Use of compounds that target and inhibit ADAMTS4 in the amnion in the preparation and screening of drugs for the prevention and treatment of preterm birth

Abstract

This invention provides the use of compounds that target and inhibit ADAMTS4 in the amniotic membrane in the preparation of drugs for screening and preventing preterm birth. This invention also provides the application of reagents for detecting ADAMTS4 in amniotic fluid or blood in the preparation of diagnostic kits for diagnosing preterm birth in pregnant women. This invention provides the use of compounds that target and inhibit the degradation of the multifunctional proteoglycan VCAN in the amniotic extracellular matrix in the preparation of drugs for screening and preventing preterm birth. This invention provides the use of compounds that target and inhibit versikine, the active degradation product of VCAN in the amniotic extracellular matrix, in the preparation of drugs for screening and preventing preterm birth. This invention also provides the application of reagents for detecting versikine in amniotic fluid or blood in the preparation of diagnostic kits for diagnosing preterm birth in pregnant women. This invention discovers that ADAMTS4-mediated degradation of the proteoglycan VCAN can serve as an important target for the prevention and treatment of preterm birth, providing new ideas and methods for future prevention and treatment of preterm birth.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to amniotic membrane ADAMTS4. Specifically, it relates to the use of compounds that target and inhibit amniotic membrane ADAMTS4 in the preparation and screening of drugs for the prevention and treatment of preterm birth. Background Technology

[0002] Preterm birth refers to delivery before 37 weeks of gestation. Preterm birth is the leading cause of death in children under five and in perinatal neonatal mortality worldwide. Although the survival rate of premature infants has improved in recent years due to advancements in medical care and nursing conditions, surviving premature infants still face a range of short-term and long-term health risks. Therefore, preventing and controlling preterm birth has profound clinical and social significance for eugenics. However, effective means of preventing and controlling preterm birth are currently lacking, primarily because the normal mechanisms of human labor initiation are not yet fully understood.

[0003] Clinically, the prediction of preterm birth mainly involves physical methods such as cervical length measurement (transabdominal / vaginal ultrasound) and laboratory molecular assessments (e.g., fetal fibronectin fFN, placental α1-microglobulin PAMG-1, phosphorylated insulin-like growth factor binding protein 1pIGFBP-1, etc.). However, since the normal mechanisms of human labor initiation are not fully understood, all methods for preventing and treating preterm birth have certain limitations.

[0004] Because the causes of preterm birth are difficult to elucidate, current methods for predicting preterm birth primarily rely on cervical length measurement, which requires highly skilled technicians and advanced equipment, making it impractical in many remote areas. Furthermore, laboratory molecular assessment methods have low molecular specificity and sensitivity, limiting their clinical use. Given the limitations of various treatment approaches, there remains an urgent need to explore the molecular mechanisms underlying the initiation of preterm / normal labor in order to identify key therapeutic targets. Summary of the Invention

[0005] In view of the above-mentioned technical problems in the prior art, the present invention provides the use of a compound that targets and inhibits ADAMTS4 of the amnion in the preparation of drugs for screening the prevention and treatment of preterm birth. The use of this compound that targets and inhibits ADAMTS4 of the amnion in the preparation of drugs for screening the prevention and treatment of preterm birth is intended to solve the technical problem of poor technical effect in preventing and treating preterm birth in pregnant women in the prior art.

[0006] This invention provides the use of compounds that target and inhibit ADAMTS4 in the amnion in the preparation of drugs for screening the prevention and treatment of preterm birth.

[0007] This invention also provides the application of reagents for detecting ADAMTS4 in amniotic fluid or blood in the preparation of diagnostic kits for diagnosing preterm birth in pregnant women.

[0008] This invention also provides the use of compounds that target and inhibit the degradation of the multifunctional proteoglycan VCAN in the extracellular matrix of amniotic cells in the preparation of drugs for screening the prevention and treatment of preterm birth.

[0009] This invention also provides the use of compounds that target and inhibit the active degradation product versikine of VCAN in the extracellular matrix of amniotic cells in the preparation of drugs for screening the prevention and treatment of preterm birth.

