Exosome-loaded polypeptide and application thereof in treatment of polycystic ovarian syndrome

Abstract

The invention discloses an exosome-loaded polypeptide and application thereof in treating polycystic ovarian syndrome. The exosome loaded polypeptide is a mesenchymal stem cell exosome loaded with 740Y-P polypeptide obtained by adding a section of anchor peptide to the tail end of a PI3K signaling pathway agonist 740Y-P polypeptide and connecting the polypeptide with a mesenchymal stem cell exosome surface mask protein. The exosome-loaded polypeptide disclosed by the invention can be used for preparing medicines for treating metabolic dysfunction and / or ovulation disorder caused by polycystic ovarian syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of exosomes, and particularly relates to an exosome-loaded polypeptide and its application in treating polycystic ovary syndrome. Background Art

[0002] Polycystic ovary syndrome (PCOS) is one of the most common endocrine and metabolic diseases among young women today. PCOS is characterized by ovulation disorders, hyperandrogenism manifestations, polycystic ovary (PCO) changes in the ovaries, insulin resistance (IR), and hyperinsulinemia. The clinical manifestations mainly include: increased body hair, irregular menstrual cycles, and even early amenorrhea. In addition to the imbalance of related reproductive hormone levels after getting the disease, the risks of miscarriage and premature birth, as well as the incidence of type 2 diabetes, also increase significantly. PCOS patients have ovulation disorders, which also makes it one of the most common reproductive and endocrine diseases among women of childbearing age. However, so far, the cause and pathogenesis of polycystic ovary syndrome are still not fully understood. At present, there is no very effective treatment method for PCOS clinically, and most treatment methods mainly focus on controlling diet and adjusting work and rest.

[0003] Exosomes (Exos) are extracellular vesicles produced by paracrine cells, with a diameter of 30 - 150 nm. They were once considered as waste produced by cells. However, with the gradual deepening of the understanding of exosomes, people have realized that the lipid outer membrane of exosomes can protect the substances inside them, enabling them to promote cell-to-cell communication. Among these substances, exosome microRNAs (miRNAs) are considered to play important roles in processes such as tumor immunity, inflammatory response, oxidative damage, apoptosis, and cancer metastasis. Exosomes derived from MSCs (MSC-Exos) have similar biological functions to MSCs and play important roles in MSC-mediated tissue repair and regeneration. Moreover, exosomes do not have tumorigenicity and immunogenicity, avoiding the risks of MSC transplantation.

[0004] Exosomes derived from human umbilical cord mesenchymal stem cells have a unique stimulating effect on primordial follicles. Previous studies have shown that HucMSCs-Exos can promote follicular activation and development in neonatal mice and improve the fertility of old female mice through the stimulating effect on primordial follicles via the phosphatidylinositol 3-kinase (PI3K) / mTOR signaling pathway. Although there have been theoretical studies on stem cell therapy for PCOS in the past two years, it is currently not clear whether exosomes derived from human umbilical cord mesenchymal stem cells (HucMSCs-Exos) can rescue the polycystic phenotype and metabolic dysfunction of the ovaries in PCOS patients.

[0005] As a phosphatidylinositol-3 kinase (PI3K) activator, 740Y-P can activate the PI3K-Akt (protein kinase B)-Foxo3a (forkhead box O3) signaling pathway in dormant oocytes. Its important role in primordial follicle activation has been extensively studied through a series of transgenic mouse models and has been clinically applied to the in vitro activation (IVA) technique of primordial follicles, which has the potential to activate residual primordial follicles in patients with diminished ovarian reserve (DOR), such as patients with premature ovarian insufficiency (POI). However, there has never been a report on the use of 740Y-P to rescue the polycystic phenotype and metabolic dysfunction of the ovaries in patients with polycystic ovary syndrome.

