Method for predicting and evaluating inflammatory bowel disease fibrosis through prostacyclin detection

Abstract

The invention relates to the technical field of biology, discloses a method for predicting and evaluating inflammatory bowel disease fibrosis through prostacyclin detection, and particularly relates to a serological biomarker for predicting and evaluating inflammatory bowel disease fibrosis and application of the serological biomarker. According to the method for predicting and evaluating inflammatory bowel disease fibrosis through prostacyclin detection, as a biomarker of inflammatory bowel disease fibrosis, PGI2 in serum can be used for preparing reagents and / or drugs for auxiliary diagnosis, curative effect evaluation and relapse monitoring of inflammatory bowel disease fibrosis, the effect is remarkable, and the specificity is good. The reagent is preferably an ELISA diagnostic kit. The invention also discloses a method for predicting and evaluating the fibrosis truncation value of the inflammatory bowel disease by using the biomarker and a method for predicting and evaluating the fibrosis truncation value of the inflammatory bowel disease by using the biomarker.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for predicting and evaluating inflammatory bowel disease fibrosis through prostacyclin detection. Background Art

[0002] Intestinal obstruction caused by intestinal fibrosis is a serious complication of Crohn's disease (Crohn's disease), a major form of inflammatory bowel disease (IBD). Intestinal fibrosis develops due to recurrent chronic inflammation and mucosal healing, resulting in extracellular matrix deposition in the mucosa and submucosa. Over a 10-year course of Crohn's disease, over 50% of patients undergo surgery for intestinal strictures and obstruction. However, surgery does not prevent the development and progression of fibrosis. While significant progress has been made in the development of anti-inflammatory drugs for IBD, which can effectively control disease activity, they are unable to prevent, alleviate, or treat fibrosis and strictures in Crohn's disease, and the incidence of strictures has not changed substantially. This suggests that intestinal inflammation may be only one component of the fibrotic mechanism, and that other fibrotic mechanisms independent of inflammation exist. Over the past few decades, studies have reported the anti-fibrotic effects of various molecules, inhibitors, and antibodies. However, to date, no therapeutic drugs have been approved for the treatment of intestinal fibrosis. This is primarily due to the lack of clear understanding of the primary and specific cellular and molecular pathways that drive fibrosis, resulting in a lack of effective and safe intervention targets. Various studies have shown that the activation of fibroblasts by various cell types and soluble mediators, leading to their production and secretion of collagen, cytokines and other ECM components, is a core factor in the occurrence and progression of intestinal fibrosis. The activation of fibroblasts by the transcriptional effector Yes-associated protein (YAP) and the transcriptional coactivator with PDZ binding motif (TAZ) of the Hippo signaling pathway has become a key step in organ fibrosis. + Intestinal stem cells are necessary for maintaining intestinal homeostasis and play an important role in stem cell recovery. Systemic inhibition of YAP / TAZ aggravates intestinal damage and inflammation in colitis mice. Prostacyclin (PGI2), as a ligand of Gαs protein-coupled prostacyclin receptor (PTGIR), has been shown in in vitro and in vivo experiments to effectively inhibit YAP / TAZ activation and alleviate the occurrence of renal and pulmonary fibrosis, but there are very few studies on the role of prostacyclin (PGI2) in fibrosis of inflammatory bowel disease. Therefore, based on clinical observations and research cohorts, developing biomarkers that effectively predict and evaluate the occurrence and progression of fibrosis in inflammatory bowel disease, and clarifying its molecular mechanisms for remodeling and maintaining intestinal homeostasis, will contribute to the development of diagnostic and therapeutic methods for intestinal fibrosis in inflammatory bowel disease. To this end, this application now proposes a method for predicting and evaluating fibrosis in inflammatory bowel disease through prostacyclin detection. Summary of the Invention

[0003] Technical Solution

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for predicting and evaluating fibrosis in inflammatory bowel disease by detecting prostacyclin, comprising detecting the level of PGI2 in the serum of patients with inflammatory bowel disease to predict the occurrence and progression of intestinal fibrosis, the method comprising:

[0005] S1. Provide samples from subjects;

[0006] S2. determining the level of prostacyclin (PGI2) of the present invention in the sample;

[0007] S3. Assess the degree of fibrosis in the subjects.