[0010] This invention also provides the application of reagents for detecting versikine in amniotic fluid or blood in the preparation of diagnostic kits for diagnosing preterm birth in pregnant women.

[0011] It is known that extracellular matrix (ECM) remodeling and inflammation are crucial components of the normal labor initiation mechanism and also contribute to some cases of preterm birth. Based on these two aspects, we identified ADAMTS4 (a disintegrin and metalloprotease domains with thrombospondins motif 4), a key target in the normal labor initiation mechanism. On one hand, pro-inflammatory factors associated with labor initiation (IL-1β, SAA1) can induce ADAMTS4 expression and secretion in amniotic fibroblasts. ADAMTS4 degrades the multifunctional proteoglycan versican (VCAN) in the amniotic ECM, leading to ECM remodeling. On the other hand, the active degradation product versikine from VCAN hydrolysis can further induce an inflammatory response, thus participating in labor initiation. Therefore, ADAMTS4-mediated degradation of the multifunctional proteoglycan VCAN can serve as an important target for the prevention and treatment of preterm birth, providing new ideas and methods for future prevention and treatment of preterm birth.

[0012] ADAMTS4 is a secreted metalloproteinase, a member of the family of depolymerizing protein-like metalloproteinases containing a type I platelet-binding protein motif. Inflammatory mediators such as TNF-α, IL-1β, and IL-6 all increase ADAMTS4 expression levels, but their pathways of action differ. It is expressed in joint tissues, ovaries, heart, lungs, and fetal membranes, and participates in the development of tumors, arthritis, cardiovascular diseases, and preeclampsia. However, the role of ADAMTS4 in the initiation of labor is currently unclear. The amino acid sequence of ADAMTS4 in amniotic fluid or blood can be found on the website: https: / / www.uniprot.org / uniprotkb / O75173 / entry The amino acid sequence of VCAN in the extracellular matrix of amniotic cells can be found on the website: https: / / www.uniprot.org / uniprotkb / P13611 / entry .

[0013] The amino acid sequence of Versikine, a VCAN activity degradation product in the amniotic extracellular matrix, is as follows (441 amino acids from the N-terminus of the human VCAN (PMID 1462) transcript NM_001164097):

[0014]

[0015] Based on transcriptome sequencing data and related studies from term labor (TL) and term non-labor (TNL) amniotic tissues obtained by our research group, we hypothesize that ADAMTS4 may play a role in labor initiation. Compared with term labor cesarean section amniotic tissues, ADAMTS4 and versikine were elevated in term labor TL tissues, while the multifunctional proteoglycan VCAN was significantly decreased. Labor initiation-related pro-inflammatory factors (IL-1β, SAA1) can induce ADAMTS4 expression in amniotic fibroblasts and inhibit its endocytosis, leading to ADAMTS4 accumulation between cells. This ADAMTS4 degrades the multifunctional proteoglycan VCAN in the amniotic ECM, resulting in ECM structural disorder. The active degradation product versikine produced by VCAN degradation can further promote the expression of inflammatory factors, chemokines, and COX-2, thus forming a positive feedback loop involved in labor initiation. In animal experiments, intra-amniotic injection of ADAMTS4-overexpressing lentivirus locally overexpressing ADAMTS4 induced premature birth in mice, leading to a decrease in VCAN and an increase in versikine in the mouse fetal membranes, disordered extracellular matrix structure, and promoted the expression of pro-inflammatory factors, chemokines, and Ptgs2 (e.g., Figure 9 (As shown).

[0016] Compared with existing technologies, the technical effects of this invention are positive and significant. Studies have shown that rupture of the amniotic membrane extracellular matrix (ECM) and aseptic inflammation are both important links in the normal labor initiation mechanism. This invention discovers that labor initiation-related pro-inflammatory factors (IL-1β, SAA1) can induce ADAMTS4 expression in amniotic fibroblasts. ADAMTS4 leads to ECM structural disorder by degrading the multifunctional proteoglycan VCAN in the amniotic extracellular matrix. Therefore, ADAMTS4 can serve as a target for the prevention and treatment of preterm birth, providing a theoretical basis for future exploration of inhibitors targeting ADAMTS4 and offering new insights into the research on the pathogenesis and prevention of preterm birth. Attached Figure Description

[0017] Figure 1 The changes of ADAMTS4 and its endocytic receptor LRP1 in the amniotic membrane tissue of a normal human delivery with spontaneous rupture of membranes were shown.