[0006] In summary, we decided to combine the PI3K activator 740Y-P and HucMSC-Exos and apply them to PCOS mice to observe whether more effective improvement effects can be achieved. Summary of the Invention

[0007] The purpose of the present invention is to provide a polypeptide-loaded exosome with high potential and harmless administration scheme for women of childbearing age with polycystic ovary syndrome. By the combined application of umbilical cord mesenchymal stem cell exosomes and the PI3K signaling pathway agonist 740Y-P, the exosomes are loaded with 740Y-P polypeptide to treat polycystic ovary syndrome.

[0008] The technical solution of the present invention is as follows:

[0009] A polypeptide-loaded exosome, a section of anchor peptide is added to the end of the PI3K signaling pathway agonist 740Y-P polypeptide, and it is combined with the surface membrane protein of mesenchymal stem cell exosomes to obtain mesenchymal stem cell exosomes loaded with 740Y-P polypeptide.

[0010] As a preference of the present invention, the mesenchymal stem cell exosomes are derived from human umbilical cord.

[0011] The 740Y-P polypeptide sequence is SDGG(p-Y)MDM, the anchor peptide sequence is CRHSQMTVTSRL, the S end of 740Y-P is connected to the L end of the anchor peptide, and the sequence is CRHSQMTVTSRLSDGG(p-Y)MDM. What binds to the surface membrane protein CD63 of the exosome is the entire anchor peptide itself. For easy observation in mice, a rhodamine dye is added to the C end of the anchor peptide, and the final sequence is Rhodamine B-CRHSQMTVTSRLSDGG(p-Y)MDM.

[0012] As a preference of the present invention, the exosomes loaded with polypeptides are prepared by the following method: Obtain human umbilical cord-derived mesenchymal stem cell exosomes by size exclusion chromatography, add an anchor peptide to the S end of the PI3K signaling pathway agonist 740Y-P so that it can bind to the exosome surface membrane protein CD63, and enable the exosomes to successfully load the 740Y-P polypeptide.

[0013] The pharmaceutical use of the exosomes loaded with polypeptides according to the present invention.

[0014] The application of the exosomes loaded with polypeptides according to the present invention in the preparation of a drug for treating polycystic ovary syndrome.

[0015] As a preference of the present invention, the application of the exosomes loaded with polypeptides in the preparation of a drug for treating metabolic dysfunction and / or ovulation disorder caused by polycystic ovary syndrome.

[0016] As a preference of the present invention, intravenous injection of the exosomes loaded with polypeptides according to the present invention can treat the metabolic dysfunction and ovulation disorder of polycystic ovary syndrome.

[0017] The beneficial effects of the present invention are as follows: Intravenous injection of HucMSC-Exos loaded with 740Y-P can restore the estrous cycle disorder, glucose metabolism disorder, and the decline of thermogenic capacity under cold conditions in mice caused by polycystic ovary syndrome. It can also activate primordial follicles, reduce the apoptosis level in the ovary, and reduce the inflammation level in the ovarian and uterine tissues of PCOS mice to improve the internal environment of the ovary and uterus and the structure and function of the ovaries in PCOS mice. In addition, the treatment with HucMSC-Exos loaded with 740Y-P can also improve the mitochondrial membrane potential of eggs and reduce the ROS level, thereby improving the reproductive ability of PCOS mice. It provides a treatment idea with high potential and harmless administration scheme for women of childbearing age with polycystic ovary syndrome, and regulates the hormone levels of women with polycystic ovary syndrome through exosomes derived from human umbilical cord mesenchymal stem cells to restore metabolism, improve the ovarian microenvironment and restore ovulation. Description of the Drawings

[0018] Figure 1 Characterization and identification of human umbilical cord mesenchymal stem cell exosomes and ovarian internalization experiment. A: Morphological characteristics of HucMSC-Exos under electron microscopy. Scale bar = 200 nm. B: Expression of exosome surface proteins CD9, Alix, and Tsg101. C: NTA results of exosomes. D: Uptake of labeled exosomes and polypeptides by mouse ovaries. Scale bar = 50 nm.