[0008] Preferably, a biomarker for predicting and evaluating fibrosis in inflammatory bowel disease and its application are characterized in that: the biomarker is prostacyclin.

[0009] Preferably, the biomarker prostacyclin PGI2 is used to prepare a reagent for monitoring the occurrence and development of inflammatory bowel disease fibrosis.

[0010] Preferably, a product for predicting and evaluating fibrosis in inflammatory bowel disease is a reagent for detecting the amount of the fatty acid derivative, and the reagent is an antibody and an antibody fragment.

[0011] Preferably, the application of a product for predicting and evaluating fibrosis in inflammatory bowel disease is an in vitro diagnostic product, and the diagnostic product is a diagnostic kit;

[0012] In one aspect, the present invention provides a biomarker for predicting and evaluating fibrosis in inflammatory bowel disease and its application, wherein the biomarker is prostacyclin (PGI2).

[0013] In one aspect, the present invention further provides a reagent for detecting the fatty acid derivative (prostacyclin PGI2) of the present invention. In a preferred embodiment, the reagent is a reagent for detecting the amount of prostacyclin (PGI2) of the present invention, particularly prostacyclin (PGI2) in serum. In a specific embodiment, the reagent is an antibody, a detection marker, or a combination thereof.

[0014] In one aspect, the present invention also provides a product for predicting and evaluating fibrosis in inflammatory bowel disease, comprising the reagent of the present invention.

[0015] In one aspect, the present invention also provides use of the prostacyclin (PGI2), reagent or product of the present invention in determining the fibrosis score of inflammatory bowel disease.

[0016] In one aspect, the present invention further provides use of the prostacyclin (PGI2) or reagent of the present invention in the preparation of a product for determining fibrosis in patients with inflammatory bowel disease.

[0017] In one embodiment, the product is an enzyme-linked immunosorbent assay (ELISA) kit. In a preferred embodiment, the product is an enzyme-linked immunosorbent assay (ELISA) kit.

[0018] In one aspect, the present invention also provides a method for predicting and evaluating the occurrence of intestinal fibrosis in a subject with inflammatory bowel disease, the method comprising: (1) providing a sample from the subject; (2) determining the level of prostacyclin (PGI2) of the present invention in the sample; and (3) evaluating the degree of fibrosis in the subject.

[0019] Compared with the existing technology, the present invention provides a method for predicting and evaluating fibrosis in inflammatory bowel disease by detecting prostacyclin, which has the following beneficial effects:

[0020] 1. The method for predicting and evaluating fibrosis in inflammatory bowel disease by prostacyclin detection. The present invention establishes an inflammatory bowel disease fibrosis assessment model and a detection method based on enzyme-linked immunosorbent assay (ELISA) technology by analyzing prostacyclin (PGI2) in the serum of patients with inflammatory bowel disease and clinical data, thereby providing assistance for improving the clinical diagnosis efficiency of fibrosis in inflammatory bowel disease.

[0021] 2. The method for predicting and evaluating fibrosis in inflammatory bowel disease by prostacyclin detection is based on the expression level of prostacyclin (PGI2) in serum samples of patients with inflammatory bowel disease of the present invention, and establishes an inflammatory bowel disease fibrosis prediction and evaluation system, which provides more efficient complication prediction and diagnosis for patients with long-term inflammatory bowel disease, and provides potential treatment approaches. For patients with low serum PGI2 expression, it can be considered that they are at high risk of fibrosis or have already developed intestinal fibrosis but have not had intestinal obstruction. In this case, anti-inflammatory treatment should be carried out in time and surgical intervention should be considered as early as possible in order to obtain the maximum clinical benefit; for patients with high serum PGI2 expression, the anti-inflammatory treatment regimen can be adjusted when histological remission is achieved to reduce the occurrence of adverse reactions and the economic burden of treatment.

