A stem cell exosome composition and its application in preparing a drug for treating diabetic complications

Abstract

The present invention provides a stem cell exosome composition and its use in the preparation of a drug for treating diabetic complications. The antimicrobial peptide has a novel amino acid structure and can inhibit the growth of multiple pathogens. The antimicrobial peptide and stem cell exosomes construct a drug composition, which can promote rapid healing of diabetic skin lesions, inhibit the secretion of inflammatory factors, and regulate inflammatory responses.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research and development, and specifically provides a stem cell exosome composition and its application in the preparation of a drug for treating diabetic complications. Background Art

[0002] Diabetes mellitus (DM) is a metabolic disease characterized by high blood sugar levels, which is caused by defects in insulin secretion or its biological effects, or both. Chronic high blood sugar levels can disrupt the balance in the body, cause oxidative stress, and induce damage to the microvasculature, nerves, and immune system, ultimately exacerbating the development of diabetic complications. Diabetic complications involve multiple human tissues, including: cardiomyopathy is induced by increased fatty acid metabolism, decreased myofilament Ca+ sensitivity, mitochondrial dysfunction, oxidative stress, apoptosis and diabetic cardiomyocyte fibrosis (see Bhatt NM, Aon MA, Tocchetti CG, et al.. Restoring Redox Balance Enhances Contractility in Heart Trabeculae From Type 2 Diabetic Rats Exposed to High Glucose. Am J Physiol Heart Circulatory Physiol (2015) 308 (4): H291–302); pathological glucose metabolism can also damage vascular structure and function, leading to microvascular apoptosis and fibrosis, inducing diabetic nephropathy, glomerular atrophy, renal fibrosis, renal insufficiency and renal failure (see un AL, Deng Jt, Guan Gj, et al.. Dipeptidyl Peptidase-IV Is a Potential Molecular Biomarker in Diabetic Kidney Disease. Diabetes Vasc DisRes (2012) 9(4):301–308); in the retina, microvascular apoptosis and paraplasm may also lead to microaneurysms and hemorrhages, which are diagnosed as diabetic retinopathy and eventually lead to vision loss (see Mohamed Q, Gillies MC, WongTY. Management of Diabetic Retinopathy: A Systematic Review. JAMA (2007) 298(8):902–316); poor blood sugar control can induce peripheral neuropathy and peripheral vascular disease with structural deformities, and environmental factors and impaired immunity can lead to the occurrence of diabetic foot or diabetic wounds that are difficult to heal (see Rebolledo FA, Soto JT, de la JE. The Pathogenesis of the Diabetic Foot Ulcer: Prevention and Management. Global Perspective Diabetic Foot Ulcerations (2011) p: 155–78).

[0003] Wound healing is a complex biological process, especially chronic skin wounds such as trauma, burns or diabetic ulcers are more difficult to heal, becoming one of the most difficult problems faced by clinicians, and also adding a heavy burden to patients physically and financially. Traditional wound care methods include skin grafting, flap transplantation, laser therapy and biological scaffolds, etc., which have the risk of atrophic scars and pigment abnormalities and are not suitable for treating large-scale trauma (see Vaghardoost R, Momeni M, Kazemikhoo N, et al. Effect of low-level laser therapy on the healing process of donor site in patients with grade 3 burn ulcer after skin graft surgery (a randomized clinical trial). Lasers Med Sci. 2018; 33 (3): 603-607). Therefore, there is an urgent need for alternative effective and safe methods to promote wound healing.

