Use of cx3cl1 as a biomarker for acute type a aortic dissection

Abstract

The application belongs to the technical field of disease diagnosis, and particularly relates to the use of CX3CL1 as a biomarker of acute type A aortic dissection. The application finds that CX3CL1 is a key inflammatory mediator of acute type A aortic dissection, and its plasma level is significantly positively correlated with neutrophil infiltration, IL-6, IL-8, IL-10, D-dimer and NT-proBNP. It is proved by characteristic curve analysis that there is a significant correlation between the CX3CL1 level and the severity of ATAAD. By evaluating the expression level of CX3CL1 protein in peripheral blood of a subject, an evaluation result related to the risk of suffering from acute type A aortic dissection is generated, non-invasive, fast and low-cost detection can be realized, and the problem that patients with renal dysfunction or contrast agent allergy cannot use CT angiography for diagnosis is solved.

Description

Technical Field

[0001] This invention belongs to the field of disease diagnosis technology, and specifically relates to the use of CX3CL1 as a biomarker for acute type A aortic dissection. Background Technology

[0002] Aortic dissection is a life-threatening condition characterized by a tear between the intima and media of the aorta, leading to interlaminar separation and intramural hematoma formation. According to the Stanford classification, aortic dissection is divided into type A and type B, a classification that guides treatment strategies. Acute type A aortic dissection (ATAAD) is a surgical emergency with an extremely high mortality rate if not treated promptly. ATAAD accounts for approximately 58%-62% of all aortic dissection cases, with an overall mortality rate of about 73%, and a pre-hospital mortality rate as high as 49%. Early suspicion and timely diagnosis are crucial for improving prognosis. Computed tomography (CT) angiography is the fastest and most reliable imaging method for diagnosing ATAAD, with excellent sensitivity and specificity. However, its application is limited in patients with renal insufficiency or contrast agent allergy, requiring the use of other diagnostic methods. Summary of the Invention

[0003] This invention discovers that C-X3-C motif chemokine ligand 1 (CX3CL1, also known as fractalkine) is a key inflammatory mediator in acute type A aortic dissection. Its plasma level is significantly positively correlated with neutrophil infiltration, specific cytokines (IL-6, IL-8, IL-10), and classic biomarkers (D-dimer, NT-proBNP). Characteristic curve analysis confirms a significant association between CX3CL1 levels and the severity of ATAAD. Based on this, this invention provides the use of CX3CL1 as a biomarker for acute type A aortic dissection.

[0004] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides the use of CX3CL1 protein as a biomarker in the preparation of products for assisting in the diagnosis of acute type A aortic dissection or for assessing its risk.

[0005] In conjunction with the first aspect of the invention, in some embodiments, the product is also used for testing: Neutrophil count; and / or, At least one cytokine selected from IL-6, IL-8, and IL-10; and / or, D-dimer; and / or, NT-proBNP.

[0006] Secondly, the present invention provides the use of a reagent for detecting the expression level of CX3CL1 protein in biological samples in the preparation of products for the auxiliary diagnosis or risk assessment of acute type A aortic dissection.

[0007] In conjunction with the first or second aspect of the present invention, in some embodiments, the product used for auxiliary diagnosis or risk assessment of acute type A aortic dissection is one or more of the following: reagent, kit, chip, system, and instrument.

[0008] In conjunction with a second aspect of the invention, in some embodiments, the biological sample is a human peripheral blood sample.

[0009] Thirdly, the present invention provides a risk assessment system for acute type A aortic dissection, comprising: The acquisition module acquires the expression level of CX3CL1 protein in the subject's biological samples; The assessment module generates assessment results related to the risk of acute type A aortic dissection based on the expression level of the CX3CL1 protein. The acquisition module and the evaluation module are connected wirelessly and / or via wired means.

[0010] In conjunction with a third aspect of the invention, in some embodiments, based on the expression level data of the CX3CL1 protein, an assessment result related to the risk of acute type A aortic dissection is generated, including: The expression level is compared with a preset reference value. If the expression level is higher than the preset reference value, it indicates that the subject has a high risk of disease.

