Biomarker related to pulmonary arterial hypertension and application thereof

Abstract

The invention relates to the technical field of biological medicine, on one hand, the invention relates to a pulmonary arterial hypertension related biomarker and application thereof, and the biomarker comprises two or three of bacteria, archaea and fungi; the bacteria comprise one or more of Blautia obeum, BlautiasspMarseilleP3087 and Coprococcomes, and the bacteria comprise one or more of the BlautiasspMarseilleP3087, the Coprococcomes and the BlautiasspMarseilleP3087; the archaea comprises the following components: Methanomethylovoranshillandica, and the archaea comprises the following components: Methanomethylovoranshillandica; the fungi comprise one or more of Sporothrix globosa, Fusariumoxyporum and Yarrowialipolytica, and the fungi comprise one or more of Sporothrix globosa, Fusariumoxyporum and Yarrowialipolytica; on the other hand, the invention relates to application of the biomarker in preparation of related products for pulmonary arterial hypertension diagnosis, prognosis evaluation and the like. Compared with a single bacterium as a biomarker, the bacterium, archaea and fungus combined as the biomarker for pulmonary arterial hypertension has higher specificity, can be used for preparing related products for pulmonary arterial hypertension diagnosis, prognosis evaluation and the like, and has important clinical significance.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a biomarker related to pulmonary arterial hypertension and application thereof. BACKGROUND

[0002] Pulmonary arterial hypertension (PAH) is a severe disease characterized by elevated resistance in the pulmonary vasculature, which, if left untreated, can lead to right heart failure and fatal consequences. Among various forms of PAH, idiopathic pulmonary arterial hypertension (IPAH) is particularly challenging due to its unknown etiology and poor prognosis. The underlying mechanisms of IPAH remain unclear, which poses significant challenges for effective treatment and management. Emerging evidence suggests a possible link between gut microbiota dysbiosis and PAH, and the gut-lung axis has become an emerging area of research in IPAH.

[0003] Gut microbiota (GM) not only includes bacteria, but also fungi and archaea, which play a fundamental role in human health and disease. Recent studies have shown that fungi and archaea in the gut play important roles in metabolic pathways, immune responses, and systemic inflammation. Studies on PAH patients have revealed significant changes in the structure and function of the intestinal bacterial community, including enrichment of bacteria involved in TMAO and purine metabolism, and reduction of butyrate and acetate-producing bacteria. Specific bacterial species in the gut are associated with the severity of PAH and clinical indicators, while patients with chronic thromboembolic pulmonary arterial hypertension (CTEPH) exhibit lower alpha diversity and higher inflammatory markers. In addition, PAH patients have lower levels of anti-inflammatory short-chain fatty acid (SCFA) genes and higher copy numbers of pro-inflammatory genes, accompanied by changes in plasma metabolite levels. Animal studies also support these findings, showing changes in the intestinal bacterial microbiota in PAH models, such as an increased Firmicutes / Bacteroidetes ratio in MCT-induced PAH rats, and intestinal pathology showing increased intestinal permeability and changes in the microbiota.

[0004] Although research on the relationship between gut microbiota and PAH has received increasing attention, there are still significant research gaps. Current research mainly focuses on bacterial components, with less attention on the role of intestinal fungi and archaea. In addition, because most existing research is observational, the causal relationship between gut microbiota and PAH is not clear. Therefore, exploring the relationship between gut microbiota and PAH and finding specific biomarkers are of great significance for the diagnosis, treatment, and prognosis evaluation of PAH.

[0005] In view of this, the present application is proposed. SUMMARY

[0006] The present application aims to provide a biomarker related to pulmonary arterial hypertension and an application thereof, and the present application uses bacteria, archaea and fungi as the biomarker related to pulmonary arterial hypertension, which is of great significance to the diagnosis, treatment and prognosis evaluation of pulmonary arterial hypertension.

[0007] In a first aspect, the present application provides a biomarker related to pulmonary arterial hypertension, which comprises two or three of bacteria, archaea and fungi; the bacteria comprises one or more of Blautia obeum, Blautia_sp_Marseille_P3087 and Coprococcus_comes; the archaea comprises Methanomethylovorans_hollandica; and the fungi comprises one or more of Sporothrix_globosa, Fusarium_oxysporum and Yarrowia_lipolytica.

[0008] Preferably, the biomarker comprises bacteria, archaea and fungi; the bacteria comprises Blautia obeum, Blautia_sp_Marseille_P3087 and Coprococcus_comes; the archaea comprises Methanomethylovorans_hollandica; and the fungi comprises Sporothrix_globosa, Fusarium_oxysporum and Yarrowia_lipolytica.

[0009] The present application screens 3 kinds of bacteria, 1 kind of archaea and 3 kinds of fungi closely related to pulmonary arterial hypertension, and simultaneously verifies that the 3 kinds of bacteria, 1 kind of archaea and 3 kinds of fungi combined as the biomarker have good diagnostic value for pulmonary arterial hypertension by using the ROC curve analysis method, and the AUC can be as high as 0.881.

[0010] The pulmonary arterial hypertension described in the present application includes but is not limited to idiopathic pulmonary arterial hypertension (IPAH).

[0011] In a second aspect, the present application provides an application of the biomarker related to pulmonary arterial hypertension in the preparation of a product for diagnosing pulmonary arterial hypertension, wherein the product is prepared by testing the relative abundance of bacteria, archaea and fungi in a sample to be tested of a subject, simultaneously performing ROC curve statistical analysis, and evaluating whether the subject has pulmonary arterial hypertension by using the ROC curve and the area AUC under the curve.

[0012] Preferably, the AUC value reaches 0.881 when the bacteria (Blautia obeum, Blautia_sp_Marseille_P3087 and Coprococcus_comes), archaea (Methanomethylovorans_hollandica) and fungi (Sporothrix_globosa, Fusarium_oxysporum and Yarrowia_lipolytica) are combined as markers.

[0013] Specifically, the sample to be tested is feces or intestinal contents.

[0014] Specifically, the product is a detection chip, a detection test paper or a detection kit, which is not strictly limited in the present application.

[0015] When the relative abundance of bacterial, archaeal and fungal strains in the sample to be tested is tested, the DNA of bacteria, archaea and fungi in the sample needs to be extracted using a kit first, therefore, the product also includes reagents required for extracting the DNA of the sample to be tested and determining the relative abundance of bacteria, archaea and fungi, including probes, primers, buffers, enzymes or mobile phases, etc.

[0016] In a third aspect of the present application, the application of the biomarker related to pulmonary arterial hypertension in the preparation of a product for evaluating the prognosis of pulmonary arterial hypertension can effectively evaluate the patient's response to treatment and the risk of future disease.