[0004] In recent years, stem cell therapy has flourished due to its pluripotency, self-renewal, and ability to promote the secretion of regenerative cytokines. Pluripotent stem cells are considered safe and overcome the ethical issues associated with embryonic stem cells. Stem cells have been proposed to promote wound healing in a paracrine manner, reduce inflammation by regulating macrophages, T cells, B cells, etc., secrete VEGF to promote angiogenesis, promote the proliferation and differentiation of fibroblasts and keratinocytes, produce anti-fibrotic cytokines, and transform into microvascular endothelial cells and keratinocytes. However, stem cell therapy may have storage and transportation problems and the risk of inducing tumorigenesis and malformations, so researchers have begun to focus on the secretory products of stem cells - exosomes (EXOs), which are one of the paracrine components and the main contributor to the efficacy of stem cells. Exosomes are microvesicles released by cells into the extracellular space, with a size of about 30-200nm. Exosomes contain nucleic acids, including single-stranded, genomic double-stranded, mitochondrial or reverse transcribed complementary DNA, RNA (including microRNA, ribosomal RNA and long non-coding RNA), which can be transferred to other cells and tissues in a functional form.

[0005] Exosomes can promote wound healing through a variety of mechanisms, including: (1) participating in the regulation of immune response and inflammation. Chronic and excessive inflammation may delay wound healing. EXOs exert immunosuppressive effects by reducing IFN-α secretion, thereby inhibiting T cell activation (see Villiger P, Ryan E, Owen R, et al.. Prevention of Bleeding After Islet Transplantation: Lessons Learned From a Multivariate Analysis of 132 Cases at a Single Institution. Am J Transplant (2005) 5(12): 2992–8); (2) promoting wound angiogenesis. Angiogenesis provides blood supply for wound healing and promotes the transport of nutrients and metabolic waste. EXOs can inhibit the expression of angiogenesis inhibitor (DLL4), thereby promoting the migration and sprouting of endothelial tip cells (see Shi H, Xu X, Zhang B, et al.. 3,3′-Diindolylmethane Stimulates Exosomal Wnt11Autocrine Signaling in Human Umbilical Cord Mesenchymal Stem Cells to Enhance Wound Healing. Theranostics (2017) 7(6): 1674); (3) Accelerate the proliferation and epithelialization of skin cells. The proliferation and epithelialization of skin cells are crucial for skin regeneration. There is evidence that EXO can upregulate 199 miRNAs and downregulate 93 miRNAs to promote the proliferation and differentiation of dermal fibroblasts, thereby accelerating skin regeneration (Xv J, Ming Q, Wang X, et al.. Mesenchymal Stem Cells Moderate Immune Response of Type 1 Diabetes. Cell Tissue Res (2017) 368(2): 239–48); (4) Regulate collagen remodeling and inhibit scar hyperplasia. EXOs can regulate collagen remodeling and inhibit scar hyperplasia.In the early stage, EXO promotes collagen remodeling by synthesizing types I and III, while in the later stage it reduces scar formation by inhibiting collagen formation (see Hao H, Liu J, Shen J, et al.. Multiple Intravenous Infusions of Bone Marrow Mesenchymal Stem Cells Reverse Hyperglycemia in Experimental Type 2 Diabetes Rats. Biochem Biophys Res Commun (2013) 436(3): 418–23).

[0006] Although a large number of studies have shown that stem cell exosomes can promote skin wound healing, their safety and effectiveness in difficult-to-heal wounds, such as diabetic skin wounds, still need to be improved. In view of this, the present invention combines stem cell exosomes with novel antimicrobial peptides to effectively promote diabetic skin wound healing, providing new ideas for the development of related drugs and clinical treatment methods. Summary of the invention

[0007] The first aspect of the present invention provides an antimicrobial peptide SMH, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] One of the important factors that make diabetic skin injuries difficult to heal is potential wound infection. Opportunistic pathogens, such as Gram-negative bacteria Pseudomonas aeruginosa or Gram-positive bacteria Staphylococcus aureus, can colonize in skin wounds, form biofilms, and cause bacterial cell aggregation (see Mancl KA, Kirsner RS, Ajdic D. Wound biofilms: lessons learned from oral biofilms. Wound Repair Regen. 2013; 21: 352–362). In addition, toxins released by bacteria promote the recruitment of immune cells, leading to excessive and destructive inflammatory responses (see Rajpaul K. Br J. Biofilm in wound care. Community Nurs. 2015; 20: S6).

