Amniotic fluid-derived polypeptide and its application in preventing and treating bronchopulmonary dysplasia

Abstract

The present invention relates to the field of biomedicine technology, and specifically to an amniotic fluid-derived polypeptide and its application in preventing and treating bronchopulmonary dysplasia, wherein the amino acid sequence of the amniotic fluid-derived polypeptide is shown in SEQ ID NO. 1. The amniotic fluid-derived polypeptide AFABP1 of the present invention has the function of promoting AT2 proliferation and differentiation to AT1. In vivo studies have found that AFABP1 can smoothly enter lung tissue and show potential for the prevention and treatment of BPD. Its mechanism of action may be related to the Wnt / β-catenin signaling pathway, and can be used to prepare drugs, health products or food additives for the prevention and treatment of BPD.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an amniotic fluid-derived polypeptide and application thereof in preventing and treating bronchopulmonary dysplasia. Background Art

[0002] With the development of perinatal medicine and the improvement of neonatal rescue technology, the survival rate of premature infants has increased significantly, and the incidence of bronchopulmonary dysplasia (BPD) has also increased. BPD is the key to the survival and clinical outcome of premature infants, and the younger the gestational age, the higher the incidence. BPD can lead to oxygen dependence, pulmonary hypertension, repeated respiratory tract infections and other diseases in the short term, and can also lead to a significantly increased risk of diseases such as nervous system dysfunction and asthma in the long term, which seriously affects the quality of life of children and brings a heavy burden to families and society. However, in current clinical practice, BPD still lacks specific prevention and treatment methods, and is mainly based on comprehensive treatment such as nutritional support, mechanical ventilation, and hormones to reduce inflammation. Therefore, exploring effective prevention and treatment methods for BPD is of great significance to improving the survival rate of newborns in my country and promoting population health.

[0003] Studies have found that amniotic fluid is an essential component for maintaining the survival and development of the fetus in the womb, and it plays a key role in the development of the fetus in the womb. In recent years, it has been reported that amniotic fluid contains a variety of active ingredients, such as extracellular vesicles, human epidermal growth factor (hEGF) and soluble bioactive factors (SBF), which can promote the development of organs such as the fetal heart, lungs and small intestine, thus inspiring the search for new active ingredients in amniotic fluid that can improve alveolar development. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies in the prior art, the object of the present invention is to provide an amniotic fluid-derived polypeptide and its application in preventing and treating bronchopulmonary dysplasia.

[0005] The objective of the present invention is achieved through the following technical solution: an amniotic fluid-derived polypeptide, the amino acid sequence of the amniotic fluid-derived polypeptide is shown as SEQ ID NO.1.

[0006] Another object of the present invention is achieved through the following technical solution: use of an amniotic fluid-derived polypeptide in the preparation of a drug for preventing and treating bronchopulmonary dysplasia.

[0007] Preferably, the drug further comprises a pharmaceutically acceptable excipient, wherein the excipient is selected from at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an antioxidant, a metal complexing agent, an inert gas, a preservative, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

[0008] Preferably, the dosage form of the drug is any one of injection, tablet, sterile powder for injection, powder, granule, capsule, oral solution, ointment and cream.

[0009] Preferably, the drug is administered by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0010] Another object of the present invention is achieved by the following technical solution: a pharmaceutical composition for preventing and treating bronchopulmonary dysplasia, the pharmaceutical composition comprising the above-mentioned amniotic fluid-derived polypeptide and pharmaceutically acceptable excipients.

[0011] Preferably, the auxiliary material is selected from at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an antioxidant, a metal complexing agent, an inert gas, a preservative, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

[0012] Preferably, the dosage form of the pharmaceutical composition is any one of injection, tablet, sterile powder for injection, powder, granule, capsule, oral solution, ointment and cream.

[0013] Preferably, the pharmaceutical composition is administered by intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

[0014] Another object of the present invention is achieved through the following technical solution: use of an amniotic fluid-derived polypeptide in the preparation of a health product or food additive for preventing and treating bronchopulmonary dysplasia.