[0017] In a fourth aspect of the present application, the application of the biomarker related to pulmonary arterial hypertension in the preparation of a product for monitoring the progression of pulmonary arterial hypertension disease can assist in identifying the progression or future possible progression of the disease, and thus realize the precise treatment of the disease.

[0018] In a fifth aspect of the present application, the application of the biomarker related to pulmonary arterial hypertension in the screening of drugs for preventing or treating pulmonary arterial hypertension provides a basis for the screening of drugs for preventing or treating pulmonary arterial hypertension.

[0019] Specifically, the drug screening includes determining whether a drug for treating or preventing pulmonary arterial hypertension is selected by judging the degree of targeting of the drug to bacterial, archaeal and fungal strains.

[0020] In a sixth aspect of the present application, a preparation method of a pulmonary arterial hypertension animal model for screening a biomarker related to pulmonary arterial hypertension is provided, including the following steps:

[0021] S1, preparing a fecal bacteria solution from feces derived from a patient with pulmonary arterial hypertension or an animal with pulmonary arterial hypertension;

[0022] S2, the recipient animal takes an antibiotic drug to eliminate the intestinal microbiota;

[0023] S3, transplanting the fecal bacteria liquid into the recipient animal to obtain a pulmonary arterial hypertension animal model.

[0024] Preferably, in step S1, the feces of the pulmonary arterial hypertension patient are prepared into the fecal bacteria liquid by the following steps:

[0025] S11, collecting the feces of the pulmonary arterial hypertension patient, mixing the feces with sterile normal saline, and stirring to obtain a mixture;

[0026] S12, filtering the mixture through a screen to remove large particulate matter, and then performing centrifugal treatment to obtain a precipitate after discarding the supernatant;

[0027] S13, adding glycerol to the precipitate to obtain the fecal bacteria liquid.

[0028] Preferably, in step S1, the feces of the pulmonary arterial hypertension animal are prepared into the fecal bacteria liquid by the following steps:

[0029] S11, collecting the feces of the pulmonary arterial hypertension animal, mixing the feces with sterile normal saline, and stirring to obtain a mixture;

[0030] S12, performing centrifugal treatment on the mixture, collecting the supernatant, and measuring the optical density of the supernatant by spectrophotometry to obtain the fecal bacteria liquid.

[0031] Preferably, in step S1, the pulmonary arterial hypertension animal is induced and constructed by intraperitoneal injection of anhydrous hypochlorite.

[0032] Preferably, in step S2, the antibiotic drugs include meclozole, vancomycin, neomycin sulfate, and ampicillin.

[0033] Preferably, the antibiotic drugs are administered orally once a day for 4 consecutive days, the administration amount of meclozole is 200 mg / kg, the administration amount of vancomycin is 100 mg / kg, the administration amount of neomycin sulfate is 200 mg / kg, and the administration amount of ampicillin is 200 mg / kg.

[0034] Preferably, in step S3, the fecal bacteria liquid is perfused into the recipient animal through a gastric tube every day for 14 consecutive days.

[0035] Preferably, the recipient animal includes a rodent.

[0036] Preferably, the rodent includes a rat and a mouse.

[0037] In a seventh aspect of the present application, a method for evaluating a pulmonary arterial hypertension animal model constructed by the method for preparing a pulmonary arterial hypertension animal model is provided, and the pulmonary arterial hypertension animal model is evaluated by echocardiography, hemodynamic measurement, histological examination, quantitative analysis of pulmonary vascular remodeling, metagenomic sequencing, and RNA-seq analysis.

[0038] In an eighth aspect of the present application, a pulmonary arterial hypertension animal model constructed by the method for preparing a pulmonary arterial hypertension animal model is provided, and the pulmonary arterial hypertension animal model is used for at least one of the following:

[0039] (I) application in screening a pulmonary arterial hypertension marker;

[0040] (II) application in preparing a product for diagnosing pulmonary arterial hypertension;

[0041] (III) application in preparing a product for prognostic evaluation of pulmonary arterial hypertension;

[0042] (IV) application in preparing a product for monitoring the progression of a pulmonary arterial hypertension disease;

[0043] (V) application in screening a drug for preventing or treating pulmonary arterial hypertension.

[0044] The pulmonary arterial hypertension-related biomarker of the present application has at least the following beneficial effects:

[0045] The bacteria, archaea and fungi closely related to pulmonary arterial hypertension screened by the present application are combined as biomarkers, have a high AUC value, and can provide efficient diagnostic performance. Compared with a single bacterium as a biomarker, the specificity is higher, which provides a new idea for using the characteristics of the microbiota for the diagnosis and prognostic evaluation of pulmonary arterial hypertension in clinical practice. The biomarkers can also be used for preparing related products such as pulmonary arterial hypertension diagnosis, prognostic evaluation and monitoring the progression of a pulmonary arterial hypertension disease, and drug screening, and have important clinical significance. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0047] Figure 1 The result graph of the intestinal bacterial, archaeal and fungal microbiota of the IPAH patient provided by the present application is changed.

[0048] Figure 2Figure showing the results of fecal microbiota transplantation in rats with pulmonary arterial hypertension phenotype and pulmonary vascular remodeling induced by pulmonary arterial hypertension in rats.

[0049] Figure 3 Figure showing the results of fecal microbiota transplantation in MCT-PAH rats inducing vascular remodeling in rats with PAH and pulmonary phenotype.

[0050] Figure 4 Figure showing the results of fecal microbiota transplantation in rats with pulmonary arterial hypertension phenotype and pulmonary vascular remodeling induced by pulmonary arterial hypertension in rats.

[0051] Figure 5 Figure showing the results of FMT restoring the dysbiosis of the intestinal microbiota in MCT-PAH rats.

[0052] Figure 6 Figure showing the results of fecal microbiota transplantation in rats with pulmonary arterial hypertension phenotype and pulmonary vascular remodeling induced by pulmonary arterial hypertension in rats.

[0053] Figure 7 Figure showing the results of RNA-seq analysis and related pathways.

[0054] Figure 8 Figure showing the diagnostic potential of bacteria, archaea and fungi in IPAH patients.

[0055] Figure 9 Figure showing the principal coordinate analysis (PCoA) plot of bacterial (a), archaeal (b) and fungal (c) communities using Adonis method to assess Bray-Curtis dissimilarity between CON and NTM groups (n=6) provided for the present invention.

