Medicine for treating skin photoaging

Abstract

The invention provides a medicine for treating skin photoaging. The medicine for treating skin photoaging comprises extracellular vesicles derived from adipose tissue. The medicine for treating skin photoaging provided by the invention can recover the activity of human dermal fibroblasts damaged by UVB, including improvement of cell proliferation and migration ability, stabilization of cell cycle, reduction of generation of reactive oxygen species (ROS) and reduction of expression of aging markers.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a medicine for treating skin photoaging. Background Art

[0002] With the improvement of living standards and the intensification of population aging, people's demand for delaying aging and treating photoaging is increasing. Skin aging is a biological phenomenon, including natural aging in the body and aging caused by external factors. Ultraviolet radiation can cause photoaging of human skin and is the most important external environmental factor. It not only affects the appearance of the skin, but also accelerates the damage and aging process of skin cells. At present, there are many strategies for delaying aging and treating photoaging, mainly including the application of chemical antioxidants, physical barrier protection and cell repair technology. However, long-term use of high-concentration antioxidants or sunscreens may cause skin irritation or allergic reactions, increase skin sensitivity, and have potential harm to the skin or human body. The research and application of cell repair technology is costly, involving complex experiments and high-end equipment, making it difficult to promote on a large scale. Therefore, developing a more natural, safe and effective product for treating skin photoaging, thereby achieving the purpose of delaying aging, not only meets market demand, but also provides important support for improving people's quality of life.

[0003] Extracellular vesicles (EVs) are heterogeneous nanoscale membrane vesicles composed of a protein-rich lipid bilayer and proteins and RNA from the cells in which they are produced. EVs are distributed throughout the body through the circulatory system and are messengers in the intercellular communication network, exchanging cargo and information between source and target cells. Since fat contains a large number of biologically active EVs and EVs have multiple effects on systemic physiological functions and homeostasis, adipose tissue-derived extracellular vesicles (AT-EVs) may mediate the aging process through a non-cell autonomous mechanism. EVs are important mediators of signal transmission between cells and between cells and the external environment. Many studies have confirmed that adipocytes have a significant impact on the function of neighboring tissues and organs through the secretion of extracellular vesicles, thereby affecting the aging process and lifespan. From a more macroscopic perspective, whether adipose tissue also regulates the youth and aging state of itself and other organs through the extracellular vesicle pathway is a question worthy of in-depth exploration. Summary of the invention

[0004] In view of the defects in the prior art, the purpose of the present invention is to provide a drug for treating skin photoaging.

[0005] The technical solution of the present invention is: The drug for treating skin photoaging provided by the present invention is characterized in that the drug for treating skin photoaging comprises extracellular vesicles derived from adipose tissue.

[0006] Furthermore, the extraction of extracellular vesicles comprises the following steps: S1. Adipose tissue extraction The collected fat tissue was rinsed, allowed to stand, and centrifuged. After centrifugation, excess water at the bottom was removed and the fat tissue in the middle was retained. The fat tissue in the middle was treated with shock waves at an intensity of 2.5par, a frequency of 4hz, and 30 rounds / cm2, and then irradiated with a red light source for 10 minutes, stirred during the process, and finally treated with ultrasound at an intensity of 1.5w / cm2 and 1.5-2.0 min. S2. Adipose tissue processing Add an equal volume of sterile saline to the adipose tissue in step S1, homogenize on ice at 20,000-25,000 rpm for a certain period of time to make the fat homogenous, discard the upper residual tissue after centrifugation, and retain the lower clear liquid; S3. Adipose tissue processing The lower clear liquid retained in step S2 is sequentially concentrated by microfiltration, ultrafiltration, and low-temperature centrifugation, and then transferred into a sterile cryopreservation tube and stored frozen.

[0007] Further preferably, in step S1, the collected adipose tissue is rinsed with sterile physiological saline.

[0008] Further preferably, in step S1, the adipose tissue is derived from autologous adipose tissue of a liposuction patient.

[0009] Further preferably, in step S1, during centrifugation, the rinsed adipose tissue is placed in a centrifuge and centrifuged at 500-2000 rpm, 37° C., for 5 min.

[0010] Further preferably, in step S2, the homogenization time of the adipose tissue on ice is 1 min.

[0011] Further preferably, in step S2, the fat is processed into a homogenous state, and then centrifuged at 1000-4000g, 4°C, for 5 min, the upper layer of residual tissue is discarded, and the lower layer of clear liquid is retained.