[0015] The beneficial effects of the present invention are as follows: the amniotic fluid-derived polypeptide AFABP1 of the present invention has the function of promoting the proliferation of AT2 and differentiation into AT1. In vivo studies have found that AFABP1 can smoothly enter lung tissue and show potential for the prevention and treatment of BPD. Its mechanism of action may be related to the Wnt / β-catenin signaling pathway, and can be used to prepare drugs, health products or food additives for the prevention and treatment of BPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The following are the effects of amniotic fluid peptides at different gestational weeks on lung epithelial cells, where A is the CCK8 experimental result and B is the real-time quantitative PCR result.

[0017] Figure 2 This is an analysis of differential peptides in amniotic fluid at different gestational weeks, where A is the significantly different peptides in amniotic fluid at different gestational weeks analyzed by mass spectrometry; B is the length range of the differential peptides; C is the molecular weight distribution of the differential peptides; and D is the isoelectric point distribution of the differential peptides.

[0018] Figure 3 The peptide AFABP1 improves the proliferation and differentiation ability of lung epithelial cells, where A is the CCK8 experimental result of peptide AFABP1 promoting the proliferation of BEAS-2B cells, B is the WB experimental result of peptide AFABP1 significantly promoting the differentiation of AT1 cells; C and D are the results of the control group SP on the proliferation and differentiation of SPBEAS-2B, respectively.

[0019] Figure 4 This is an electron microscopic image of the peptide AFABP1 entering lung epithelial cells.

[0020] Figure 5 This is an immunofluorescence detection diagram of Cy5-labeled peptide AFABP1 entering lung tissue.

[0021] Figure 6 This is the target analysis of the peptide AFABP1, where A is the mass spectrometry detection diagram and B is the Western blot result diagram.

[0022] Figure 7 Western blot was used to detect the expression of related proteins, where A shows the up-regulation of Wnt3a expression in BEAS-2B cells after AFABP1 intervention, B shows the phosphorylation of β-catenin downstream of the Wnt signaling pathway after AFABP1 intervention, and C shows the nuclear translocation of β-catenin downstream of the Wnt signaling pathway after AFABP1 intervention. DETAILED DESCRIPTION

[0023] In order to facilitate the understanding of those skilled in the art, the following embodiments and attached Figure 1-7 The present invention is further described, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0024] Example 1 Effects of amniotic fluid polypeptides at different gestational weeks on lung epithelial cells

[0025] After recognizing the potential therapeutic prospects of small molecule peptides, our research team has conducted preliminary exploration of amniotic fluid-derived peptides to improve the self-renewal of AT2. By collecting amniotic fluid from premature infants of different gestational ages (less than 25 weeks, 26-32 weeks, 33-36 weeks), small molecule peptides were extracted using the Covaris method plus ultrafiltration. We added the collected peptides to lung epithelial cells BEAS-2B: CCK8 results showed that amniotic fluid small molecule peptides can significantly promote the proliferation of BEAS-2B cells, and the relationship is increasing with gestational age ( Figure 1 A); Real-time quantitative PCR showed that AT1 marker AQP-5 was significantly upregulated ( Figure 1 B), indicating that the differentiation ability of AT1 cells is enhanced. Therefore, we have obtained preliminary experimental evidence that small molecule peptides derived from amniotic fluid promote the proliferation of AT2 and differentiation into AT1.

[0026] Example 2 Screening of differential peptides in amniotic fluid at different gestational weeks

[0027] We further used iTRAQ labeling combined with LC-MS / MS technology to identify the small molecule peptides separated and collected above. As a result, a total of 210 differentially expressed peptides were identified, of which 128 were highly expressed and 82 were lowly expressed (Fold change ≥ 5, p < 0.01) ( Figure 2 A). We analyzed the basic characteristics of all detected peptides and found that: the length of the peptides mainly ranged from 6 to 17 amino acids; the molecular weight (Mw) was mainly concentrated in the range of 0.6 to 1.5 kDa; the isoelectric point (pI) was mainly in the range of 3.0 to 9.0 ( Figure 2 B-2D). Next, we screened and obtained 5 peptides for further verification based on mass spectrometry signal intensity, difference fold and structural stability, as shown in Table 1. For ease of description, according to their potential functions in the prevention and treatment of BPD, we named them AFABP1-5 (Amniotic Fluid Anti-BPD Peptide 1-5), and their amino acid sequences are shown in SEQ ID NO.1-5, respectively.