[0056] Figure 10 Figure showing the diversity of the CON, MCT and NTM groups on bacteria, fungi and archaea provided for the present invention; A-B are the alpha diversity indices at the species level for the bacterial communities of the CON, MCT and NTM groups: Chao1 (A) and Shannon index (B); C-D are the alpha diversity indices at the species level for the archaeal communities of the CON, MCT and NTM groups: Chao1 (C) and Shannon index (D); E-F are the alpha diversity indices at the species level for the fungal communities of the CON, MCT and NTM groups: Chao1 (E) and Shannon index (F). DETAILED DESCRIPTION

[0057] It should be noted that the following detailed description is illustrative only and is intended to provide further description in addition to the disclosure provided in the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0058] It is to be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, "including," "has," "having," "comprising," "comprises" or "comprised of," when preceding the permutation of a listing of elements such as a jurisdictional element, are used to mean "including but not limited to," and is not intended to (and does not) exclude other moieties, elements, components, or steps. Throughout this application, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. As used herein, the term "or" as used herein, without additional language when used in a list of two or more items, means any of the items in the list.

[0059] The technical solutions of the present application will be described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0060] Embodiments

[0061] Materials and methods

[0062] Human study cohort

[0063] This study prospectively recruited 31 patients with idiopathic pulmonary arterial hypertension (IPAH) in Beijing Chaoyang Hospital, Capital Medical University from November 2023 to April 2024. The diagnosis followed the standard criteria in the latest guidelines. Exclusion criteria included: 1) age outside 18 to 70 years old; 2) positive response to acute pulmonary vasodilator testing in right heart catheterization; 3) severe comorbidities such as coronary heart disease, pneumonia, diabetes, or chronic kidney disease; 4) family history of autoimmune diseases, cancer, or other serious genetic diseases; 5) history of antibiotic use within the past three months. In addition, 31 gender-matched healthy control (HC) subjects were also recruited from Chaoyang Hospital.

[0064] All participants signed a written informed consent form before data and biological sample collection. This study was ethically approved by the Ethics Committee of Beijing Chaoyang Hospital, Capital Medical University (Approval No.: 2023-ke-726).

[0065] Animals

[0066] This study used 5-week-old male Sprague-Dawley (SD) rats weighing between 180-210 grams, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were raised under specific pathogen-free (SPF) conditions at Beijing Chaoyang Hospital, Capital Medical University. The rats were placed in an environment with a 12-hour light-dark cycle, the temperature was maintained at 25°C, and food and water were provided without restriction. To prevent cross-interference between groups, each group of rats was placed in a separate cage, and the cages were cleaned regularly.

[0067] This study followed the ethical standards set by the Beijing Animal Ethics Committee of Capital Medical University (Approval Number: AEEI-2022-011) and strictly abided by the current National Institutes of Health (NIH) guidelines on animal welfare and experimental protocols.

[0068] (ii) Human fecal transplant (HST) to rats

[0069] Fecal donors were from a human research cohort. Fresh fecal samples were collected and mixed with an appropriate amount of sterile pre-cooled normal saline, stirring evenly. The mixture was filtered through a screen to remove large particulate matter, then transferred to a centrifuge tube. The sample was centrifuged at 3000 rpm for 20 minutes at 4°C. After discarding the supernatant, the bacterial precipitate was weighed. An equal volume of pre-cooled glycerol was added to the precipitate (glycerol volume was 20% of the bacterial precipitate volume). The mixture was aliquoted into 15 ml centrifuge tubes, 5 ml per tube, and stored at -80°C to obtain the fecal bacteria solution.

[0070] Recipient rats were orally administered antibiotics (Metronidazole 200 mg / kg, Vancomycin 100 mg / kg, Neomycin Sulfate 200 mg / kg, and Ampicillin 200 mg / kg) daily for four consecutive days to eliminate the intestinal microbiota (GM). The fecal bacteria solution was infused into the rats through a gastric tube daily for two consecutive weeks. Rats receiving fecal transplant (FMT) from healthy controls were designated as the HTN group, while rats receiving FMT from IPAH patients were designated as the PTN group. Eight weeks after FMT treatment, echocardiography was performed, followed by anesthesia of the rats using sodium pentobarbital (40 mg / kg) and euthanasia for hemodynamic measurements.

[0071] (iii) Rat fecal transplant (FMT) to rats

[0072] After adapting to the controlled environment, pulmonary arterial hypertension (PAH) was induced by intraperitoneal injection of monocrotaline (MCT) (60 mg / kg, Sigma-Aldrich). The MCT powder was dissolved in a solution of ethanol and normal saline (2:8 ratio). Rats were randomly divided into a control group (CON group, injected with solvent) and an MCT-PAH group (MCT group, injected with MCT). For rats receiving FMT, fresh fecal samples (10 grams each time) from CON and MCT group rats were collected every morning by using sterile tubes. The fecal samples were mixed with 20 mL of sterile normal saline at 37°C for 1 minute using a standard stirrer. The mixture was then centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant was collected, and its optical density (OD) was measured at 620 nm by spectrophotometry to obtain the fecal bacteria solution.

[0073] Before FMT, the recipient rats were orally administered antibiotics (Vancomycin 100 mg / kg, Neomycin Sulfate 200 mg / kg, Metronidazole 200 mg / kg, and Ampicillin 200 mg / kg) once daily for 4 consecutive days to clear the gut microbiota (GM). Each rat in the NTN group was daily gavaged with fecal microbiota liquid (containing 2 x 10 9 9 9 Eight weeks after FMT, echocardiography was performed. Subsequently, rats were anesthetized using sodium pentobarbital (40 mg / kg) and euthanized for hemodynamic measurements.

[0074] (iv) Fecal microbiota transplantation treatment

[0075] Rats were randomly divided into two groups: CON group (n = 6, received solvent injection) and MCT group (n = 12, received MCT injection). Subsequently, MCT-induced PAH rats were further randomly divided into MCT group and normal transplanted microbiota (NTM) group, which received fecal microbiota transplantation (FMT) from the CON group (n = 6). Fresh fecal samples (10 grams) were collected daily from the CON group, mixed with 20 mL of sterile normal saline at 37 °C, and then centrifuged at 1000 rpm for 5 minutes at 4 °C to collect the supernatant. The optical density (OD value) of the supernatant was measured at 620 nm. Each rat in the NTM group was gavaged with fecal microbiota liquid containing 2 x 10 9 9

[0076] Four weeks after MCT injection, echocardiography was performed, followed by hemodynamic measurements after rats were anesthetized using sodium pentobarbital (40 mg / kg) and euthanized.

[0077] (v) Echocardiography

[0078] Echocardiography was performed by an expert operator using the VisualSonics VEVO 2100 system. The assessment of right ventricular (RV) function included the following measurements: tricuspid annular plane systolic excursion (TAPSE), right ventricular free wall thickness (RVWT), right ventricular end-diastolic dimension (RVEDD), and pulmonary artery acceleration time (PAT). Data were collected from 10 consecutive heartbeats to normalize variability.