[0011] In conjunction with a third aspect of the invention, in some embodiments, the preset reference value is determined based on the expression level of CX3CL1 protein in biological samples from healthy control subjects.

[0012] In conjunction with a third aspect of the present invention, in some embodiments, the preset reference value is the statistical average of the CX3CL1 protein expression level in the healthy controls.

[0013] Fourthly, the present invention provides an instrument for assessing the risk of acute type A aortic dissection, which employs the acute type A aortic dissection risk assessment system described in the third aspect.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: This invention assesses the expression level of CX3CL1 protein in the peripheral blood of subjects to generate an assessment result related to the risk of acute type A aortic dissection. It can achieve non-invasive, rapid and low-cost detection, and solve the problem that patients with renal insufficiency or contrast agent allergy cannot be diagnosed by CT angiography. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Elevated CX3CL1 levels were associated with neutrophil abundance in ATAAD patients; (A, B) frequency (A) and absolute count (B) of neutrophils in peripheral blood of ATAAD patients and healthy controls (n = 20 per group); (C) representative Wright-Giemsa staining of peripheral blood neutrophils in ATAAD patients and controls; scale bar: 10 µm; (D) plasma CX3CL1 concentration in ATAAD patients and controls (n = 20) as detected by ELISA; (E, F) correlation analysis of plasma CX3CL1 levels in ATAAD patients (n = 20) with neutrophil frequency (E) or count (F); data are expressed as mean ± standard deviation. This means P < 0.05. This means P < 0.01. This represents P < 0.001; Figure 2 The study aimed to investigate the association between CX3CL1 levels and neutrophil-related cytokines in patients with acute tachycardia (ATAAD). Specifically, the study included: (AF) plasma concentrations of IL-6 (A), IL-8 (B), IL-10 (C), IL-17A (D), IFN-γ (E), and IL-1β (F) in ATAAD patients and controls (n=20); and (GL) correlation analysis of plasma CX3CL1 levels with IL-6 (G), IL-8 (H), IL-10 (I), IL-17A (J), IFN-γ (K), and IL-1β (L) in ATAAD patients (n=20). Data are presented as mean ± standard deviation. This means P < 0.01. P < 0.001, ns indicates no statistical significance; Figure 3 The correlation between CX3CL1 levels and established diagnostic markers for ATAAD was as follows: (A) Plasma D-dimer levels in ATAAD patients and controls; (B) Correlation between plasma CX3CL1 and D-dimer levels in ATAAD patients; (C) Plasma NT-proBNP levels in ATAAD patients and controls; (D) Correlation between plasma CX3CL1 and NT-proBNP levels in ATAAD patients. Data are expressed as mean ± standard deviation (n=20). This represents P < 0.001; Figure 4 Diagnostic efficacy of CX3CL1 for ATAAD severity: Receiver operating characteristic (ROC) curves for assessing plasma CX3CL1 levels in predicting ATAAD severity.

[0017] Figure 5 : Results from the external verification queue. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0019] According to existing technology reports, CX3CL1 can bind to its receptor CX3CR1, mediating leukocyte adhesion and migration. CX3CL1 has been detected in aortic aneurysm tissue. + With CX3CR1 + Cells, suggesting their involvement in the recruitment of inflammatory cells. In allergic airway inflammation, the CX3CL1 / CX3CR1 axis regulates neutrophil infiltration by modulating pro-inflammatory cytokines and neutrophil-specific chemokines. However, the association between the CX3CL1 signaling pathway, neutrophil activity, and ATAAD remains unclear.

[0020] This invention discovers and verifies that CX3CL1 can serve as a biomarker for acute type A aortic dissection, and its plasma level is significantly positively correlated with neutrophil infiltration, IL-6, IL-8, IL-10, D-dimer, and NT-proBNP. Assessing the expression of CX3CL1 in biological samples from ATAAD patients may provide a new perspective for early diagnosis and risk stratification.

