Application of serum marker for intestinal mucosa inflammation

Abstract

The invention relates to application of an intestinal mucosa inflammation serum marker in preparation of a diagnosis and treatment product for treating the intestinal mucosa inflammation state of a patient suffering from Crohn disease and predicting the treatment effect of infliximab, and belongs to the technical field of biological medicine, and the intestinal mucosa inflammation serum marker is matrix remodeling related 5 protein (MXRA5). When the matrix remodeling related 5 protein (MXRA5) is used as a target spot for serum protein detection, high sensitivity and specificity are achieved, and therefore the MXRA5 protein can be used as a screening target spot for predicting the intestinal mucosa inflammation state of a patient suffering from the Crohn disease and the treatment effect of the infliximab.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, specifically to the application of intestinal mucosal inflammation serum markers. Background Art

[0002] Crohn's disease (CD) is a chronic intestinal inflammatory disease with a complex pathogenesis. Its prevalence and incidence are on the rise in both industrialized and developing countries. Infliximab (IFX) is widely used in the treatment of CD, but the prediction of treatment response remains challenging.

[0003] Currently, the disease activity can be evaluated through clinical symptoms, endoscopic manifestations, imaging examinations, or histological assessments. The core goal of treatment is to achieve and maintain endoscopic remission. However, monitoring the treatment response through colonoscopy or imaging requires a repair cycle of 3 - 6 months, followed by re - evaluation of mucosal healing and adjustment of the treatment plan. Although this is the current optimal strategy, using repeated colonoscopy as an endpoint in routine clinical practice or clinical trials still faces challenges: poor patient tolerance, high operation risks, the need for anesthesia, and high costs. Therefore, there is an urgent need to develop new biomarkers that can accurately reflect the endoscopic disease activity. Despite significant progress in multi - omics biomarker research, for example, markers such as C - reactive protein (CRP) have certain value in assessing disease susceptibility, activity, and phenotype, but their specificity is low (it can increase due to various inflammatory states), and the correlation with endoscopic activity is limited (the sensitivity for detecting endoscopic active CD is only 49%). Alternatively, fecal calprotectin is the most reliable diagnostic protein biomarker in inflammatory bowel disease (IBD). However, randomized trials have shown that early treatment intervention based on fecal calprotectin can improve the prognosis of CD, but fecal testing has limitations in clinical applications. Therefore, no marker has been translated into routine clinical tests yet.

[0004] Therefore, it is of great significance to search for candidate serum markers related to Crohn's disease, so as to provide new biomarkers for the diagnosis of Crohn's disease and the treatment effect of infliximab. Summary of the Invention

[0005] The object of the present invention: To overcome the defects of the prior art, the present invention provides an intestinal mucosal inflammation serum marker and applies it to the diagnostic and prognostic prediction products for the intestinal mucosal inflammation state of Crohn's disease patients and the treatment effect of infliximab.

[0006] To achieve the above object, the present invention provides the following technical solutions: Application of intestinal mucosal inflammation serum markers in the diagnosis of intestinal mucosal inflammation status and the therapeutic effect of infliximab in Crohn's disease patients, wherein the serum marker is Matrix Remodeling Associated 5 protein (MXRA5).

[0007] MXRA5, Matrix Remodeling Associated 5 protein, is a secreted glycoprotein containing 7 leucine-rich repeats and 12 immunoglobulin-like C2 domains, and plays an important role in regulating cell adhesion and extracellular matrix remodeling. Studies have shown that the extracellular matrix remodeling process is significantly enhanced during the active phase of CD. Therefore, this target can be used in the diagnosis of intestinal mucosal inflammation status and the therapeutic effect of infliximab in Crohn's disease patients, so as to provide a basis for physicians to formulate the next diagnosis and treatment plan.

[0008] The present invention will be further described below with reference to the accompanying drawings. Description of the Drawings

[0009] Figure 1 It is a volcano plot of differential protein expression in Crohn's disease intestinal tissue of the present invention; Figure 2 It is a comparison chart of the expression levels of differentially expressed proteins in inflammatory and non-inflammatory tissues of the present invention; Figure 3 It is a graph of the expression levels of four proteins in peripheral blood during the active and remission phases of Crohn's disease of the present invention; Figure 4 It is a longitudinal analysis graph of the protein expression levels of Crohn's disease patients (paired samples of active and remission phases) of the present invention; Figure 5 It is a Receiver Operating Characteristic (ROC) curve graph. The area under the curve (AUC) values of MXRA5, CRP and their combined model in predicting active Crohn's disease and remission were evaluated Detailed Embodiments

[0010] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention. The experimental methods without specific conditions and the reagents without specific formulations in the examples are all in accordance with the conventional conditions in the art. Example 1

[0011] Screen markers for the inflammatory activity of CD patients.

