Use of ninj1 protein in preparation of products for early diagnosis, treatment or prognosis detection of kawasaki disease

Abstract

The application belongs to the technical field of biotechnology and medicine, and particularly discloses application of NINJ1 protein in preparation of a product for early diagnosis, treatment or prognosis detection of Kawasaki disease; the application finds that, compared with a healthy control group, the plasma NINJ1 of an acute Kawasaki disease patient in a Kawasaki disease patient group is significantly increased, the application finds that the expression level of the plasma NINJ1 protein can effectively diagnose the Kawasaki disease, the diagnosis threshold for diagnosing the Kawasaki disease is 50.44 pg / mL, the sensitivity is 60.2%, and the specificity is 91.3%, so the NINJ1 protein provided in the application can be used as a marker for early diagnosis of the Kawasaki disease, and provides important application value for early diagnosis of the Kawasaki disease; the application also finds that the expression level of the plasma NINJ1 is significantly related to coronary artery damage of the Kawasaki disease, and the NINJ1 protein has good sensitivity as an index for predicting the coronary artery damage of the Kawasaki disease, and can be another predictive index for the coronary artery damage of the Kawasaki disease.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, and specifically discloses the application of NINJ1 protein in the preparation of products for early diagnosis, treatment or prognosis detection of Kawasaki disease. Background Technology

[0002] Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is an acute febrile rash illness characterized by systemic vasculitis. It is prevalent in Asia, primarily affecting male infants under 5 years of age. The main clinical manifestations include fever, conjunctival congestion in both eyes, flushed and cracked lips, strawberry tongue, diffuse congestion of the oral mucosa, rash, erythema on the palms and soles, redness, induration, and desquamation of the fingertips and toes, and cervical lymphadenopathy. This disease can cause systemic inflammatory damage to multiple organs, centered on vasculitis, and can affect the heart. Kawasaki disease primarily harms the cardiovascular system, leading to coronary artery damage, including coronary arteritis, coronary artery dilation, coronary aneurysm, and coronary embolism, seriously endangering the health of infants and young children. Kawasaki disease is also a significant cause of coronary artery disease in young and middle-aged adults. Early intravenous high-dose intravenous immunoglobulin (IVIG) combined with oral aspirin can reduce the incidence of coronary artery injury from 25% to about 4%. Currently, diagnosis relies mainly on clinical symptoms and general inflammatory markers. However, incomplete Kawasaki disease still exists clinically, with indistinct clinical features, making diagnosis difficult. Currently, there is a lack of specific and sensitive laboratory biomarkers for Kawasaki disease. Therefore, finding a sensitive laboratory marker for early diagnosis of Kawasaki disease is of great significance for reducing coronary artery injury and improving the prognosis of Kawasaki disease.

[0003] NINJ1 is a 16-kDa plasma membrane protein, evolutionarily conserved and present in all higher eukaryotes. During inflammasome-driven pyroptosis, the cell death executive protein gasdermin D (GSDMD) is activated by caspase-dependent cleavage, subsequently oligomerizing with higher-order NINJ1, causing pores in the plasma membrane and inducing downstream membrane rupture. This rupture is accompanied by cell death and the release of large amounts of inflammatory factors. When the body is infected, NINJ1 oligomerizes to cause cell pores, leading to the release of cellular contents and inflammatory factors. These characteristics make NINJ1 a promising candidate for the development and application of disease diagnosis and treatment methods. However, to date, most NINJ1 research has focused on lipid membrane pores that induce inflammatory cell death. Literature reports that plasma NINJ1 may be used to diagnose diseases including liver cancer, ovarian cancer, coronary artery disease, and atrial fibrillation. There are currently no reports of NINJ1 being used as a molecular marker for the diagnosis of Kawasaki disease. Summary of the Invention

[0004] To address the aforementioned issues, this invention discloses the application of NINJ1 protein in the preparation of products for early diagnosis, treatment, or prognosis testing of Kawasaki disease.