[0022] 3. Compared with the current inflammatory bowel disease fibrosis assessment and prediction methods, the method for predicting and evaluating inflammatory bowel disease fibrosis by prostacyclin detection has the advantage that the PGI2 expression level in the serum of patients with inflammatory bowel disease is highly correlated with the occurrence of fibrosis, which is helpful for the development of diagnostic and therapeutic methods for intestinal fibrosis in inflammatory bowel disease. Another advantage of the present invention is that it provides a method for predicting and evaluating inflammatory bowel disease fibrosis. By detecting serological markers in patients with inflammatory bowel disease, it has the characteristics of simple method, high sensitivity, high specificity, and high correlation with fibrosis. It can be used as a supplementary method other than endoscopy, imaging examination, and inflammatory marker detection to further predict and evaluate the occurrence of fibrosis in patients with inflammatory bowel disease and take intervention measures as early as possible to achieve maximum clinical treatment benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the collection of serum PGI2 levels from patients with inflammatory bowel disease and healthy controls for the present invention;

[0024] Figure 2 Schematic diagram of the PGI2 expression cutoff value for predicting the occurrence of inflammatory bowel disease fibrosis through the ROC curve of the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] A method for predicting and evaluating fibrosis in inflammatory bowel disease by detecting prostacyclin, comprising detecting the level of PGI2 in the serum of patients with inflammatory bowel disease to predict the occurrence and progression of intestinal fibrosis, the method comprising:

[0027] S1. Provide samples from subjects;

[0028] S2. determining the level of prostacyclin (PGI2) of the present invention in the sample;

[0029] S3. Assess the degree of fibrosis in the subjects.

[0030] Preferably, a biomarker for predicting and evaluating fibrosis in inflammatory bowel disease and its application are characterized in that: the biomarker is prostacyclin.

[0031] Preferably, the biomarker prostacyclin PGI2 is used to prepare a reagent for monitoring the occurrence and development of inflammatory bowel disease fibrosis.

[0032] Preferably, a product for predicting and evaluating fibrosis in inflammatory bowel disease is a reagent for detecting the amount of the fatty acid derivative, and the reagent is an antibody and an antibody fragment.

[0033] Preferably, the application of a product for predicting and evaluating fibrosis in inflammatory bowel disease is an in vitro diagnostic product, and the diagnostic product is a diagnostic kit.

[0034] The diagnostic product of the present invention:

[0035] In another aspect, the present invention relates to a reagent for detecting the expression level of prostacyclin (PGI2) and use thereof in preparing a detection / diagnosis product.

[0036] In a preferred embodiment, the reagent is used to detect the amount of the target antigen (e.g., PGI2 secretion of the present invention) in serum. In one embodiment, the reagent is a reagent for detecting the amount of antibodies bound to prostacyclin (PGI2). In another embodiment, the reagent is a reagent for detecting the absorbance of a marker bound to the above-mentioned antibody.

[0037] In a preferred embodiment, the reagent is an antibody, a detection marker, or a combination thereof, which can bind to the target antigen (e.g., PGI2 of the present invention) to form a complex. Preferably, the antibody has a high specificity for the target antigen, and the antibody and detection marker can be artificially synthesized.

[0038] In one embodiment, the reagent is an enzyme-linked immunosorbent assay (ELISA) kit. In one embodiment, the reagent is used to detect the amount of the target antigen (PGI2 of the present invention). Preferably, the reagent is an antibody, antibody fragment or affinity protein that can specifically bind to the target antigen. More preferably, the reagent is an antibody or antibody fragment that can specifically bind to the target antigen. The antibody, antibody fragment or affinity protein can also carry a label for detection, such as an enzyme (such as horseradish peroxidase), a radioactive isotope, a fluorescent marker (such as Alexa Fluor dye, FITC, Texas Red, Cy3, Cy5, etc.), a chemiluminescent substance (such as luminol), biotin and quantum dot labels (Qdot), etc. Therefore, in a preferred embodiment, the reagent is an antibody or antibody fragment that can specifically bind to the target antigen, and optionally carries a label for detection. In one embodiment, the label is selected from an enzyme, a radioactive isotope, a fluorescent marker, a chemiluminescent substance, biotin and quantum dot labels. In one embodiment, the reagent is used to prepare a detection / diagnosis product, which is an enzyme-linked immunosorbent assay (ELISA) kit.

