Application of PAI-1 protein in detection and treatment of NEC patients and FNEC patients

Abstract

The application provides application of PAI-1 protein in detection and treatment of NEC patients and FNEC patients. The inventor of the application, in combination with years of experience and a large amount of research screening, obtains a biomarker capable of quickly assisting in diagnosis of NEC patients and identifying FNEC patients and Non-FNEC patients, and the biomarker is PAI-1 protein. The PAI-1 protein can be used for auxiliary diagnosis of NEC patients and early screening of FNEC patients, and FNEC patients are identified in time, so that intervention measures are taken in time, and mortality is reduced. Moreover, inhibition of expression of the PAI-1 protein can effectively relieve intestinal villus injury of a NEC model animal, and significantly reduce mortality. Therefore, the PAI-1 protein can be used as a potential target for treating NEC patients, and an agent for inhibiting expression of the PAI-1 protein can be used as a potential drug for treating NEC patients.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, in particular relates to the diagnosis and treatment of NEC, and particularly relates to the application of PAI-1 protein in the detection and treatment of NEC patients and FNEC patients. BACKGROUND

[0002] Necrotizing enterocolitis (NEC) is a severe digestive system disease in newborns, especially premature infants, which is characterized by intestinal mucosal necrosis and gas formation in the intestinal lumen, leading to complications such as intestinal perforation and sepsis. It is an important cause of death in newborns. About 30% of low birth weight premature infants die of NEC, and the NEC mortality rate of very low birth weight premature infants is as high as 50.9%.

[0003] Fulminant necrotizing enterocolitis (FNEC) is the most severe subtype of NEC, accounting for about 10% of NEC cases, and leading to rapid progression of the disease and ultimately death. Its characteristic is that the time from diagnosis to death is less than 48 hours, accompanied by massive, nearly complete intestinal ischemia and necrosis. Even after surgical treatment, the mortality rate is still as high as 98%.

[0004] Meanwhile, the lack of typical imaging features and abdominal signs in the differential diagnosis of FNEC makes it very challenging to identify NEC children in the early stages of differential diagnosis. However, there is currently no effective method to predict the rapid progression of FNEC, so finding a simple and efficient diagnostic indicator to identify newborns with rapid progression of the disease in NEC children has great potential for clinical application. SUMMARY

[0005] Therefore, the purpose of the present application is to provide the application of PAI-1 protein in the detection and treatment of NEC patients and FNEC patients. PAI-1 protein can quickly detect NEC patients and effectively distinguish FNEC patients from Non-FNEC patients.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions.

[0007] The first aspect of the present application provides the application of PAI-1 protein as a biomarker in the detection of NEC patients.

[0008] The second aspect of the present application provides the application of PAI-1 protein as a biomarker in the differentiation of FNEC patients and Non-FNEC patients.

[0009] The third aspect of the present application provides use of a reagent for detecting the level of PAI-1 protein in a biological sample in the preparation of a product for detecting a patient with necrotizing enterocolitis in a newborn.

[0010] The fourth aspect of the present application provides use of a reagent for detecting the level of PAI-1 protein in a biological sample in the preparation of a product for distinguishing a patient with an outbreak of necrotizing enterocolitis in a newborn from a patient with non-outbreak necrotizing enterocolitis in a newborn.

[0011] In some embodiments, the reagent comprises a reagent for ELISA detection, POCT, chemiluminescence detection.

[0012] In some embodiments, the reagent comprises a specific binding antibody of PAI-1 protein; preferably, the antibody is a monoclonal antibody.

[0013] In some embodiments, the product is a kit.

[0014] The fifth aspect of the present application provides use of a reagent for inhibiting the expression of PAI-1 protein in the preparation of a drug for treating a patient with necrotizing enterocolitis in a newborn.

[0015] In some embodiments, the reagent for inhibiting the expression of PAI-1 protein is Tiplaxtinin.

[0016] The sixth aspect of the present application provides a drug for treating a patient with necrotizing enterocolitis in a newborn, wherein the main active ingredient of the drug comprises a reagent for inhibiting the expression of PAI-1 protein.

[0017] In some embodiments, the reagent for inhibiting the expression of PAI-1 protein is Tiplaxtinin.

[0018] Compared with the prior art, the present application has the following beneficial effects.