[0012] Further preferably, in step S3, the lower clear liquid is filtered using a filter, placed in an ultrafiltration centrifuge tube with a 100KD membrane, concentrated by centrifugation at 3000g and 4°C to an inner tube volume of 500 μL, transferred into a sterile cryopreservation tube, and transferred to a -80°C refrigerator for cryopreservation.

[0013] More preferably, in step S3, the lower clear liquid is filtered using a 0.22 μm filter.

[0014] Further preferably, the drug for treating skin photoaging includes tablets, capsules, granules, injections, ointments, hydrogels, water injections, powder injections or oral solutions.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a drug for treating skin photoaging, which has at least the following functions: restoring the activity of human dermal fibroblasts damaged by UVB, including improving cell proliferation and migration ability, stabilizing the cell cycle, reducing the generation of reactive oxygen species (ROS) and reducing the expression of aging markers; promoting the synthesis of collagen (Col-1) and inhibiting the expression of matrix metalloproteinase-3 (MMP-3); restoring the normal function of aging cells by regulating multiple aging-related signal pathways such as AMPK, MAPK, autophagy, mitochondrial function and oxidative stress.

[0016] 2. The present invention provides a drug for treating skin photoaging, which includes extracellular vesicles derived from adipose tissue. By conducting in vivo and in vitro experiments on young AT-EVs and old AT-EVs, it is confirmed that young AT-EVs show great potential for treating skin photoaging by regulating cell metabolism and energy balance. These research works have laid a good working foundation for screening new drugs related to the treatment of skin photoaging from extracellular vesicles derived from adipose tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: FIG1 is a schematic diagram of AT-EVs purified from young (20±2.3 years old) and old (55±2.9 years old) human fat provided in the experimental examples of the present invention, and the results of characterizing AT-EVs using TEM, NTA, Western blotting and AT-EVs cell uptake experiments provided in the experimental examples of the present invention (young AT-EVs refers to: young AT-EVs, extracellular vesicles derived from young human adipose tissue; old AT-EVs refers to: old AT-EVs, extracellular vesicles derived from old human adipose tissue; Figure 1 a: TEM images of young AT-EVs and old AT-EVs, scale bar: 200 nm; Figure 1 b: Schematic diagram of the size distribution and concentration of young AT-EVs and old AT-EVs measured using NTA; Figure 1 c: Schematic diagram of NTA quantitative analysis results (n = 3); Figure 1 d: Schematic diagram of the expression results of young AT-EVs and old AT-EVs markers CD63, CD81, TSG101 and GM130 detected by Western blotting; Figure 1 e: Schematic diagram of the cellular uptake experimental results of young AT-EVs and old AT-EVs. Immunofluorescence showed that both young AT-EVs and old AT-EVs were internalized into fibroblasts after 12 hours of incubation); FIG2 is a schematic diagram of the effects of young AT-EVs and old AT-EVs on cell aging phenotypes provided by the experimental examples of the present invention: (control refers to the control group, i.e., no intervention is used; UVB refers to the photoaging group, i.e., photoaging modeling is performed using UVB irradiation; UVB+young AT-EVs: photoaging + young AT-EVs treatment; UVB+old AT-EVs: photoaging + old AT-EV treatment; HDFs refers to human dermal fibroblasts; Figure 2 a: Schematic diagram of the cell survival rate of HDFs cultured with different concentrations of young AT-EVs, the culture time was 72 hours (n = 3), UVB+50ug / mL refers to: photoaging group + 50ug / mL young AT-EVs treatment; UVB+100ug / mL refers to: photoaging group + 100ug / mL young AT-EVs treatment; UVB+150ug / mL refers to: photoaging group + 150ug / mL young AT-EVs treatment; UVB+200ug / mL refers to: photoaging group + 200ug / mL young AT-EVs treatment; UVB+250ug / mL refers to: photoaging group + 250ug / mL young AT-EVs treatment; UVB+300ug / mL refers to: photoaging group + 300ug / mL young AT-EVs treatment;; Figure 2 b: Schematic diagram of the protein expression results of Col1 and MMP3 in the four groups: control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs; Figure 2 c: Schematic diagram of protein expression results of P53 and P16 in the four groups: control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs; Figure 2 d: When the concentration of AT-EVs is 200 μg / mL, the migration results of HDFs in the four groups of control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs after 12 hours of culture are shown in the figure. Scale bar: 250 μm. Figure 2 e: Schematic diagram of the HDFs migration results after 12 hours of culture in the four groups: control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs; Figure 2f: Schematic diagram of SA-β-Gal staining results. Young AT-EVs and old AT-EVs improved the aging of HDFs as assessed by SA-β-Gal staining results. Scale bar = 250 μm. Figure 2 g: Schematic