Application of goat milk extracellular vesicles in preparation of products for preventing and / or treating aging

Abstract

The invention belongs to the technical field of medicines and cosmetics, and particularly relates to application of goat milk extracellular vesicles in a product for preventing and / or treating aging. The goat milk extracellular vesicles are used as active ingredients, the content of I-type collagen can be adjusted by adjusting expression of MMP-1, MMP-3 and TIMP-1 in cells, and H2O2-induced cell senescence is further improved by adjusting the content of the I-type collagen, the content of III-type collagen and the content of elastin; in addition, the goat milk extracellular vesicles can also promote proliferation of human skin fibroblasts (HSFs) and inhibit apoptosis, and counteract negative effects of H2O2 on a TGF-beta1 / SMAD pathway, so that generation of collagen in senescent cells is promoted, and growth, migration and repair of the senescent cells are effectively promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and cosmetics, and specifically relates to the application of goat milk extracellular vesicles in preparing products for preventing and / or treating aging. Background Art

[0002] Aging is a continuous process of change. The international mainstream believes that free radicals damage human cells, causing many diseases and accelerating human aging. From generation to quenching, free radicals have a profound impact on the formation of lipofuscin, mutation of mitochondrial DNA, induction of cell apoptosis and protein synthesis, the relationship between free radicals and aging, and the diseases that free radicals can cause. Therefore, it is proposed how to delay aging, which provides a certain scientific basis for humans to slow down their own aging. Various biological molecules in the system have a large number of active groups, which must interact with each other to react chemically so that the biological molecules slowly cross-link to tend to the stability of chemical activity. As time goes by, the degree of cross-linking continues to increase, the active groups of biological molecules continue to consume and reduce, and the original molecular structure gradually changes. The accumulation of these changes will gradually cause biological tissues to gradually show aging phenomena. Oxygen free radicals are the number one killer of accelerated aging of the skin and body.

[0003] Extracellular vesicles (EVs) are nano-sized vesicles secreted by cells. They are vesicle-like structures surrounded by a double layer of phospholipids and carry unique bioactive substances (such as lipids, proteins, miRNAs and secondary metabolites, etc.). In theory, any type of cell can secrete exosomes. However, the function of EVs is closely related to its specific source. The exosomes currently being studied in the field of anti-aging are mainly derived from human induced pluripotent stem cells (IPSC), bone marrow mesenchymal stem cells (MSC) and human skin fibroblasts (HSFs). There is no record of the anti-aging effect of extracellular vesicles from goat milk cells. Summary of the invention

[0004] The purpose of the present invention is to provide the use of goat milk extracellular vesicles in products for preventing and / or treating aging, so as to improve aging induced by oxidative stress, especially to improve H2O2-induced aging of human skin fibroblasts (HSFs).

[0005] The present invention provides application of goat milk extracellular vesicles in products for preventing and / or treating aging.

[0006] Preferably, the prevention and / or treatment of aging includes the prevention and / or treatment of skin aging.

[0007] Preferably, the prevention and / or treatment of aging comprises preventing and / or treating H2O2-induced aging of human skin fibroblasts.

[0008] Preferably, the product for preventing and / or treating aging includes a product that promotes the growth, migration and repair of human skin fibroblasts.

[0009] Preferably, the product for preventing and / or treating aging includes a product that promotes the production of collagen in senescent cells.

[0010] Preferably, the product for preventing and / or treating aging includes a product for promoting the proliferation of human skin fibroblasts in senescent cells.

[0011] Preferably, the method for preparing the goat milk extracellular vesicles comprises: purifying goat milk by sucrose density gradient centrifugation, collecting fractions between 20wt.% and 45wt.% sucrose layers, and obtaining the goat milk extracellular vesicles.

[0012] Preferably, the product comprises cosmetics and / or medicines.

[0013] The present invention also provides an anti-aging product, wherein the active ingredients of the product include goat milk extracellular vesicles.

[0014] Preferably, the concentration of goat milk extracellular vesicles in the product is 0.03-0.07 mg / mL.