[0012] Ten patients with active CD who were hospitalized in the Department of Gastroenterology, the Second Affiliated Hospital of Wenzhou Medical University and diagnosed [Simple Endoscopic Score of Crohn’s disease (SES-CD) ≥ 3] were enrolled. Inflammatory biopsy tissues were obtained from the margins of the ulcers at the terminal ileum of the patients, as well as non-inflammatory biopsy tissues that were macroscopically normal around the ulcers at the terminal ileum. Biopsy specimens were collected in the endoscopy room according to the standardized procedure, and then quickly frozen and stored in liquid nitrogen.

[0013] After processing these samples, proteomic studies were performed on the Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) platform technology. The specific processing procedures are as follows: Extract and quantify proteins using the Micro BCA kit (Thermo Scientific Pierce).

[0014] After the samples were reduced (10 mM DTT, 95°C, 10 minutes), alkylated (40 mM iodoacetamide, 20 minutes in the dark at 24°C), and precipitated with acetone (-20°C, 2 hours), the precipitate was dissolved in 50 mM ammonium bicarbonate buffer containing 5% acetonitrile, and trypsin (protein:trypsin mass ratio 70:1) was added for digestion overnight at 37°C.

[0015] Desalting was performed using an Oasis HLB column (Waters). The digested peptides were separated on a C18 column (Thermo Scientific) at a flow rate of 300 nL / min with a gradient elution condition: 0.1% formic acid (solvent A) and 80% acetonitrile / 0.1% formic acid (solvent B). The proportion of solvent B was increased from 8% to 28% within 70 minutes, then to 35% within 10 minutes, and then to 100% within 6 minutes and maintained for 4 minutes. Each sample was analyzed 4 times in duplicate. Mass spectrometry data were acquired on an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) using the top-10 data-dependent acquisition mode. The full scan (m / z 350–1500) had a resolution of 120,000, and the tandem mass spectrometry after HCD fragmentation (collision energy 30%) had a resolution of 30,000. The data were processed by Protein Discoverer 2.4 (Uniprot human database, false discovery rate FDR < 1%). The proteome data were processed by multidimensional protein identification technology (MudPIT) combined with the Sequest HT search engine (Proteome Discoverer 1.4). Protein intensity was quantified based on the mean of the top three highly abundant peptides, and the statistical thresholds included FDR < 1%, detection in at least half of the samples (≥5 cases), and median normalization. Missing values were filled with random numbers from a normal distribution. Statistical analysis was performed using Perseus software.

[0016] By comparing the protein expression profiles of inflamed intestinal tissues and non-inflamed normal tissues in CD patients, a total of 5252 proteins were quantitatively detected, among which 384 were differentially expressed between inflamed and non-inflamed tissues (screening criteria: fold change of inflammation / non-inflammation ≥ 2 or ≤ 0.5, and P < 0.05). Among them, 196 proteins were upregulated and 188 were downregulated in the inflamed tissues (such as Figure 1 : showing the differential protein expression between the inflamed and non-inflamed regions of the intestine in Crohn's disease patients; significantly upregulated proteins (orange-red, including the labeled MXRA5, AZU1 / HBP, CRYAB, DEFA3) and downregulated proteins (blue) were highlighted. The horizontal axis was the log2-transformed fold change, and the vertical axis was -log10 p-value. Four novel secreted proteins (MXRA5, AZU / HBP, CRYAB, DEFA3) were focused on and further verified in subsequent serum samples. The expression differences of these proteins between the inflamed and non-inflamed regions are shown as Figure 2 (comparison of the expression of four selected proteins in inflamed and non-inflamed tissues). Example 2

[0017] Verify the predictive value of the serum expression level of the biomarker for the inflammatory activity of CD patients.

[0018] A total of 74 serum samples from 68 CD patients (43 active stage and 31 remission stage, among which 6 samples were from the same patient at different times) hospitalized in the Department of Gastroenterology, the Second Affiliated Hospital of Wenzhou Medical University from September 2019 to March 2021 were included for subsequent enzyme-linked immunosorbent assay (ELISA) detection. Patients in the remission stage were defined as those with SES-CD score < 3, and patients in the active stage were defined as those with SES-CD score ≥ 3. Serum was stored at -80 °C for later use after centrifugation of fasting venous blood (2000×g, 20 minutes). The operation was strictly carried out according to the reagent instructions to detect the expression levels of four secreted proteins (MXRA5, AZU1 / HBP, CRYAB, and DEFA3).