diagram of the statistical results of SA-β-Gal staining); Figure 3 Schematic diagram of the effects of young AT-EVs and old AT-EVs on cell cycle, oxidative stress, metabolic pathways, etc. provided in the experimental examples of the present invention (control: control group, i.e., no intervention was used; UVB: photoaging group, i.e., photoaging model was established using UVB irradiation; UVB+young AT-EVs: after photoaging with UVB irradiation, young AT-EVs were used for treatment; UVB+old AT-EVs: after photoaging with UVB irradiation, old AT-EVs were used for treatment; Figure 3 a: Volcano plot, genes significantly upregulated (red dots) and significantly downregulated (blue dots) in HDFs after UVB irradiation; Figure 3 b: Schematic diagram of KEGG pathway enrichment analysis results of differentially expressed genes (DEGs); Figure 3 c: Volcano plot, proteins that are significantly upregulated (red dots) and significantly downregulated (blue dots) in HDFs after UVB irradiation; Figure 3 d: Schematic diagram of KEGG pathway enrichment analysis results of differentially expressed proteins; Figure 3 e: Schematic diagram of the expression of ROS in HDFs cells in the four groups of control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs under a fluorescence microscope; Figure 3 f: Schematic diagram of the statistical results of ROS expression in the four groups: control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs; Figure 3 g: Schematic diagram of the results of detecting ROS levels of cells using flow cytometry in the four groups: control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs; Figure 3 h: Schematic diagram of the cell cycle distribution results detected by flow cytometry in the four groups: control, UVB, UVB+young AT-EVs, and UVB+old AT-EVs; Figure 3 i: control, UVB, UVB+young AT-EVs, UVB+old AT-EVs, schematic diagram of the proportion of cells in the four groups at each cycle); Figure 4Schematic diagram of the effect of young AT-EVs on the proteome of HDFs provided in the experimental examples of the present invention (Control: young HDFs; UVB: HDFs cells irradiated with UVB at the same time; UVB+young AT-EVs: HDFs cells treated with young AT-EVs after UVB irradiation; Figure 4 a: Schematic diagram of trend analysis results in the order of Control > UVB > young AT-EVs. All data were divided into 10 modules by mathematical models, and 2 modules were screened out after processing. Among them, the proteins in cluster4 and cluster6 showed obvious recovery to normal levels; Figure 4 b: Schematic diagram of KEGG pathway analysis results of differentially expressed proteins, with the 20 most enriched terms displayed in the form of bubble charts; Figure 4 c: Schematic diagram of GO functional enrichment analysis results of differentially expressed proteins classified by “biological process”, “cellular component” and “molecular function”); Figure 5 Schematic diagram of the changes in miRNA content and enrichment analysis results in exosomes derived from adipose tissue during aging provided by the experimental examples of the present invention ( Figure 5 a: Volcano plot showing the miRNAs that are significantly upregulated (red dots) and downregulated (blue dots) in old AT-EVs relative to young AT-EVs; Figure 5 b: Heat map showing the differentially expressed miRNAs in AT-EVs in old (n=4) and young (n=3) adipose tissue (P<0.05 and FC>1.5); Figure 5 c: Schematic diagram of the quantitative changes of miRNAs in old AT-EVs relative to young AT-EVs, with blue boxes indicating lower relative expression levels and red boxes indicating higher relative expression levels; Figure 5 d: Schematic diagram of the third-level results of KEGG pathway analysis of DEGs, showing the ten most enriched terms; Figure 5 e: Schematic diagram of the second-level GO analysis results of predicted DEGs based on the identified differentially expressed miRNAs, classified by “biological process”, “cellular component” and “molecular function”; Figure 5 f: Schematic diagram of the Reactome pathway analysis results of DEGs, showing the twenty most enriched terms in the form of a bubble chart); Figure 6The experimental examples provided in the present invention are provided with a trend analysis in the order of Control > UVB > young AT-EVs, and a schematic diagram of the effects of young AT-EVs on proteins and genes (Control: young HDFs; UVB: HDFs cells irradiated with UVB at the same time; UVB+youngAT-EVs: HDFs cells treated with young AT-EVs after UVB irradiation; Figure 6 a: Schematic diagram of the protein spectrum module results that were significantly recalled by young AT-EVs after HDFs cells were irradiated with UVB; Figure 6 b: Figure 6 In a, the protein spectrum module that was significantly recalled by young AT-EVs after HDFs cells were irradiated with UVB, and the heat map of the specific proteins; Figure 6 c: Schematic diagram of the gene modules that were significantly recalled by young AT-EVs after HDFs cells were irradiated with UVB; Figure 6 d: Figure 6 c, heat map of specific genes in the gene module that was significantly called back by young AT-EVs after HDFs cells