[0015] Beneficial effects:

[0016] The present invention uses goat milk extracellular vesicles as active ingredients, which can regulate the content of type I collagen by regulating the expression of MMP-1, MMP-3 and TIMP-1 in cells, and further improve H2O2-induced cell senescence by regulating the content of type I collagen, type III collagen and elastin; goat milk extracellular vesicles can also promote the production of collagen in adult skin fibroblasts, improve cell senescence based on the TGF-β1 / SMAD signaling pathway, effectively promote the growth and migration of senescent cells, and help their repair process. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 The results of viability test of HSFs cells treated with different concentrations of H2O2;

[0019] Figure 2 The results of viability test of HSFs cells treated with different concentrations of GMEVs-1 or GMEVs-2; Compared with the H2O2 group, * p<0.05, ** p<0.01, *** p<0.001;

[0020] Figure 3 The results of HSFs cells' uptake of GMEVs; the scale bar is 50 μm;

[0021] Figure 4 The results of β-galactosidase staining in different treatment groups; the scale bar is 50 μm;

[0022] Figure 5 Fluorescence images of reactive oxygen species in cells of different treatment groups; the scale bar is 50 μm;

[0023] Figure 6 The collagen-I levels in cells of different treatment groups; collagen-I is red, DAPI-stained cell nuclei are blue, and the scale bar is 100 μm;

[0024] Figure 7 Statistical results of collagen-I levels in cells of different treatment groups; compared with the H2O2 group, ** p<0.01, *** p<0.001;

[0025] Figure 8 The Ki-67 levels in cells of different treatment groups; Ki-67 is red, the cell nucleus stained with DAPI is blue, and the scale bar is 100 μm;

[0026] Fig. 9 Statistical results of Ki-67 levels in cells of different treatment groups; compared with the H2O2 group, ** p<0.01, *** p<0.001; compared with GMEVs-1 and GMEVs-2, # p<0.01;

[0027] Fig.10 is the concentration of MMP-1 in cells of different treatment groups; compared with the H2O2 group, ** p<0.01;

[0028] Fig.11 is the concentration of MMP-3 in cells of different treatment groups; compared with the H2O2 group, * p<0.05, **** p<0.01; compared with GMEVs-1 and GMEVs-2, #### p<0.0001;

[0029] Fig.12 is the concentration of TIMP-1 in cells of different treatment groups; compared with the H2O2 group, **** p<0.0001;

[0030] Fig.13 The results of Western Blot detection of type III collagen and elastin in cells of different treatment groups;

[0031] Fig.14 The statistical results of elastin in cells of different treatment groups; compared with the H2O2 group, ** p<0.01, **** p<0.01; compared with GMEVs-1 and GMEVs-2, ### p<0.01;

[0032] Fig.15 The statistical results of type III collagen and elastin in cells of different treatment groups are shown in Figure 2. Compared with the H2O2 group, * p<0.05, ** p<0.01;

[0033] Fig.16 Western Blot detection results of caspase expression in cells of different treatment groups;

[0034] Fig.17 The statistical results of caspase-6 expression in cells of different treatment groups; compared with the H2O2 group, * p<0.05, ** p<0.01; compared with GMEVs-1 and GMEVs-2, ## p<0.01;

[0035] Fig.18 The statistical results of caspase-7 expression in cells of different treatment groups; compared with the H2O2 group, * p<0.05, ** p<0.01;

[0036] Fig.19 The statistical results of caspase-8 expression in cells of different treatment groups; compared with the H2O2 group, * p<0.05;

[0037] Fig. 20 Western Blot detection results of TGF-β1 / SMAD pathway expression in cells of different treatment groups;

[0038] Fig.21 The statistical results of TGF-β1 expression in cells of different treatment groups; compared with the H2O2 group, ** p<0.01;

[0039] Fig. 22The statistical results of SMAD7 expression in cells of different treatment groups; compared with the H2O2 group, * p<0.05, ** p<0.01;

[0040] Fig.23 Statistical results of SMAD2 / 3 expression in cells of different treatment groups; compared with the H2O2 group, * p<0.05, ** p<0.01; compared with GMEVs-1 and GMEVs-2, # p<0.05;

[0041] Fig.24 The microscopic observation results of different treatment groups at 0h and 6h of incubation;

[0042] Fig.25 Cell migration rate of different treatment groups after 6 hours of culture; Compared with the H2O2 group, ** p<0.01; compared with GMEVs-1 and GMEVs-2, # p<0.05. DETAILED DESCRIPTION

[0043] The present invention provides the use of goat milk extracellular vesicles in the preparation of products for preventing and / or treating aging. In the present invention, the prevention and / or treatment of aging preferably includes the prevention and / or treatment of skin aging, and more preferably the prevention and / or treatment of H2O2-induced HSFs aging. The products of the present invention preferably include cosmetics and / or medicines. The products of the present invention for preventing and / or treating aging are preferably anti-aging products.