[0018] Figure 2 The changes of the multifunctional proteoglycan VCAN in the amniotic membrane tissue of a normal human delivery with spontaneous rupture of membranes were shown.

[0019] Figure 3 The study showed that ADAMTS4 degradation of VCAN caused ECM structural disorder.

[0020] Figure 4 The results showed that IL-1β and SAA1 promoted the expression of ADAMTS4 and the shedding of LRP1 in amniotic fibroblasts.

[0021] Figure 5 This demonstrates that LRP1 regulates the endocytosis of ADAMTS4.

[0022] Figure 6 The study showed that versikine, a VCAN degradation product, caused an inflammatory response in the fetal membranes.

[0023] Figure 7 The study showed that the levels of ADAMTS4, VCAN, and versikine in mouse fetal membranes varied with the number of days of gestation.

[0024] Figure 8 This study demonstrated that intraamniotic injection of a lentiviral vector overexpressing ADAMTS4 induced premature birth in mice.

[0025] Figure 9 This study demonstrates the working mechanism of ADAMTS4-mediated VCAN degradation in spontaneous rupture of membranes during term delivery. Detailed Implementation

[0026] Example 1

[0027] The procedure for detecting ADAMTS4 in maternal blood and amniotic fluid using enzyme-linked immunosorbent assay (ELISA) is as follows:

[0028] 1. Collect 5ml of blood from women who have experienced spontaneous rupture of membranes at full term and those who have undergone cesarean section at full term.

[0029] 2. Centrifuge the blood at 3000 rpm at room temperature for 15 minutes, and collect the clear yellow liquid (serum) from the top layer.

[0030] 3. Collect 50ml of amniotic fluid from mothers who have experienced spontaneous rupture of membranes at full term or who have undergone a full-term cesarean section (avoid blood contamination as much as possible).

[0031] 4. Centrifuge the amniotic fluid at 12,000 rpm for 10 minutes, collect the supernatant, and remove dead cells and impurities.

[0032] 5. Add ADAMTS4 capture antibody to a 96-well microplate according to the ELISA kit (ADAMTS4:R&D,DY4307-05) and incubate overnight for coating.

[0033] 6. After washing the 96-well plate three times with detergent, add 100 μl of 1% bovine serum albumin and block at room temperature for 1 hour.

[0034] 7. After washing the 96-well plate three times with detergent, add 100 μl of serum, amniotic fluid and standard to the 96-well plate and incubate at room temperature for 2 hours.

[0035] 8. After washing the 96-well plate three times with detergent, add 100 μl of ADAMTS4 detection antibody to perform antigen-antibody reaction and incubate at room temperature for 2 hours.

[0036] 9. After washing the 96-well plate three times with detergent, add 100 μl of HRP colorimetric solution and incubate at room temperature in the dark for 20 minutes.

[0037] 10. After washing the 96-well plate three times with detergent, add 100 μl of substrate reaction solution and incubate at room temperature for 20 minutes. Finally, add 100 μl of reaction stop solution, place the plate in a microplate reader, and read the absorbance value (OD value). Calculate the specific concentration of the sample based on the standard curve.

[0038] The procedure for detecting versikine in maternal blood and amniotic fluid using enzyme-linked immunosorbent assay (ELISA) is as follows:

[0039] 1. Collect 5ml of blood from women who have experienced spontaneous rupture of membranes at full term and those who have undergone cesarean section at full term.

[0040] 2. Centrifuge the blood at 3000 rpm at room temperature for 15 minutes, and collect the clear yellow liquid (serum) from the top layer.

[0041] 3. Collect 50ml of amniotic fluid from mothers who have experienced spontaneous rupture of membranes at full term or who have undergone a full-term cesarean section (avoid blood contamination as much as possible).