[0028] Table 1 Basic characteristics of candidate peptides AFABP1-5

[0029] Name Sequence Fold Change Conf index (%) P-value AFABP1 MVTDGRLPMRSVQQPDCR 6.9 99.0 0.0014 AFABP2 GDGGFDSGGVELAER 5.3 99.1 0.0041 AFABP3 PMTELGEFVPGFFSSESRGS -9.3 99.2 0.0091 AFABP4 YSKSSYQFNRGDSTSSTTGR -11.6 98.3 0.0056 AFABP5 FFSYSKQFTSENESRTCDS 7.3 97.5 0.0087

[0030] Example 3 Peptide AFABP1 improves the proliferation and differentiation ability of lung epithelial cells

[0031] Based on the potential of AFABP1-5 to promote AT2 proliferation and differentiation to AT1, we successfully chemically synthesized the above five peptides and added them to lung epithelial cells BEAS-2B: ①CCK8 results showed that peptides AFABP1 and AFABP2 had the effect of significantly promoting BEAS-2B cell proliferation, among which peptide AFABP1 showed concentration-dependent effect ( Figure 3 A); ②WB experimental results showed that the peptide AFABP1 significantly promoted the upregulation of AT1 cell marker AQP-5 ( Figure 3 B). For the sake of rigor, we also synthesized a scrambled peptide (SP) with a disrupted sequence as a control experiment: using the same concentration to intervene in BEAS-2B cells, the results showed that the control group had no significant effect on cell proliferation and differentiation ability ( Figure 3 C-3D). So far, we have successfully identified the peptide AFABP1 as a peptide that has the function of promoting AT2 proliferation and differentiation into AT1.

[0032] Example 4 Electron microscopic observation of the entry of polypeptide AFABP1 into lung epithelial cells

[0033] We used the ProtParam tool to analyze AFABP1 online and found that it is composed of 18 amino acids, with a molecular weight of 1182.31Da, a small molecule polypeptide; isoelectric point 5.23; instability coefficient -11.94, a stable polypeptide; fat coefficient 78.97, hydrophilicity 0.436, a lipophilic polypeptide. This small molecule polypeptide can exert its function by binding to cell surface receptors or directly enter cells to perform its function. We synthesized FITC-labeled AFABP1 and observed under a microscope that AFABP1 can smoothly enter BEAS-2B cells ( Figure 4 ), which is mainly expressed in the cytoplasm, suggesting that AFABP1 may exercise its biological activity in the cytoplasm. In summary, the small molecule peptide AFABP1 derived from amniotic fluid has the characteristics of stable structure, high lipophilicity and easy cell entry, and has good potential for the prevention and treatment of BPD. We have consulted multiple databases (such as CNKI, Wanfang, PubMed, etc.) and have not found any similar functional reports, so the research on AFABP1 is original and innovative.

[0034] Example 5 Immunofluorescence detection of Cy5-labeled peptide AFABP1 entering lung tissue

[0035] Given that the small molecule peptide AFABP1 has the characteristics of promoting AT2 proliferation and differentiation to AT1, can AFABP1 enter the lung tissue? To this end, we conducted animal experiments to answer this question. We administered Cy5-labeled AFABP1 to BALB / c mice through the nasal cavity to evaluate the tissue localization of AFABP1. The mice were killed 3 hours after administration, and the lung tissue was taken for frozen section. Under the microscope, it was found that the mouse lung tissue showed red fluorescence ( Figure 5 ), indicating that AFABP1 can enter the lung tissue smoothly. The above studies found that the small molecule peptide AFABP1 not only has the characteristics of low molecular weight, good stability, high lipophilicity, and easy entry into cells, but also can enter the lung tissue smoothly through nasal administration, which provides the possibility for us to carry out in vivo experimental research in the future.

