Preparation method and application of fresh periplaneta americana nano vesicles
Through efficient isolation and characterization of fresh American cockroach, nano chestic vesicles with significant healing effects were prepared, which solved the problem of insufficient application of insect-derived exosome-like vesicles in skin wound healing and achieved significant promotion of wound healing Advantages.
Patent Information
- Application Number
- CN202411687916.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-25
- Publication Date
- 2025-02-18
AI Technical Summary
The prior art is difficult to effectively utilize exosome-like vesicles from insect-derived sources, especially in the process of skin wound healing, and lacks efficient preparation methods and application research.
Fresh American cockroach nano-chestic vesiculus suspension was obtained by crushing fresh American cockroach and separating under different centrifugation conditions, and its components and potential healing were characterized by proteomics, metabolites and Lnc-RNA-omics analysis. mechanism.
Fresh American cockroach nano-chestic vesicles were successfully prepared, which significantly promoted wound healing in mice, involving the promotion of wound inflammation inhibition, collagen deposition and epithelial regeneration.
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Figure CN119454763A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine and relates to a preparation method and application of fresh American cockroach nano-vesicles. Background Art
[0002] As the largest organ in the human body, the skin plays a barrier role in protecting the body from external physical, chemical and biological factors, and is widely involved in a variety of physiological processes such as regulating body temperature, sensory transmission and immune response. Skin trauma, including abrasions, lacerations, cuts, burns, etc., is a common event in daily life. It not only directly damages the quality of life of individuals, but also may affect psychological and even social burdens. The economic burden and demand for medical resources for the treatment and management of skin trauma are increasing worldwide. However, the healing process of skin trauma is complex, involving multiple stages such as hemostasis, inflammation, cell proliferation, and matrix remodeling. Disorders in any stage may lead to delayed healing, infection, scar formation and even chronic wounds. Therefore, timely intervention and delay of deterioration in the early stage of trauma are of great significance.
[0003] Extracellular vesicles (EVs) are a type of nano-sized vesicles secreted by cells with a spherical closed structure and a double-layer lipid membrane structure. EVs can encapsulate and carry a variety of signal molecules such as RNA, DNA, proteins, lipids, etc., and play an important role in intercellular communication, material transport and signal transmission. In recent years, in addition to EVs from mammals, vesicles from other species (also called nanovesicles, NVs) have gradually become a hot topic of research due to their unique biological characteristics and potential clinical application value. For example, natural vesicles from plants such as ginseng and lemon have the advantages of low immunogenicity, high stability and easy large-scale preparation. After injection into the body, they can effectively curb the growth of melanoma and gastric cancer by regulating immunity and cell apoptosis, and have great application prospects. However, plants with medicinal value generally have a long planting cycle and are greatly affected by natural environmental factors, so obtaining EVs from them is not the best choice. Among animal-derived natural vesicles, especially insect-derived vesicles, they have the characteristics of fast reproduction, simple reproduction conditions, and low economic cost, and have unique advantages in the development and treatment of diseases. For example, exosome-like vesicles in honey bee gland secretions are involved in antibacterial properties and are internalized by human MSCs, affecting their migration behavior. Dried American cockroach natural vesicles PAELN significantly promote wound healing in diabetic mice. However, there are currently very few studies on insect-derived EVs, and more comprehensive and extensive research is urgently needed.