[0019] The inventors of the present application obtain a biomarker that can quickly assist in the diagnosis of NEC patients and distinguish FNEC patients and Non-FNEC patients in combination with years of experience and a large amount of research screening, and the biomarker is PAI-1 protein. Compared with healthy control newborns, the content of PAI-1 protein in the biological sample of NEC patients is significantly increased; and compared with Non-FNEC patients, the content of PAI-1 protein in the biological sample of FNEC patients is further significantly increased. The ROC curve analysis result shows that when PAI-1 protein is used for diagnosing NEC patients, the AUC is 0.9773, the sensitivity is 90.91%, and the specificity is 91.67%; when PAI-1 protein is used for distinguishing FNEC patients and Non-FNEC patients, the AUC is 0.9667, the sensitivity is 100%, and the specificity is 83.33%. The results show that PAI-1 protein can be used for assisting in the diagnosis of NEC patients, and further distinguishing FNEC patients and Non-FNEC patients, and has high sensitivity and specificity. Therefore, PAI-1 protein can be used for the auxiliary diagnosis of NEC patients, and the early screening of FNEC patients, and timely identification of FNEC patients, so as to take intervention measures in time and reduce the mortality.

[0020] Further, the present application further finds that the increase of PAI-1 protein has a strong correlation with NEC occurrence through NEC animal model experiments, and inhibiting the expression of PAI-1 protein can effectively alleviate the intestinal villus injury of NEC model animals and significantly reduce the mortality, indicating that PAI-1 protein can be used as a potential target for treating NEC patients, and an agent for inhibiting the expression of PAI-1 protein can be used as a potential drug for treating NEC patients. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The results of SMART-Seq sequencing analysis of the intestinal tissues of the control group children, Non-FNEC children and FNEC children.

[0022] Figure 2 The results of PAI-1 protein content detection in the plasma of the control group children and NEC children, Non-FNEC children and FNEC children.

[0023] Figure 3 The ROC curve in Example 1.

[0024] Figure 4 The results of PAI-1 protein content detection in the plasma of the control group children and NEC children, Non-FNEC children and FNEC children.

[0025] Figure 5 The results of PAI-1 protein content detection in the plasma of the control group children and NEC children, Non-FNEC children and FNEC children.

[0026] Figure 6 Figure 6. Immunofluorescence staining images of intestinal pathological sections of control, Non-FNEC and FNEC children.

[0027] Figure 7 Figure 7. Therapeutic effect of PAI-1 Inhibitor on NEC model animals.

[0028] Figure 8 Figure 8. Elisa detection of serum PAI-1 protein, Plasmin protein, IL-6 protein expression levels and correlation analysis results of Control group, NEC-Vehicle group and NEC-PAI-1 Inhibitor group. DETAILED DESCRIPTION

[0029] The experimental methods in the following examples of the present application, unless otherwise specified, are generally carried out according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. The various common chemical reagents used in the examples are commercially available.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0031] The terms "comprising" and "having" and any variations thereof used herein are intended to cover a non-exclusive inclusion. For example, a process, method, article, or apparatus that comprises a list of steps or modules is not necessarily limited to those listed steps or modules, but can optionally include additional steps or modules not expressly listed or can optionally include steps or modules inherent to such process, method, article, or apparatus.

[0032] The following is described in conjunction with specific embodiments.

[0033] PAI-1 (Serine protease inhibitor 1, also known as SERPINE1) is a key regulatory factor in the fibrinolytic system. Studies have shown that it is closely related to cell immune response, tissue repair, inflammation regulation, etc., and its expression level is closely related to the development of many diseases (such as cardiovascular disease, tumor and infection, etc.).

[0034] Example 1

[0035] 1. Sample grouping

[0036] NEC group: According to Bell staging criteria of NEC revised edition of Practical Neonatology (4th edition), children diagnosed as NEC in Guangzhou First People's Hospital and Guangzhou Women and Children's Medical Center were selected.

[0037] FNEC group and Non-FNEC group: The NEC children included were further divided into FNEC group and Non-FNEC group. The NEC children with clinical symptoms of leukocyte, neutrophil and lymphocyte count reduction, thrombocytopenia, and a significant increase in the ratio of immature cells to mature cells in leukocytes, accompanied by massive, nearly complete intestinal ischemia and necrosis, were classified into FNEC group, and other NEC children were classified into Non-FNEC group.

[0038] Control group: Non-NEC children (mainly physiological diarrhea children) with the same gestational age, sex and birth weight as the NEC group were selected for matching.

[0039] All samples included in the study were signed by the parents of the children and approved by the ethics committee.

[0040] 2. Gene transcriptomic analysis

[0041] The intestinal tissue removed from the included children during surgery was stored in Trizol reagent and stored in a -80°C refrigerator for SMART-Seq gene transcriptomic analysis of gene expression changes, and the gene transcriptomic analysis was entrusted to Hangzhou Liancun Biotechnology Co., Ltd.