[0019] Figure 2Improvement of estrous cycle in PCOS mice by HucMSC-Exos and 740Y-P-loaded exosomes. A: Vaginal epithelial cell staining in proestrus, estrus, metestrus, and diestrus. B: Change curve of estrous cycle in each group of mice after 21 days of treatment. C: Disorder rate of estrous cycle in each group of mice after 21 days of treatment.

[0020] Figure 3 Improvement of metabolic characteristics in PCOS mice by HucMSC-Exos and 740Y-P-loaded exosomes. A: Change curve of body weight in each group of mice after 21 days of treatment. B: Glucose tolerance test. C: Insulin tolerance test. D: Detection of the number of offspring after co-housing each group of mice. E: Statistical analysis of the number of offspring.

[0021] Figure 4 Improvement of ovarian morphology and function in PCOS mice by HucMSC-Exos and 740Y-P-loaded exosomes. A: Analysis of ovarian appearance morphology in each group of mice. Scale bar = 1 mm. B: Ratio of ovarian weight to body weight in each group of mice. C: HE staining of ovarian morphology. Scale bar = 50 μm.

[0022] Figure 5 Activation of primordial follicles in the ovaries of PCOS mice by HucMSC-Exos and 740Y-P-loaded exosomes. A: Proportion of follicles at each stage in mouse ovaries. B: Statistical analysis of primordial follicles, corpora lutea, cystic follicles, and atretic follicles in the ovaries of each group of mice. C: Immunohistochemical staining of Foxo3A in the ovaries of each group of mice. Scale bar = 50 μm. D: Expression of proteins related to the PI3K-AKT-MTOR signaling pathway in the ovaries of each group of mice.

[0023] Figure 6 Improvement of ovarian microenvironment in PCOS mice by HucMSC-Exos and 740Y-P-loaded exosomes. A: TUNEL staining of ovaries in each group of mice. Scale bar = 100 μm. B: Statistical analysis of TUNEL fluorescence intensity. C: Immunofluorescence staining of iNOS and CD206 in the ovaries of each group of mice. D: Statistical analysis of the fluorescence intensity of iNOS and CD206. Scale bar = 100 μm. Detailed implementation manners

[0024] The present invention will be described in detail below in conjunction with embodiments, but the embodiments provided herein are for illustrative purposes only and are not intended to limit the present invention.

[0025] Example 1: Identification of human umbilical cord mesenchymal stem cell-derived exosomes and preparation of 740Y-P-loaded exosomes.

[0026] After the human umbilical cord mesenchymal stem cells were subcultured to passage 7 (P7), the culture medium was replaced with serum-free medium and the cells were cultured for 48 h. The cell supernatant was collected, and exosomes were isolated by size exclusion chromatography, and their various characteristics were analyzed and identified. Under transmission electron microscopy, the structure of exosomes was observed to be round, and the bilayer membrane structure of vesicles was visible. The WB results showed that exosomes expressed the tetraspanin CD9 and the vesicle-associated proteins Alix and Tsg101. The NTA results indicated that the particle size of exosomes mainly concentrated in: 125.7 ± 5.6 nm.

[0027] After exosomes enter the blood circulation, they are rapidly eliminated from the blood vessels and enter the parenchymal organs, and the plasma half-life is only 2 - 4 min. In order to enable the 740Y-P polypeptide to quickly enter the target organs and utilize the therapeutic effects of mesenchymal stem cell exosomes in injury and inflammatory responses, a linker peptide is needed to link 740Y-P to the exosomes. The 740Y-P polypeptide is a polypeptide sequence containing 8 amino acids, with the sequence SDGG(p-Y)MDM. The selected linker peptide sequence is CRHSQMTVTSRL, and the S-terminus of 740Y-P is connected to the L-terminus of the linker peptide; at the same time, in order to monitor exosomes in mice, we added rhodamine to the C-terminus of the linker peptide, and the final sequence is Rhodamine B-CRHSQMTVTSRLSDGG(p-Y)MDM. The linker peptide can recognize and specifically bind to the surface membrane protein CD63 of exosomes. The synthesized polypeptide was co-incubated with HucMSC-Exos at 4 °C for 12 hours to prepare HucMSC-Exos carrying 740Y-P.