[0079] (vi) Hemodynamic measurements and tissue processing

[0080] After echocardiographic evaluation, right ventricular systolic pressure (RVSP) was measured by right heart catheterization. Subsequently, the heart and lung were extracted and weighed. The weight of the right ventricular free wall (RV) and the left ventricle plus septum (LV+S) was measured and the ratio of the right ventricle to the left ventricle plus septum (RV / LV+S) was calculated.

[0081] (vii) Histological examination

[0082] Lung tissue was first fixed in 10% neutral buffered formalin solution for 72 hours, then embedded in paraffin and cut into 4 pm-thick sections. Lung tissue sections were stained by Elastica-van Gieson (EVG) and Hematoxylin and Eosin (HE) staining to evaluate vascular remodeling.

[0083] For 40-60 intra-alveolar vessels less than 100 pm, the distribution of smooth muscle layer was classified as no smooth muscle (NPA), partial smooth muscle (PPA) and complete smooth muscle (MPA). By classifying these vessels, the degree of vascular smooth muscle was evaluated. The degree of smooth muscle was expressed by the ratio of NPA, PPA and MPA, with the numerator being the number of each type of vessel and the denominator being the total number of vessels.

[0084] (viii) Quantitative analysis of pulmonary vascular remodeling

[0085] Elastica-van Gieson (EVG) staining was used to evaluate the thickness of the pulmonary artery media and measure the degree of remodeling of the pulmonary artery. The degree of vascular smooth muscle was evaluated by two independent blinded reviewers, and the inter-person variability of their evaluation results was less than 10%. The degree of obstruction in the pulmonary vessels (less than 100 pm) was calculated by the following formula:

[0086] Obstruction degree = (outer layer vessel area - inner layer vessel area) outer layer vessel area Obstruction degree = (outer layer vessel area - inner layer vessel area) outer layer vessel area

[0087] The pulmonary artery was divided into small pulmonary arterioles (<50 pm) and medium pulmonary arteries (>50 pm) according to the outer diameter. Four vessels (usually round or oval) were randomly selected for measurement from each animal, and the average value was calculated.

[0088] (ix) Metagenomic sequencing

[0089] Total DNA was extracted with cetyltrimethylammonium bromide (CTAB) and sequenced by Oriental Yikang Medical Technology Co., Ltd. (Beijing, China) using the Illumina Novasek 6000 platform, generating 2 × 150-bp paired-end reads. The quality of the extracted DNA was assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), a Qubit 2.0 fluorometer (Invitrogen), and agarose gel electrophoresis (AGE). Raw sequencing data were used for bioinformatics analysis.

[0090] (10) RNA-seq analysis

[0091] Total RNA was extracted from six lung samples from each of the MCT and NTM groups. The quantity and integrity of RNA were assessed using K5500 (Beijing Kaiao, China) and Agilent 2200 Tape Station (Agilent Technologies, USA). mRNA was enriched using a The oligo-dT method of Poly(A) mRNA magnetic separation module (NEB, USA) was used, and then it was cleaved to about 200 bp. The RNA fragments were synthesized into first and second strand cDNA, connected with adapters and enriched at low cycle rate. Ultra TM RNA library preparation kit (Illumina). The purified library product was quality checked using an Agilent 2200 Tape Station and Qubit (Thermo Fisher Scientific, USA). Sequencing was performed at Ribobio Co., Ltd. (Ribobio, China) using Illumina 150 bp paired-end sequencing technology.

[0092] Raw data were cleaned by removing adapter sequences, poly-N sequences, and low-quality reads. Cleaned reads were aligned to the rat reference genome mm10 using HISAT2 with default parameters. Aligned short sequences were converted to read counts per gene model using HTSeq. Differential expression analysis was performed using DESeq2, and differentially expressed genes were selected based on a fold change > 2 and a p-value < 0.05. All differentially expressed genes were analyzed using volcano plots and Kyoto Encyclopedia of Genes and Genomes (KEGG) ontology enrichment, with a significance level set at p < 0.05.

[0093] (11) Statistical analysis

[0094] Hemodynamic parameters between two groups were compared using two-tailed Student's t-test, one-way ANOVA test was used to compare the differences between three groups. Mann-Whitney U test was used to compare the differences between two groups of microbial communities, Kruskal-Wallis test was used to compare the significant differences between three groups. Dilution curves were used to evaluate the sequencing depth of each sample. The Alpha diversity of GM was evaluated using Chao1 and Shannon indices. Based on Bray-Curtis distance, PCoA was used to analyze Beta diversity, and the relative abundance of archaea, fungi and bacterial species was considered, and the analysis was performed using the vegan R package. Adonis analysis based on Bray-Curtis dissimilarity was used to test the differences in microbial community composition between groups. The muscularization and middle layer thickness percentage of pulmonary vessels were compared using two-way ANOVA test. The relationship between the difference microbial community and the hemodynamic parameters of IPAH patients and rat models was studied by Spearman correlation analysis. The area under the receiver operating characteristic curve (AUC) was determined using multivariate logistic regression method. All statistical analyses were performed using R (v4·2·2) and GraphPad Prism (version 9·0). p values < 0·05 were considered statistically significant, and the results were expressed as mean ± standard deviation (S.D.).

[0095] (D) Results

[0096] (1) Intestinal bacterial, archaeal and fungal microbiota of IPAH patients are altered

[0097] The present invention collected fecal samples from 31 healthy controls (HC) and 31 idiopathic pulmonary arterial hypertension (IPAH) patients. The basic clinical data are shown in Table 1. There was no significant difference in mean age (p = 0.164) and gender (p = 0.446) between the HC group and the IPAH group. The mean pulmonary arterial pressure (mPAP) of IPAH patients was 46.8 ± 9.4 mmHg, the mean right ventricular systolic pressure (RVSP) was 72.3 ± 14.9 mmHg, the mean 6-minute walk distance (6MWD) was 363.3 ± 97.6 m, the cardiac output was 2.6 ± 0.5 L / min / m 2 .

[0098] The present invention analyzed the changes of IPAH patients' multi-domain intestinal microbiota, including bacteria, archaea and fungi, using shotgun metagenomics. After quality filtering, the total number of clean reads was 4,487,697,472, and the average number of reads per sample was 72,382,217. Based on clean reads, the present invention detected the number of bacteria, archaea and fungi at each taxonomic level.

[0099] The present application further describes the changes in bacterial, archaeal and fungal community structure at the species level by alpha and beta diversity indices. Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity showed significant differences in the intestinal taxonomic composition between IPAH and HC groups in bacterial, archaeal and fungal communities (Bacteria: Adonis, R 2 = 0.05, P = 0.001; Archaea: Adonis, R 2 = 0.03, P = 0.049; Fungi: Adonis, R 2 = 0.04, P = 0.045; see a-c in Figure 1 ). Only an increase in the richness index (Chao1) of fungi was observed in the IPAH group compared to the HC group (see d-i in Figure 1 ).