[0021] To address the issue of patients with renal insufficiency or contrast agent allergies being unable to undergo CT angiography for diagnosis, this invention provides the use of CX3CL1 protein as a biomarker or a reagent for detecting CX3CL1 protein expression levels in biological samples in the preparation of products for assisting in the diagnosis of acute type A aortic dissection or assessing its risk. These products can be diagnostic reagents, kits, chips, systems, instruments, etc., used to assist in the diagnosis of acute type A aortic dissection or assess its risk.

[0022] Studies have shown that established risk factors for aortic dissection include hereditary connective tissue diseases, arterial hypertension, and atherosclerosis. Furthermore, mounting evidence suggests that immune cells, such as neutrophils, macrophages, and lymphocytes, play a crucial role in the pathogenesis of aortic dissection, with immune activation now considered a core mechanism. Neutrophil activation, along with vascular inflammation, contributes to the formation of a unique pro-inflammatory and chemotactic microenvironment in the aortic wall. Neutrophilic inflammation is associated with elevated levels of interleukin (IL)-17A, pro-inflammatory cytokines (such as TNF-α, IL-1α, and IL-1β), and neutrophil-targeting chemokines CCL3 and CCL4. Moreover, the expression of pro-inflammatory cytokines IL-6 and IL-8, and the anti-inflammatory cytokine IL-10, is upregulated in the aortic tissue of patients with dissection compared to healthy individuals. This cytokine dysregulation promotes the degradation of extracellular matrix proteins, particularly collagen and elastin, leading to weakening of the aortic wall structure. Neutrophil aggregation further exacerbates vascular damage, promoting progressive aortic dilation and rupture. This invention confirms that the expression level of CX3CL1 protein is strongly positively correlated with the established diagnostic markers D-dimer and NT-proBNP. Figure 3 Based on this, the above-mentioned products are also designed to detect: neutrophil infiltration, IL-6, IL-8, IL-10, D-dimer, and NT-proBNP. This allows CX3CL1 to be used in conjunction with existing biomarkers to corroborate each other, forming a more sensitive and specific diagnostic model and providing clinicians with more comprehensive risk assessment information.

[0023] In this embodiment of the invention, the concentration of CX3CL1 protein in peripheral blood plasma of ATAAD patients was detected and analyzed, and systematically compared with that of healthy controls and other disease-related indicators. This ultimately confirmed a significant correlation between CX3CL1 protein expression levels and acute type A aortic dissection. Therefore, the preferred biological sample for the above product testing is human peripheral blood, which can more accurately reflect the pathophysiological process of the disease. Furthermore, using human peripheral blood as the test sample allows for non-invasive, rapid, and low-cost detection.

[0024] This invention also provides a risk assessment system for acute type A aortic dissection, comprising: The acquisition module acquires the expression level of CX3CL1 protein in the subject's biological samples; The assessment module generates assessment results related to the risk of acute type A aortic dissection based on the expression level of the CX3CL1 protein. The acquisition module and the evaluation module are connected wirelessly and / or via wired means.

[0025] This acute type A aortic dissection risk assessment system is a standardized and operational clinical assessment tool. The data acquisition module handles data input, drawing from direct readings of automated immunoassay analyzers, manually entered test reports by healthcare professionals, and data automatically acquired through integration with other laboratory information systems. The assessment module processes the data and determines the risk based on a built-in algorithm. This system enables rapid and objective assisted diagnosis. The assessment results can be used not only for preliminary diagnosis but also to reflect the severity of acute type A aortic dissection, helping physicians to stratify patients based on risk and develop more individualized treatment and monitoring plans.