[0044] In the present invention, the method for preparing the goat milk extracellular vesicles preferably comprises: purifying goat milk by sucrose density gradient centrifugation, collecting the fractions between 20wt.% and 45wt.% sucrose layers, and obtaining the goat milk extracellular vesicles. The present invention preferably uses 45wt.%, 30wt.%, 20wt.% and 5wt.% sucrose solutions to establish a sucrose density gradient, and collects the fractions between 20wt.% and 45wt.% sucrose layers after ultracentrifugation, and further preferably collects the fractions between 20wt.% and 30wt.% sucrose layers, or collects the fractions between 30wt.% and 45wt.% sucrose layers. The speed of the ultracentrifugation of the present invention is preferably 100000g, and the time is preferably 60min.

[0045] The present invention uses goat milk extracellular vesicles as active ingredients, which can regulate the content of type I collagen by regulating the expression of MMP-1, MMP-3 and TIMP-1 in cells, and further improve aging by regulating the content of type I collagen, type III collagen and elastin, especially improve H2O2-induced cell aging; goat milk extracellular vesicles can also promote the proliferation of HSFs induced by H2O2 and inhibit cell apoptosis, offset the negative effect of H2O2 on TGF-β1 / SMAD pathway, thereby promoting the production of collagen in aging cells, effectively promoting the growth and migration of aging cells, and helping their repair process.

[0046] The present invention also provides an anti-aging product, wherein the active ingredient of the product includes goat milk extracellular vesicles. In the present invention, the concentration of goat milk extracellular vesicles in the product is preferably 0.03-0.07 mg / mL, and more preferably 0.05-0.07 mg / mL.

[0047] In order to further illustrate the present invention, the application of goat milk extracellular vesicles provided by the present invention in products for preventing and / or treating aging is described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0048] The chemicals and reagents used in the present invention are as follows:

[0049] Fresh goat milk was obtained from Northwest Agriculture and Forestry University (Xianyang, China); HSFs cells were human skin fibroblasts, i.e., primary cells isolated from the superficial layer of human skin tissue; fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline (PBS), 100× penicillin-streptomycin solution, and 0.25% trypsin solution were purchased from Thermo Fisher Science (MA, American), respectively; chymosin, cell counting kit 8 (CCK-8), and ROS detection kit were purchased from China Solar Energy Technology Co., Ltd., Beijing, respectively; Dio and DAPI were purchased from Sigma-Aldrich (Steinheim, Germany); MMP-1, MMP-3, and TIMP-1 Elisa detection kits were purchased from Uping Biotechnology Co., Ltd.; other chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0050] All data of the present invention were input and analyzed using SPSS22.0 software (SPSS Inc., Chicago, IL, United States). After the data were tested for normal distribution, one-way analysis of variance (ANOVA) and Tukey post hoc test were used to compare the differences between the entire groups. All tests were performed three times, and statistical means and standard deviations were calculated.

[0051] Example 1

[0052] 1. Isolation of Goat Milk Extracellular Vesicles (GMEVs)

[0053] (1) Fresh goat milk samples (n=30) were centrifuged at 4000 g for 15 min at 4°C, the middle layer of the liquid was extracted, treated with 0.0002 g / L chymosin, and incubated at 37°C for 1 h. Subsequently, the supernatant was filtered through a 0.22 μm pore size filter and ultracentrifuged at 135,000 g for 90 min at 4°C. The resulting precipitate was washed with PBS and ultracentrifuged at 135,000 g for 60 min at 4°C to collect the precipitate.

[0054] (2) A sucrose density gradient was established using 45%, 30% and 5% sucrose solutions (mass concentration), and the precipitate obtained in step (1) was placed on the top of the sucrose density gradient layer and centrifuged horizontally at 100000 g (60 min, 4° C.). The fractions between the 45 wt.% to 30 wt.% and 30 wt.% to 20 wt.% sucrose layers were collected, respectively, and named GMEVs-1 and GMEVs-2, and stored at −80° C.

