRP catabolism plays an important role in maintaining normal physiology and rapid adaptation to stress conditions. Endogenously produced TRP metabolites have been well studied. There are three main pathways for TRP metabolism: (1) the KYN pathway, involving IDO1, IDO2, and tryptophan 2,3-dioxygenase (TDO2); (2) the 5-HT production pathway, involving TPH; and (3) the indole-3-pyruvate production pathway. The KYN pathway is the major pathway. Recent studies have shown a link between the KYN pathway of TRP metabolism and certain cardiovascular diseases. For example, KYN is a valuable biomarker for CHF and can be used to predict mortality.

[0163] There is increasing evidence that the gut microbiota is associated with the onset and progression of HF. The complex mechanisms of the gut-heart axis involve physiological and pathological processes, including immune, metabolic, inflammatory, and blood circulation processes. In this example, preliminary 16S rDNA sequencing was performed on fecal samples. It revealed that the composition and function of the gut microbiota in the HF rat model were profoundly altered, as evidenced by the different clustering patterns in the PCoA plot and the significant changes represented by the community histogram. Through rigorous screening, four bacterial genera significantly associated with HF were identified: Erysipelatoclosdium, Parabacteroides, Turicibacter and Dielma . These findings highlight the complex interaction between gut microbial dysbiosis and the development or progression of HF. The KYN pathway is closely related to the gut microbiota in cardiovascular disease. The KYN pathway and gut microbiota can affect the occurrence and development of cardiovascular disease by regulating the body's immunity. To determine the relationship between the gut microbiota and the KYN pathway, a differentially expressed bacterium with a significant negative correlation with the KYNA / TRP ratio was further screened, namely PD Subsequently, validation experiments were performed to verify the involvement of the KYN pathway in HF progression. PD The relationship between.

[0164] The KYN pathway is initiated by IDO1, IDO2, or TDO2, producing the first stable product KYN. KYN can be further metabolized to 3-HK, 3-hydroxyaminobenzoic acid (3-HAA), quinolinic acid, or KYNA. To further study the KYN pathway and PD strain interactions in HF progression, using 1-MT to inhibit the KYN pathway. Notably, this intervention significantly improved cardiac function, thus confirming the key role of KYN pathway activation in the onset and progression of HF. In addition, 1-MT administration significantly increased PD strain abundance, indicating that the KYN pathway affects the composition of the gut microbiota in the context of HF, especially PD Overall, the findings of the present invention highlight the importance of KYN pathway activation for PD Therefore, targeting the KYN pathway in the management of HF may provide a new therapeutic strategy to regulate the gut microbiota through the heart-gut axis, especially PD levels, ultimately helping to improve HF.

[0165] In order to elucidate the role of the KYN pathway in the cardiogut axis during HF progression PD The complex relationship between strains, administered by gavage to HF rats PD The results showed that PDIntervention alleviated KYN pathway dysregulation and improved cardiac function and myocardial tissue pathology. These findings suggest that the heart-gut axis PD The strain can modulate the KYN pathway and exert therapeutic effects on HF, highlighting its potential as a novel therapeutic target.

[0166] In conclusion, this paper first discovered that KYN pathway dysregulation is associated with HF progression. PD The validation analysis not only confirmed that the KYN pathway is associated with PD A bidirectional relationship was also identified between PD Targeting these pathways could treat HF ​​through a bidirectional loop. This new insight provides a promising therapeutic target and approach for HF treatment.

[0167] 4. Conclusion

[0168] In summary, our results suggest that the KYN pathway is strongly activated in the circulation and cardiac tissues of HF rats. In addition, the microbial profiles of HF rats detected by 16S rDNA sequencing technology also showed significant differences in taxonomy and composition. PD Administration of the IDO enzyme inhibitor 1-MT to HF rats showed that inhibition of the KYN pathway could alleviate LAD-induced cardiac function damage in HF rats and increase intestinal microbiota. PD Expression. PD Treatment with KYN pathway disturbances was suppressed and HF was improved. These results suggest that KYN- PD This provides new insights into the mechanism of interaction between the microbiota and the heart-gut axis in HF and suggests a new potential target for the treatment of HF.

[0169] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

Claims

1. Use of the reagent in preparing a drug for treating or improving heart failure, characterized in that: The reagent includes Parabacteroides distichum ( Parabacteroides distasonis or a culture thereof, and / or 1-methyltryptophan.

2. The use according to claim 1, characterized in that The treatment or improvement includes curing, alleviating, alleviating, and preventing the occurrence or development of heart failure.

3. Use of a reagent for displaying Parabacteroides distichous in the preparation of a product for predicting, detecting or diagnosing heart failure.

4. The use according to claim 3, characterized in that: The products include kits, diagnostic devices and / or computer systems.

5. The use according to claim 3, characterized in that: The prediction, detection or diagnosis comprises the following steps: (1) measuring the amount of Parabacteroides distichoui in a sample collected from a subject using a detection reagent to obtain a measurement value; (2) comparing the measured value with a standard value; and (3) when the measured value is lower than the standard value, the subject is predicted to suffer from heart failure, or is predicted to be at risk of suffering from heart failure; when the measured value is higher than the standard value, the subject is predicted to not suffer from heart failure, or is predicted to not be at risk of suffering from heart failure; The standard value is a value obtained from a biological sample of a normal subject of the same age as the subject.

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