[0026] Figure 1 D showed that the expression level of CX3CL1 protein in ATAAD patients was significantly higher than that in healthy controls. Figure 4 The ROC curve demonstrates its good diagnostic predictive ability (AUC=0.736). In some embodiments of the present invention, based on the expression level data of the CX3CL1 protein, an assessment result related to the risk of acute type A aortic dissection is generated, including: comparing the expression level with a preset reference value; if the expression level is higher than the preset reference value, it indicates that the subject has a high risk of disease. The preset reference value is determined based on the expression level of CX3CL1 protein in the biological samples of healthy controls. For example, the preset reference value is the mean or median of the healthy group, the 95% reference interval of the healthy group, or the optimal diagnostic cutoff value obtained from ROC curve analysis. In the embodiments of the present invention, the preset reference value is the statistical average of the CX3CL1 protein expression level in age-matched healthy individuals.

[0027] This invention also provides an instrument for assessing the risk of acute type A aortic dissection, employing the aforementioned acute type A aortic dissection risk assessment system. This instrument is a physical diagnostic tool that can be used quickly and conveniently in the clinical setting, eliminating reliance on large imaging equipment and complex laboratory testing procedures. It has significant auxiliary diagnostic value when imaging examinations are limited, and possesses clear prospects for clinical translation.

[0028] The technical solution provided by the present invention will be described in detail below with reference to the embodiments. The experimental methods used in the embodiments are as follows: 1) Study Subjects: This invention was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University. Written informed consent is waived according to national regulations and institutional policies. A total of 20 patients diagnosed with acute type A aortic dissection at the First Affiliated Hospital of Guangzhou Medical University were included. All patients were diagnosed by computed tomography and underwent surgical treatment within four days of admission. Age-matched healthy controls were also recruited, all without a history of hematologic disorders, infections, or other systemic diseases. Peripheral blood samples were collected from all subjects. The baseline demographics and clinical characteristics of the study cohort are summarized in Table 1.

[0029] 2) CX3CL1 Enzyme-Linked Immunosorbent Assay: Peripheral blood samples were centrifuged at 3,000 rpm for 10 minutes at 4°C to separate plasma. The plasma was aliquoted and stored at -80°C until analysis. CX3CL1 concentration was measured using a commercial ELISA kit (Fine Biotech) strictly following the manufacturer's instructions.

[0030] 3) Neutrophil analysis: The frequency and absolute count of peripheral blood neutrophils were determined using a fully automated blood analyzer (Sysmex XN-2800). This analysis combined semiconductor laser optical detection with flow cytometry to identify and quantify neutrophils.

[0031] 4) Inflammatory Cytokine Detection: Using a commercially available cytokine detection kit (Cell-Genebio), the levels of IL-1β, IL-6, IL-8, IL-10, IL-17A, and interferon-γ (IFN-γ) in plasma were detected according to the manufacturer's instructions. The simplified procedure is as follows: Peripheral blood samples were centrifuged at 800 × g for 20 minutes to obtain the supernatant; an equal volume of microsphere buffer was added to the supernatant, vortexed, and incubated in the dark for 15 minutes; after vortexing again, 25 µL of the microsphere mixture was mixed with 25 µL of the supernatant and the fluorescent detection reagent, and incubated at room temperature in the dark for 2.5 hours; then 1 mL of phosphate buffer was added, and the mixture was centrifuged at 200 × g for 5 minutes; the supernatant was discarded, the microspheres were resuspended, and the microspheres were detected using a Novocyte D3000 flow cytometer (Agilent).

[0032] 5) D-dimer assay: Peripheral blood samples were centrifuged at 3,000 rpm for 10 minutes at 4°C to separate plasma. D-dimer levels were quantitatively detected using the VIDAS 30 immunoassay system (BioMérieux).

[0033] 6) NT-proBNP detection: The concentration of NT-proBNP in plasma samples was determined using a Roche Cobas 8000 E602 analyzer (Roche Diagnostics) strictly following the manufacturer's guidelines.

[0034] 7) Statistical Analysis: All statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software). Normally distributed data were analyzed using a two-tailed Student's t-test, while non-normally distributed data were analyzed using the Mann-Whitney U test. Spearman rank correlation was used for nonparametric correlation analysis. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve was calculated to assess predictive power. Data are expressed as mean ± standard deviation. Statistical significance was defined as: This means P < 0.05. This means P < 0.01. This means P < 0.001.