[0005] This invention includes the following technical solutions:

[0006] This invention discloses for the first time the application of NINJ1 protein as a diagnostic biomarker for early Kawasaki disease.

[0007] Furthermore, this invention discloses the application of NINJ1 protein in the preparation of products for early diagnosis, treatment, or prognosis testing of Kawasaki disease.

[0008] Furthermore, in the above applications, the product is used to detect the expression level of NINJ1 protein.

[0009] Furthermore, in the above applications, the NINJ1 protein is present in plasma.

[0010] Furthermore, in the above applications, the product is a product for detecting the expression level of NINJ1 protein in plasma.

[0011] Furthermore, in the above applications, the product is an ELISA kit.

[0012] The present invention also discloses a kit for early diagnosis, treatment or prognosis detection of Kawasaki disease, the kit containing a reagent for detecting the expression level of NINJ1 protein in plasma.

[0013] Furthermore, the kit described above is a kit for predicting coronary artery damage in patients with Kawasaki disease.

[0014] Furthermore, the kit described above is a kit for predicting the sensitivity of Kawasaki disease patients to IVIG treatment.

[0015] The present invention has the following beneficial effects:

[0016] 1. The NINJ1 molecular marker provided by this invention has been found through research to significantly increase the plasma NINJ1 level in patients with acute Kawasaki disease compared to healthy controls and patients with Kawasaki disease. Therefore, the NINJ1 marker provided by this invention can be used as a marker for the early diagnosis of Kawasaki disease, providing certain application value for the early diagnosis of Kawasaki disease.

[0017] 2. This invention found that plasma NINJ1 can effectively predict Kawasaki disease. The optimal diagnostic threshold for predicting Kawasaki disease is 50.44 pg / mL, with a sensitivity of 60.2% and a specificity of 91.3%, suggesting that NINJ1 may serve as a diagnostic biomarker for Kawasaki disease.

[0018] 3. This invention found that plasma NINJ1 concentration is significantly correlated with coronary artery damage in Kawasaki disease, and has good sensitivity as an indicator for predicting coronary artery damage in Kawasaki disease, and can be used as another predictive indicator for coronary artery damage in Kawasaki disease.

[0019] 4. This study found that plasma NINJ1 has a predictive effect on the sensitivity to IVIG treatment and can be used as a reference indicator for the treatment of Kawasaki disease. Attached Figure Description

[0020] Figure 1 The graph shows the expression level of NINJ1 in plasma during the acute phase of Kawasaki disease. ****P < 0.0001;

[0021] Figure 2 ROC curve of NINJ1 in plasma for diagnosing Kawasaki disease;

[0022] Figure 3 The expression levels of NINJ1 in plasma were plotted between the immunoglobulin-sensitive Kawasaki disease group and the immunoglobulin-unresponsive Kawasaki disease group. **P < 0.01;

[0023] Figure 4 The expression levels of plasma NINJ1 in Kawasaki disease patients without coronary artery injury and those with coronary artery injury are shown in the figure. *P<0.05;

[0024] Figure 5 The graph shows the expression levels of plasma NINJ1 in the acute and recovery phases of Kawasaki disease. **P < 0.01. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, all reagents or instruments used in the embodiments of this invention are commercially available conventional reagent products.

[0027] The technical solution of this patent will be further described in detail below with reference to specific embodiments.

[0028] I. Experimental Materials and Methods

[0029] 1.1 Case Selection

[0030] 1.1.1 Diagnostic criteria

[0031] The diagnosis was confirmed as Kawasaki disease based on the diagnostic criteria proposed by the Japanese MCLS Research Association.