[0019] The results showed that the level of MXRA5 in the peripheral blood of active stage CD patients was significantly lower than that in the remission stage ( Figure 3 showed the expression levels of four proteins (MXRA5, AZU1 / HBP, CRYAB, and DEFA3) in the peripheral blood of active and remission stages of Crohn's disease. The box plot showed the relative abundance of each protein and compared the differences between the active and remission stages.). However, there were no significant differences in AZU, CRYAB, and DEFA3. Among the 6 patients who had serum samples collected simultaneously in the active and remission stages, MXRA5 also showed low expression in the active stage, Figure 4 showed a longitudinal analysis of the protein expression levels of some Crohn's disease patients (paired samples of active and remission stages). The bar chart showed the changes in the expression level of MXRA5 over time in the same individual. ROC curve analysis showed that the AUC of serum MXRA5 in distinguishing active stage CD from remission stage was 0.8 (cut-off value: 309 ng / ml, sensitivity 83.7%, specificity 77.4%, positive predictive value 72.7%, negative predictive value 86.8%). Combining with serum CRP (AUC = 0.86) could further improve the prediction efficiency (combined AUC = 0.89). Binary Logistic regression analysis confirmed (see Table 1, univariate and multivariate logistic regression analysis of risk factors related to Crohn's disease patients) that the AUC of the combined prediction of mucosal complete healing by MXRA5 and CRP reached 0.89 (see Figure 5 : The receiver operating characteristic (ROC) curve graph evaluated the area under the curve (AUC) values of MXRA5, CRP, and their combined model in predicting active Crohn's disease and remission stage). Example 3

[0020] Predictive value of the biomarker MXRA5 for the therapeutic effect of infliximab in CD patients.

[0021] All 43 active CD patients described in Example 2 received infliximab treatment, and the regimen was standard intravenous administration (5 mg / kg, induction treatment at weeks 0, 2, and 6, followed by maintenance treatment every 8 weeks). Treatment response was defined as endoscopic response (SES-CD reduction ≥50%) at 24 weeks after the first dose, and non-response was defined as no improvement or deterioration in endoscopic appearance or symptoms. The results showed that 26 cases (60%) achieved endoscopic response at 24 weeks. The baseline serum MXRA5 level in the non-response group was significantly lower than that in the response group (184.00 [170.00, 206.00] vs 270.50 [207.00, 305.75], P<0.001). Univariate analysis showed that disease phenotype (OR=0.27, 95% CI: 0.07–1.00) and serum MXRA5 level (OR=1.019, 95% CI: 1.00–1.03) were associated with endoscopic response or non-response after infliximab treatment. After multivariate correction, low expression level of MXRA5 (OR=1.018, 95% CI: 1.00–1.03) and perianal lesions (OR=0.17, 95% CI: 0.03–0.97) were independent predictors of reduced endoscopic response at 24 weeks.

[0022] In the present invention, the methods for detecting serum MXRA5 level include enzyme-linked immunosorbent assay (ELISA method), immunoturbidimetry, chemiluminescence immunoassay, fluorescence immunoassay, and electrochemiluminescence.

[0023] To sum up, in this study, proteomic analysis based on the liquid chromatography-tandem mass spectrometry platform was first used to compare the protein expression levels in inflammatory and non-inflammatory intestinal tissue samples of 10 active CD patients, and the differentially expressed secreted proteins were screened out; further, the ELISA method was used to compare the expression differences of these proteins in serum samples of 43 active and 31 remission CD patients. In addition, the predictive value of serum MXRA5 level for the treatment effect of CD patients and infliximab was analyzed. ROC analysis and logistic regression were used to evaluate the diagnostic and predictive potential of MXRA5.

[0024] Results: Proteomic analysis identified 384 proteins with significantly different expressions between the inflamed mucosa and non-inflamed intestinal mucosa of CD patients, and MXRA5 was one of the secreted proteins (fold change 6.65, P value 0.005). Further ELISA detection found that the level of MXRA5 in the peripheral blood of active CD patients was significantly lower than that in the remission phase. With a cut-off value of 309 ng / ml, the sensitivity was 83.7%, the specificity was 77.4%, the positive predictive value was 72.7%, and the negative predictive value was 86.8%. ROC analysis showed that serum MXRA5 levels could distinguish active from remission CD (AUC = 0.8), and combined detection with CRP could increase the AUC to 0.89. Logistic regression analysis indicated that low serum MXRA5 levels were associated with endoscopic non-response to IFX, and low MXRA5 expression levels and perianal lesions were independent predictors of poor endoscopic response.

[0025] Conclusion: Serum MXRA5 levels can predict intestinal inflammation and the therapeutic effect of IFX in CD patients and are potential biomarkers. Its combined application with CRP can further improve the ability to distinguish disease stages and predict mucosal healing. Serum MXRA5 levels can be used as an important indicator to predict endoscopic response to IFX, providing clinical reference for individualized treatment of CD.

Claims

1. Use of an intestinal mucosal inflammation serum marker in a diagnosis and treatment product for judging the intestinal mucosal inflammation state and the treatment effect of infliximab in patients with Crohn's disease, wherein the serum marker is matrix remodeling-associated 5 protein.

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