[0028] Example 2: Localization of human umbilical cord mesenchymal stem cell-derived exosomes loaded with 740Y-P in the ovary.

[0029] To verify the ability of ovarian tissue in PCOS mice to uptake exosomes, we fluorescently labeled exosomes with PKH67 dye, which expresses green fluorescence at a wavelength of 488 nm; while the polypeptide 740Y-P loaded exosomes carry rhodamine and express red fluorescence at a wavelength of 594 nm. The injection volume for each mouse via the tail vein was 3.0×10 8 partical, and the dose of the polypeptide 740Y-P loaded was 100 μg. 48 h after tail vein injection, green fluorescence and red fluorescence were observed in the ovarian stromal blood vessels and follicles of PCOS mice, and almost complete co-localization was observed, indicating that by means of tail vein injection, HucMSC-Exos carrying 740Y-P were localized to the ovarian tissue of PCOS mice through blood circulation ( Figure 1 ).

[0030] Example 3: Improvement of the estrous cycle of PCOS mice by HucMSC-Exos loaded with 740Y-P.

[0031] According to the method in Example 1, after 21 days of tail vein injection of mice with HucMSC-Exos loaded with 740Y-P of the present invention, the estrous cycles of each group of mice were monitored. The vaginal smears were stained with alkaline methylene blue solution for about 8 minutes, and the histological changes of the smears were observed under a microscope to determine the stage of the estrous cycle. As Figure 2 shown, the control group of mice regularly showed each stage of the estrous cycle, the estrous cycle of PCOS mice was significantly disordered, and after treatment with either HucMSC-Exos or HucMSC-Exos loaded with 740Y-P, the estrous cycle was significantly restored, and the treatment effect of the polypeptide-loaded group was the best.

[0032] Example 4: Improvement of metabolic phenotypes of PCOS mice by HucMSC-Exos and 740Y-P-loaded HucMSC-Exos.

[0033] According to the method in Example 1, continuous body weight records were made 21 days after tail vein injection of mice with HucMSC-Exos loaded with 740Y-P of the present invention. It was found that compared with the PCOS group, the body weights of the mice in the groups injected with HucMSC-Exos and HucMSC-Exos loaded with 740Y-P gradually returned to normal. Each group of mice was fasted for 16 (4) hours in advance and drank normal water. After the fasting ended, the basal blood glucose was measured, and the blood glucose change curve was drawn. Through glucose and insulin tolerance experiments, as Figure 3 shown, the insulin sensitivity of the mice in the groups injected with HucMSC-Exos and HucMSC-Exos loaded with 740Y-P was significantly improved, and the polypeptide-loaded group was superior to the HucMSC-Exos group. Further fertility detection experiments showed that the number of offspring born to the two groups of mice was significantly increased.

[0034] Example 5: Restoration of ovarian function in PCOS mice by 740Y-P-loaded HucMSC-Exos

[0035] According to the method in Example 1, the appearance of the ovaries was observed under a stereomicroscope 21 days after tail vein injection of mice with HucMSC-Exos loaded with 740Y-P of the present invention. The results are as Figure 4 shown. Compared with the control group, the ovarian volume of PCOS mice was significantly increased, the weight was increased, and cystic follicle protrusions could be observed on the surface. However, the number of cystic follicles in the ovaries of the mice in the groups injected with HucMSC-Exos and HucMSC-Exos loaded with 740Y-P was significantly reduced, the follicles resumed development, and dominant follicles and corpora lutea could be seen, indicating the restoration of ovarian function.