[0100] In addition, the present application also analyzed the taxonomic abundance of bacteria, fungi and archaea at the species level. In the bacterial community, the top five species identified were Faecalibacterium prausnitzii, Blautia wexlerae, Eubacterium rectale, Bacteroides uniformis and Escherichia coli (see j in Figure 1 ). In the archaeal community, Methanobrevibacter smithii, Nitrosopumilus SGB14899, Methanobrevibacter oralis, Methanolaciniapetrolearia and Methanomethylovorans SGB40959 were the top five species (see k in Figure 1 ). In the fungal community, the top five species were Schizosaccharomyces pombe, Fusarium pseudograminearum, Thermothielavioidesterrestris, Colletotrichum higginsianum and Lachanceakluyveri (see l in Figure 1 ).

[0101] These data suggest that IPAH patients have a unique intestinal microbiota (GM) structure and composition, including bacteria, archaea and fungi, compared to healthy controls.

[0102] (2) IP AH patient fecal microbiota transplantation induces phenotypic changes in rat pulmonary arterial hypertension

[0103] To explore the causal relationship between gut microbiota (GM) dysbiosis and pulmonary arterial hypertension (PAH), we used two different fecal microbiota transplantation (FMT) models: an IPAH patient-to-rat FMT model ( Figure 2 Figure 5 (a) and PAH rat-to-rat FMT model. Prior to FMT, recipient Sprague Dawley (SD) rats received four days of antibiotic treatment to remove the original intestinal microbiota and promote the colonization of new microorganisms. Fecal microbiota from healthy controls or IPAH patients were transplanted into recipient rats (HTN group and PTN group) and transplanted continuously by gavage for 2 weeks.

[0104] After 8 weeks of FMT, rats in the PTN group showed a significant increase in right ventricular systolic pressure (RVSP) ( Figure 2 bc in the figure), indicating increased pulmonary artery pressure. Echocardiography showed a significant decrease in pulmonary artery acceleration time (PAT), indicating increased pulmonary vascular resistance. In addition, the tricuspid annular plane contraction amplitude (TAPSE) decreased, the right ventricular end-diastolic diameter (RVEDD) increased, and the right ventricular free wall thickness (RVWT) increased, indicating changes in right ventricular morphology and function ( Figure 2 in de).

[0105] Consistent with the observed hemodynamic changes, the thickness and muscularization of the pulmonary vascular wall increased in the PTN group, as measured by HE and Verhoeff's Van Gieson (EVG) staining ( Figure 2 In the HTN group, most of these vessels showed a non-muscular phenotype. In contrast, in the PTN group, the proportion of non-muscular vessels was significantly reduced, as shown in Figure 2. Figure 2 In addition, the thickness of the media of pulmonary arteries with a diameter of less than 50 μm was significantly increased in the PTN group ( Figure 2 h) in the text.

[0106] Consistent with these hemodynamic changes, rats in the PTN group showed increased pulmonary vascular wall thickness and vascular smooth muscle formation, as observed by hematoxylin-eosin (HE) staining and Verhoeff-Van Gieson (EVG) staining ( Figure 2 In the HTN group, the main vessels generally showed a non-smooth muscle phenotype, while in the PTN group, the proportion of non-smooth muscle vessels was significantly reduced ( Figure 2 In addition, the medial thickness of pulmonary arteries with a diameter of less than 50 μm was significantly increased in the PTN group ( Figure 3 h) in the text.

[0107] These results indicate that fecal microbiota from IPAH patients can induce phenotypic changes of pulmonary hypertension after transplantation into rats via FMT, suggesting that dysbiosis of the intestinal microbiota may play a role in the pathogenesis of PAH.

[0108] (3) Fecal microbiota transplantation in MCT-PAH rats induces phenotypic changes in pulmonary hypertension in rats

[0109] Next, the fecal microbiota of normal control rats and monocrotaline (MCT)-induced PAH rats were transplanted into recipient rats (NTN group and MTN group) respectively, and the transplantation was continued for 2 weeks by gavage. Figure 3 After 8 weeks of FMT, the right ventricular systolic pressure (RVSP) of rats in the MTN group was significantly increased compared with that in the NTN group ( Figure 3 However, there was no significant difference in right ventricular index (RVHI) between the NTN and MTN groups ( Figure 3 Pulmonary artery acceleration time (PAT), right ventricular free wall thickness (RVWT), tricuspid annular plane contraction amplitude (TAPSE), and right ventricular end-diastolic dimension (RVEDD) were significantly worse in the MTN group ( Figure 3 in de).

[0110] Corresponding to the hemodynamic changes, FMT exacerbated pulmonary vascular remodeling, as shown by HE staining and EVG staining analysis ( Figure 3 f), in both groups, pulmonary arterioles with a diameter less than 100 μm mainly showed a non-smooth muscle phenotype, but in the MTN group, the number of partially smooth muscled pulmonary arterioles increased significantly ( Figure 3 In addition, the thickness of the pulmonary artery media with a diameter of less than 50 μm was also significantly increased in the MTN group ( Figure 4 h) in the text.

[0111] Taken together, these results suggest that the gut microbiota (GM) plays a role in regulating pulmonary vascular remodeling via FMT, further supporting a potential role for the GM in the pathogenesis of PAH.

[0112] The results of (2)-(3) show that the present invention successfully constructed an animal model of pulmonary hypertension through the method of fecal transplantation. Compared with the existing monocrotaline induction, the operation is simple and can simulate the characteristics of the intestinal flora of human PAH patients. It can be used to develop pulmonary hypertension markers, prepare products for diagnosing pulmonary hypertension, prepare products for prognosis assessment of pulmonary hypertension, prepare products for monitoring the progression of pulmonary hypertension, and screen drugs for prevention or treatment of pulmonary hypertension, which is of great significance to the clinical research of pulmonary hypertension.

[0113] (4) Reversal of pulmonary arterial pressure and vascular remodeling in PAH rats by fecal microbiota transplantation treatment

[0114] To fully explore the effects of FMT on PAH, the present invention established a therapeutic FMT model that mimics the clinical situation when a human is diagnosed with the disease. Given the effects of antibiotic treatment on hypoxia-induced PAH, the present invention developed a fecal microbiota transplantation model that does not use antibiotics, aiming to test whether specific intestinal microbiota can slow the progression of PAH. In this protocol, the intestinal microbiota from normal control group (CON group) was transplanted into MCT-induced PAH rats (NTM group) and continued for 3 weeks after injection of 60 mg / kg body weight of MCT. Meanwhile, rats in the MCT group received MCT injection and placebo treatment by gavage for 3 weeks (MCT group). All rats were sacrificed on day 28 Figure 4 a) of FIG. 1.