[0004] American cockroach (AC) is an insect of the genus Periplaneta, order Blattaria, family Blattidae, class Insecta. It has the characteristics of wide distribution, large number, fast reproduction, strong vitality, cheap and easy to obtain. Modern research shows that American cockroach has multiple pharmacological effects such as anti-inflammatory, anti-tumor, promoting tissue repair, liver protection, and antioxidant. Traditional Chinese medicines such as Xinmailong injection, Ganlong capsule, and Kangfuxin liquid, which are made of American cockroach, have been developed and marketed. Among them, Kangfuxin liquid is widely used in clinical practice, especially in promoting wound repair, eliminating inflammation, and promoting tissue regeneration. The American cockroach extract obtained by different solvents and different extraction methods contains active ingredients such as nucleosides, amino acids, polypeptides, and polysaccharides, which have the effects of promoting angiogenesis, inhibiting inflammation, and increasing the expression of growth factors. In addition, the polypeptide components in American cockroach have antibacterial, neuroprotective, host defense, antiviral, and anti-cancer activities. This shows that the American cockroach is an insect with extremely high medicinal value, and it is of great significance to explore its potential clinical uses from different levels. Although there are many studies on the chemical composition and pharmacological effects of the American cockroach, most of them are carried out on chemical extracts of dried American cockroaches, which may destroy the biologically active components such as proteins and lipids of the cockroaches and lose some medicinal functions. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preparing fresh American cockroach nano vesicles, prepare fresh American cockroach nano vesicles, and the present invention also provides the use of fresh American cockroach nano vesicles in cell migration and proliferation and promoting wound healing in mice.
[0006] In order to achieve the above object, the present invention adopts the following technical scheme to achieve it: a method for preparing fresh American cockroach nano vesicles, which comprises crushing the fresh American cockroach, and then centrifuging at 1000×g to 10000×g, where g is the gravitational acceleration, to remove impurities and cell debris, and then further centrifuging at 120,000×g, discarding the supernatant, and resuspending the precipitate in a buffer solution to obtain a fresh or dried American cockroach nano vesicle suspension.
[0007] Further preferably, fresh American cockroaches are crushed in a PBS solution to obtain a suspension; subsequently, the suspension is centrifuged at 1000-2000×g for 10-20 min, then at 4000-5000×g for 20 min, and finally at 1000-2000×g for 30 min to remove impurities and cell debris, and further centrifuged twice continuously at 120,000-150,000×g for 70 min using an ultracentrifuge, the supernatant is discarded, and the precipitate is resuspended in a PBS solution to obtain a fresh American cockroach nanovesicle suspension, which is stored at -80°C.
[0008] The present invention also provides the use of the fresh American cockroach nano-vesicle-like vesicle in preparing a drug for cell migration, proliferation or skin wound healing. In particular, the use of the fresh American cockroach nano-vesicle-like vesicle in preparing a drug for skin wounds caused by mechanical injuries, burns, scalds, and diabetes. In particular, the use of the fresh American cockroach nano-vesicle-like vesicle in preparing a drug for promoting proliferation and migration of human umbilical vein endothelial cells and human skin keratinocytes, or repairing skin wounds.
[0009] Compared with the prior art, the present invention has the following beneficial effects:
[0010] The present invention successfully isolated exosome-like nanoparticles from dried and fresh American cockroaches, and conducted pharmacodynamic studies on both. Finally, fresh American cockroaches with better effects were selected for subsequent experiments. Proteomics, metabolomics, and lnc-RNAomics analysis were used to characterize the American cockroach nanovesicles, suggesting that the potential wound healing mechanism is related to these miRNA and protein cargoes. In addition, the topical application of American cockroach nanovesicles showed significant advantages in promoting wound healing in mice, which involved wound inflammation inhibition, collagen deposition, and promotion of epithelial re-epithelialization. In summary, given the inherent amputation regeneration properties of American cockroaches and the benefits of promoting wound healing, American cockroach nanovesicles will show potential wound healing effects by delivering these intrinsic macromolecular cargoes. This study provides a new method for treating skin wounds and a new idea for the development of medicinal insects such as American cockroaches. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 Transmission electron microscopy morphology of dried American cockroach nanovesicles (D-ACNVs) and fresh American cockroach nanovesicles (F-ACNVs).
[0012] Figure 2 Particle size distribution and concentration of dried American cockroach nanovesicles (D-ACNVs).
[0013] Figure 3 Particle size distribution and concentration of fresh American cockroach nanovesicles (F-ACNVs).
[0014] Figure 4 Zeta potential measurement of dried American cockroach nanovesicles (D-ACNVs) and fresh American cockroach nanovesicles (F-ACNVs), n=3.
[0015] Figure 5 CCK-8 assay was used to detect the promoting effect of 5μg / mL and 50μg / mL D-ACNVs and F-ACNV on the proliferation of HACAT cells, ***P<0.001, ****P<0.0001, n=6.
