Polypeptide aiuap for preventing, assisting in preventing or treating formation of intrauterine adhesions and application thereof

Abstract

The application discloses a polypeptide AIUAP for preventing or assisting in preventing formation of intrauterine adhesion and application thereof. The polypeptide AIUAP for preventing, assisting in preventing or treating formation of intrauterine adhesion has an amino acid sequence of TFGGAPGFPLGSPLSSPVFPR (SEQ ID NO. 1), and is composed of 21 amino acids. The application discloses application of the polypeptide AIUAP in preparation of a medicine for preventing, assisting in preventing or treating intrauterine adhesion. In the research, a method of tandem mass spectrometry is used to screen endogenous polypeptides differentially expressed in intrauterine adhesion tissues and normal proliferative endometrial tissues, and it is found that the polypeptide AIUAP highly expressed in the intrauterine adhesion tissues can inhibit formation of fibrosis. Since the AIUAP has the characteristics of small molecular weight, self-source and small toxicity, the AIUAP can become an important auxiliary medicine for preventing and treating intrauterine adhesion.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a polypeptide AIUAP for the prevention, adjuvant prevention or treatment of intrauterine adhesions and its application. Background Technology

[0002] Intrauterine adhesions (IUA) are caused by damage to the endometrium due to pregnancy or non-pregnancy factors. Normal endometrial tissue is replaced by fibrotic tissue, resulting in reduced menstrual flow or amenorrhea, periodic lower abdominal pain, infertility, embryonic arrest, or habitual abortion [1]. Many patients have permanently lost their fertility as a result, which seriously damages women's fertility and quality of life. IUA is currently a difficult gynecological disease to treat clinically. How to reduce the occurrence of IUA and improve the physiological and fertility of IUA patients is an important clinical issue.

[0003] The main clinical pathological feature of IUA is endometrial fibrosis. Clinically, the treatment of IUA includes hysteroscopic cold knife or electrocautery to cut or remove adhesions and restore the volume of the uterine cavity; oral estrogen after surgery, and local use of physical barriers and anti-adhesion drugs in the uterine cavity to prevent the occurrence of re-adhesion and promote endometrial regeneration[2]. However, for patients with severe IUA, the prognosis of existing clinical treatments is not good. Due to the lack of normal endometrium, poor intrauterine environment, and easy recurrence of adhesions, it is difficult for patients to conceive naturally or receive embryo transfer. Many studies have hoped to achieve endometrial repair and regeneration through bone marrow mesenchymal, endometrial or embryonic stem cell transplantation. There are also reports of successful pregnancy after stem cell therapy, but problems such as low stem cell survival rate, difficulty in directional induction differentiation and tumorigenicity still need to be overcome. There are still many limitations in large-scale clinical application[3-5]. Inhibiting the formation and recurrence of IUA is a prerequisite for promoting endometrial regeneration[6]. Therefore, it is of great significance to prevent, reduce or even reverse endometrial fibrosis from the source.

[0004] The specific pathogenesis of IUA is still unclear. The main etiological mechanism of IUA is the active fibroblast proliferation theory, that is, any damage to the basal layer of the endometrium can lead to the production of a large number of various pro-fibrotic factors, which stimulate fibroblast proliferation and excessive deposition of a large amount of ECM (Extracellular matrix), which in turn leads to the proliferation of fibrous connective tissue and scar formation [7]. Research on the mechanism of IUA has covered multiple aspects such as cytokines [8-11], extracellular matrix-related factors [12-13] and non-coding RNA [14-15], and has made a lot of research progress, but there is no effective clinical application at present.

[0005] With the continuous development of peptidomics, peptide drugs have become a new hot spot in pharmaceutical research and development. Small molecule peptides have been reported in fibrosis research. Decorin, a component of the ECM matrix, can form different peptides under different pathological conditions. Among them, the peptide in the C-terminal 335-359 region can bind to CTGF and inhibit the formation of fibrosis, while the N-terminal 42-71 peptide can inhibit the activity of Smad2 / 3 in a dose-dependent manner by inhibiting myostatin

[16] . The anti-fibrotic peptide AcSDKP naturally present in plasma can inhibit the Smad signaling pathway in glomerular mesangial cells

[17] . The new host defense peptide (HDP) fragment RP-832c targeting the CD206 receptor can significantly reduce the effect of pulmonary fibrosis and significantly reduce the expression of inflammatory cytokines and fibrosis markers TGF-β1 and MMP-13

[18] . These results indicate that functional peptide molecules do exist in the body and participate in the regulation of fibrosis formation. Peptide drugs have unique advantages that other drugs cannot match due to their low toxicity, high targeting, and the ability to control their half-life through chemical modification. However, no reports have been found in IUA-related studies. Therefore, we believe that exploring the pathogenesis of IUA from a peptide perspective and investigating bioactive peptides with therapeutic potential is scientifically sound and feasible.

