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

Abstract

The invention provides application of PAI-1 protein in detection and treatment of NEC patients and FNEC patients. The inventor of the invention combines years of experience and a large amount of research screening to obtain a biomarker capable of rapidly 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 the FNEC patients can be recognized in time, so that intervention measures are taken in time, and the death rate is reduced. In addition, by inhibiting the expression of the PAI-1 protein, intestinal villus injury of NEC model animals can be effectively relieved, and the death rate is remarkably reduced. Therefore, the PAI-1 protein can be used as a potential target spot for treating the NEC patient, and a reagent for inhibiting the expression of the PAI-1 protein can be used as a potential medicine for treating the NEC patient.

Description

Technical Field

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

[0002] Neonatal necrotizing enterocolitis (NEC) is a severe digestive system disease in neonates, especially premature infants. Its clinical manifestations include intestinal mucosal necrosis, gas formation in the intestinal lumen, and subsequent complications such as intestinal perforation and sepsis, which is an important cause of neonatal death. Approximately 30% of low-birth-weight premature infants die from NEC, and the NEC mortality rate of extremely 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 a rapid progression of the disease course and ultimately death. Its characteristic is that the time from diagnosis to death of the patient is less than 48 hours, accompanied by large-scale and nearly complete intestinal ischemia and necrosis. Even after surgical treatment, the mortality rate is still as high as 98%.

[0004] At the same time, due to the lack of typical imaging features and abdominal signs for the differential diagnosis of FNEC, it poses a great challenge in the early differential diagnosis of NEC children. However, there is currently no effective method to predict the rapid progression of FNEC. Therefore, finding a simple and efficient diagnostic index to identify neonates with a rapid progression of the disease course among NEC children has great clinical application potential. Summary of the Invention

[0005] Based on this, the purpose of the present invention is to provide the application of PAI-1 protein in the detection and treatment of NEC patients and FNEC patients. The PAI-1 protein can rapidly detect NEC patients and effectively distinguish FNEC patients from Non-FNEC patients.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions.

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

[0008] The second aspect of the present invention provides the application of PAI-1 protein as a biomarker in distinguishing fulminant neonatal necrotizing enterocolitis patients from non-fulminant neonatal necrotizing enterocolitis patients.

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

[0010] The fourth aspect of the present invention provides the use of a reagent for detecting the level of PAI-1 protein in a biological sample in the preparation of a product for differentiating between patients with fulminant neonatal necrotizing enterocolitis and non-fulminant neonatal necrotizing enterocolitis.

[0011] In some embodiments, the reagent includes reagents for ELISA detection, POCT, and chemiluminescence detection.

[0012] In some embodiments, the reagent includes a specific binding antibody to 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 invention provides the use of a reagent for inhibiting the expression of PAI-1 protein in the preparation of a drug for treating patients with neonatal necrotizing enterocolitis.

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

[0016] The sixth aspect of the present invention provides a drug for treating patients with neonatal necrotizing enterocolitis, and the main active ingredient of the drug includes 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 invention has the following beneficial effects.

[0019] The inventors of the present invention, combining their own years of experience and a large number of research screenings, obtained a biomarker that can quickly assist in the diagnosis of NEC patients and distinguish FNEC patients from Non-FNEC patients. The biomarker is PAI-1 protein. Compared with healthy control neonates, the content of PAI-1 protein in the biological samples of NEC patients is significantly increased; and compared with Non-FNEC patients, the content of PAI-1 protein in the biological samples of FNEC patients is further significantly increased. The results of ROC curve analysis show that when PAI-1 protein is used to diagnose NEC patients, the AUC is 0.9773, the sensitivity is 90.91%, and the specificity is 91.67%; when PAI-1 protein is used to distinguish FNEC patients from Non-FNEC patients, the AUC is 0.9667, the sensitivity is 100%, and the specificity is 83.33%. The results indicate that PAI-1 protein can be used to assist in the diagnosis of NEC patients and further distinguish FNEC patients from 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, timely identify FNEC patients, so as to take intervention measures in time and reduce the mortality rate.

[0020] Furthermore, through experiments on NEC animal models, the present invention also found that the increase of PAI-1 protein is strongly correlated with the occurrence of NEC, and inhibiting the expression of PAI-1 protein can effectively relieve the intestinal villus damage of NEC model animals and significantly reduce the mortality rate, indicating that PAI-1 protein can be used as a potential target for treating NEC patients, and reagents that inhibit the expression of PAI-1 protein can be used as potential drugs for treating NEC patients. Description of the Drawings

[0021] Figure 1 It is the result of SMART-Seq sequencing analysis of the intestinal tissues of children in the control group, Non-FNEC children and FNEC children.