[0042] 4. Centrifuge the amniotic fluid at 12,000 rpm for 10 minutes, collect the supernatant, and remove dead cells and impurities.

[0043] 5. Add 100 μl of serum, amniotic fluid, and standard to the ELISA plate and incubate at room temperature for 2 hours.

[0044] 6. After washing three times with PBS, add 100 μL of 1% bovine serum albumin to a 96-well microplate and block at room temperature for 1 hour. Then discard the liquid.

[0045] 7. After washing the 96-well plate three times with detergent, add 100 μl of versikine detection antibody (Thermo Fisherscientific, #PA1-1748A) to carry out the antigen-antibody reaction and incubate at room temperature for 2 hours.

[0046] 8. After washing the 96-well plate three times with detergent, add 100 μl of HRP colorimetric solution and incubate at room temperature in the dark for 20 minutes.

[0047] 9. After washing the 96-well plate three times with detergent, add 100 μl of substrate reaction solution and incubate at room temperature for 20 minutes.

[0048] 10. Finally, add 100 μl of reaction stop solution, place it in an ELISA reader and read the absorbance value (OD value). Calculate the specific concentration of the sample based on the standard curve.

[0049] Example 2: Changes of ADAMTS4 and its endocytic receptor LRP1 in amniotic tissue from spontaneous rupture of membranes during normal human delivery.

[0050] Previous studies on fetal membrane ECM remodeling have mainly focused on MMP family-mediated extracellular matrix degradation, while the role of the ADAMTS family in labor initiation has been less reported. Analysis of our group's previous amniocentesis transcriptome data (NCBI GEO ID GSE166453) showed that ADAMTS4 was the only ADAMTS family member significantly increased in TL amniocentesis compared to the TNL group. Figure 1 A), and confirmed by qRT-PCR and Western blotting. Figure 1 B and C). Furthermore, transcriptome sequencing, qRT-PCR, and Western blotting analysis showed that, compared to TNL amniotic tissue, the mRNA and protein levels of the endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP1) of ADAMTS4 were significantly reduced in the TL group. Figure 1 (C and D). Our single-cell sequencing data, qRT-PCR, and immunohistochemical staining of human amniotic tissue showed that ADAMTS4 was mainly present in amniotic fibroblasts and rarely present in epithelial cells, while LRP1 was present in both fibroblasts and epithelial cells. Figure 1 Therefore, we chose amniotic fibroblasts as the main cells for our study (EG).

[0051] Example 3: Changes of the multifunctional proteoglycan VCAN in the amniotic membrane tissue of a human woman experiencing spontaneous rupture of membranes during normal childbirth.

[0052] ADAMTS4 degrades using four hyaluronic acid proteoglycans: aggrecan (ACAN), versican (VCAN), neurocan (NCAN), and brevican (BCAN). Analysis of our previously published transcriptome sequencing data (NCBI GEO number GSE166453) showed that VCAN is highly expressed in human amniotic tissue, while ACAN, NCAN, and BCAN are almost undetectable in human amniotic tissue. Figure 2 A). Therefore, the multifunctional proteoglycan VCAN is the focus of our subsequent research. The abundance of VCAN mRNA did not differ between the TNL and TL groups ( Figure 2 B). However, compared with the TNL group, the abundance of VCAN protein in human amniotic tissue of the TL group was significantly reduced, while the abundance of versikine protein was significantly increased. Figure 2 (C and D). These data indicate that VCAN is the major hyaluronic acid proteoglycan in human amniotic tissue. VCAN degradation increases during spontaneous rupture of membranes at term.

[0053] Example 4: ADAMTS4 degradation of VCAN causes ECM structural disorder

[0054] Treatment of cultured human amniotic fibroblasts / human amniotic tissue explants with exogenous human ADAMTS4 (rhADAMTS4, 50 ng / mL, 24 hours) significantly reduced VCAN abundance while increasing versikine content. Figure 3 Furthermore, transmission electron microscopy results showed that collagen fibrils in the amniotic ECM were deformed after rhADAMTS4 treatment. Figure 3 D). However, Masson's trichrome (blue) and Sirius red (red) staining showed that the abundance of total collagen was not affected by ADAMTS4 treatment. Figure 3 E). These data suggest that ADAMTS4 may primarily induce amniotic ECM remodeling by inducing VCAN protein hydrolysis.