[0006] References

[0007] [1]March CM.Asherman's syndrome.Semin Reprod Med.2011;29(2):83-94.

[0008] [2] Gargett CE, Healy DL. Generating receptive endometrium in Asherman's syndrome. J Hum Reprod Sci. 2011; 4(1): 49-52.

[0009] [3] Bosteels J, Weyers S, Kasius J, Broekmans FJ, Mol BWJ, D'HoogheTM. Anti-adhesion therapy following operative hysteroscopy for treatment of offemale subfertility. Cochrane database of systematic reviews (Online), 2015; 11(11): CD011110.

[0010] [4]Jing Z,Qiong Z,Yonggang W,Yanping L.Rat bone marrow mesenchymalstem cells improve regeneration of thin endometrium in rat.FertilSteril.2014;101(2):587-594.

[0011] [5]Nagori CB,Panchal SY,Patel H.Endometrial regeneration usingautologous adult stem cells followed by conception by in vitro fertilizationin a patient of severe Asherman’s syndrome.J Hum Reprod Sci.2011;4(1):43-48.

[0012] [6]Ye L,Mayberry R,Lo CY,Britt KL,Stanley EG,Elefanty AG,GargettCE.Generation of human female reproductive tract epithelium from humanembryonic stem cells.PLoS One.2011;6(6):e21136.

[0013] [7]March CM.Management of Asherman’s syndrome.ReproductiveBiomedicine Online,2011;23(1):63-76.

[0014] [8]Li J,Cen B,Chen S,He Y.MicroRNA-29b inhibits TGF-β1-inducedfibrosis via regulation of the TGF-β1 / Smad pathway in primary humanendometrial stromal cells.Mol Med Rep.2016;13(5):4229-4237.

[0015] [9]Hu J,Zeng B,Jiang X,Hu L,Meng Y,Zhu Y,Mao M.The expression ofmarker for endometrial stem cell and fibrosis was increased in intrauterineadhesious.Int J Clin Exp Pathol.2015;8(2):1525-1534.

[0016]

[10] Zhu Y,Hu J,Yu T,Ren Y,Hu L.High Molecular Weight Hyaluronic AcidInhibits Fibrosis of Endometrium.Med Sci Monit.2016;22:3438-3445.

[0017]

[11] Wang X,Ma N,Sun Q,Huang C,Liu Y,Luo X.Elevated NF-κB signaling inAsherman syndrome patients and animal models.Oncotarget.2017;8(9):15399-15406.

[0018]

[12] Hu S,Li Y,Meng WJ,Tan SQ.Effects of Fukang oral liquid on theprevention of intrauterine adhesion and expressions of TGF-beta1,PAI-1andMMP-9in endometrium of rats.Sichuan Da Xue Xue Bao Yi Xue Ban.2013;44(4):540-544.

[0019]

[13] Ries C.Cytokine functions of TIMP-1.Cell Mol Life Sci.2014;71(4):659-672.

[0020]

[14] Liu X,Duan H,Zhang HH,Gan L,Xu Q.Integrated Data Set of microRNAsand mRNAs Involved in Severe Intrauterine Adhesion.Reprod Sci.2016;23(10):1340-1347.

[0021]

[15] Ning J,Zhang H,Yang H.MicroRNA-326inhibits endometrial fibrosisby regulating TGF-β1 / Smad3 pathway in intrauterine adhesions.Mol MedRep.2018;18(2):2286-2292.

[0022]

[16] Vial C,Gutiérrez J,Santander C,Cabrera D,Brandan E.Decorininteracts with connective tissue growth factor(CTGF) / CCN2 by LRR12 inhibitingits biological activity.J Biol Chem.2011;286(27):24242-24252.