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

[0023] Figure 3 It is the ROC curve in Example 1.

[0024] Figure 4 It is the detection result of the content of PAI-1 protein in the plasma of children in the control group and NEC children in Example 2 and the ROC curve.

[0025] Figure 5 It is the detection result of the content of PAI-1 protein in the plasma of FNEC children and Non-FNEC children in Example 2 and the ROC curve.

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

[0027] Figure 7 Results of the study on the therapeutic effect of PAI-1 Inhibitor on NEC model animals.

[0028] Figure 8 Results of the expression levels of serum PAI-1 protein, Plasmin protein, and IL-6 protein and correlation analysis detected by Elisa in the Control group, NEC-Vehicle group, and NEC-PAI-1Inhibitor group. Specific implementation manners

[0029] In the following examples of the present invention, the experimental methods without specific conditions are usually carried out according to conventional conditions, such as the conditions 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. All common chemical reagents used in the examples are commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0031] The terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, device, product, or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, products, or equipment.

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

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

[0034] Example 1

[0035] 1. Sample grouping

[0036] NEC group: According to the Bell staging criteria of the NEC revised version in "Practical Neonatology" (Fourth Edition), children diagnosed with 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 included NEC children were further divided into FNEC group and Non-FNEC group. Among the NEC children, those with clinical symptoms of decreased white blood cells, neutrophils, lymphocytes, thrombocytopenia, and a significantly increased ratio of immature cells to mature cells in white blood cells, accompanied by large-scale and nearly complete intestinal ischemia and necrosis, were classified into the FNEC group, and other NEC children were classified into the Non-FNEC group.

[0038] Control group: Non-NEC children (mainly children with physiological diarrhea, excluding children with inflammatory diseases, digestive tract malformations, and genetic metabolism) with corrected gestational age, gender, and birth weight matched to those in the NEC group during the same period of hospitalization were selected.

[0039] Informed consent forms were signed with the parents of all samples included in the study and ethical review was passed.

[0040] 2. Gene transcriptomics analysis

[0041] The intestinal tissues resected from the included children during the surgical treatment were placed in Trizol reagent and stored in a -80°C refrigerator for SMART-Seq gene transcriptomics analysis of gene expression changes. Hangzhou Lianchuan Biotechnology Co., Ltd. was commissioned for gene transcriptomics analysis.

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

[0043] 3. Proteomics analysis

[0044] Based on the results of gene transcriptomics analysis, we further carried out proteomics.

[0045] Fresh peripheral blood from all included children was collected into EDTA anticoagulant tubes, and the plasma was centrifuged and stored in a -80°C refrigerator for proteomics analysis of plasma protein expression levels. Hangzhou Lianchuan Biotechnology Co., Ltd. was commissioned for proteomics analysis.

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

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

[0048] Example 2

[0049] This example verifies the results of Example 1.

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

[0051] This example included a total of 6 control group children and 15 NEC children; among them, among the NEC children, there were 6 FNEC children and 9 Non-FNEC children.

[0052] Fresh peripheral blood of each group of children was collected again into EDTA anticoagulant tubes, centrifuged to collect plasma and stored in a -80°C refrigerator, and the plasma protein expression level was analyzed by ELISA method (Invitrogen Human PAI1 ELISA Kit BMS2033), and the operation was carried out strictly according to the instructions.

[0053] The analysis results of the control group children and the NEC group children are as Figure 4 shown. As Figure 4 It can be seen that compared with the children in the control group (Ctrl), the content of PAI-1 protein in the peripheral blood of the NEC group was significantly increased. The further ROC curve analysis results showed that the AUC was 0.9333, the sensitivity was 93.33%, and the specificity was 83.33%.

[0054] The analysis results of children in the FNEC group and those in the Non-FNEC group are as follows Figure 5 shown. Compared with the Non-FNEC group, the content of PAI-1 protein in the peripheral blood of children in the FNEC group increased significantly (p = 0.0005). The further ROC curve analysis results showed that when PAI-1 protein was used to distinguish children with FNEC from those with Non-FNEC, the AUC value was 0.9074, the best cut-off corresponded to a sensitivity of 83.33%, and the specificity was 88.89%.

[0055] The above results further verified that PAI-1 protein could be used as a specific diagnostic marker for children with NEC, and could further distinguish children with FNEC from those with Non-FNEC, with high specificity and sensitivity.