[0039] Therefore, in another aspect, the present invention provides a product (preferably a kit) that can be used to determine the onset and progression of inflammatory bowel disease fibrosis. The product (preferably a kit) comprises a reagent of the present invention. The product can be an enzyme-linked immunosorbent assay (ELISA) diagnostic kit.

[0040] In one embodiment, the product is an in vitro diagnostic product. In a specific embodiment, the product is a diagnostic kit.

[0041] In one embodiment, the product is used to determine the onset and progression of fibrosis in inflammatory bowel disease.

[0042] The diagnostic product of the present invention (preferably in the form of a kit) also preferably comprises an apparatus for extracting a test sample from a subject; for example, an apparatus for extracting tissue or blood from a subject, preferably any blood collection needle, syringe, etc. The subject is a mammal, preferably a human, particularly a patient suffering from inflammatory bowel disease.

[0043] The method and application of the present invention:

[0044] In another aspect, the present invention also relates to a method for predicting and evaluating the occurrence and progression of inflammatory bowel disease fibrosis, the method comprising

[0045] (1) provide samples from subjects,

[0046] (2) determining the level of prostacyclin (PGI2) of the present invention in the sample, and

[0047] (3) Assess the degree of fibrosis in the subjects.

[0048] The methods of the present invention may be used for diagnostic or non-diagnostic purposes.

[0049] The subject used in the method of the present invention is a mammal, preferably a human, in particular a patient with inflammatory bowel disease.

[0050] The sample used in step (1) is not particularly limited, as long as the expression level of prostacyclin (PGI2) can be obtained therefrom. For example, serum or tissue homogenate of a subject (e.g., an inflammatory bowel disease patient) can be extracted from the sample. The sample is preferably a blood, plasma, or serum sample, preferably a serum sample. In a preferred embodiment, the sample is a serum sample.

[0051] Step (2) can be achieved by detecting the amount of the target antigen (PGI2 of the present invention). The detection can be achieved using the reagents described above and techniques known in the art, including but not limited to enzyme-linked immunosorbent assay (ELISA), magnetic microsphere Luminex single-factor sandwich method, etc.

[0052] In a preferred embodiment, step (2) can be achieved by detecting the amount of prostacyclin (PGI2). The detection can be achieved using the reagents described above and techniques known in the art, including but not limited to enzyme-linked immunosorbent assay (ELISA), magnetic microsphere Luminex single-factor sandwich, etc., preferably enzyme-linked immunosorbent assay (ELISA). More preferably, the detection is achieved using the reagents of the present invention.

[0053] In one embodiment of the present invention, step (3) can be accomplished by statistically analyzing the prostacyclin (PGI2) levels in the sample obtained in step (2). Prediction and assessment of fibrosis in inflammatory bowel disease can be performed based on established serum prostacyclin (PGI2) level cutoffs established in the inventors' research. Analysis of the prostacyclin (PGI2) expression data obtained in step (2) can be used to predict and assess the onset and progression of fibrosis.

[0054] Accordingly, the present invention also provides the use of prostacyclin (PGI2) to assess the occurrence and progression of fibrosis in inflammatory bowel disease. The present invention also provides the use of prostacyclin (PGI2) or a reagent for detecting the expression level of prostacyclin (PGI2) in the preparation of a product for assessing fibrosis in inflammatory bowel disease. In a preferred embodiment, the product is a detection / diagnostic kit. In one embodiment, the product is an in vitro diagnostic product. The reagent is as described above. According to the method or application of the present invention, the occurrence and progression of fibrosis in patients with inflammatory bowel disease can be assessed.