[0032] 1.2 Case Data

[0033] This study collected 103 Kawasaki disease patients eligible for inclusion from July 2023 to April 2024, all from Nanjing Children's Hospital. The patients' ages ranged from 0.5 to 13 years, with a median age of 3.77 years. A healthy control group of 46 patients, aged 1 to 14 years (median age 6.76 years), also from Nanjing Children's Hospital, was included. The study protocol complied with ethical standards for human trials and was approved by the ethics committee. All participants were informed and provided written consent before the study. NINJ1 was measured using ELISA at Nanjing Children's Hospital. Color Doppler ultrasound was used to measure the diameter of the left and right coronary arteries in the children at rest to determine whether coronary arteries were dilated.

[0034] 1.3 Group Design

[0035] Kawasaki disease group and healthy control group.

[0036] 1.4 Main Reagents and Consumables

[0037] NINJ1 Enzyme-Linked Immunosorbent Assay (ELISA) Kit (Wuhan Huamei Biotechnology).

[0038] 1.5 Experimental Methods and Techniques

[0039] 1.5.1 Collection of materials

[0040] Clinical observations were recorded according to the specific clinical observation form used in this study. After admission and confirmation of diagnosis, blood samples were collected from Kawasaki disease patients on an empty stomach. 4-5 mL of blood was collected by trained personnel from the research team, centrifuged at 3,000 rpm for 10 minutes at room temperature within 1 hour, and the supernatant plasma was collected and stored in liquid nitrogen in cryovials before being transferred to a -80°C freezer for later use. Samples were removed before experiments and thawed on ice; all blood samples underwent a single freeze-thaw cycle.

[0041] 1.5.2 Detection of plasma NINJ1 (Gene ID: 4814) expression level

[0042] 1.5.2.1 Experimental Procedure

[0043] Enzyme-linked immunosorbent assay (ELISA) kit (batch number: CSB-EL015808HU, brand: CUSABIO) for determining NINJ1 expression in plasma.

[0044] The experimental steps are as follows:

[0045] 1. Allow all reagents to reach room temperature (18-25℃) for at least 30 minutes to equilibrate. Prepare the reagents according to the instructions and set aside.

[0046] 2. Sample addition: Set up separate wells for standard and test samples. Add 100 μl of standard or test sample to each well, gently shake to mix, cover with a plate label, and incubate at 37°C for 2 hours;

[0047] 3. Discard the liquid, spin dry, no washing required;

[0048] 4. Add 100 μl of biotin-labeled antibody working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour;

[0049] 5. Discard the liquid in the wells, spin dry, and wash the plate 3 times. Each time, soak for 2 minutes, using 200 μl per well, then spin dry.

[0050] 6. Add 100 μl of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate, and incubate at 37°C for 1 hour;

[0051] 7. Discard the liquid in the wells, spin dry, and wash the plate 5 times. Each time, soak for 2 minutes, using 200 μl per well, then spin dry.

[0052] 8. Add 90 μl of substrate solution to each well in sequence and develop color at 37°C in the dark for 15-30 minutes;

[0053] 9. Add 50 μl of the stop solution to each well in sequence to terminate the reaction;

[0054] 10. Within 5 minutes after the reaction is terminated, use an ELISA reader to measure the optical density (OD value) of each well sequentially at a wavelength of 450 nm.

[0055] Note: The model of the microplate reader is: BioTek Synergy H1 Multifunctional Microplate Reader

[0056] 1.6 Statistical Analysis

[0057] Data analysis in this study was performed using SPSS software. Before analysis, the Kolmogorov-Smirnov method was used to test the distribution characteristics of each group. Skewed data were logarithmically transformed before analysis. Independent samples t-tests were used for pairwise comparisons of continuous data; chi-square tests were used for pairwise comparisons of categorical data. ROC curve analysis was used to assess the predictive diagnostic value, sensitivity, and specificity of plasma NINJ1 for Kawasaki disease. A p-value < 0.05 was considered statistically significant.

[0058] Example 1

[0059] Kawasaki disease patients have higher plasma NINJ1 levels during the acute phase.