[0035] Example 1: Elevated CX3CL1 levels are associated with neutrophil abundance in ATAAD patients Previous studies have shown that neutrophils play a crucial role in triggering acute aortic dissection, and elevated neutrophil levels are associated with increased diagnostic potential and poorer clinical prognosis. To further explore this association, this invention performed complete blood cell count analysis to assess the proportion and absolute number of neutrophils in ATAAD patients determined by multiple methods, including computed tomography angiography (CTA), transesophageal echocardiography (TEE), and magnetic resonance angiography (MRA). The results showed that, compared with healthy controls, the frequency and count of neutrophils in the peripheral blood of ATAAD patients were significantly increased (…). Figure 1 (A and 1B).

[0036] To assess the relationship between neutrophil levels and treatment response, patients with ATAAD underwent emergency surgery and anticoagulation therapy. Following treatment, both the neutrophil percentage and count significantly decreased. Figure 1 Table 1 summarizes the detailed demographic and clinical characteristics of the participants. Furthermore, Wright-Giemsa staining confirmed the typical neutrophil morphology in peripheral blood samples from both ATAAD patients and healthy controls. Figure 1 C). These results collectively indicate that neutrophil levels are significantly elevated in ATAAD patients. This invention then investigates whether CX3CL1 levels are correlated with neutrophil abundance in ATAAD patients. Plasma concentrations of the chemokine receptor ligand CX3CL1 in peripheral blood samples from patients were measured using ELISA. The results showed that CX3CL1 levels were significantly elevated in ATAAD patients compared to the control group (C). Figure 1 D). Furthermore, correlation analysis showed that in ATAAD patients, CX3CL1 levels were strongly positively correlated with both the neutrophil percentage and absolute neutrophil count. Figure 1 E and 1F). These data demonstrate that CX3CL1 expression is closely related to neutrophil abundance in ATAAD.

[0037] Table 1. Clinical characteristics of ATAAD patients and healthy controls Note: In Table 1, ATAAD is the abbreviation for Acute Type A Aortic Dissection.

[0038] Example 2: Association between CX3CL1 and neutrophil-associated cytokines in ATAAD patients Several cytokines, including interleukin-6 (IL-6), IL-8, IL-10, IL-17A, interferon-γ (IFN-γ), and IL-1β, are known to mediate neutrophil recruitment, chemotaxis, autophagy, and the formation of neutrophil extracellular traps. This invention shows that, compared with healthy controls, plasma levels of IL-6, IL-8, IL-10, and IL-17A were significantly elevated in ATAAD patients. Figure 2 A-2D). In contrast, there was no significant difference in IFN-γ and IL-1β levels between ATAAD patients and controls. Figure 2 E and 2F). This invention further evaluated the relationship between CX3CL1 and these neutrophil-associated cytokines. Correlation analysis showed that in ATAAD patients, CX3CL1 levels were positively correlated with IL-6, IL-8, and IL-10 (E and 2F). Figure 2 G-2I), but not significantly correlated with IL-17A ( Figure 2 J). Furthermore, no significant correlation was observed between CX3CL1 and IFN-γ or IL-1β (J). Figure 2 These results indicate that CX3CL1 levels are closely associated with specific neutrophil-associated cytokines—namely IL-6, IL-8, and IL-10—in patients with ATAAD.