were irradiated with UVB); Figure 7 Schematic diagram of the regulatory mechanism of miRNA in young AT-EVs on aged cells after UVB irradiation provided by the experimental example of the present invention ( Figure 7 a: Schematic diagram of the target relationship between characteristic miRNAs in old AT-EVs and young AT-EVs and trend genes in transcriptome sequencing; Figure 7 b: Venn diagram of the overlap between target genes of characteristic miRNAs in old AT-EVs and young AT-EVs and trend genes in transcriptome sequencing; Figure 7 c: Network diagram of the relationship between trend genes and trend proteins in AMPK, MAPK, and autophagy signaling pathways; Figure 7 d: Schematic diagram of the third-level KEGG pathway analysis results of DEGs; Figure 7 e: Schematic diagram of the second-level GO analysis results of DEGs predicted based on the identified differentially expressed miRNAs, classified by “biological process”, “cellular component”, and “molecular function”; Figure 7 f: Trend analysis was performed in the order of Control > UVB > UVB+young AT-EVs. Schematic diagram of trend protein results in different signaling pathways. Control: young HDFs; UVB: HDFs cells irradiated with UVB at the same time; UVB+youngAT-EVs: HDFs cells treated with young AT-EVs after UVB irradiation;); Figure 8Schematic diagram of the gene regulation mechanism of miRNA in young AT-EVs on aged cells after UVB irradiation provided by the experimental example of the present invention ( Figure 8 g: Target trend genes in different signaling pathways, schematic diagram of trend analysis results in the order of Control > UVB > UVB+young AT-EVs; Figure 8 h: Schematic diagram of the results of young AT-EVs improving the effect of UVB on SIRT1 and AKT3 in the skin, Control: skin not irradiated with UV light; UVB: skin irradiated with UVB at the same time; UVB+young AT-EVs: skin treated with young AT-EVs after UVB irradiation; UVB+old AT-EVs: skin treated with old AT-EVs after irradiation); Fig. 9 Schematic diagram of the results of young AT-EVs improving UVB-induced skin photoaging provided by the experimental example of the present invention ( Fig. 9 a: Schematic diagram of establishing an in vivo model of skin photoaging by irradiating the back of nude mice with UV light for 8 weeks; Fig. 9 b: Flow chart of the extraction of AT-EVs from young and old adipose tissues and injection into the back of nude mice once a week for 8 weeks; Fig. 9 c: Schematic diagram of the test results under dermatoscope. After 8 weeks of UV light exposure, the epidermis of the skin photoaging model in vivo was obviously rough and covered with wrinkles, blood vessels in the dermis were visible, and the skin became thinner; Fig. 9 d: Schematic diagram of wrinkle scoring results. The wrinkle scoring results show that the in vivo photoaging model has obvious wrinkles; Fig. 9 e: Schematic diagram of the results of the in vivo model of skin photoaging after 8 weeks of treatment with young AT-EVs and old AT-EVs, respectively. Young AT-EVs significantly improved the degree of keratinization, thickness, and roughness of the skin, but old AT-EVs had no significant effect; Fig. 9 f: Schematic diagram of wrinkle scoring results after 8 weeks of treatment with young AT-EVs and old AT-EVs); Fig.10 Schematic diagram of the staining results of skin tissue sections of animals provided in the experimental examples of the present invention (Control: skin of nude mice without UVB irradiation; UVB: skin of nude mice irradiated with UVB; UVB+young AT-EVs: skin of nude mice treated with young AT-EVs after irradiation; UVB+old AT-EVs: skin of nude mice treated with old AT-EVs after irradiation. Fig.10 a: Schematic diagram of HE tissue staining results of epidermis and dermis; Fig.10 b: Schematic diagram of Masson staining results of epidermis and dermis; Fig.10c: Schematic diagram of the epidermal thickness results of the four experimental groups; Fig.10 d: Schematic diagram of the dermis thickness results of the Control group, UVB group, UVB+young AT-EVs group, and UVB+old AT-EVs group; Fig.10 e: Schematic diagram of the results of staining skin tissue with β-galactosidase; Fig.10 f: Schematic diagram of the expression results of the number of β-galactosidase-positive cells in the Control group, UVB group, UVB+young AT-EVs group, and UVB+old AT-EVs group; Fig.10 g: Schematic diagram of PGP9.5 staining of tissues to observe changes in peripheral nerves; Fig.10 h: Schematic diagram of the expression results of the mean fluorescence intensity of PGP9.5 in the Control group, UVB group, UVB+young AT-EVs group, and UVB+old AT-EVs group; Fig.10 i: Schematic diagram of the results of anti-PCNA immunohistochemical staining to detect cell proliferation in the epidermis and dermis; Fig.10 j: Schematic diagram of the number of PCNA-positive cells in the Control group, UVB group, UVB+young AT-EVs group, and UVB+old AT-EVs group). DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with specific embodiments, and the following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several changes and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention.