[0023]

[17] Kanasaki K,Koya D,Sugimoto T,Isono M,Kashiwagi A,Haneda M.N-Acetyl-seryl-aspartyl-lysyl-proline inhibits TGF-beta-mediated plasminogenactivator inhibitor-1expression via inhibition of Smad pathway in humanmesangial cells.J Am Soc Nephrol.2003;14(4):863-872.

[0024]

[18] Ghebremedhin A, Salam AB, Adu-Addai B, Noonan S, Stratton R, AhmedMSU, Khantwal C, Martin GR, Lin H, Andrews C, Karanam B, Rudloff U, Lopez H, JaynesJ, Yates CA Novel CD206 Targeting Peptide Inhibits Bleomycin InducedPulmonary Fibrosis in Mice.bioRxiv[Preprint].2020;2020.07.27.218115. Summary of the Invention

[0025] The purpose of this invention is to address the shortcomings of existing anti-adhesion measures in the clinical diagnosis and treatment of intrauterine adhesions by providing a polypeptide AIUAP (anti-intrauterine adhesion peptide) for the prevention or adjuvant prevention of intrauterine adhesion formation.

[0026] Another object of the present invention is to provide the application of the polypeptide AIUAP.

[0027] The present invention discloses a polypeptide AIUAP for preventing, assisting in the prevention or treatment of intrauterine adhesions, the amino acid sequence of which is: TFGGAPGFPLGSPLSSPVFPR (SEQ ID NO.1), and the polypeptide consists of 21 amino acids.

[0028] The application of the polypeptide AIUAP described in this invention in the preparation of drugs for the prevention, adjunctive prevention, or treatment of intrauterine adhesions.

[0029] A pharmaceutical composition for the prevention, adjunctive prevention or treatment of intrauterine adhesions, characterized in that it contains the polypeptide AIUAP described in this invention.

[0030] The polypeptide AIUAP described in this invention can serve as a potential target for the development of other drugs for the treatment of fibrosis-related diseases.

[0031] Beneficial effects

[0032] This study used tandem mass spectrometry to screen differentially expressed endogenous peptides in intrauterine adhesion tissue and normal proliferative endometrial tissue, and found that the peptide AIUAP, which is highly expressed in intrauterine adhesion tissue, can inhibit fibrosis formation. Due to its small molecular weight, natural origin, and low toxicity, AIUAP may become an important adjuvant drug for the prevention and treatment of intrauterine adhesions. Attached Figure Description

[0033] Figure 1 Overall situation of the two peptide groups

[0034] A: Number of detected peptides; B: Number of unique peptides detected; C: Bar chart of peptide length distribution; D: Bar chart of peptide molecular weight distribution; E: Bar chart of peptide isoelectric point distribution.

[0035] Figure 2 Volcano plot of differentially expressed peptides in IUA and normal endometrial tissue

[0036] Red dots indicate peptides that are significantly upregulated in IUA tissues; green dots indicate peptides that are significantly downregulated in IUA tissues.

[0037] Figure 3 Site analysis of differentially expressed peptides

[0038] A: Enzyme cleavage sites at the C-terminus and N-terminus of upregulated and downregulated peptides. Each series, from left to right, represents the C-terminal amino acid of the upregulated peptide, the N-terminal amino acid of the upregulated peptide, the C-terminal amino acid of the downregulated peptide, and the N-terminal amino acid of the downregulated peptide, respectively. B: The top ten precursor proteins containing the most peptides.

[0039] Figure 4 Gene and protein expression of fibrosis-related markers in endometrial stromal cells treated with different peptides. A: Relative gene expression levels of α-SMA, COL1A1, VIM, CTGF, N-cadherin, and FN in endometrial stromal cells of the control group and each peptide treatment group. B: Relative protein expression levels of α-SMA and COL1A1 in endometrial stromal cells of the control group and the T1–T6 treatment group. C: Immunohistochemical staining of COL1A1 in endometrial stromal cells of the control group and the T1–T6 treatment group.