[0115] The results showed that FMT significantly improved the PAH phenotype of MCT-PAH rats Figure 4 b-h) of FIG. 1. Compared with the CON group, MCT group rats showed a significant increase in right ventricular systolic pressure (RVSP) and a decrease in pulmonary acceleration time (PAT), suggesting an increase in pulmonary vascular resistance Figure 4 b, e) of FIG. 1. Changes in right ventricular function and morphology were manifested as an increase in right ventricular index (RVHI), right ventricular end-diastolic dimension (RVEDD), and right ventricular free wall thickness (RVWT) in MCT group rats, accompanied by a shortening of tricuspid annular plane systolic excursion (TAPSE) Figure 4 c-e) of FIG. 1. However, FMT treatment reversed these phenotypic changes in the NTM group Figure 4 b-e) of FIG. 1. Similar to the observed hemodynamic changes, FMT also alleviated pulmonary vascular remodeling in MCT group rats Figure 4 f) of FIG. 1. Although FMT treatment did not significantly affect the proportion of smooth muscle in pulmonary vessels, it significantly reversed the remodeling of pulmonary microvessels, especially in pulmonary arteries with a diameter less than 50 pm Figure 5 g-h) of FIG. 1.

[0116] These results indicate that, unlike the intestinal microbiota of IPAH models that promote the progression of PAH, the intestinal microbiota of normal control group rats promote the recovery of PAH by slowing down pulmonary vascular remodeling.

[0117] (5) Fecal microbiota transplantation treatment improves intestinal bacterial, archaeal, and fungal microbiota dysbiosis in PAH rats

[0118] To elucidate the mechanism by which FMT treatment restores the gut microbiota (GM) in PAH, we performed shotgun metagenomic sequencing on rats in the CON, MCT, and NTM groups. A total of 785,394,069 reads were obtained, with an average of 43,633,004 reads per sample. Based on the cleaned reads, we counted the number of bacteria, archaea, and fungi at each taxonomic level.

[0119] The present invention uses α diversity and β diversity indices to characterize the ecological characteristics of the microbiome at the species level. Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity showed that there were significant differences in the structure and composition of the microbiome between the MCT group and the CON group (ADONIS bacteria: R 2 =0.32, P=0.002; Archaea: R 2 =0.21, P=0.009; fungi: R 2 =0.19, P=0.096; Figure 9 In contrast, the microbiota structure and composition of the NTM group, including bacteria, archaea, and fungi, were similar to those of the CON group, suggesting that the intestines of recipient rats were successfully colonized with the donor microbiota after FMT ( Figure 10 AC in). According to the alpha diversity analysis of Chao1 and Shannon index, there was no significant difference in the diversity of bacteria, fungi and archaea among the CON, MCT and NTM groups ( Figure 5 AF in ).

[0120] The present invention hypothesizes that the success of FMT treatment may be through the establishment of beneficial microbial communities and the elimination of pathogenic microorganisms, thereby slowing the progression of PAH. To elucidate the therapeutic mechanism of FMT, the present invention conducted a linear discriminant analysis effect size (LEFSE) analysis to explore the differential microbial communities at the bacterial, archaeal and fungal levels between the CON group, MCT group and NTM group. To fully understand the role of the microbiome in PAH, the present invention used heat map visualization to display the significantly different microbial taxa identified by LEFSE analysis. The present invention focused on taxa with significant changes in abundance at the species level in the MCT group, and these changes were reversed in the NTM group. The changes in these microorganisms may reveal their potential mechanisms of action ( Figure 5 d) in the above.

[0121] These results indicate that FMT treatment can not only restore the intestinal microbiota structure of PAH rats, but also reverse the progression of PAH by adjusting the composition of the microbiota, providing new insights into the microbiota-targeted treatment of PAH.

[0122] The present application identified 78 different bacterial species in the microbial community, of which 49 species were enriched in the CON group and the NTM group, and 29 species were enriched in the MCT group. For the archaeal community, of the 4 different archaeal species, 2 species were enriched in the CON group and the NTM group, and the other 2 species were enriched in the MCT group. Similarly, in the fungal community, 5 different fungal species were identified, of which 3 species were enriched in the CON group and the NTM group, and 2 species were enriched in the MCT group.

[0123] Further Spearman correlation analysis showed that there were significant correlations between the hemodynamic parameters such as right ventricular systolic pressure (RVSP), right ventricular index (RVHI), pulmonary artery acceleration time (PAT), right ventricular free wall thickness (RVWT), and right ventricular end-diastolic dimension (RVEED) and the characteristics of the intestinal microbiota (including bacteria, archaea, and fungi) Figure 6 These findings highlight the significant changes in the composition and function of the intestinal microbiota in PAH.

[0124] These data suggest that the dysbiosis of the intestinal microbiota in PAH rats is closely related to the changes in hemodynamics, and FMT treatment can alleviate the progression of PAH by restoring the structure of the intestinal microbiota, and provide a new microbial-targeted intervention approach for the treatment of clinical PAH.

[0125] (6) FMT treatment changes the serum metabolite composition of PAH rats

[0126] Since the intestinal microbiota (GM) affects target organs through metabolites, thereby affecting disease progression, the present application performed non-targeted metabolomics analysis on the serum samples of each group of rats. Principal component analysis (PCA) showed that there were significant differences in the metabolic profiles between the CON group, the MCT group, and the NTM group Figure 6 a) of the above. The bar chart shows that the main metabolites in the serum include lipids and lipid analogs, organic acids and derivatives, phenylpropanoids, and polyketides Figure 6 b) of the above. The volcano plot further demonstrates the differences in metabolite abundance between the CON group and the MCT group, and the MCT group and the NTM group Figure 6 c-d) of the above. Specifically, according to the criteria of (log2|fold-change|≥0.58 and p<0.05), the present application found that 153 metabolites were up-regulated between the CON group and the MCT group, and 61 metabolites were down-regulated; between the MCT group and the NTM group, 128 metabolites were up-regulated, and 47 metabolites were down-regulated. Based on these difference analysis results, the present application selected the metabolites that changed under the condition of PAH, and showed the reversing effect of FMT on these metabolites through a heat map Figure 7 e) of the above.

[0127] To further explore the relationship between GM, serum metabolites and PAH, Spearman correlation analysis was performed to analyze the association between differential microbiota, serum differential metabolites and hemodynamic parameters, and these correlations were visualized by Sankey. The results of the present application revealed the close correlation between GM, metabolites and PAH-related indicators. Blautia wexlerae in bacteria, Thermococcus pacificus in archaea and Sporothrix globosa in fungi showed stronger correlation with serum metabolites than other species in their respective categories. In addition, a-ketoisovalerate, as the most strongly correlated metabolite with PAH among serum metabolites, also showed significant association with PAH.