[0016] Figure 6CCK-8 was used to detect the promoting effect of different concentrations of D-ACNVs and F-ACNV on the proliferation of HACAT cells, ****P<0.0001, n=6.
[0017] Figure 7 Representative images of HUVECs transwell migration assay (left) and quantitative analysis (right), n=6.
[0018] Figure 8 (A) Representative photos of mouse wounds; (B) Quantitative analysis of wound healing; n=6, **P<0.01.
[0019] Fig. 9 H&E and Masson staining analysis of skin tissue.
[0020] Fig.10 Transcriptomic analysis of skin tissue.
[0021] Fig.11 To analyze the expression levels of angiogenesis-related factors. DETAILED DESCRIPTION
[0022] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.
[0023] Adult American cockroaches were collected from a farm in Yunnan. Coomassie brilliant blue fast staining solution was purchased from Beyotime Biotechnology (Shanghai, China). DL-dithiothreitol and iodoacetamide were purchased from Adamas Pharmaceuticals, Inc. RIPA lysis buffer, BCA protein concentration determination kit, 5× reducing protein loading buffer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit were purchased from Servicebio (Wuhan, China). Dulbecco's modified Eagle's medium (DMEM) and penicillin-streptomycin (PS) were purchased from Gibco. Fetal bovine serum (FBS) was purchased from PAN. DILLINOLYL Dil (cell membrane orange-red fluorescent probe), DAPI staining solution, and DIO cell membrane green fluorescent probe were all purchased from US Everbright Corporation.
[0024] Fresh American cockroach nanovesicles (F-ACNVs) were isolated and obtained by ultracentrifugation. American cockroaches were crushed in 1× phosphate buffered saline (PBS) solution at 4°C to obtain a suspension; then, the suspension was centrifuged at 2000×g for 10 min, g is the acceleration of gravity, then at 4000×g for 20 min, and finally at 1000×g for 30 min to remove impurities and cell debris. In order to obtain F-ACNVs, a MAX-XP tabletop ultracentrifuge (BeckmanCoulter Life Sciences, California, USA) was used for further continuous centrifugation at 120,000×g for 70 min twice, the supernatant was discarded, and the precipitate was resuspended in 1× PBS to obtain a fresh American cockroach nanovesicle (F-ACNVs) suspension, which was stored at -80°C.
[0025] Dried American cockroach nanovesicles (D-ACNVs) were isolated and obtained by differential centrifugation. Dried American cockroach powder was soaked in PBS solution (w / v (g / ml), 1 / 10) at 4°C for 4h. Subsequently, the suspension was centrifuged at 3000×g for 10min and then at 10000×g for 60min to remove impurities and cell debris. In order to obtain D-ACNVs, a MAX-XP tabletop ultracentrifuge (Beckman Coulter Life Sciences, California, USA) was used for 2 consecutive centrifugations at 120,000×g for 70min, the supernatant was discarded, the precipitate was resuspended in 1×PBS solution, and the resulting D-ACNVs suspension was stored at -80°C.
[0026] Physical characterization of F-ACNVs and D-ACNVs: Morphological observations of F-ACNVs and D-ACNVs were performed using transmission electron microscopy (TEM). Sample preparation was performed by fixing the American cockroach nanovesicles with 4% paraformaldehyde (4 g of solid paraformaldehyde dissolved in 100 ml of PBS). 10 μL of the American cockroach nanovesicle suspension was placed on a formvar / carbon-coated copper grid and placed at 25°C for 10 min. It was then stained with a 3% phosphotungstic acid aqueous solution for 35 s to enhance the contrast of the extracellular membrane. After staining, the grids were air-dried and examined using TEM (Tecnai 12; Philips, Amsterdam, the Netherlands). The size distribution of the American cockroach nanovesicles was analyzed using NanoSight NS300 (Malvern Instruments, Malvern, UK), which was equipped with high-speed video capture capabilities and particle tracking software. In addition, the Zeta potential of the nanovesicles was measured using a Zetasizer nanoparticle size analyzer (Malvern-Panalytic).