[0040] Figure 5 Effects of different concentrations of AIUAP on the expression of fibrosis genes and proteins in endometrial stromal cells. A: Relative gene expression levels of α-SMA, COL1A1, VIM, CTGF, N-cadherin, and FN in endometrial stromal cells of the control group and groups treated with different concentrations of AIUAP. B: Relative protein expression of α-SMA and COL1A1 in endometrial stromal cells of the control group and groups treated with different concentrations of AIUAP. C: Immunofluorescence staining of α-SMA in endometrial stromal cells of the control group and groups treated with different concentrations of AIUAP. Detailed Implementation Plan

[0041] Example 1: Screening of IUA-differential endogenous peptides

[0042] 1.1 Sample Collection: Three patients hospitalized at the Obstetrics and Gynecology Hospital of Nanjing Medical University were selected. Three cases each of severe intrauterine uterine bleeding (IUA) tissue and proliferative endometrial tissue from patients undergoing total hysterectomy due to cervical lesions were collected. Both IUA and normal proliferative full-thickness endometrial tissues were collected using a cold knife in the operating room. Immediately after collection, the tissues were placed in an ice box, trimmed to approximately 5mm*5mm*5mm in size, and then immediately transferred to liquid nitrogen at -80℃ for preservation. This study was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital of Nanjing Medical University, and written informed consent was obtained from the patients.

[0043] 1.2 Peptide Extraction: The sample tissue was placed in a mortar and ground into powder under liquid nitrogen conditions. The collected powder was placed in a tube, and 50% protein lysis buffer was added. After mixing, the mixture was incubated on ice for 5 min. DTT was added to a final concentration of 10 mM, and the mixture was sonicated on ice for 15 min. The mixture was centrifuged at 4°C and 13,000 rpm for 30 min, and the supernatant was transferred to a new centrifuge tube. DTT was added to the centrifuge tube to a final concentration of 10 mM, and the mixture was subjected to a reduction reaction at 56°C for 30 min. IAM was added to a final concentration of 55 mM, and the mixture was placed at room temperature in the dark for an alkylation reaction for 30 min. The mixture was then processed using a 10 kD ultrafiltration tube, centrifuged at 4°C and 10,000 rpm for 30 min, and the collected permeate was the peptide sample.

[0044] 1.3 Mass Spectrometry Analysis: Mass spectrometry data acquisition was performed using a TripleTOF 5600+ LC-MS system (SCIEX). Peptide samples were dissolved in 2% acetonitrile / 0.1% formic acid, followed by systematic analysis using a TripleTOF 5600plus mass spectrometer coupled to an Eksigent nanoLC system (SCIEX, USA). The peptide solution was added to a C18 capture column, and gradient elution was performed on the C18 analytical column at a flow rate of 300 nL / min with a time gradient of 90 min. The two mobile phases were buffer A (2% acetonitrile / 0.1% formic acid / 98% H2O) and buffer B (98% acetonitrile / 0.1% formic acid / 2% H2O). IDA (Information-Dependent Acquisition) was used, scanning the primary mass spectrum with an ion accumulation time of 250 ms, and simultaneously acquiring the secondary mass spectra of 30 precursor ions with an ion accumulation time of 50 ms. MS1 spectra were acquired in the range of 350-1500 m / z, and MS2 spectra were acquired in the range of 100-1500 m / z. The precursor ion dynamic exclusion time was set to 15 s. The mass spectrometry data were matched with theoretical mass spectrometry data simulated in the database to obtain protein identification results. A total of 5416 peptides of 786 precursor proteins were identified in both IUA and normal endometrial tissue groups. The peptide lengths were mainly distributed between 10-30 AA, with an average length of 19.25 AA; the molecular weights were mainly between 1000-4000 Da; and the isoelectric points of the peptides were 3-14, mainly distributed between 4-5. A total of 321 peptides of 155 precursor proteins were differentially expressed. In IUA tissue, 70 peptides of 56 precursor proteins were upregulated, and 251 peptides of 120 precursor proteins were downregulated. Figure 1-3 (and Table 1).

[0045] Table 1

[0046]

[0047] Example 2: Functional validation of differentially expressed endogenous peptides

[0048] 2.1 Peptide Synthesis: All screened peptides were synthesized by Jiangsu GenScript Biotech Co., Ltd., with a purity ≥95%. All peptides were standard transacetate peptides, unmodified. The peptides were dissolved in sterile water at 20 mM and diluted to 10 μM, 20 μM, 50 μM, and 100 μM as needed for the experiment.

[0049] 2.2 Cell line: hESC (Human endometrial stromal cells) were provided by Shanghai Xinyu Biotechnology Co., Ltd. Cell culture conditions were as follows: Culture medium: DMEM incomplete high-glucose medium (1×) + 10% fetal bovine serum (FBS) + 1% penicillin-dextrose antibody; Digestion solution: 0.25% Trypsin-EDTA (1×); Cryopreservation solution: FBS: dimethyl sulfoxide (DMSO) = 9:1.