[0042] A total of 7 control children, 3 Non-FNEC children and 3 FNEC children were collected for SMART-Seq gene transcriptomic analysis. The results are shown in Figure 1 Compared with the control children, the PAI-1 gene expression in the FNEC children was significantly increased (p = 0.0003); compared with the Non-FNEC children, the PAI-1 gene expression level in the FNEC children was also significantly increased (p = 0.0089).

[0043] 3. Proteomic analysis

[0044] Based on the results of gene transcriptomic analysis, we further performed proteomic analysis.

[0045] Fresh peripheral blood of all included children was collected in EDTA anticoagulant tubes, centrifuged to collect plasma and stored in a -80°C refrigerator for proteomic analysis of plasma protein expression level, and the proteomic analysis was entrusted to Hangzhou Liancun Biotechnology Co., Ltd.

[0046] The results are shown in Figure 2As shown, by Figure 2 It was found that the level of PAI-1 protein in the peripheral blood of children with NEC was significantly higher than that of the control group (Ctrl). Compared with the Non-FNEC group, the level of PAI-1 protein in the peripheral blood of children with FNEC was significantly increased. The results suggest that PAI-1 protein can serve as a biomarker for the detection of children with NEC and can further distinguish between children with FNEC and Non-FNEC.

[0047] To evaluate the value of PAI-1 protein in detecting NEC in children and distinguishing between FNEC and Non-FNEC, we performed ROC curve analysis. The results are as follows: Figure 3 As shown in the figure, when used to differentiate between control group children and children with NEC, the AUC of PAI-1 protein was 0.9773, with a sensitivity of 90.91% and a specificity of 91.67%. When used to differentiate between children with FNEC and Non-FNEC, the AUC of PAI-1 protein was 0.9667, with a sensitivity of 100% and a specificity of 83.33%. These results indicate that PAI-1 protein can serve as a specific diagnostic molecular marker for NEC and for differentiating between FNEC and Non-FNEC.

[0048] Example 2

[0049] This embodiment verifies the results of embodiment 1.

[0050] Children diagnosed with NEC at Guangzhou First People's Hospital and Guangzhou Women and Children's Medical Center, as well as control group children, were selected according to the same criteria as in Example 1. The included NEC children were divided into FNEC group and Non-FNEC group.

[0051] This embodiment included 6 control group children and 15 NEC children; among the NEC children, there were 6 FNEC children and 9 Non-FNEC children.

[0052] Fresh peripheral blood was collected from each group of children in EDTA anticoagulant tubes, and plasma was collected by centrifugation and stored at -80°C. Plasma protein expression levels were analyzed by ELISA (Invitrogen Human PAI1 ELISA Kit BMS2033), and the procedure was strictly performed in accordance with the instructions.

[0053] The analysis results of the control group and the NEC group are as follows: Figure 4 As shown, Figure 4 It was found that the level of PAI-1 protein in the peripheral blood of the NEC group was significantly higher than that of the control group (Ctrl). Further ROC curve analysis showed that the AUC was 0.9333, the sensitivity was 93.33%, and the specificity was 83.33%.

[0054] The analysis results of the FNEC group and the Non-FNEC group are as follows: Figure 5 As shown, compared with the Non-FNEC group, the level of PAI-1 protein in the peripheral blood of children in the FNEC group was significantly increased (p = 0.0005). Further ROC curve analysis showed that when PAI-1 protein was used to distinguish between children with FNEC and Non-FNEC, the optimal cutoff value of 0.9074 corresponded to a sensitivity of 83.33% and a specificity of 88.89%.

[0055] The above results once again confirm that PAI-1 protein can serve as a specific diagnostic marker for children with NEC, and can further distinguish between children with FNEC and Non-FNEC, demonstrating high specificity and sensitivity.

[0056] Example 3

[0057] In this embodiment, intestinal tissues removed during surgical treatment of control group children, Non-FNEC children, and FNEC children were collected, soaked in 4% paraformaldehyde solution, and the expression of PAI-1 protein was detected.