[0009] A large number of studies have shown that antimicrobial peptides (AMPs) can promote skin wound healing. For example, hBD-3 has been shown to exhibit in vitro and in vivo anti-inflammatory activity by inhibiting the TLR signaling pathway in immune cells and thus inhibiting the transcription of pro-inflammatory genes (see Semple F, Webb S, Li HN, et al. Human beta-defensin 3 has immunosuppressive activity in vitro and in vivo. Eur J Immunol. 2010; 40: 1073–1078); LL-37 is involved in the regulation of angiogenesis and can stimulate proliferation and new blood vessel formation (see Koczulla R, von Degenfeld G, Kupatt C, et al. An angiogenic role for the human peptide antibiotic LL-37 / hCAP-18. J Clin Invest. 2003; 111: 1665–1672). The present invention provides a novel antimicrobial peptide that can inhibit the growth of multiple pathogens and promote the healing of refractory skin windows.

[0010] The second aspect of the present invention provides a pharmaceutical composition for promoting the healing of skin injuries, wherein the pharmaceutical composition comprises the antimicrobial peptide SMH and stem cell exosomes.

[0011] Furthermore, the stem cell exosomes are mesenchymal stem cell exosomes.

[0012] Furthermore, the stem cell exosomes are adipose-derived mesenchymal stem cell exosomes.

[0013] Furthermore, the method for preparing stem cell exosomes includes: culturing adipose-derived mesenchymal stem cells, replacing the complete culture medium with a serum-free basal culture medium, starving the cells for 48 hours, collecting the cell supernatant, and collecting the exosomes by gradient centrifugation.

[0014] Furthermore, the gradient centrifugation method includes: centrifugation at 1000g / min for 10min, collecting the supernatant; centrifugation at 10000g / min for 30min, collecting the supernatant; passing the supernatant through a 0.22μm filter membrane, ultracentrifuging at 100000g for 90min, removing the supernatant and collecting the precipitate; the precipitate is washed once with 4°C pre-cooled PBS, and ultracentrifuged at 100000g for 30min; removing the supernatant and resuspending the precipitate to obtain exosomes EXO.

[0015] The third aspect of the present invention provides a use of the pharmaceutical composition in the preparation of a drug for treating diabetic complications.

[0016] Furthermore, the diabetic complication is diabetic skin damage.

[0017] Beneficial Effects

[0018] The present invention provides a stem cell exosome composition and its use in preparing a drug for treating diabetic complications, with the following specific beneficial effects:

[0019] (1) Provides a new antimicrobial peptide that can inhibit the growth of multiple pathogens;

[0020] (2) The antimicrobial peptide combined with stem cell exosomes can promote rapid healing of diabetic skin lesions;

[0021] (3) Antimicrobial peptides and stem cell exosomes can inhibit the secretion of inflammatory factors and regulate the inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :Transmission electron microscopy images of exosomes;

[0023] Figure 2 : Wound healing in rats;

[0024] Figure 3 : HE staining of skin tissue, A is the EXO group, B is the EXO+SMH group, and C is the control group;

[0025] Figure 4 : TNF-α expression level in serum;

[0026] Figure 5 : Expression level of IL-2 in serum. 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 Screening and identification of antimicrobial peptide SMH

[0029] 1.1 Isolation and screening of antimicrobial peptides

[0030] The sources of antimicrobial peptides are quite extensive, including plants, insects, animals, microorganisms, etc., and my country has a wide range of medicinal plants and in-depth research. It has been reported in the prior art that a variety of medicinal plants have antibacterial effects, including garlic, cloves, cinnamon, pepper seeds, mulberry leaves, white striata, honeysuckle, coptis chinensis, etc. Among them, coptis chinensis has a strong bactericidal effect and has a clear "heat-clearing and detoxifying" effect. Therefore, in this embodiment, coptis chinensis is used as a raw material to extract antimicrobial peptides.