[0055] (3) GMEVs-1 and GMEVs-2 were tested according to the guidelines established by the International Society for Extracellular Vesicles (MISEV2018). The results showed that the average particle sizes of GMEVs-1 and GMEVs-2 were approximately 174.3±1nm and 213.8±3nm, and the polydispersity index (PDI) was 0.144 and 0.108. For GMEVs-1, the zeta potential was -9.11mV, and for GMEVs-2, the zeta potential was -10.15mV. Under transmission electron microscopy, both showed different lipid bilayer membrane structures, characterized by a "cup-and-disc" morphology, which is consistent with the morphological characteristics of EVs. In addition, they both contained marker proteins of HSP70 and TSG101. These findings indicate that the isolated GMEVs-1 and GMEVs-2 are EVs.

[0056] 2. Uptake of GMEV by HSFs

[0057] 2.1. Cell culture and passaging

[0058] HSFs cells adhered to the wall and were cultured in complete medium (DMEM medium containing 10v / v% FBS) in an incubator at 37°C and 5% CO2 saturated humidity. The culture medium was replaced every 1-2 days. When the cell confluence was about 70%-80%, the cells were subcultured (1:3-1:6), and a small amount of culture medium was left in the culture bottle. The cells were digested with TrypLETm Select enzyme (1×) for 2 minutes, centrifuged at 1000g for 5 minutes, the supernatant was discarded, and the complete medium was added and mixed by pipetting, and then cultured in a cell culture incubator.

[0059] 2.2. Cell cryopreservation

[0060] Collect the cells and centrifuge them according to the method in 2.1, add freezing solution (FBS:DMSO=9:1) to the cell pellet, mix them by gently pipetting, and then dispense them into cryopreservation tubes, 1-1.5 mL per tube. Seal them tightly with sealing film, and write the cell name, generation, freezing time and cryopreservation on the cryopreservation tubes; put the cryopreservation tubes into isopropanol gradient freezing boxes, freeze them at -80℃ overnight, and then transfer them to liquid nitrogen tanks for long-term storage.

[0061] 2.3. Cell recovery

[0062] Take out the frozen cells from the liquid nitrogen tank and put them into a 37°C constant temperature water bath. Keep the top of the cryotube above the liquid level to avoid contamination. Thaw them quickly and transfer them to a 15mL centrifuge tube. Centrifuge at 1000g for 5 min at room temperature and discard the supernatant. Add complete culture medium and mix thoroughly by pipetting to form a single cell suspension. Transfer them to a cell culture flask and culture them in a cell culture incubator.

[0063] Example 2

[0064] 1. Cell Viability Detection

[0065] (1) HSFs cells in the logarithmic growth phase were inoculated in a 96-well plate at 100 μL / well, with a cell concentration of 1.0×10 5 Cells / mL, different concentrations of H2O2 (0 (blank), 100, 200, 300, 400, 500, 600, 700, 800 and 900 μmol / L) were added to the cell culture wells, and 6 replicates were set up in each well. After 3 h of cell culture, cell viability was detected by CCK-8 assay. The results are shown in Figure 1 shown.

[0066] (2) HSFs cells in the logarithmic growth phase were inoculated in a 96-well plate at 100 μL / well, with a cell concentration of 1.0×10 5Cells / mL, different concentrations of GMEVs-1 or GMEVs-2 (0, 30, 60, 90, and 120 μg / mL) were added to the cell culture wells, and 6 replicates were set up in each well. After 24 h of cell culture, cell viability was detected by CCK-8 assay. The results are shown in Figure 2 shown.

[0067] according to Figure 1 and Figure 2 It can be seen that H2O2 treatment reduced cell viability in a concentration-dependent manner. A H2O2 concentration of 900 μmol / L was selected to induce oxidative damage in HSF cells, resulting in a decrease in cell viability of approximately 50%. In addition, GMEVs-1 and GMEVs-2 did not show toxic effects on HSF cell viability, therefore, 60 μg / mL of GMEVs-1 and 40 μg / mL of GMEVs-2 were selected for subsequent studies.