[0027] Transmission electron microscopy analysis showed that F-ACNVs and D-ACNVs had a unique cup-shaped morphology ( Figure 1 ). The average particle sizes of F-ACNVs and D-ACNVs were 107.4 nm and 104.3 nm, respectively. In addition, the concentrations of these American cockroach nanovesicles were found to be 1.49×10 12 ±2.11×10 10 Number of particles / mL( Figure 2 ) and 6.57×107±1.02×106 particles / mL( Figure 3 The zeta potential of F-ACNVs and D-ACNVs was measured by dynamic light scattering (DLS), and the values were -13.88±1.08mV and -14.32±1.12mV, respectively. Figure 4 ).
[0028] Cell culture: Human immortalized keratinocytes (HACAT) and human umbilical vein endothelial cells (HUVEC) were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. These cells were stored in a 37°C incubator containing 5% CO2.
[0029] Cell proliferation assay: Cell counting kit-8 (CCK-8) was used to detect the effect of different concentrations of F-ACNVs on the proliferation capacity of HUVEC and HACAT. The cells were seeded in a 96-well plate at a density of 5×103 cells per well and cultured overnight in an incubator at 37°C with a volume fraction of 5% CO2. The next day, the old culture medium was discarded and replaced with complete culture medium containing 0.008-50μg / mL F-ACNVs for 24 hours. After completion, 10μL of CCK-8 solution was added to each well, and the culture dish was placed in an incubator for 2 hours. The absorbance of each well was measured at 450nm using a microplate reader to quantify cell proliferation.
[0030] In vitro cell proliferation experiments showed that F-ACNVs were significantly better than D-ACNVs in promoting HACAT cell proliferation. Figure 5 ) These results suggest that F-ACNVs and D-ACNVs have similar physical properties, but nanovesicles from fresh American cockroaches have superior human immortalized keratinocyte (HACAT) proliferation-promoting activity.
[0031] Cell migration assay: To quantitatively evaluate the effect of F-ACNVs on HUVEC migration, a transwell migration assay was performed using a 24-well plate and a polycarbonate membrane with a pore size of 8.0 μm. HUVECs were suspended in serum-free DMEM medium and seeded into the upper chamber of the transwell at a density of 1×104 cells / cell. 1×PBS or DMEM containing 50 μg / mL F-ACNVs was added to the lower chamber. After 24 h, the non-migrated cells remaining on the upper surface of the membrane were gently removed with a cotton swab. The migrated cells were fixed to the lower surface of the cell membrane and stained with 0.5% crystal violet solution (0.5 g crystal violet dissolved in 100 ml distilled water, g / ml) for 15 min, washed 3 times with PBS solution, and the stained cells were observed under an inverted microscope to obtain images of the stained cells.
[0032] RT-qPCR experiment: Total RNA was extracted from skin tissue using the Biog RNA tissue kit (Biog, Changzhou, China). First-strand cDNA was synthesized using the PrimeScriptTM FAST RT kit (TaKaRa, RR092A) using oligo-dT as the reverse transcription primer. Real-time PCR was performed using an Applied Biosystems instrument (Thermo Fisher Scientific, Singapore). qPCR SYBR Green Master Mix (Yeasen, 11201ES08) was used for quantitative PCR (qPCR). The target mRNA Ct value was normalized to the value of GAPDH (ΔCt) by the comparative threshold (Ct) method to determine the relative expression level of the target gene.
[0033] The sequences of the primers are shown in Table 1 below. -ΔΔCt The fold changes of gene expression were calculated.
