Stem cell exosome composition for treating erectile dysfunction and application thereof

Abstract

The invention provides a stem cell exosome composition for treating erectile dysfunction and application thereof, the stem cell exosome composition comprises stem cell exosome and vasoactive intestinal peptide, and the amino acid sequence of the vasoactive intestinal peptide is shown as SEQ ID NO: 2. The amino acid structure of the vasoactive intestinal peptide is modified, so that the biological stability can be improved; the vasoactive intestinal peptide is combined with the adipose-derived mesenchymal stem cell exosome for use, so that the ED symptom can be relieved; the vasoactive intestinal peptide and the adipose tissue-derived stem cell exosome are combined for use, so that the expression level of cell factors can be adjusted, and the microenvironment of penis tissues can be improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a stem cell exosome composition for treating erectile dysfunction and its use. Background Art

[0002] Erectile dysfunction (ED) refers to the inability to achieve or maintain an erection sufficient for satisfactory sexual performance, which in turn affects the male sexual life. ED is a common medical problem that seriously affects men's health. 150 million men worldwide suffer from ED to varying degrees. Men over 40 are more likely to suffer from ED. It is estimated that by 2025, there will be more than 322 million men worldwide (see Burnett AL, Nehra A, Breau RH, et al.. Erectile dysfunction: AUA guideline. J Urol. (2018) 200(3): 633–41). In the past, people generally believed that mental illness was an important factor in the development of ED, but many studies have shown that organic causes account for more than 80% of ED cases, and cardiovascular disease, diabetes, dyslipidemia, hypogonadism and nerve damage are independent risk factors for ED. More importantly, ED is no longer simply related to sexual dysfunction. It may also indicate underlying endothelial dysfunction and is an early indicator of cardiovascular disease (see Gandaglia G, Briganti A, Jackson G, et al. A systematic review of the association between erectile dysfunction and cardiovascular disease. Eur Urol. (2014) 65(5): 968–78). Selective phosphodiesterase type 5 inhibitors (PDE5i), such as sildenafil citrate, have been widely used as first-line treatments for ED because they enhance erectile function in 63% of patients and work based on the role of nitric oxide (NO) in relaxation of corpus cavernosum smooth muscle (see Xu W, Jiang H, Liu J, et al. Non-coding RNAs: New dawn for diabetes mellitus induced erectile dysfunction. Front Mol Biosci (2022) 9: 888624). However, due to the complexity of the pathways that regulate penile erection, up to 35% of patients do not respond to drug treatment. Since current treatments do not provide the greatest benefit to patients, it is crucial to study new strategies for treating ED.

[0003] Extracellular vesicles are membrane structures derived from cells with a diameter of 40nm to 1000nm. Exosomes (EXO) are nanosized particles with a diameter of about 40-160nm secreted by various cells under physiological or pathological conditions. Exosomes contain many components, such as metabolites, proteins, lipids and nucleic acids. Exosomes are involved in a variety of physiological and pathological processes in the human body and can serve as molecules and signal carriers for intercellular communication. Exosomes may be involved in cardiovascular and metabolic diseases, play a role in the pathogenesis of neurological diseases, and dynamically affect the growth of cancer (see Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science (2020) 367 (6478): eaau6977).

[0004] The effect of exosomes on improving ED was first analyzed in 2017. Chen et al. established a rat model of type 2 diabetes and isolated exosomes from adipose-derived stem cells (ADSCs) by ultracentrifugation. They found that ADSC-derived exosomes promoted the recovery of erectile function by increasing the ratio of maximum intracavernous pressure (ICP) to mean arterial pressure (MAP), increasing endothelial and smooth muscle content, and reducing apoptosis of endocavernous endothelial cells and smooth muscle cells (see Chen F, Zhang H, Wang Z, et al.. Adipose-derived stem cell-derived exosomes ameliorate erectile dysfunction in a rat model of type 2 diabetes. J Sex Med (2017) 14(9): 1084–94). Exosomes isolated from ADSCs and BMSCs can be used not only to treat diabetic erectile dysfunction, but also to treat BCNI-induced erectile dysfunction. Ouyang et al. found that after four weeks of injection of exosomes into the corpus cavernosum of rats with BCNI-induced erectile dysfunction, erectile function was significantly improved by inhibiting apoptosis in CCSMCs (see Ouyang X, Han X, Chen Z, et al. MSC-derived exosomes ameliorate erectile dysfunction by alleviation of corpus cavernosum smooth muscle apoptosis in arat model of cavernous nerve injury. Stem Cell Res Ther (2018) 9 (1): 246). However, exosomes also show uncertainty in efficacy in the treatment of ED, such as the therapeutic effects of exosomes obtained from different cell sources and different culture conditions vary greatly.