[0068] 2. Cellular Uptake of GMEVs

[0069] Under light-proof conditions, GMEVs-1 and GMEVs-2 obtained in Example 1 were resuspended in PBS to a concentration of 100 μg / mL, and PKH26 dye was added at a final concentration of 5 μM. The mixture was incubated at 37°C on a shaker for 20 min, centrifuged at 120,000 g, 4°C for 1 h, the supernatant was discarded (to remove excess dye), resuspended in PBS, filtered with a 0.22 μm filter membrane, and incubated with HSFs cells. The cell nuclei were stained with DAPI fluorescent dye. The uptake of HSFs was detected at different time points (3 h and 6 h), and the cells were observed under an electric inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan). The results showed that DiO-labeled GMEVs gradually entered the cell membrane and transferred to the area around the cell nucleus ( Figure 3 ), indicating that GMEVs can be taken up by HSF cells.

[0070] Example 3

[0071] HSFs cells in the logarithmic growth phase were seeded in 6-well plates at a concentration of 200 μL / well and a cell density of 1.0 × 10 5 The cells were cultured at 37°C and 5% CO2 for 24 h until the cells adhered to the wall and randomly divided into 4 treatment groups. The specific treatment steps are as follows:

[0072] Control group: only culture medium was added;

[0073] Aging model group (H2O2): 900 μmol / L H2O2 was added to the culture medium;

[0074] Experimental group 1 (H2O2+GMEVs-1): 900 μmol / L H2O2 and 40 μg / mL GMEVs-1 obtained in Example 1 were added to the culture medium; GMEVs were treated for 24 h and H2O2 for 3 h;

[0075] Experimental group 2 (H2O2+GMEVs-2): 900 μmol / L H2O2 and 60 μg / mL GMEVs-2 obtained in Example 1 were added to the culture medium; GMEVs were treated for 24 h and H2O2 was treated for 3 h.

[0076] 1. β-Galactosidase staining

[0077] β-Galactosidase staining is a method for staining senescent cells or tissues based on the upregulation of SA-β-Gal activity in senescent cells. The cells in the above-mentioned treatment groups were stained according to the instructions of the kit. Specifically, the treated cells in the 6-well plate were added to the tissue fixative for 15 minutes, washed with PBS, and 1 ml of freshly prepared SA-β-gal staining solution (containing 10 μL β-galactosidase staining solution A and B, 930 μL solution C and 50 μL X-Gal solution) was added to each well, incubated at 37°C overnight, and the cells were observed under an electric inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan). The results showed that the intervention of GMEVs-1 and GMEVs-2 led to a decrease in the proportion of SA-β-GAL positive cells, indicating a decrease in aging-related β-galactosidase activity ( Figure 4 ).

[0078] 2. ROS Determination

[0079] Serum-free medium containing 10 μM DCFH-DA was added to each well of the above treatment groups for ROS detection. The cells were then observed using an intelligent motorized inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan). The results showed that treatment with GMEVs-1 and GMEVs-2 reduced the level of reactive oxygen species (ROS) induced by H2O2 ( Figure 5 GMEVs can improve H2O2-induced cell senescence.

[0080] Example 4

[0081] HSFs cells in the logarithmic growth phase were inoculated in a 12-well plate at 1 mL / well, with a cell concentration of 1×10 5 The cells were cultured at 37°C and 5% CO2 for 24 h until the cells adhered to the wall and randomly divided into 4 treatment groups. The specific treatment steps are as follows:

[0082] Control group: only culture medium was added;

[0083] Aging model group (H2O2): 900 μmol / L H2O2 was added to the culture medium;

[0084] Experimental group 1 (H2O2+GMEVs-1): 900 μmol / L H2O2 and 60 μg / mL GMEVs-1 obtained in Example 1 were added to the culture medium; GMEVs were treated for 24 h and H2O2 for 3 h;

[0085] Experimental group 2 (H2O2+GMEVs-2): 900 μmol / L H2O2 and 40 μg / mL GMEVs-2 obtained in Example 1 were added to the culture medium; GMEVs were treated for 24 h and H2O2 was treated for 3 h.