[0034]
[0035] Western blot experiments: Skin tissues were collected and homogenized in pre-cooled RIPA buffer (Solarbio, Beijing, China) supplemented with protease inhibitors (Solarbio, Beijing, China). Protein concentration was determined using a BCA assay kit (Pierce, Rockford, IL, USA). Protein samples were separated by 10% SDS-PAGE (CD31) or 6% SDS (Figf) and transferred to 0.22 μm PVDF membranes (Bio-Rad, CA, USA). After blocking with 5% BSA in 1× TBST at 25°C for 2 h, the membranes were incubated with primary antibodies against Figf (1:1000, Proteintech, Chicago, USA), CD31 (1:2000, Proteintech, Chicago, USA) or GADPH (1:4000, Servicebio, Wuhan, China) at 4°C overnight. The membrane was then washed with TBST and probed with HRP-conjugated secondary antibody (1:10000, Servicebio, Wuhan, China) for 1 h at 25° C. Immunoreactive bands were detected using the ChemiDocTM XRS+ imaging system (Bio-Rad, CA, USA) with an enhanced chemiluminescence (ECL) detection kit (Biosharp, Hefei, China).
[0036] Animals: Babl / c male mice (6-8 weeks old, 18-20 g) were purchased from Nanjing Koris Animal Co., Ltd. and were adaptively raised for one week. After the hair on the back of the mouse was shaved short with a razor, a depilatory cream was applied to the back. After the depilatory cream took effect, the remaining hair was shaved with a cotton swab containing 75% alcohol by volume, the skin was cleaned, and the mouse was completely anesthetized with isoflurane, and a circular wound with a diameter of about 1 cm was cut on the back. After the model was completed, 40 μL of F-ACNVs with a concentration of 2.5 mg / m was suspended in physiological saline and evenly dripped onto the wound site to ensure that the F-ACNVs suspension covered all areas of the wound; the control group mice were only given 1× PBS solution. All mice were housed in individual cages, and the wound area was photographed on days 0, 2, 4, and 6 after surgery, and the wound healing rate was calculated using ImageJ1.46r software.
[0037] Skin tissue pathological analysis: The mice were killed on the 12th day after modeling, and the skin wound tissues were collected, fixed in 4% polyformaldehyde solution at 25°C, embedded in paraffin, and sectioned. They were stained with hematoxylin-eosin (H&E) and Masson's trichrome.
[0038] Quantitative and statistical analysis: All data are expressed as standard deviation ± mean, and GraphPad Prism version 9.0 was used for graphical analysis. The data were analyzed by student t-test. P < 0.05 was considered statistically significant. During the wound healing cascade, fibroblasts undergo mitotic proliferation, secrete a large amount of collagen fibers and extracellular matrix components, and generate granulation tissue together with new blood vessels to effectively fill and repair injured tissue defects. Therefore, the proliferation of fibroblasts and the formation of new blood vessels are important for wound healing. Different concentrations of F-ACNVs were co-incubated with HUVEC and HACAT, respectively. The results showed that cell proliferation was significantly accelerated under the treatment of 16.67μg / mL and 50μg / mL F-ACNVs concentrations ( Figure 6 ). Further flow cytometry analysis of cell proliferation showed that compared with the control group, the proliferation rate of HACAT was significantly increased within 1-24 hours after F-ACNVs treatment.
[0039] The repair of damaged tissues during wound repair is closely related to the contractile activity of fibroblasts and the process of re-epithelialization. In order to study the role of F-ACNVs in these processes, Transwell assay was used for analysis. Figure 7 As shown, 50 μg / mL F-ACNVs significantly promoted the 3D migration ability of HUVEC. This result shows that F-ACNVs can significantly promote the migration of HUVEC at the three-dimensional level, suggesting that F-ACNVs plays an important role in promoting skin wound healing.
[0040] In order to further evaluate the in vivo wound healing effect of F-ACNVs, a skin injury model was established by skin excision. Compared with the control group, the wound healing of mouse skin was significantly accelerated after 100 μg F-ACNVs was dripped into the wound every day ( Figure 8 A and 8B). H&E staining showed that the skin tissue of the control mice still showed incomplete epidermal layer formation on the 10th day after wounding, while the wound skin tissue fibroblast layer of the mice in the F-ACNVs treatment group was tightly arranged, with less inflammatory cell infiltration and fewer bubbles, indicating late epithelialization and skin appendage formation, and almost complete regeneration of the epidermis ( Fig. 9 ). Collagen is an important structural component of the extracellular matrix and plays an important role in wound repair. Masson trichrome staining showed that the collagen in the skin tissue of the control group was loosely and sparsely distributed, while the collagen deposition in the F-ACNVs treatment was more orderly and extensive ( Fig. 9 ). These observations suggest that F-ACNVs significantly enhance collagen deposition at the wound site, thereby accelerating the wound healing process.