[0039] Example 3: Association between CX3CL1 and diagnostic biomarkers in ATAAD patients To assess the clinical relevance of CX3CL1 in ATAAD, this invention examined its relationship with established diagnostic biomarkers. D-dimer has been considered an effective "exclusion" marker in suspected cases of acute aortic dissection. Consistent with previous studies, plasma D-dimer levels in ATAAD patients were significantly higher than in healthy controls in this invention. Figure 3 A). Furthermore, in the peripheral blood of ATAAD patients, CX3CL1 levels were strongly positively correlated with D-dimer levels (A). Figure 3B). Elevated levels of another easily detectable biomarker, NT-proBNP, are associated with increased in-hospital mortality in patients with ATAAD. Consistent with this, the present invention observed significantly elevated NT-proBNP levels in patients with ATAAD ( Figure 3 C). Importantly, CX3CL1 levels were also significantly correlated with NT-proBNP levels (C). Figure 3 D). In summary, these findings indicate that CX3CL1 levels are positively correlated with key diagnostic markers -D-dimer and NT-proBNP- in ATAAD, suggesting that CX3CL1 may serve as a novel biomarker for monitoring disease progression.

[0040] Example 4: Predictive value of CX3CL1 for ATAAD severity To further explore the association between plasma CX3CL1 levels and ATAAD, this invention performed receiver operating characteristic (ROC) curve analysis to evaluate its diagnostic utility. The analysis yielded an area under the curve of 0.736 ( ). Figure 4 This indicates that CX3CL1 has valuable predictive ability for the diagnosis of ATAAD. In summary, this finding reveals a significant association between CX3CL1 levels and the severity of ATAAD, suggesting that it may play a role in disease progression.

[0041] Example 5: External validation queue, predictive value of CX3CL1 for ATAAD severity To verify the generalizability of the above results, an external validation cohort was recruited independently of Example 1. This cohort included 183 patients with ATAAD diagnosed by multiple methods including CTA, TEE, and MRA, and 179 age- and sex-matched healthy controls. The inclusion and exclusion criteria were the same as in Example 1. After collecting peripheral blood samples, plasma CX3CL1 concentrations were measured according to the method described in Example 1. Receiver operating characteristic (ROC) curve analysis showed that the area under the curve (AUC) was 0.753 (…). Figure 5 This indicates that CX3CL1 has valuable predictive ability for the diagnosis of ATAAD. In summary, this finding reveals a significant association between CX3CL1 levels and the severity of ATAAD, suggesting that it may play a role in disease progression.

[0042] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of CX3CL1 protein as a biomarker in the preparation of products for the auxiliary diagnosis of acute type A aortic dissection or for assessing its risk.

2. The use according to claim 1, characterized in that, The product is also used for testing: Neutrophil count; and / or, At least one cytokine selected from IL-6, IL-8, and IL-10; and / or, D-dimer; and / or, NT-proBNP.

3. The use of reagents for detecting CX3CL1 protein expression levels in biological samples in the preparation of products for the auxiliary diagnosis or risk assessment of acute type A aortic dissection.

4. The use according to claim 1 or 3, characterized in that: Products used for the auxiliary diagnosis or risk assessment of acute type A aortic dissection include one or more of the following: reagents, kits, chips, systems, and instruments.

5. The use according to claim 3, characterized in that: The biological sample is a human peripheral blood sample.

6. A risk assessment system for acute type A aortic dissection, characterized in that, include: The acquisition module acquires the expression level of CX3CL1 protein in the subject's biological samples; The assessment module generates assessment results related to the risk of acute type A aortic dissection based on the expression level of the CX3CL1 protein. The acquisition module and the evaluation module are connected wirelessly and / or via wired means.

7. The acute type A aortic dissection risk assessment system according to claim 6, characterized in that: Based on the CX3CL1 protein expression level data, an assessment of the risk of acute type A aortic dissection was generated, including: The expression level is compared with a preset reference value. If the expression level is higher than the preset reference value, it indicates that the subject has a high risk of disease.

8. The acute type A aortic dissection risk assessment system according to claim 7, characterized in that: The preset reference value is determined based on the expression level of CX3CL1 protein in biological samples from healthy control subjects.

9. The acute type A aortic dissection risk assessment system according to claim 8, characterized in that: The preset reference value is the statistical average of the CX3CL1 protein expression level in the healthy controls.

10. A risk assessment instrument for acute type A aortic dissection, characterized in that, The acute type A aortic dissection risk assessment system as described in claim 8 or 9 is adopted.

Images