[0005] Vasoactive intestinal polypeptide (VIP) is a novel peptide with strong vasoactivity isolated from porcine intestinal extract by Said and Mutt. Subsequently, the biological effects of VIP and its signal transduction pathways have been widely studied. Various in vitro and in vivo studies have shown that VIP expressed in intrinsic non-adrenergic non-cholinergic (NANC) neurons is a potent regulator of gastrointestinal (GI) motility, water absorption and ion flux, mucus secretion and immune homeostasis. These VIP effects are believed to be mediated mainly through interaction with highly expressed VPAC 1 receptors and the production of nitric oxide (NO) (see Abad C, Martinez C, Juarranz MG, et al. Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn's disease. Gastroenterology. (2003) 124: 961–71). In addition, studies have shown that the combination of VIP and phentolamine mesylate can treat moderate to severe ED, and VIP has a strong effect on the venous occlusive mechanism and can also reduce the incidence of penile pain (see Dinsmore W, Wyllie M. Vasoactive intestinal polypeptide / phentolamine for intracavernosal injection in erectile dysfunction, BJU Int, (2008) 102 (8): 933-7). Although VIP has a good effect in inhibiting the occurrence of ED, its biological stability is poor, which is not conducive to clinical application.

[0006] In order to overcome the defects of the above-mentioned ED therapeutic drugs, the present invention provides a stem cell exosome composition for treating erectile dysfunction and its use, which modifies the VIP amino acid structure to improve stability and bioavailability. The composition is used in combination with adipose mesenchymal stem cell exosomes to significantly alleviate ED symptoms, thus providing an effective ED treatment method. Summary of the invention

[0007] A first aspect of the present invention provides a stem cell exosome composition for treating erectile dysfunction, comprising stem cell exosomes and vasoactive intestinal peptide, wherein the amino acid sequence of the vasoactive intestinal peptide is shown in SEQ ID NO:2.

[0008] In the present invention, the amino acid sequence of vasoactive intestinal peptide is modified, so that its stability is significantly increased, the in vivo bioavailability is improved, and it is more suitable for the slightly alkaline environment in the body; and it is combined with stem cell exosomes to treat erectile dysfunction, which can improve the penile tissue microenvironment, regulate cytokine secretion, and effectively treat erectile dysfunction.

[0009] Furthermore, the stem cells are mesenchymal stem cells.

[0010] Furthermore, the mesenchymal stem cells are selected from at least one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells.

[0011] Furthermore, the mesenchymal stem cells are adipose-derived mesenchymal stem cells, and their preparation method includes: cutting the adipose tissue into pieces, filtering with a 100-mesh filter, adding type IV collagenase, mixing and digesting for 1 hour; collecting the cells by centrifugation at 1000 rpm for 5 minutes; washing the cells again with PBS and centrifuging at 1000 rpm for 5 minutes to collect the cells; resuspending in complete culture medium, culturing at 37°C and 5% CO2, changing the medium once every 2 days, and collecting the cells by trypsin digestion when the density reaches 80%.