[0058] Intestinal tissue soaked in 4% paraformaldehyde solution for one day was dehydrated, paraffin-impregnated, and embedded to prepare paraffin blocks, which were then sectioned using a paraffin microtome. Immunofluorescence staining of the paraffin sections was performed using PAI-1 Antibody (Affinity, Human, AF5176) as follows: Collected intestinal tissue was immediately placed in 4% paraformaldehyde fixative and fixed at 4°C for 24 hours to maintain tissue morphology and antigenicity. After fixation, the tissue was dehydrated using graded alcohols, then cleared with xylene and embedded in paraffin. After embedding, the tissue was sectioned into 4μm sections using a paraffin microtome, spread on adhesive slides, dried, and then used for staining. Before immunofluorescence staining, the tissue was dewaxed with xylene and hydrated with graded alcohols, followed by antigen retrieval using citrate buffer or EDTA, releasing antigen sites through heating. The sections were then blocked with blocking solution (5% bovine serum albumin solution) at room temperature for 2 hours to reduce nonspecific binding. After blocking, PAI-1 antibody (1:500 dilution) was added to the tissue site and incubated overnight at 4°C. After washing, fluorescently labeled secondary antibody was added, and the tissue was incubated at room temperature for 2 hours in the dark. After staining, the tissue was washed three times with PBS solution, and then DAPI solution was added and incubated at room temperature for 10 minutes to stain the nuclei. The tissue was then mounted with anti-fluorescence quenching mounting medium. Finally, the results were observed and recorded under a fluorescence microscope.

[0059] like Figure 6It can be seen that compared with the control group of children (Control), the green fluorescence in the intestinal tissue of Non-FNEC children increased, indicating that the expression of PAI-1 protein in the intestinal tissue of Non-FNEC children increased; At the same time, it can also be clearly seen that the fluorescence expression level of the intestinal tissue of the FNEC group of children is higher than that of the Non-FNEC group of children, that is, the expression level of PAI-1 protein increases.

[0060] Example 4

[0061] 1. NEC disease model construction and experimental grouping

[0062] NEC group: Take C57BL / 6 mice of appropriate age with a body weight of about 3.5-4.5g at 7 days after birth, use LPS (lipopolysaccharide) to feed at a dose of 30mg / kg per day to induce intestinal inflammation, twice a day in the morning and evening; At the same time, high-osmotic milk (45%) mixed with human milk powder and dog milk powder is fed, once in the morning, noon and evening; And accept 5% O2 concentration hypoxic treatment for 10 minutes, and cold stimulation for 10 minutes, twice a day, for three days, which can construct a mouse NEC disease model.

[0063] NEC-Vehicle group: Based on the above construction of mouse NEC disease model, feed PBS 100μL / day.

[0064] NEC-PAI-1Inhibitor group: Based on the above construction of mouse NEC disease model, feed PAI-1 inhibitor (Tiplaxtinin) 1mg / kg / day.

[0065] Control group: Take C57BL / 6 mice of appropriate age with a body weight of about 3.5-4.5g at 7 days after birth, normally breastfeed for three days, and do not do anything.

[0066] The schematic diagram of NEC disease animal model construction and grouping is shown in Figure 7 A.

[0067] 2. Detection index

[0068] (1) HE staining

[0069] On the fourth day of modeling, intestinal tissue was collected from the experimental animals, paraffin sections were prepared, and hematoxylin and eosin (HE) staining was performed. The method was as follows: After collecting the mouse intestinal tissue, it was immediately rinsed with PBS to remove surface impurities and contents. The tissue was then placed in 4% paraformaldehyde fixative and fixed at room temperature for 24 hours to preserve the tissue structure. After fixation, the tissue was dehydrated with a gradient of alcohols, then cleared in xylene, and embedded in paraffin. After embedding, the tissue was cut into 4 μm thick sections using a paraffin microtome. The sections were spread on preheated glass slides, dried, and then stained with HE. For staining, the tissue was first dewaxed with xylene, then hydrated sequentially with a gradient of alcohols and water, then stained with hematoxylin, and finally thoroughly washed with running water to remove excess stain. Next, the sections were treated with differentiation and bluing solutions, stained with eosin, and washed again with running water. Finally, after dehydration, clearing, and mounting, HE-stained sections for microscopic observation were obtained.

[0070] (2) Pathological scoring

[0071] The pathological scores were determined based on the HE staining results, and the scoring criteria are shown in Table 1.

[0072] Table 1. Chiu's Grading Criteria for Small Intestinal Mucosal Injury

[0073]

[0074] (3) Survival rate

[0075] The mouse survival rate was calculated using the following formula: (Number of mice surviving on day 4 of modeling / Number of mice surviving on day 0) × 100%.

[0076] (4) Serum expression levels of PAI-1, Plasmin and IL-6 proteins

[0077] On the fourth day of modeling, blood from each group was collected into 1.5 mL anticoagulant tubes. After centrifugation, serum was collected, and the expression levels of PAI-1, Plasmin, and IL-6 proteins in the serum were detected by ELISA (Ruixin Biotech, Mouse, RXW202553M, RXW200350M, RXW203048M). The operation was strictly performed in accordance with the instructions.