[0056] Example 3

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

[0058] The intestinal tissues soaked in 4% paraformaldehyde solution for one day were dehydrated, infiltrated with wax, and embedded to prepare paraffin blocks, and paraffin sections were prepared using a paraffin slicer. Immunofluorescence staining of the paraffin sections was performed using PAI-1 Antibody (Affinity, Human, AF5176). The method was as follows: The collected intestinal tissues were immediately placed in 4% paraformaldehyde fixative and fixed at 4°C for 24 hours to maintain tissue morphology and antigenicity. After fixation, dehydration was performed using gradient alcohol, followed by clearing with xylene and infiltration into paraffin for embedding. After embedding, the tissues were cut into 4-μm sections using a paraffin slicer, spread on adhesive glass slides, and dried for staining. Before immunofluorescence staining, the paraffin was removed with xylene and hydrated with gradient alcohol, and then antigen retrieval was performed using citrate buffer or EDTA to release antigen sites by heating. Subsequently, the tissues were blocked with a blocking solution (5% bovine serum albumin solution) at room temperature for 2 hours to reduce non-specific binding. After blocking, PAI-1 antibody (diluted 1:500) was added dropwise at the tissue position and incubated overnight at 4°C. After washing, a fluorescently labeled secondary antibody was added and incubated at room temperature for 2 hours in the dark. After staining, the tissues were washed three times with PBS solution, DAPI solution was added dropwise and incubated at room temperature for 10 minutes to stain the nuclei, and the sections were sealed with an anti-fluorescence quenching mounting medium. Finally, the results were observed and recorded under a fluorescence microscope.

[0059] As Figure 6It can be seen that compared with the children in the control group (Control), the cyan fluorescence in the intestinal tissues of children with Non-FNEC increased, indicating an increase in the expression of PAI-1 protein in the intestinal tissues of children with Non-FNEC; at the same time, it was also clearly visible that the fluorescence expression level in the intestinal tissues of children in the FNEC group was higher than that of children in the Non-FNEC group, that is, the expression level of PAI-1 protein increased.

[0060] Example 4

[0061] 1. Construction of NEC disease model and experimental grouping

[0062] NEC group: Newborn 7-day-old C57BL / 6 mice weighing about 3.5 - 4.5 g were used and fed with LPS (lipopolysaccharide) at a dose of 30 mg / kg per day to induce intestinal inflammation, once in the morning and once in the evening; at the same time, they were fed with hypertonic milk (45%) after mixing human milk powder and dog milk powder, once in the morning, once at noon, and once in the evening; and they were subjected to hypoxia treatment for 10 minutes under an environment with a concentration of 5% O 2 and cold stimulation for 10 minutes, twice a day, for three consecutive days to construct a mouse NEC disease model.

[0063] NEC-Vehicle group: On the basis of the above construction of the mouse NEC disease model, an additional 100 μL / day of PBS was fed.

[0064] NEC-PAI-1Inhibitor group: On the basis of the above construction of the mouse NEC disease model, an additional 1 mg / kg / day of PAI-1 inhibitor (Tiplaxtinin) was fed.

[0065] Control group: Newborn 7-day-old C57BL / 6 mice weighing about 3.5 - 4.5 g were breastfed normally for three days without any treatment.

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

[0067] 2. Detection indicators

[0068] (1) HE staining

[0069] On the fourth day after modeling, the experimental animals were sacrificed (intestinal tissues were collected), paraffin sections were prepared, and HE staining was performed. The method is as follows: After the intestinal tissues of the mice were collected, they were immediately rinsed thoroughly with PBS to remove the impurities and contents attached to the surface. Subsequently, the tissues were placed in 4% paraformaldehyde fixative and fixed at room temperature for 24 hours to preserve the tissue structure. After fixation, the tissues were dehydrated with gradient ethanol, then cleared in xylene, and embedded in paraffin. After embedding, the tissues were cut into sections with a thickness of 4 μm using a paraffin slicer. The sections were spread on preheated glass slides, dried and fixed, and then subjected to HE staining. During staining, first, the paraffin was removed with xylene, then hydrated successively with gradient ethanol and water, then stained with hematoxylin, and subsequently, the excess stain was thoroughly washed off with running water. Next, the sections were treated in differentiating solution and bluing solution, 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] Pathological scoring was performed according to the HE staining results, and the scoring criteria are shown in Table 1.

[0072] Table 1 Chiu's grading scoring criteria for small intestinal mucosal injury

[0073]

[0074] (3) Survival rate

[0075] The survival rate of the mice was calculated according to the following formula: the number of surviving mice on the fourth day after modeling / the number of surviving mice on day 0 × 100%.

[0076] (4) Protein expression levels of PAI-1, Plasmin, and IL-6 in serum

[0077] On the fourth day after modeling, blood was collected from each group into 1.5 mL anticoagulant tubes. After centrifugation, the serum was aspirated, and the protein expression levels of PAI-1, Plasmin, and IL-6 in the serum were detected by the Elisa method (Ruixin Bio, Mouse, RXW202553M, RXW200350M, RXW203048M). The operation was performed strictly according to the instructions.