[0128] Overall, the data of the present application provided evidence to support the role of GM in the occurrence and progression of PAH by regulating serum metabolites, indicating that FMT might further alleviate the pathological process of PAH by affecting the composition of metabolites.

[0129] (7) FMT treatment modulates PAH-related core signaling pathways

[0130] To explore the mechanism of FMT in alleviating PAH, the present application performed RNA-seq analysis on the lung tissues of MCT and NTM groups of rats Figure 7 ). Differentially expressed genes (DEGs) were screened using the criteria of log2fold change≥1 and p≤0.05. The analysis results showed that compared with the MCT group, 737 genes were differentially expressed in the NTM group, of which 394 genes were up-regulated and 340 genes were down-regulated Figure 7 a) in the present application.

[0131] To further understand the signaling pathways involved in these DEGs, KEGG pathway enrichment analysis was performed. Through FMT treatment, the enriched KEGG pathways were related to vascular inflammation and vascular remodeling, which are the basic pathological mechanisms of PAH. The relevant pathways include leukocyte transendothelial migration, cytokine-cytokine receptor interaction and cell adhesion molecules Figure 8 b) in the present application. In addition, pathways related to vascular smooth muscle contraction were also significantly enriched. These results suggest that FMT therapy can slow down the progression of PAH by reducing vascular inflammation, vascular remodeling and alleviating pulmonary vascular contraction.

[0132] (8) Correlation between pulmonary arterial hypertension and microbiota

[0133] Subsequently, the present invention identified 7 microbial species whose expression trends were consistent with the changes in the FMT treatment group, including 3 bacterial, 1 archaea and 3 fungal species. The present invention used a heat map to visualize the relative abundance of these differential microbial populations ( Figure 8 Then, the present invention explored the efficacy of single boundary markers and combination of multiple boundary markers in diagnosing IPAH by receiver operating characteristic curve (ROC) analysis ( Figure 8 b). The present invention found that the diagnostic performance of any two-boundary combination was better than that of a single population. When markers from all three boundaries were combined, the diagnostic efficacy was the strongest, with an AUC value of 0.881.

[0134] The pulmonary hypertension-related biomarkers provided by the present invention can be used to prepare products for diagnosing pulmonary hypertension, preparing products for prognostic assessment of pulmonary hypertension, preparing products for monitoring the progression of pulmonary hypertension, and screening drugs for the prevention or treatment of pulmonary hypertension, which is of great significance to the clinical research of pulmonary hypertension.

[0135] Given the excellent performance of microbial communities in diagnosis, the present invention further explored their correlation with hemodynamic parameters. The results showed that Blautia obeum was negatively correlated with pulmonary vascular resistance (PVR) and right ventricular systolic pressure (RVSP) in patients ( ​ These findings further validate the present invention's observations in animal experiments, indicating that the microbiome plays an important role in the occurrence and progression of PAH and may serve as a potential diagnostic marker.

[0136] The present invention explores the role of the multi-kingdom gut microbiota (GM), including bacteria, fungi, and archaea, in pulmonary arterial hypertension (PAH). The present invention found that the gut microbiota of IPAH patients underwent significant changes, manifested by changes in α- and β-diversity indices and alterations in specific microbial species. FMT from IPAH patients or MCT-induced PAH rats was able to induce a PAH phenotype in the recipient rats. Conversely, FMT from healthy rats could improve PAH, restore microbiota composition, and reverse PAH-associated serum metabolite changes. In addition, the present invention found a strong correlation between the microbiota, metabolites, and PAH-related indicators. Finally, the model using multi-kingdom markers of bacterial, archaeal, and fungal communities showed superior performance in diagnosis. These findings emphasize the key role of the entire microbiota in PAH and highlight the therapeutic and diagnostic potential of microbiota-based interventions.

[0137] Previous studies have reported dysbiosis in PAH patients, characterized by decreased bacterial diversity and increased pro-inflammatory taxa. The present study confirmed these findings, showing that the bacterial community in IPAH patients significantly changed in both a- and b-diversity indices, indicating a disruption of the intestinal bacterial community structure in IPAH patients. However, previous studies mainly focused on intestinal bacterial populations, often neglecting the role of archaea and fungi. This gap in the literature overlooks the important contributions of these microbial populations in health and disease. Recent studies have begun to explore the role of intestinal archaea and fungi, revealing their involvement in various metabolic pathways, immune responses, and inflammatory processes.

[0138] These findings suggest that the diversity and composition of the intestinal microbiota have important implications for the development and progression of PAH. In particular, dysbiosis of the microbiota can play a key role in the pathophysiological processes of PAH by altering metabolic pathways, affecting the immune system, and promoting inflammatory responses. Therefore, the repair or reconstruction of the microbiota could become a new strategy for the treatment of PAH.

[0139] In addition, the present study also demonstrated the potential of microbiota markers in the diagnosis of PAH. The combined use of multi-kingdom microbiota markers, including bacteria, archaea, and fungi, provided efficient diagnostic performance with high AUC values, offering a new approach for the early diagnosis of PAH using microbiota characteristics in clinical practice. These results not only open up new directions for the treatment and diagnosis of PAH, but also emphasize the clinical application value of microbiota as potential biomarkers.

[0140] Although many archaea are known to survive in extreme conditions, some thermophilic species have been found in the human gut. These stable commensal archaea are involved in various physiological processes, such as trimethylamine metabolism, immune regulation, methane production, and heavy metal transformation. Previous studies have shown changes in archaeal communities in diseases such as colorectal cancer, Crohn's disease, and asthma. For example, a decrease in archaeal diversity in patients with colorectal cancer suggests their potential involvement in tumorigenesis. Other studies have shown that certain archaea can be good indicators of the chronicity and activity of Crohn's disease. These studies on the influence of archaeal populations on metabolic and inflammatory processes provide a broader context for understanding their potential role in PAH.

[0141] Although the abundance of intestinal fungi is lower than that of bacteria, they play a crucial role in maintaining gut health and participating in disease processes. Intestinal fungi are involved in regulating the host immune system and maintaining the integrity of the intestinal barrier. Changes in intestinal fungal communities have been observed in other diseases, such as hypertension, coronary artery disease, and alcohol-related liver disease. These findings highlight the potential influence of intestinal fungi on inflammatory and metabolic pathways, which can also be of great significance to the pathogenesis of PAH.