[0086] 1. Immunofluorescence staining of collagen-Ⅰ and Ki-67 cells

[0087] After intervention in each treatment group, HSFs cells on 12-well cover slips were fixed with 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.5% TritonX-100 in PBS for 20 min. After blocking with immunofluorescence blocking solution, the cells were incubated with anti-Ki67 (1:500) and anti-Collagen-Ⅰ antibodies (1:500) at 4°C overnight and then incubated with fluorescent secondary antibodies (488 nm) for 1 h at room temperature in the dark. Anti-fading mounting medium containing DAPI was applied, and the samples were analyzed using an intelligent electric inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan). The results showed that in the H2O2-induced aging model, type I collagen showed lower fluorescence intensity compared with the control group. In the experimental group, GMEVs-1 and GMEVs-2 promoted the expression of type I collagen in senescent cells ( Figure 6 and Figure 7 ); Ki-67 showed reduced fluorescence intensity levels compared to the control group. In the experimental groups, the addition of GMEVs-1 and GMEVs-2 was associated with upregulation of Ki-67 expression in the senescent cell population (P<0.01)( Figure 8 and Fig. 9 ).

[0088] 2. Detection of MMP-1, MMP-3 and TIMP-1

[0089] According to the instructions of the kit, the enzyme-linked immunosorbent assay (ELISA) was used to detect the content of MMP-1, MMP-3, and TIMP-1 in the cells of each treatment group. The results showed that compared with the control group, the concentrations of MMP-1 and MMP-3 in the cell aging model group increased significantly (P < 0.01), while TIMP-1 decreased significantly (P < 0.01). In the experimental group, after the intervention of GEMVs-1 and GMEVs-2, the levels of MMP-1 and MMP-3 gradually decreased, while the concentration of TIMP-1 in the cells increased significantly (P < 0.01). Therefore, GEMVs-1 and GMEVs-2 can regulate the content of type I collagen by regulating the expression of MMP-1, MMP-3, and TIMP-1 in cells ( Figures 10-12 ).

[0090] 3. Western Blot

[0091] (1) Total protein extraction: Take an appropriate amount of sample from each treatment group, add an equal volume of RIPA lysis buffer (RIPA: PMSF: phosphatase inhibitor = 100:1:1), mix by pipetting and place on ice for 30 min, and oscillate on a vortex mixer for 30 s every 10 min. Centrifuge at 12000g for 10 min at 4°C, and transfer the supernatant to a new eppendorf tube to obtain the GMEVs total protein product.

[0092] (2) Protein quantification: The extracted total protein was quantified according to the instructions of the BCA kit.

[0093] (3) Western Blot: Prepare different concentrations of separation gel (lower layer) according to the molecular weight of the protein. When the separation gel has not solidified, quickly add the concentrated gel (upper layer) and quickly insert it into the electrophoresis. Let it stand for 25 to 40 minutes to fully solidify. Pull out the comb, fix the glass plate on the gel making rack and put it into the electrophoresis tank, add electrophoresis buffer to immerse the gel, and load the sample equally; according to the instructions of the electrophoresis buffer, wait for the maker to run away and the sample to migrate to the bottom of the gel to end the electrophoresis, cut the gel, activate the PVDF membrane with methanol in advance for 1 to 2 minutes, soak the filter paper in the transfer solution, and wet transfer at a constant current of 400mA for 25 to 30 minutes (determine the transfer time according to the protein molecular weight): Place the transferred PVDF membrane in a rapid blocking solution and block it on a shaker at room temperature for 5-15 minutes; after blocking, wash the membrane with the membrane washing solution (PB ST / TBST) for 6 times, 5 min each time, and after absorbing the water, put it in the corresponding protein primary antibody at 4°C overnight; take out the membrane incubated with the primary antibody, wash it 6 times with the membrane washing solution (PBST / TBST), 5 min each time, absorb the water and put it in the corresponding secondary antibody, and incubate it on a shaker at room temperature for 1 hour; after the secondary antibody incubation, wash the membrane 6 times with the membrane washing solution (PBST / TBST), 5 min each time, absorb the water with absorbent paper, and evenly cover the membrane with luminescent liquid, use gel imaging system imaging and ImageJ software for image acquisition and grayscale analysis. The results are shown in Figure 9-10 shown.