[0078] 3. Experimental results

[0079] As Figure 7 shown, the HE staining images of the intestinal pathological sections and the statistical graphs of pathological scores of the Control group, NEC-Vehicle group, and NEC-PAI-1 Inhibitor group showed that LPS successfully induced the occurrence of NEC in the NEC-Vehicle group: severe intestinal villus damage, reduced goblet cells, and damaged intestinal mucosa (Figure 7 In group B), compared with the NEC-Vehicle group, the intestinal villus injury in the NEC-PAI-1 Inhibitor group was significantly alleviated ( Figure 7 in group B), and the pathological score was significantly reduced ( Figure 7 in group C). From the mortality rate of the experimental animals ( Figure 7 in group D), the final survival rate of the NEC-Vehicle group was only 60% (n = 10), while the survival rate of the NEC-PAI-1 Inhibitor group was as high as 90% (n = 10). The above results indicate that inhibiting the expression of PAI-1 can effectively relieve intestinal villus injury in NEC mice, reduce the pathological score, and effectively reduce the mortality rate of the NEC disease model mice.

[0080] As Figure 8 shown in Figure A, the expression level of PAI-1 protein in the NEC-Vehicle group was significantly higher than that in the Control group (p < 0.05), while it was significantly decreased in the NEC-PAI-1 Inhibitor group compared with the NEC-Vehicle group. The results indicate that the expression level of PAI-1 increases in the NEC disease model, and the PAI-1 inhibitor can inhibit the expression of PAI-1. Plasmin, as the most direct downstream signaling molecule of PAI-1, is negatively regulated by PAI-1, and the two are involved in the activation of the fibrinolytic system and the formation of thrombi. The expression levels of PAI-1 and Plasmin proteins were analyzed for their correlation with the NEC disease. As Figure 8 shown in Figure B and Figure 8 Figure E, the correlation analysis results showed that R 2 = 0.56198, and the Pearson coefficient was -0.74965, indicating that PAI-1 and its downstream signaling molecule Plasmin were negatively correlated and had a good correlation with the progression of NEC. IL-6, as a classical inflammatory signaling molecule, has a positive regulatory effect on the progression of most inflammatory diseases. The expression level of IL-6 protein in the NEC-Vehicle group was significantly higher than that in the Control group (p < 0.0001), while the expression level of IL-6 in the NEC-PAI-1 Inhibitor group decreased, as Figure 8 shown in Figure C. The results indicate that the PAI-1 inhibitor can effectively reduce the NEC inflammatory level; considering the association between the two in the progression of NEC, the expression levels of PAI-1 and IL-6 proteins were analyzed for their correlation with the NEC disease. As Figure 8 shown in Figure D, the correlation analysis results showed that R 2= 0.62382, the Pearson coefficient was 0.78982. The results showed that the PAI-1 expression level was positively correlated with the degree of NEC inflammation, and the expression level of PAI-1 could affect the inflammation level of NEC. The results indicated that PAI-1 was involved in the progression of NEC disease and was positively correlated with the inflammation level of NEC.

[0081] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0082] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

Claims

1. Application of PAI-1 protein as a biomarker in detecting patients with neonatal necrotizing enterocolitis.

2. Application of PAI-1 protein as a biomarker in distinguishing patients with fulminant neonatal necrotizing enterocolitis from patients with non-fulminant neonatal necrotizing enterocolitis.

3. Use of a reagent for detecting PAI-1 protein levels in a biological sample in the preparation of a product for detecting patients with neonatal necrotizing enterocolitis.

4. Use of a reagent for detecting the level of PAI-1 protein in a biological sample in the preparation of a product for distinguishing patients with fulminant neonatal necrotizing enterocolitis from patients with non-fulminant neonatal necrotizing enterocolitis.

5. The use according to claim 3 or 4, characterized in that The reagents include reagents for ELISA detection, POCT, and chemiluminescence detection.

6. The use according to claim 5, characterized in that The reagent includes an antibody that specifically binds to the PAI-1 protein; preferably, the antibody is a monoclonal antibody.

7. The use according to any one of claims 3 to 6, characterized in that: The product is a test kit.

8. Use of an agent that inhibits the expression of PAI-1 protein in the preparation of a drug for treating patients with neonatal necrotizing enterocolitis.

9. A drug for treating patients with neonatal necrotizing enterocolitis, characterized in that: The main active ingredient of the drug includes an agent that inhibits the expression of PAI-1 protein.

10. The use according to claim 8 or the medicine according to claim 9, characterized in that: The reagent for inhibiting the expression of PAI-1 protein is Tiplaxtinin.

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