[0055] Example 1:

[0056] Serum samples from patients with inflammatory bowel disease and healthy controls were collected to detect PGI2 levels.

[0057] Methods: Serum samples and clinical data of 118 Crohn's disease patients and 50 healthy controls were collected from Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Ruijin Hospital Affiliated to Shanghai Jiao Tong University, and the Tenth People's Hospital Affiliated to Tongji University. Serum PGI2 levels were measured by enzyme-linked immunosorbent assay (ELISA). Stratified analysis of patients' disease behavior was performed based on clinical data to determine the correlation between downregulated PGI2 expression and fibrosis in inflammatory bowel disease.

[0058] Results: Compared with the healthy control group, the serum PGI2 level of patients with inflammatory bowel disease was significantly lower. Compared with patients without intestinal stricture, the serum PGI2 level of patients with inflammatory bowel disease with intestinal stricture was further reduced. The serum PGI2 level of patients with inflammatory bowel disease with penetrating complications showed an upward trend, which indicates that the PGI2 level changes dynamically during the progression of the disease (Appendix Figure 1 ).

[0059] Example 2:

[0060] PGI2 expression cutoff value for predicting fibrosis in inflammatory bowel disease using ROC curve

[0061] The PGI2 detection data obtained in Example 1 were analyzed in combination with the inflammatory bowel disease with clinical information collected in Example 1, and the Youden Index was used to determine the PGI2 expression level cutoff value for predicting the occurrence of fibrosis.

[0062] Results: When the serum PGI2 cutoff value was 272.9 pg / ml, the maximum value of Youden index was 0.3724, the sensitivity was 73.47%, and the specificity was 63.77% (Appendix Figure 2 ).

[0063] Example 3:

[0064] Serum PGI2 expression cutoff value for risk factor analysis of fibrosis in inflammatory bowel disease

[0065] Based on the PGI2 detection data obtained in Example 1 and using the serum PGI2 critical value in Example 2, the inflammatory bowel disease cases with clinical information collected in Example 1 were statistically analyzed for risk factors using one-way analysis of variance and multiple regression analysis.

[0066] Results: Based on the serum PGI2 cutoff value of 272.9 pg / ml, 61 samples (51.7%) and 57 samples (48.3%) were divided into the low PGI2 level group and the high PGI2 level group, respectively. One-way analysis of variance and multiple regression analysis showed that serum PGI2 levels were significantly associated with intestinal stricture (OR = 4.772, 95% CI = 1.981-11.494, p < 0.001) (Table 1)

[0067] Example 4:

[0068] Enzyme-linked immunosorbent assay (ELISA) kit for determining fibrosis in inflammatory bowel disease

[0069] Based on the principle of antigen-antibody interaction and using enzyme and colorimetric detection to quantify the target molecule, a PGI2 detection kit in serum was designed. This kit uses the competitive ELISA method. The PGI2 antigen is coated on an enzyme-labeled plate. During the experiment, the PGI2 in the sample (or standard) competes with the coated PGI2 for the binding sites on the biotin-labeled anti-PGI2 monoclonal antibody, and the free components are washed away. Horseradish peroxidase-labeled avidin is added, and biotin specifically binds to the avidin to form an immune complex, and the free components are washed away. A chromogenic substrate (TMB) is added, and TMB exhibits a blue color under the catalysis of horseradish peroxidase, which turns yellow after adding the stop solution. The OD value is measured at a wavelength of 450nm using an enzyme reader. The PGI2 concentration is inversely proportional to the OD450 value, and the PGI2 concentration in the sample is calculated by drawing a standard curve. The method for using the kit to assess fibrosis in inflammatory bowel disease through ELISA detection is described as follows.

[0070] Experimental method: The serum of patients with inflammatory bowel disease was collected and the expression level of PGI2 was detected by ELISA technique.