[0094] according to Fig. 9 and Fig.10 It can be seen that compared with the control group, the levels of type III collagen and elastin in the cell senescence model group were significantly decreased (P < 0.05). GMEVs-1 and GMEVs-2 promoted the expression of type III collagen and elastin in H2O2-induced senescent cells (P < 0.05) ( Figures 13-15 ). Therefore, GMEVs can improve aging by regulating the content of type I collagen, type III collagen, and elastin. Compared with the cell aging model group, the levels of these caspases in the GMEVs-treated group were significantly reduced (P<0.05). However, in the control group and GMEVs-treated group, the expression of caspase-8 was reduced compared with the cell aging model group, and GMEVs-2 intervention significantly reduced the expression of caspase-8 (P<0.01); H2O2 treatment inhibited the expression of intracellular TGF-β1, SMAD 2 / 3, and SMAD7. However, GMEVs treatment reversed these inhibitory effects and restored the expression of these proteins ( Figures 16 to 23 These results suggest that GMEVs promoted the proliferation and inhibited apoptosis of HSFs cells in H2O2-induced senescence, and could offset the negative effects of H2O2 on the TGF-β1 / SMAD pathway, thereby promoting collagen production in senescent cells.

[0095] Example 5

[0096] Cell wound scratch assay

[0097] The scratch test was performed to analyze the migration ability of HSFs treated with GMEVs-1 or GMEVs-2. The specific steps were as follows: HSFs in the logarithmic growth phase were inoculated with 1.0×10 5 The cells were inoculated into 6-well plates at a concentration of 10 cells / well. After 24 h of culture, a 10 μL pipette tip was used to create cell scratches perpendicular to the plate, and the width of each scratch was as consistent as possible. Then, each well was washed twice with PBS to remove cell debris and randomly divided into 4 treatment groups. The specific treatment steps were as follows:

[0098] Control group (Control): only serum-free medium was added;

[0099] Aging model group (H2O2): serum-free medium containing 900 μmol / L H2O2 was added;

[0100] Experimental group 1 (H2O2+GMEVs-1): serum-free medium containing 900 μmol / L H2O2 and 60 μg / mL GMEVs-1 obtained in Example 1 was added;

[0101] Experimental group 1 (H2O2+GMEVs-1): serum-free culture medium containing 900 μmol / L H2O2 and 40 μg / mL GMEVs-2 obtained in Example 1 was added.

[0102] After 6 h of treatment in each treatment group, cell migration was observed using an intelligent electric inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan) (magnification, ×20), and the scratch area was analyzed using ImageJ software. The cell migration rate was calculated according to the following formula. The results are shown in Fig.24 and Fig.25 shown.

[0103]

[0104] according to Fig.24 and Fig.25 It can be seen that the mobility of the H2O2-induced senescence model group was lower than that of the control group (P<0.01). The addition of GMEVs-1 and GMEVs-2 significantly increased the mobility of senescent cells, bringing it close to the level observed in the control group. This suggests that GMEVs can effectively promote the growth and migration of senescent cells and help their repair process.

[0105] Based on the above content, it can be seen that goat milk extracellular vesicles have the effect of preventing and / or treating aging, especially the ability to improve H2O2-induced HSFs aging.

[0106] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of goat milk extracellular vesicles in the preparation of products for preventing and / or treating aging.

2. The use according to claim 1, characterized in that: The prevention and / or treatment of aging includes the prevention and / or treatment of skin aging.

3. The use according to claim 1, characterized in that: The prevention and / or treatment of aging includes preventing and / or treating H2O2-induced aging of human skin fibroblasts.

4. The use according to claim 1, characterized in that: The products for preventing and / or treating aging include products for promoting the growth, migration and repair of human skin fibroblasts.

5. The use according to claim 1, characterized in that: The products for preventing and / or treating aging include products that promote the production of collagen in senescent cells.

6. The use according to claim 1, characterized in that: The products for preventing and / or treating aging include products that promote the proliferation of human skin fibroblasts in senescent cells.

7. The use according to claim 1, characterized in that: The method for preparing goat milk extracellular vesicles comprises: purifying goat milk by sucrose density gradient centrifugation, collecting fractions between 20wt.% and 45wt.% sucrose layers, and obtaining the goat milk extracellular vesicles.

8. The use according to any one of claims 1 to 7, characterized in that: The products include cosmetics and / or pharmaceuticals.

9. An anti-aging product, characterized in that: The active ingredients of the product include goat milk extracellular vesicles.

10. The product according to claim 9, characterized in that The concentration of goat milk extracellular vesicles in the product is 0.03-0.07 mg / mL.

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