[0055] Example 5: IL-1β and SAA1 promote ADAMTS4 expression and LRP1 shedding in amniotic fibroblasts.

[0056] We investigated whether inflammation can modulate ADAMTS4 and its receptor in the human amnion. We found that pro-inflammatory factors IL-1β and SAA1, which are recognized as being involved in the inflammatory response during labor, can induce ADAMTS4 expression in human amniotic fibroblasts. Figure 4 A and B), but failed to affect LRP1 generation ( Figure 4C). Conversely, IL-1β and SAA1 can induce LRP1 shedding from amniotic fibroblasts: increased LRP1 abundance in cell culture medium corresponds to decreased intracellular LRP1 abundance. Figure 4 D).

[0057] Example 6: LRP1 regulates the endocytosis of ADAMTS4

[0058] Knockdown of LRP1 in amniotic fibroblasts led to an increase in ADAMTS4 abundance in the culture medium and a decrease in VCAN content. Figure 5 A), and immunofluorescence showed reduced colocalization of ADAMTS4 and the early endosome marker EEA1 in amniotic fibroblasts (A). Figure 5 B). Since IL-1β and SAA1 can induce ADAMTS4 expression and inhibit ADAMTS4 endocytosis by enhancing LRP1 shedding (Figure C), intercellular accumulation of ADAMTS4 promotes VCAN degradation. Figure 5 (D and E).

[0059] Example 7: The degradation product versikine caused an inflammatory reaction in the fetal membranes.

[0060] We found that versikine was overexpressed in amniotic fibroblasts. Figure 6 A) induced the expression of multiple pro-inflammatory factors, including COX-2 ( Figure 6 B) Pro-inflammatory cytokines (IL1B, IL6, TNF) and chemokines (CCL2, CCL3, CCL20) Figure 6 C) This suggests that versikine released after ADAMTS cleaves the VCAN may also act as a novel injury-associated pattern molecule to induce an inflammatory response in the fetal membranes.

[0061] Example 8: Changes in ADAMTS4, VCAN, and versikine levels in mouse fetal membranes as a function of gestational age

[0062] The role of ADAMTS5-mediated VCAN degradation in fetal membrane ECM remodeling and labor initiation was further investigated in a mouse model. The mouse fetal membrane consists of the amnion and yolk sac membrane. Immunohistochemical staining of the mouse fetal membrane showed that ADAMTS4 was present in the yolk sac and amnion layer. Figure 7 A). Furthermore, Western blotting / immunofluorescence showed that with increasing gestational age, ADAMTS4 and versikine levels increased, while VCAN levels decreased in mouse fetal membranes. Figure 7 (B and C).

[0063] Example 9: Intraamnional injection of a lentiviral vector overexpressing ADAMTS4 induced premature birth in mice.

[0064] In mice at day 16 of gestation, intraamniotic injection of a lentivirus overexpressing Adamts4 (1×10⁶ PFU / 10 μl / each gestation sac) was performed under anesthesia. The preterm birth rate in mice was 77.8%. Figure 8 AC). Compared with the control group, VCAN was decreased and versikine was increased in the fetal membranes of mice overexpressing Adamts4. Figure 8 D), disordered ECM structure ( Figure 8 F), and induces the expression of Ptgs2 (encoding COX-2), I11b, I16, Cc12, Cc13 and Ccl20. Figure 8 E).

[0065] Animal experiments have shown that ADAMTS4-mediated degradation of the multifunctional proteoglycan VCAN serves as a novel target for the prevention and treatment of preterm birth, providing new ideas and methods for future prevention and treatment of preterm birth.

Claims

1. The use of compounds that target and inhibit the degradation of VCAN (multifunctional proteoglycan) in the extracellular matrix of amniotic cells in screening drugs for the prevention and treatment of preterm birth.

2. The use of compounds that target and inhibit the activity of versikine, a degradation product of VCAN in the extracellular matrix of amniotic cells, in screening drugs for the prevention and treatment of preterm birth.

Images