[0142] FMT from IP AH patients or MCT-induced PAH rats was able to induce the PAH phenotype in recipient rats, confirming the causal relationship between gut microbiota dysbiosis and PAH. These experimental evidences support previous observations showing significant differences in gut bacterial structure between PAH patient populations and controls, and between PAH animal models and their respective controls. In contrast, FMT from healthy rats significantly improved PAH symptoms in the PAH rat model, restored gut microbiota composition, and reversed PAH-associated serum metabolite changes. This is consistent with previous studies showing the therapeutic potential of microbiota restoration in various diseases, such as hypertension, Parkinson's disease, and Clostridium difficile infection. In this study, the remission effect of FMT in the PAH model suggests that microbiota-based therapy can have similar potential in treating PAH, however, clinical trials are still needed to validate these findings.

[0143] This study further deepens the understanding of the key role of the multi-domain gut microbiota in the pathogenesis of PAH. Through this experimental model of microbiota transplantation, the present invention confirms the relationship between microbiota dysbiosis and the PAH phenotype, and provides a new perspective for clinical microbiota therapy.

[0144] FMT treatment improved gut bacterial, archaeal, and fungal dysbiosis in PAH rats. There were significant differences in GM structure between the MCT and CON groups, while the microbiota composition of the NTM group was similar to that of the CON group, indicating that FMT successfully restored a healthy gut microbiota. The correlation between gut microbiota features and hemodynamic and right heart ultrasound indices (such as RVSP, RVHI, PAT, RVWT, RVEED) further highlights the important role of the gut microbiota in the pathophysiology of PAH.

[0145] The present study demonstrates that FMT effectively alters the disrupted metabolism in PAH, highlighting the potential mechanisms by which the gut microbiota influences disease progression through metabolites. Previous studies have shown that the gut microbiota influences disease progression through metabolites that act on target organs. Key metabolites, including lipids, organic acids, phenolic acrylates, and polyketides, were restored following FMT treatment. 5-hydroxyindoleacetic acid (5-HIAA), a metabolic byproduct of serotonin, was significantly elevated in MCT-PAH rats and was closely associated with changes in the gut microbiota. Recent studies have shown that 5-HIAA can act as a ligand for GPR35, promoting the recruitment of neutrophils to sites of inflammation, thereby exerting a pro-inflammatory effect. The recruitment of inflammatory cells around the pulmonary vessels is also a key pathological feature of IPAH. Proline, as an amino acid, is also a metabolite in the gut that plays a crucial role in the development of pulmonary hypertension. Proline is essential for the formation of collagen, which is the main component of vascular structure. Changes in collagen metabolism are the basis of PAH vascular pathology. These findings suggest that FMT improves PAH symptoms by restoring the gut microbiota and modulating metabolites.

[0146] The underlying pathological mechanisms of PAH include vascular remodeling, perivascular inflammation, and pulmonary vasoconstriction, and FMT treatment significantly modulated these mechanisms, affecting the expression of related signaling pathways. The modulation of pathways such as cytokine-cytokine receptor interactions, leukocyte transendothelial migration, and cell adhesion molecules suggests that FMT may improve PAH by reducing inflammation and vascular remodeling pathways. FMT may help alleviate inflammatory cell infiltration and endothelial dysfunction, thereby reducing the inflammatory burden on pulmonary vessels. In addition, changes in pathways related to vascular smooth muscle contraction are particularly important, as sustained vasoconstriction plays a crucial role in PAH. The effects of FMT on these signaling pathways suggest that it may reduce pulmonary arterial pressure by alleviating vascular smooth muscle contraction, perivascular inflammation, and vascular remodeling, thereby improving the development of PAH. These findings further explore the mechanisms by which the gut microbiota influences the development of PAH.

[0147] The present study identified seven microbial species, including three bacteria, one archaea, and three fungi, whose expression trends were consistent with those observed in the CON and NTM groups, with significant diagnostic potential. The combination of bacterial, archaeal, and fungal multi-domain microbiota provided the highest diagnostic performance, highlighting the important role of fungi and archaea beyond bacteria. In particular, Blautia obeum was associated with key hemodynamic parameters, supporting its role in IPAH. These findings highlight the importance of the gut microbiota in the pathophysiology of PAH and suggest that combining microbial markers can improve the accuracy of diagnosis.

[0148] The study made several important contributions to the pathogenesis and treatment of PAH. First, it established a causal relationship between intestinal dysbiosis and PAH, a link that had been proposed but not empirically confirmed. Second, by including archaea and fungi in the analysis, the invention provided a more comprehensive view of the role of the intestinal microbiota in PAH. Finally, the discovery of the potential of FMT treatment highlighted a promising avenue for microbiota-based non-pharmacological treatment of PAH, underscoring the need for further clinical studies.

[0149] The study of the invention highlights the key role of the multi-kingdom intestinal microbiota (including bacteria, archaea, and fungi) in the pathogenesis and treatment of PAH. By demonstrating the efficacy of FMT in improving PAH symptoms, the invention opens the way for innovative microbiota-based treatments, offering new hope for patients with this disease.

[0150] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A biomarker associated with pulmonary hypertension, characterized in that: The biomarkers include: two or three of bacteria, archaea and fungi; the bacteria include: one or more of Blautia obeum, Blaut ia_sp_Marseille_P3087 and Coprococcus_comes; the archaea include: Methanomethylovorans_hollandica; the fungi include: one or more of Sporothrix_globosa, Fusarium_oxysporum and Yarrowia_lipolytica.

2. The biomarker related to pulmonary hypertension according to claim 1, characterized in that The biomarkers include bacteria, archaea and fungi; the bacteria include Blautia obeum, Blautia_sp_Marseille_P3087 and Coprococcus_comes; the archaea include Methan omethylovorans_hollandica; the fungi include Sporothrix_globosa, Fusarium_oxysporum and Yarrowia_lipolytica.

3. Use of the pulmonary hypertension-related biomarker according to claim 1 in the preparation of a product for diagnosing pulmonary hypertension, characterized in that: The product tests the relative abundance of bacteria, archaea and fungi in the test samples of the subjects, and performs ROC curve statistical analysis at the same time, and evaluates whether the subjects have pulmonary hypertension through the ROC curve and the area under the curve AUC.

4. The use according to claim 3, characterized in that When the bacteria, archaea and fungi were used as markers, the AUC value reached 0.

881.

5. The use according to claim 3, characterized in that The sample to be tested is feces or intestinal contents.

6. The use according to claim 3, characterized in that The product is a detection chip, a detection test paper or a detection kit.

7. The use according to claim 3, characterized in that The product also includes reagents required for extracting DNA from the sample to be tested and determining the relative abundance of bacteria, archaea and fungi.

8. Use of the pulmonary hypertension-related biomarker according to claim 1 in the preparation of a product for prognostic assessment of pulmonary hypertension.

9. Use of the pulmonary hypertension-related biomarker according to claim 1 in the preparation of a product for monitoring the progression of pulmonary hypertension.

10. Use of the pulmonary hypertension-related biomarker according to claim 1 in screening drugs for preventing or treating pulmonary hypertension.

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