[0012] Furthermore, the method for preparing stem cell exosomes includes: culturing adipose-derived mesenchymal stem cells in a complete medium without exosome serum for 48 hours and collecting the cell supernatant; centrifuging at 4°C and 2000g for 20 minutes, collecting the supernatant, and discarding the precipitate; centrifuging the supernatant at 4°C and 10000g for 40 minutes, collecting the supernatant, and discarding the precipitate; filtering the supernatant with a 0.22μm filter and collecting the filtrate; ultracentrifuging the filtrate at 4°C and 100000g for 70 minutes, discarding the supernatant, and retaining the precipitate; resuspending the precipitate with PBS, and ultracentrifuging the resuspended solution at 4°C and 100000g for 70 minutes, discarding the supernatant, and retaining the exosome precipitate; resuspending the exosome precipitate with an appropriate amount of PBS and storing it at -80°C.

[0013] Furthermore, the mass ratio of the stem cell exosomes to vasoactive intestinal peptide is 1:5 to 5:1.

[0014] Furthermore, the mass ratio of the stem cell exosomes to vasoactive intestinal peptide is 2:1.

[0015] The second aspect of the present invention provides a use of the composition in the preparation of erectile dysfunction drugs.

[0016] The erectile dysfunction includes vascular erectile dysfunction, diabetic erectile dysfunction, organic erectile dysfunction and neurogenic erectile dysfunction, etc.

[0017] Beneficial Effects

[0018] The present invention provides a stem cell exosome composition for treating erectile dysfunction and its use, with the following specific beneficial effects:

[0019] (1) Provide a novel vasoactive intestinal peptide, the amino acid structure of which is modified to improve biological stability;

[0020] (2) The combination of vasoactive intestinal peptide and adipose-derived mesenchymal stem cell exosomes can alleviate ED symptoms;

[0021] (3) The combined use of vasoactive intestinal peptide and adipose-derived mesenchymal stem cell exosomes can regulate cytokine expression levels and improve the penile tissue microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : ICP / MAP detection in rats;

[0023] Figure 2 : Expression level of eNOS in rat penile tissue;

[0024] Figure 3 : NGF expression level in rat penile tissue;

[0025] Figure 4 : VEGF expression level in rat penile tissue. DETAILED DESCRIPTION

[0027] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents, biological materials, and detection kits are all commercially available unless otherwise specified.

[0028] Example 1 Preparation of muVIP

[0029] Natural VIP is an endogenous neuropeptide composed of 28 amino acids, and its amino acid sequence is shown in SEQ ID NO: 1. It has multiple physiological activities and has a good therapeutic effect on ED, but its in vivo stability is poor, its half-life is long, and it is not conducive to in vitro storage, preparation and transportation. Studies have shown that VIP is stable in an acidic environment, but slowly degrades in an alkaline environment, and rapidly degrades when the pH value is > 11 (see Cui Xu, Han Xuan, Wang Zhimin, et al. Study on the chemical and biological stability of vasoactive intestinal peptide, Chinese Journal of New Drugs. (2011) 20 (19): 1922-1925).

[0030] To this end, the present invention has modified its amino acid sequence, replaced some amino acids with basic amino acids, thereby improving its stability, and the modified muVIP amino acid sequence is shown in SEQ ID NO: 2. Using the solid phase synthesis method, MBHA resin is selected, and muVIP is obtained according to the amino acid sequence order through the steps of resin swelling, deprotection, condensation reaction, polypeptide cleavage, extraction, etc.

[0031] Native VIP and muVIP were treated at pH 7, 8, 9, 10, 11, and 12 for 1 h, and then the characteristic peaks of the target protein were detected by HPLC, as shown in Table 1. The pH stability of muVIP was clearly increased, and it could still maintain some activity at pH 11 or 12.

[0032] Table 1 VIP stability test

[0033]

[0034] Note: +++, with obvious and clear characteristic peaks of the target protein and no obvious impurity peaks; ++, with obvious characteristic peaks, but with partial degradation and a small amount of impurity peaks; +, with characteristic peaks of the target protein, but with increased degradation and obvious impurity peaks; -, with no obvious characteristic peaks of the target protein.