[0071] Here are the steps:

[0072] Step 1: Take a blood sample from the subject. Place the whole blood sample at room temperature for 1 hour or at 2-8°C overnight, then centrifuge at 2-8°C, 1000×g for 20 minutes, and take the supernatant as the original material.

[0073] Step 2: Preparation of standard solution: The following concentrations are recommended: 2000, 1000, 500, 250, 125, 62.5, 31.25, 0 pg / mL. Serial dilution method: Take 7 EP tubes and add 500 μg / mL to each tube.

[0074] Pipette 500 μL of the 2000 pg / mL standard working solution into the first EP tube and mix thoroughly to make a 1000 pg / mL standard working solution. Repeat this procedure to mix thoroughly.

[0075] Step 3: Set up standard wells, blank wells, and sample wells. Add 50 μL of serially diluted standard to the standard wells, 50 μL of standard and sample diluent to the blank wells, and 50 μL of the test sample to the remaining wells. (It is recommended that all test samples and standards be set up in duplicate; it is recommended to determine the dilution factor of the test sample through preliminary experiments or by consulting technical support.) Immediately add 50 μL of the prepared biotinylated antibody working solution to each well. Cover the plate with film and incubate at 37°C for 45 minutes.

[0076] Step 4: Discard any liquid from the wells and pat dry on clean absorbent paper. Add 350 μL of wash buffer to each well and soak for 1 minute. Aspirate or discard any liquid from the plate and pat dry. Repeat this wash step three times. After washing, proceed immediately to the next step; do not allow the microplate to dry.

[0077] Step 5. Add 100 μL of HRP enzyme conjugate working solution to each well, cover the ELISA plate with film, and incubate at 37°C for 30 minutes.

[0078] Step 6. Shake off all liquid in the wells and wash the plate 5 times using the same method as step 4.

[0079] Step 7. Add 90 μL of substrate solution (TMB) to each well, cover the plate with film, and incubate at 37°C in the dark for approximately 15 minutes. This time can be shortened or extended as needed depending on the color development, but should not exceed 30 minutes. Stop the assay when a clear gradient appears in the standard wells (a clear blue gradient appears in the first four color-developing wells). Preheat the plate 15 minutes in advance.

[0080] Step 8. Add 50 μL of stop solution to each well to terminate the reaction. Tip: The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution.

[0081] Step 9. Immediately measure the optical density (OD value) of each well using a microplate reader at a wavelength of 450 nm.

[0082] Step 10: Statistical analysis of the results: statistical analysis of the obtained sequencing results.

[0083]

[0084]

[0085] Table 1. Univariate and multivariate analyses of risk factors for Crohn's disease stricture

[0086] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for predicting and evaluating fibrosis in inflammatory bowel disease by detecting prostacyclin, characterized in that: The method includes predicting the occurrence and progression of intestinal fibrosis by detecting the level of PGI2 in the serum of patients with inflammatory bowel disease, the method comprising: S1. Provide samples from subjects; S2. determining the level of prostacyclin (PGI2) of the present invention in the sample; S3. Assess the degree of fibrosis in the subjects.

2. According to the method for predicting and evaluating fibrosis in inflammatory bowel disease by detecting prostacyclin according to claim 1, a biomarker for predicting and evaluating fibrosis in inflammatory bowel disease and its application are proposed, characterized in that: The biomarker is prostacyclin.

3. The method for predicting and evaluating fibrosis in inflammatory bowel disease by prostacyclin detection according to claim 2, characterized in that: The biomarker prostacyclin PGI2 is used to prepare a reagent for monitoring the occurrence and development of inflammatory bowel disease fibrosis.

4. A product for predicting and evaluating inflammatory bowel disease fibrosis applied to a method for predicting and evaluating inflammatory bowel disease fibrosis by prostacyclin detection according to claims 1-3, which is a reagent for detecting the amount of the fatty acid derivative, and the reagent is an antibody and an antibody fragment.

5. Use of a product for predicting and evaluating fibrosis in inflammatory bowel disease according to claim 4, wherein the product is an in vitro diagnostic product, and the diagnostic product is a diagnostic kit.

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