[0036] Example 6: Activation of primordial follicles in the ovaries of PCOS mice by HucMSC-Exos and 740Y-P-loaded HucMSC-Exos.

[0037] According to the method in Example 1, HucMSC-Exos loaded with 740Y-P of the present invention were injected into the tail vein of mice. After 21 days, the ovarian tissues of each group of mice were collected, fixed and sliced, and the number of follicles at each stage was counted after H&E. The results are as follows: Figure 5 As shown. After treatment with peptide-loaded exosomes, the number of primordial follicles in the mouse ovaries decreased significantly, the number of primary and secondary follicles increased significantly, and the number of Gref follicles and corpus luteum also increased significantly. At the same time, the number of atretic follicles and cystic follicles in the ovaries also decreased. FoxO3a is very important for maintaining the dormant state of primordial follicles. By immunohistochemistry, it can be found that in the primordial follicles of the ovaries of PCOS mice, FoxO3a is accumulated in the nuclei of primordial oocytes in large quantities. After 48 hours of exosome and peptide treatment, the nuclear outflow of FoxO3a was observed in the primordial follicles. At the same time, WB of mouse ovaries showed that the PI3K-AKT-MTOR signaling pathway was strongly activated. The phosphorylation levels of AKT, mTOR and RPS6 in the ovaries of the HucMSC-Exos group loaded with 740Y-P were significantly higher than those in the group treated with exosomes alone. The above results show that the primordial follicles in the ovaries of PCOS mice were activated in large quantities after treatment with exosomes and peptides.

[0038] Example 6: Improvement of the ovarian environment of PCOS mice by HucMSC-Exos and 740Y-P loading.

[0039] TUNEL staining results of ovaries ( Figure 6 ) showed that HucMSC-Exos and HucMSC-Exos loaded with 740Y-P could significantly improve the apoptosis of follicles in the ovarian tissue of PCOS mice. The immunofluorescence results of ovarian macrophages showed that the expression of M1 and M2 macrophage markers in the ovarian tissue of PCOS mice was significantly increased. After treatment with the two groups of exosomes, the fluorescence intensity of iNOS and CD206 was significantly reduced. This indicates that the level of inflammation in the ovarian tissue of PCOS mice decreased after treatment.

[0040] Matters not covered in the present invention are known technologies. The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A polypeptide-loaded exosome, characterized in that a targeting peptide is added to the end of the PI3K signaling pathway agonist 740Y-P polypeptide, and it is connected to the surface membrane of mesenchymal stem cell exosomes to obtain mesenchymal stem cell exosomes loaded with 740Y-P polypeptide.

2. The polypeptide-loaded exosome according to claim 1, characterized in that the mesenchymal stem cell exosomes are derived from human umbilical cord.

3. The polypeptide-loaded exosome according to claim 1, characterized in that the targeting peptide sequence is CRHSQMTVTSRL.

4. The polypeptide-loaded exosome according to claim 3, characterized in that the 740Y-P polypeptide sequence with the added targeting peptide is CRHSQMTVTSRLSDGG(p-Y)MDM.

5. The polypeptide-loaded exosome according to claims 1 to 4, characterized in that the polypeptide-loaded exosome is prepared by the following method: obtaining mesenchymal stem cell exosomes derived from human umbilical cord by size exclusion chromatography, and binding the PI3K signaling pathway agonist 740Y-P polypeptide with the targeting peptide added at the end to the surface protein CD63 of the exosomes, so that the exosomes are successfully loaded with 740Y-P polypeptide.

6. The pharmaceutical use of the polypeptide-loaded exosome according to claims 1 to 4.

7. The application of the polypeptide-loaded exosome according to claims 1 to 4 in the preparation of a drug for treating polycystic ovary syndrome.

8. The application according to claim 7, characterized in that the polypeptide-loaded exosome according to claims 1 to 3 is used in the preparation of a drug for treating metabolic dysfunction and / or ovulation disorder caused by polycystic ovary syndrome.

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