[0078] 3. Experimental Results

[0079] like Figure 7 As shown, HE-stained images of intestinal pathological sections from the Control group, NEC-Vehicle group, and NEC-PAI-1 Inhibitor group, along with pathological score statistics, demonstrate that LPS successfully induced NEC in the NEC-Vehicle group: severe intestinal villus damage, reduced goblet cells, and damaged intestinal mucosa.Figure 7 (B) The NEC-PAI-1 Inhibitor group showed significantly less intestinal villus damage compared to the NEC-Vehicle group. Figure 7 (B) and the pathological score was significantly reduced ( Figure 7 (C). From the mortality rate of laboratory animals ( Figure 7 In the NEC-Vehicle group, the final survival rate was only 60% (n=10), while the survival rate of the NEC-PAI-1 Inhibitor group was as high as 90% (n=10). These results indicate that inhibiting PAI-1 expression can effectively alleviate intestinal villus damage in NEC mice, reduce pathological scores, and effectively reduce the mortality rate of NEC disease model mice.

[0080] like Figure 8 As shown in Figure A, the PAI-1 protein expression level in the NEC-Vehicle group was significantly higher than that in the Control group (p < 0.05), while the expression level in the NEC-PAI-1 Inhibitor group was significantly lower than that in the NEC-Vehicle group. These results indicate that PAI-1 expression is elevated in the NEC disease model, and that PAI-1 inhibitors can suppress PAI-1 expression. Plasmin, as the most direct downstream signaling molecule of PAI-1, is negatively regulated by PAI-1, and both participate in the activation of the fibrinolytic system and the formation of thrombi. Correlation analysis was performed between PAI-1 and Plasmin protein expression levels and NEC disease, as shown in Figure A. Figure 8 China B and Figure 8 As shown in Figure E, the correlation analysis results show that R 2 =0.56198, Pearson coefficient -0.74965, indicating a negative correlation between PAI-1 and its downstream signaling molecule Plasmin, and a good correlation with NEC progression. IL-6, as a classic inflammatory signaling molecule, plays a positive regulatory role in the progression of most inflammatory diseases. The IL-6 protein expression level in the NEC-Vehicle group was significantly higher than that in the Control group (p < 0.0001), while the IL-6 expression level in the NEC-PAI-1 Inhibitor group was decreased. Figure 8 As shown in Figure C. The results indicate that PAI-1 inhibitors can effectively reduce inflammation levels in NEC; considering the association between the two in NEC progression, a correlation analysis was performed between PAI-1 and IL-6 protein expression levels and NEC disease, as shown in Figure C. Figure 8 As shown in Figure D, the correlation analysis results show that R 2= 0.62382, and the Pearson coefficient is 0.78982, which indicates that the expression level of PAI-1 is positively correlated with the inflammation level of NEC, and the expression level of PAI-1 can affect the inflammation level of NEC. The results show that PAI-1 is involved in the disease progression of NEC and is positively correlated with the inflammation level of NEC.

[0081] Any combination of the technical features of the above-described embodiments can be made, and in order to make the description simple, all possible combinations of the technical features in the above embodiments are not described, however, as long as the combination of the technical features does not exist, it should be considered as the scope of the description.

[0082] The above-described embodiments only express several implementation manners of the present application, the description is more specific and detailed, but it should not be understood as the limitation of the patent scope of the present application. It should be pointed out that, for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of a reagent for detecting the level of PAI-1 protein in a biological sample in the preparation of a product for detecting a patient with necrotizing enterocolitis in a newborn.

2. Use of a reagent for detecting the level of PAI-1 protein in a biological sample in the preparation of a product for distinguishing a patient with explosive necrotizing enterocolitis in a newborn from a patient with non-explosive necrotizing enterocolitis in a newborn.

3. Use according to claim 1 or 2, characterized in that, The reagent includes a reagent for ELISA detection, POCT, chemiluminescence detection.

4. The use according to claim 3, wherein the compound is ###0002### The reagent includes a specific binding antibody of PAI-1 protein.

5. The use according to claim 4, wherein the compound is ###0002### The antibody is a monoclonal antibody.

6. Use according to claim 1 or 2, wherein the compound is ###0002### The product is a kit.

7. Use of an agent that inhibits the expression of PAI-1 protein for the manufacture of a medicament for the treatment of a patient suffering from necrotizing enterocolitis in a neonate, characterized in that, The reagent for inhibiting the expression of PAI-1 protein is Tiplaxtinin.

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