[0035] Example 2 Preparation of adipose-derived mesenchymal stem cell exosomes

[0036] Adipose tissue of healthy volunteers was obtained, rinsed three times with PBS containing penicillin and streptomycin, cut into pieces, filtered with a 100-mesh filter, added with 1 mg / mL type IV collagenase, mixed and digested for 1 hour; centrifuged at 1000 rpm for 5 minutes to collect cells; washed cells again with PBS and centrifuged at 1000 rpm for 5 minutes to collect cells; resuspended in α-MEM medium containing 10% fetal bovine serum, cultured at 37°C and 5% CO2, and the medium was changed every 2 days. When the density reached 80%, adipose-driven stem cells (ADSCs) were collected by trypsin digestion for subsequent experiments.

[0037] ADSCs were cultured in complete medium without exosome serum for 48 hours, and the cell supernatant was collected. Exosomes were extracted from the collected stem cell culture medium by ultracentrifugation. The culture medium was centrifuged at 4°C and 2000g for 20 minutes, the supernatant was collected, and the precipitate was discarded. The supernatant was centrifuged at 4°C and 10000g for 40 minutes, the supernatant was collected, and the precipitate was discarded. The supernatant was filtered with a 0.22μm filter and the filtrate was collected. The filtrate was ultracentrifuged at 4°C and 100000g for 70 minutes, the supernatant was discarded, and the precipitate was retained. The precipitate was resuspended with a large amount of PBS, and the resuspended solution was ultracentrifuged at 4°C and 100000g for 70 minutes, the supernatant was discarded, and the exosome precipitate was retained. The exosome precipitate was resuspended with an appropriate amount of PBS and stored at -80°C for subsequent experiments. The microscopic morphology of exosomes was observed using a transmission electron microscope, and the size distribution of exosome particles was measured. The exosomes were found to be spherical vesicles with a diameter of about 100 nm.

[0038] Example 3 muVIP and stem cell exosomes in the treatment of ED in rats

[0039] 3.1 Preparation of rat ED model and drug administration

[0040] SPF healthy male SD rats with a body weight of 200-220 g were selected and kept at room temperature (21±2)℃, relative humidity 50%-60%, and light and dark 12h / 12h environment, and the rats were allowed to eat and drink freely. The rats were adaptively raised for 1 week. The ED model was established by bilateral internal iliac artery ligation. SD rats were anesthetized with 3% sodium pentobarbital, and the abdominal midline incision was made to separate the abdominal wall muscle tissue, expose the abdominal aorta and common iliac artery. Under the surgical magnifying glass, the common iliac artery was carefully separated until the internal iliac artery, and the bilateral internal iliac artery was ligated with surgical thread. Rats in the sham operation group received sham surgery, and the same incision and separation were performed according to the modeling steps, but no ligation was performed, and then each layer of tissue was sutured in turn. The APO experiment was used to screen rats with successful modeling as subsequent experimental subjects.

[0041] Two days after modeling, 40 ED rats were selected and randomly divided into 4 groups, 10 rats in each group, namely: muVIP group, 50 mg / kg muVIP was injected into the corpus cavernosum twice a week (day 1 and day 4); exosome group (EXO group), 100 mg / kg stem cell exosomes were injected into the corpus cavernosum twice a week (day 1 and day 4); combined group, 50 mg / kg muVIP and 100 mg / kg stem cell exosomes were injected into the corpus cavernosum twice a week (day 1 and day 4); control group, the same amount of normal saline was injected into the corpus cavernosum twice a week (day 1 and day 4). In addition, 10 rats in the sham operation group were injected with the same amount of normal saline into the corpus cavernosum twice a week (day 1 and day 4). Each group was treated for a total of 8 weeks.

[0042] 3.2ICP / MAP detection

[0043] The right common carotid artery and corpus cavernosal of anesthetized rats were exposed, and a PE-50 tube filled with heparin solution was inserted into the proximal end of the right common carotid artery along the incision to record the mean arterial pressure (MAP) level. Another PE-50 tube filled with heparin solution was inserted into the corpus cavernosal. The cavernous nerve of the penis was electrically stimulated (voltage 5V, frequency 20Hz, pulse 1.2ms, duration 60s) to record the maximum intracavernosal pressure (ICPmax) value under erection. Figure 1 As shown in the figure, the maxICP / MAP level of rats decreased significantly after modeling, but recovered after treatment. Among them, the maxICP / MAP level of the combined group recovered the most and was close to the normal level.