[0031] After 50g of Coptis chinensis was crushed, passed through a 200-mesh sieve, and soaked in 200mL PBS buffer for 24h. After stirring and homogenizing, the mixture was filtered through gauze, centrifuged at 2500r / min for 30min at 4℃, the supernatant was taken and ammonium sulfate was added to make its saturation reach 80%, stirred at 200r / min for 30min, and allowed to stand at 4℃ overnight; centrifuged at 10000r / min for 60min, the supernatant was discarded and the precipitate was collected to obtain a crude antimicrobial peptide. After the crude antimicrobial peptide was separated by HPLC, a pure antimicrobial peptide was obtained, named antimicrobial peptide SMH, and after freeze-drying, it was stored in a -80℃ refrigerator for use. After identification, its amino acid sequence is shown in SEQ ID NO:1.

[0032] 1.2 Detection of antimicrobial peptide activity

[0033] The inhibitory effect of the antimicrobial peptides provided in the present invention on pathogenic microorganisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, Salmonella, Acinetobacter baumannii, Vibrio parahaemolyticus, and Vibrio vulnificus was investigated. The minimum inhibitory concentration (MIC) value of the peptide was quantitatively determined by the microdilution method. The pathogens were cultured to the logarithmic growth phase and 1×10 5 CFU / mL of pathogens were inoculated in a 96-well plate; the peptides were gradiently diluted into a 100 μL series of phosphate buffer solutions with a final concentration of 64.0 to 2.0 μmol / L using the microdilution method, and then added to the 96-well ELISA plate containing the pathogens; the plates were shaken and cultured at 37°C at 150 r / min for 18 h, the OD630 value was measured with an ELISA reader, and the MIC values ​​of the peptides for different strains were calculated.

[0034] The results are shown in Table 1. The antimicrobial peptides provided in the present invention have obvious antibacterial effects on a variety of pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae, and Vibrio parahaemolyticus.

[0035] Table 1 Antibacterial effect of antimicrobial peptide SMH

[0036]

[0037]

[0038] Example 2 Preparation of adipose-derived mesenchymal stem cell exosomes

[0039] Obtain adipose tissue from healthy volunteers, chop it with sterile surgical scissors, wash it three times with sterile PBS, let it stand for 5 minutes, and discard the upper aqueous phase; add 0.5 mg / mL type I collagenase solution at a volume ratio of 1:2, place on a constant temperature shaker, and digest at 37°C, 250 r / min for 30 minutes; collect the digested tissue fluid, centrifuge at 2000 r / min for 10 minutes, and collect the cell precipitate; resuspend the cells with complete culture medium, inoculate them in a cell culture dish, and perform primary culture in a 37°C, 5% CO2 incubator; replace the cell culture medium according to the culture conditions, and perform subculture when the cell fusion degree reaches more than 80%, for a total of 3-5 generations.

[0040] Take the above-mentioned subcultured cells, replace the complete medium with serum-free basal medium, starve and culture for 48 hours, collect the cell supernatant; centrifuge at 1000g / min for 10 minutes, collect the supernatant, remove cells and cell debris; centrifuge at 10000g / min for 30 minutes, collect the supernatant, remove cell debris and liposomes; pass the supernatant through a 0.22μm filter membrane, ultracentrifuge at 100000g for 90 minutes, remove the supernatant and collect the precipitate; wash the precipitate once with 4°C precooled PBS, ultracentrifuge at 100000g for 30 minutes; remove the supernatant, resuspend the precipitate with 4°C precooled PBS, and obtain exosomes EXO. Use transmission electron microscopy to observe the exosomes, such as Figure 1 As shown, the obtained exosomes have a uniform particle size distribution, with a diameter of about 80-120 nm. The obtained exosomes are stored at -80°C.

[0041] Example 3: Adipose-derived mesenchymal stem cell exosomes and antimicrobial peptides for the treatment of diabetic skin lesion animal model 3.1 Animal model preparation and drug administration

[0042] SPF male Wistar rats aged 8 weeks, weighing 250±20g, were fed adaptively for 1 week at a temperature of 22±5℃, a humidity of 55%±5%, and a light period of 7:00-19:00. Subsequently, high-fat diet was continuously fed for 10 weeks. After fasting for one night, the rats were intraperitoneally injected with streptozotocin 55mg / kg. After 48h, the fasting tail vein blood glucose level was measured. Rats with fasting blood glucose ≥16.7mmol / L were considered diabetic rats.