[0050] 2.3 Differential endogenous peptide analysis

[0051] We further screened differentially expressed endogenous peptides (PIs) by using bioinformatics analysis, identifying six PIs most closely associated with fibrosis. Detailed peptide information is shown in Table 2. Peptide 1 (T1), peptide 2 (T2), and peptide 3 (T3) were specifically downregulated in IUA tissues; peptide 4 (T4) was downregulated in IUA tissues; while peptide 5 (T5) and peptide 6 (T6) were upregulated in IUA tissues.

[0052] Table 2

[0053]

[0054] 2.4 Functional Validation of Endogenous Peptides: We chemically synthesized six selected peptides and co-cultured them (final concentration 50 μM) with TGF-β1 (final concentration 5 ng / ml) and hESC for 48 h. The effects of endogenous peptides on endometrial stromal cell fibrosis were evaluated at both the gene and protein levels using qRT-PCR, immunohistochemical staining, and Western blot. Results showed that compared with the control group, T6-treated endometrial stromal cells exhibited significant downregulation of fibrosis-related markers α-SMA, COL1A1, VIM, N-cadherin, and FN at the gene level (with statistically significant differences), while CTGF expression decreased (with no statistically significant difference). The protein expression of α-SMA and COL1A1 was also significantly reduced, with the most significant downregulation of fibrosis markers among the six peptides (see [link to relevant documentation]). Figure 4 No functional reports have been found for the DESM-derived endogenous peptide T6. For the convenience of subsequent research, we have named this DESM-derived endogenous peptide AIUAP.

[0055] Example 3: Inhibitory effect of different concentrations of AIUAP on endometrial stromal cell fibrosis

[0056] Different concentrations (final concentrations of 20 μM, 50 μM, and 100 μM) of AIUAP and its disordered peptide (peptide amino acid sequence: PGSGRFPGLSTLPFVAGPSPF, final concentration 100 μM) were co-cultured with TGF-β1 (final concentration 5 ng / ml) and hESC for 72 h. The effects of different concentrations of endogenous peptide AIUAP on fibrosis-related indicators of endometrial stromal cells were evaluated at the RNA level using qRT-PCR. The results showed that at a peptide concentration of 50 μM, the relative expression levels of fibrosis-related indicators α-SMA, COL1A1, VIM, CTGF, N-cadherin, and FN were significantly decreased compared to the control group, with statistically significant differences. However, at peptide concentrations of 20 μM or 100 μM, the relative expression levels of α-SMA, VIM, CTGF, N-cadherin, and FN did not significantly decrease compared to the control group, and the differences were not statistically significant. Different concentrations (final concentrations of 10 μM, 20 μM, 50 μM, and 100 μM) of AIUAP and its disordered peptide (PGSGRFPGLSTLPFVAGPSPF, final concentration 100 μM) were co-cultured with TGF-β1 (final concentration 5 ng / ml) and hESC for 72 h. Western blot and immunofluorescence staining were used to evaluate the effects of different concentrations of endogenous peptide AIUAP on endometrial stromal cell fibrosis-related indicators at the protein level. Results showed that compared with the control group, the protein expression of α-SMA was significantly decreased in the 20 μM, 50 μM, and 100 μM AIUAP treatment groups, with the most significant decrease observed in the 100 μM AIUAP group. COL1A1 protein expression was significantly decreased in the 50 μM AIUAP treatment group, with no significant difference in COL1A1 protein expression among the 10 μM, 20 μM, and 100 μM AIUAP groups. Figure 5 ). sequence list <110> Nanjing Maternal and Child Health Hospital <120> AIUAP, a polypeptide for the prevention, adjuvant prevention, or treatment of intrauterine adhesions, and its application. <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty one <212> PRT <213> Artificial Sequence <400> 1 Thr Phe Gly Gly Ala Pro Gly Phe Pro Leu Gly Ser Pro Leu Ser Ser 1 5 10 15 Pro Val Phe Pro Arg 20

Claims

1. The use of the polypeptide AIUAP shown in SEQ ID NO.1 in the preparation of drugs for the prevention, adjunctive prevention or treatment of intrauterine adhesions.

Images