[0041] To explore the potential mechanism of F-ACNVs in promoting skin wound repair, we performed transcriptome sequencing analysis on healing skin tissues. The results showed that the expression of 407 genes was significantly different between the F-ACNVs treatment group and the control group, with a threshold of |log2-fold change|>1 and a q value of <0.05. Among them, 331 genes were upregulated and 76 genes were downregulated ( Fig.10 A and 10B). KEEG enrichment analysis showed that F-ACNVs treatment resulted in significant upregulation of pathways associated with wound healing, including complement and coagulation cascades, cell adhesion molecules, and the renin-angiotensin system ( Fig.10 C). GO enrichment analysis showed that the down-regulated genes were mainly involved in the negative regulation of gap junction channel activity, cell communication and wound healing ( Fig.10 D). In contrast, the upregulated genes were mainly related to the immune system, immune response and positive regulation of blood vessels ( Fig.10 E). Notably, key angiogenesis-related genes Ccr3 (9.95-fold), Mmp9 (5.71-fold), Ccl11 (2.92-fold), Serpine1 (2.57-fold), Cma1 (2.39-fold), Pgf (2.34-fold), Hmox1 (2.22-fold), and Figf (2.21-fold) were significantly upregulated in the F-ACNVs-treated group ( Fig.10 F). These results suggest that F-ACNVs may exert their wound healing effects by regulating the expression of angiogenesis-related genes, thereby creating a favorable environment for effective skin regeneration and wound healing.
[0042] The expression of angiogenesis-related genes was further verified by RT-qPCR. Fig.10 F), After F-ACNVs treatment, the expression levels of Figf, Pgf, Mmp9 and Hmox1 genes in injured skin tissues increased significantly ( Fig.11 AD). In addition, Cmal, Serpine1, Ccl11, and Ccr3 showed similar trends in expression, although they did not reach significance ( Fig.11 EH). Given the important role of Figf in lymphangiogenesis and angiogenesis, and the established function of CD31 as an endothelial cell marker involved in the angiogenesis process, we used Western blotting to further study its expression level. Our results showed that compared with the control group, the expression of Figf in the F-ACNVs treatment group was significantly increased, and the expression of CD31 was increased ( Fig.11 IL), and Figure 5 These data are consistent with those shown above. These data ultimately prove that F-ACNVs can upregulate the expression of angiogenesis-related genes, thereby promoting wound repair.
[0043] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.
Claims
1. A method for preparing fresh American cockroach nano-vesicles, characterized in that: Fresh American cockroaches are crushed and then centrifuged at 1000×g to 10000×g, where g is the acceleration due to gravity, to remove impurities and cell debris. The mixture is then further centrifuged at 120,000×g, the supernatant is discarded, and the precipitate is resuspended in a buffer solution to obtain a fresh American cockroach nanovesicle suspension.
2. The method for preparing fresh American cockroach nano-vesicles according to claim 1, characterized in that: Fresh American cockroaches were crushed in PBS solution to obtain a suspension; Subsequently, the suspension was centrifuged at 1000-2000×g for 10-20 min, then at 4000-5000×g for 20-30 min, and finally at 1000-2000×g for 20-30 min to remove impurities and cell debris, and further centrifuged twice continuously at 120,000-150,000×g for 70-90 min using an ultrahigh-speed centrifuge, the supernatant was discarded, and the precipitate was resuspended in PBS solution to obtain a fresh American cockroach nanovesicle suspension.
3. Application of fresh American cockroach nanovesicles in the preparation of drugs for cell migration, proliferation or promoting skin wound healing.
4. Application of fresh American cockroach nanovesicles in the preparation of drugs for skin wounds caused by mechanical, burns, scalds and diabetes.
5. Application of fresh American cockroach nanovesicles in the preparation of drugs for promoting proliferation and migration of human umbilical vein endothelial cells, human skin keratinocytes, or repairing skin wounds.