[0043] Thirty rats with successful modeling were anesthetized with 7% chloral hydrate intraperitoneal injection. After removing the hair on the skin on the left side of the dorsal midline, a circular skin wound with a diameter of 20 mm was prepared with surgical scissors, deep to the fascia layer. The experimental animals were randomly divided into three groups: EXO group, 20 mg / kg stem cell exosomes were injected at multiple points around the skin wound every day; EXO+SMH group: 20 mg / kg stem cell exosomes and 100 mg / kg antimicrobial peptide SMH were injected at multiple points around the skin wound every day; control group: an equal volume of normal saline was injected at multiple points around the skin wound every day.

[0044] The wound healing status was observed daily, and the wound healing percentage was calculated on the 7th and 14th days, where the wound healing percentage = wound area on the day / wound area on the first day*100%. Figure 2 As shown, EXO+SMH can significantly promote skin wound healing, and its healing rate is significantly higher than that of the EXO group and the control group.

[0045] 3.2 Histological observation

[0046] The experimental animals were killed 14 days after administration, and the skin tissue was separated and fixed in paraffin at 55-65°C. The skin tissue was cut into 10 μm slices using a paraffin slicer. HE staining was used and histological observation was performed under a microscope. Figure 3 As shown, there was a large amount of inflammatory cell infiltration in the control group, the wound recovery was slow, and the skin tissue stratification was disordered; after EXO treatment, especially the combined treatment of EXO and SMH, the inflammatory cells decreased, the skin tissue was arranged neatly, and the physiological function gradually recovered.

[0047] 3.3 Plasma cytokine detection

[0048] After 14 days of administration, blood was collected from the rats and serum was collected by centrifugation. ELISA kits (purchased from Nanjing Jiancheng Company) were used to detect the levels of TNF-α and IL-2 in the serum. The specific steps were carried out according to the instructions of the kit.

[0049] The results are as follows Figure 4 , Figure 5 As shown in the data, the expression levels of TNF-α and IL-2 were higher in the control group, which was consistent with the histological observation of a large number of inflammatory cell infiltrations. After treatment with exosomes or a combination of exosomes and antimicrobial peptides, the expression level of inflammatory factors was reduced, which was conducive to the rapid healing of the wound.

[0050] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the spirit or basic features of the present invention, the present invention can be implemented in other specific forms and various changes and improvements can be made. These changes and improvements all fall within the scope of the present invention to be protected. Therefore, the scope of protection of the present invention is defined by the attached claims and their equivalents, rather than the above description.

Claims

1. An antimicrobial peptide SMH, characterized in that: The amino acid sequence of the antimicrobial peptide SMH is shown in SEQ ID NO:

1.

2. A pharmaceutical composition for promoting the healing of skin damage, characterized in that: The pharmaceutical composition comprises the antimicrobial peptide SMH described in claim 1 and stem cell exosomes, wherein the stem cell exosomes are adipose mesenchymal stem cell exosomes, and the preparation method thereof comprises: culturing adipose mesenchymal stem cells, replacing the complete culture medium with a serum-free basal culture medium, starving and culturing for 48 hours, collecting the cell supernatant, and collecting the exosomes by gradient centrifugation; the gradient centrifugation comprises: centrifuging at 1000g for 10 minutes to collect the supernatant; centrifuging at 10000g for 30 minutes to collect the supernatant; passing the supernatant through a 0.22 μm filter membrane, ultracentrifuging at 100000g for 90 minutes, removing the supernatant and collecting the precipitate; washing the precipitate once with PBS precooled at 4°C, and ultracentrifuging at 100000g for 30 minutes; removing the supernatant, and resuspending the precipitate to obtain the exosomes.

3. Use of the pharmaceutical composition according to claim 2 in the preparation of a drug for treating diabetic complications, wherein the diabetic complication is diabetic skin damage.

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