[0044] 3.4 Detection of eNOS expression level

[0045] After completing the above test, the rats in each group were killed, and part of the corpus cavernosum tissue was taken to make a tissue homogenate, centrifuged at 3000rpm for 15min, and the supernatant was taken and stored for later use. According to the instructions of the ELISA kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.), the erection-related bioactive factor eNOS in the corpus cavernosum tissue of the rats in each group was determined.

[0046] like Figure 2 As shown, compared with the sham operation group, the eNOS expression level in the control group was significantly reduced, while the eNOS expression level recovered to varying degrees after treatment, among which the muVIP group was higher than the EXO group, but lower than the combined group, indicating that the combined treatment of muVIP and EXO has a better effect.

[0047] 3.5 Detection of cytokine expression levels

[0048] The occurrence and development of ED is associated with abnormal expression of multiple cytokines. In the present invention, an ELISA kit (purchased from Shanghai ELISA Biotechnology Co., Ltd.) was used to detect the expression levels of NGF and VEGF in the homogenate of the corpus cavernosum of the penis. The specific steps were carried out according to the instructions of the kit.

[0049] like Figure 3 , Figure 4 As shown in the figure, compared with the normal level, the expression levels of NGF and VEGF in the model group decreased significantly, indicating that the secretion of the above two factors was significantly inhibited. After treatment, the expression levels of NGF and VEGF were restored, and this trend was more obvious in the combined group.

[0050] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A vasoactive intestinal peptide, characterized in that: The amino acid sequence of the vasoactive intestinal peptide is shown in SEQ ID NO:

2.

2. A stem cell exosome composition for treating erectile dysfunction, comprising stem cell exosomes and vasoactive intestinal peptide, wherein the amino acid sequence of the vasoactive intestinal peptide is shown in SEQ ID NO: 2, and the stem cells are mesenchymal stem cells.

3. The composition according to claim 2, characterized in that The mesenchymal stem cells are selected from at least one of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and adipose mesenchymal stem cells.

4. The composition according to claim 3, characterized in that The mesenchymal stem cells are adipose-derived mesenchymal stem cells, and the preparation method thereof comprises: cutting adipose tissue into pieces, filtering with a 100-mesh filter, adding type IV collagenase, mixing and digesting for 1 hour; collecting cells by centrifugation at 1000 rpm for 5 minutes; washing the cells again with PBS and centrifuging at 1000 rpm for 5 minutes to collect the cells; resuspending the cells in complete culture medium, culturing at 37° C. and 5% CO2, changing the medium once every 2 days, and collecting the cells by digesting with trypsin when the density reaches 80%.

5. The composition according to claim 4, characterized in that The method for preparing stem cell exosomes comprises: culturing adipose-derived mesenchymal stem cells in a complete medium without exosome serum for 48 hours and collecting the cell supernatant; centrifuging at 4°C and 2000g for 20 minutes, collecting the supernatant, and discarding the precipitate; centrifuging the supernatant at 4°C and 10000g for 40 minutes, collecting the supernatant, and discarding the precipitate; filtering the supernatant with a 0.22 μm filter and collecting the filtrate; ultracentrifuging the filtrate at 4°C and 100000g for 70 minutes, discarding the supernatant, and retaining the precipitate; resuspending the precipitate with PBS, and ultracentrifuging the resuspended solution at 4°C and 100000g for 70 minutes, discarding the supernatant, and retaining the exosome precipitate; resuspending the exosome precipitate with an appropriate amount of PBS and storing it at -80°C.

6. The composition according to claim 2, characterized in that The mass ratio of the stem cell exosomes to the vasoactive intestinal peptide is 1:5 to 5:

1.

7. The composition according to claim 2, characterized in that The mass ratio of the stem cell exosomes to vasoactive intestinal peptide is 2:

1.

8. Use of the composition according to any one of claims 2 to 7 in the preparation of a drug for erectile dysfunction.

Images