[0060] The results of NINJ1 expression level detection in plasma samples from healthy control group and Kawasaki disease group were as follows: the relative expression level of NINJ1 in the control group was 29.83±16.52 pg / mL, and in the Kawasaki disease group it was 75.42±54.59 pg / mL. Statistical analysis of the relative expression level of NINJ1 in each group showed a significant difference in plasma NINJ1 between the normal group and the Kawasaki disease group (p<0.0001). Plasma NINJ1 level was statistically significant with KD and has diagnostic value for KD. Figure 1 ).

[0061] Using healthy individuals as the control group and the Kawasaki disease group as the disease group, ROC curve analysis was performed on the relative expression level of NINJ1, with an AUC value of 0.788. The optimal diagnostic threshold for NINJ1 was 50.44 pg / mL, with a diagnostic sensitivity of 60.2% and a specificity of 91.3%. Figure 2 ).

[0062] The results above indicate that plasma NINJ1 can be used as a potential biomarker for differentiating healthy children from Kawasaki disease. If the relative expression level of Kawasaki disease in the plasma of the tested individual is greater than or equal to 50.44 pg / mL, the individual is considered or a candidate for Kawasaki disease.

[0063] Example 2

[0064] Plasma NINJ1 levels were significantly elevated in patients with Kawasaki disease who were not immunoglobulin-responsive. Figure 3 ).

[0065] The results of plasma NINJ1 expression levels in the gamma globulin-sensitive Kawasaki disease group and the non-responder group were as follows: The mean ± standard deviation of the relative plasma NINJ1 expression level in the gamma globulin-sensitive Kawasaki disease group was 70.29 ± 48.45 pg / mL, while that in the non-responder group was 129.02 ± 84.63 pg / mL. There was a significant difference in plasma NINJ1 levels between the gamma globulin-sensitive Kawasaki disease group and the non-responder group (**P < 0.01).

[0066] Example 3:

[0067] Plasma NINJ1 levels were significantly elevated in patients with Kawasaki disease and coronary artery injury. Figure 4 ).

[0068] The results of plasma NINJ1 expression levels in the Kawasaki disease group without coronary artery injury and the Kawasaki disease group with coronary artery injury were as follows: the mean ± standard deviation of plasma NINJ1 expression level in the Kawasaki disease group without coronary artery injury was 70.95 ± 52.87 pg / mL, while that in the Kawasaki disease group with coronary artery injury was 101.68 ± 58.87 pg / mL. There was a significant difference in plasma NINJ1 levels between the two groups (*P < 0.05).

[0069] Example 4

[0070] Plasma NINJ1 levels in Kawasaki disease patients decreased significantly during the recovery period. Figure 5 )

[0071] The results of plasma NINJ1 expression levels in the acute and convalescent phases of Kawasaki disease were as follows: the mean ± standard deviation of relative NINJ1 expression levels in the acute phase group was 62.24 ± 43.91 pg / mL, while that in the convalescent phase group was 42.21 ± 29.50 pg / mL. There was a significant difference in plasma NINJ1 levels between the acute and convalescent phases of Kawasaki disease (**P < 0.01).

[0072] In summary, NINJ1 can be detected in the plasma of patients with acute Kawasaki disease, and its expression level is significantly higher than that of normal children, indicating its potential for diagnosing Kawasaki disease. The expression level of plasma NINJ1 in Kawasaki disease patients is significantly correlated with coronary artery damage in Kawasaki disease patients; the expression level of plasma NINJ1 in Kawasaki disease patients is correlated with responsiveness to gamma globulin therapy; and high expression of plasma NINJ1 is an independent prognostic factor for Kawasaki disease patients. The diagnostic efficacy of plasma NINJ1 markers demonstrates that NINJ1 has the advantages of high sensitivity and accuracy.

[0073] The above embodiments illustrate and describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. Application of products for detecting the expression level of NINJ1 protein in plasma in the preparation of diagnostic kits for Kawasaki disease.

2. The application according to claim 1, characterized in that, The product in question is an ELISA kit.

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