[0036] Example 6 Study on the Target of Peptide AFABP1

[0037] The mechanism by which the peptide AFABP1 exerts its BPD protective effect has become the focus we need to explore. Studies have shown that peptides usually exert their biological functions after binding to proteins, and can regulate the activity of target proteins positively or negatively. In order to find the direct target of AFABP1, we chemically synthesized the Biotin-labeled peptide AFABP1 (MVTDGRLPMRSVQQPDCR) and the control peptide (Scrambled peptide, SP, PMVTDGRLPMDRSVQQCR); the above peptides were co-incubated and eluted with the BEAS-2B cell lysate treated with hyperoxia, and then mass spectrometry was performed. The results showed that a total of 15 differential proteins were identified between the two groups (Fold change ≥ 5, FDR < 0.05). Among them, the peptide coverage of the Wnt3a protein was the highest, and the difference multiple was the largest ( Figure 6 A). We then performed Western blot experiments and identified the protein Wnt3a in the eluate ( Figure 6 B), based on this, we speculate that Wnt3a is very likely to be the direct target of AFABP1.

[0038] Example 7 Study on the mechanism of action of polypeptide AFABP1

[0039] To further clarify the mechanism of action of the peptide AFABP1, we conducted an in-depth literature review and preliminary experimental studies. The literature review found that: Wnt signaling plays a key role in alveolar damage and repair by regulating the proliferation and differentiation of type II pneumonic epithelial cells (AT2), among which AT2 is the main site of Wnt3a activation; in the mouse lung injury model, Wnt3a expression is downregulated, which inhibits the activation of the Wnt / β-catenin pathway, thereby affecting the differentiation of AT2 to AT1. When Wnt3a is overexpressed, the Wnt / β-catenin pathway is reactivated to promote the proliferation and differentiation of alveolar cells; after Wnt3a is activated, it promotes β-catenin phosphorylation and nuclear translocation, thereby activating the expression of downstream related pathway gene proteins and regulating cell proliferation and differentiation. Experimental detection found that after high oxygen treatment of BEAS-2B cells, Wnt3a expression was downregulated, and after the addition of AFABP1 intervention, Wnt3a expression was upregulated in BEAS-2B cells ( Figure 7 A), suggesting that the role of AFABP1 may be related to the activation of Wnt / β-catenin signaling; we further detected that after AFABP1 intervention, β-catenin phosphorylation and β-catenin nuclear translocation occurred in the downstream of the Wnt signaling pathway ( Figure 7 B), which once again confirmed that the function of AFABP1 may be related to the Wnt / β-catenin signaling pathway. Based on this, we have more evidence to support the speculation that AFABP1 may activate the Wnt / β-catenin signaling pathway by binding to Wnt3a and play a mechanistic role in improving BPD.

[0040] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention may also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. An amniotic fluid-derived polypeptide, characterized in that: The amino acid sequence of the amniotic fluid-derived polypeptide is shown in SEQ ID NO.

1.

2. Use of an amniotic fluid-derived polypeptide as claimed in claim 1 in the preparation of a medicament for preventing and treating bronchopulmonary dysplasia.

3. The use according to claim 2, characterized in that: The drug also includes pharmaceutically acceptable excipients, which are selected from at least one of diluents, wetting agents, adhesives, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH regulators, and isotonic or isotonic regulators.

4. The use according to claim 2, characterized in that: The dosage form of the drug is any one of injection, tablet, sterile powder for injection, powder, granule, capsule, oral solution, ointment and cream.

5. The use according to claim 2, characterized in that: The administration route of the drug is intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

6. A pharmaceutical composition for preventing and treating bronchopulmonary dysplasia, characterized in that: The pharmaceutical composition comprises the amniotic fluid-derived polypeptide according to claim 1 and pharmaceutically acceptable excipients.

7. A pharmaceutical composition for preventing and treating bronchopulmonary dysplasia according to claim 6, characterized in that: The auxiliary material is selected from at least one of diluents, wetting agents, adhesives, disintegrants, lubricants, color and flavor regulators, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH regulators, and isotonic or isotonic regulators.

8. The pharmaceutical composition for preventing and treating bronchopulmonary dysplasia according to claim 6, characterized in that: The dosage form of the pharmaceutical composition is any one of injection, tablet, sterile powder for injection, powder, granule, capsule, oral solution, ointment and cream.

9. The pharmaceutical composition for preventing and treating bronchopulmonary dysplasia according to claim 6, characterized in that: The administration route of the pharmaceutical composition is intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration.

Images