[0019] Example This embodiment provides a drug for treating skin photoaging. The drug for treating skin photoaging in this embodiment includes extracellular vesicles derived from adipose tissue. The extraction of the extracellular vesicles includes the following steps: S1. Adipose tissue extraction The collected fat tissue was rinsed, allowed to stand, and centrifuged. After centrifugation, excess water at the bottom was removed and the fat tissue in the middle was retained. The fat tissue in the middle was treated with shock waves with an intensity of 2.5 par, a frequency of 4 Hz, and 30 rounds / cm 2 Then, the red light source was irradiated for 10 minutes, stirring during the process, and finally ultrasonic treatment was used with an ultrasonic intensity of 1.5w / cm 2 , ultrasound for 1.5-2.0 min. The use of shock waves, infrared irradiation, and ultrasound to treat adipose tissue can accelerate the secretion of more extracellular vesicles.

[0020] The collected adipose tissue was rinsed with sterile saline.

[0021] The adipose tissues were derived from the autologous adipose tissue of liposuction patients.

[0022] During centrifugation, the rinsed adipose tissue was placed in a centrifuge and centrifuged at 500-2000 rpm, 37°C, for 5 min.

[0023] S2. Adipose tissue processing Add an equal volume of sterile physiological saline to the adipose tissue from which water has been removed in step S1, homogenize on ice at 20,000-25,000 rpm for a certain period of time to homogenize the fat, discard the upper residual tissue after centrifugation, and retain the lower clear liquid.

[0024] The adipose tissue was homogenized on ice for 1 min to make the fat homogenous, and then centrifuged at 1000-4000g, 4°C, for 5 min, the upper residual tissue was discarded, and the lower clear liquid was retained.

[0025] S3. Adipose tissue processing The lower clear liquid retained in step S2 is sequentially concentrated by microfiltration, ultrafiltration, and low-temperature centrifugation, and then transferred into a sterile cryopreservation tube and stored frozen.

[0026] The lower clear liquid was filtered through a 0.22 μm filter, placed in an ultrafiltration centrifuge tube with a 100KD membrane, concentrated by centrifugation at 3000 g and 4° C. to an inner tube volume of 500 μL, transferred into a sterile cryopreservation tube, and transferred to a -80° C. refrigerator for freezing.

[0027] The medicine for treating skin photoaging provided by the present invention includes tablets, capsules, granules, injections, ointments, hydrogels, water injections, powder injections or oral solutions.

[0028] Experimental example This experimental example uses the following experiments to prove that the AT-EVs contained in the drug for treating skin photoaging provided in the example provide new experimental basis for restoring the activity of UVB-damaged human dermal fibroblasts (HDFs / FB) and revealing the role of AT-EVs in treating skin photoaging.

[0029] 1. Experimental part 1.1) Transmission electron microscopy experiment Dissociated fresh AT-EVs samples (20 μL) were loaded onto carbon-coated copper EM grids for 5 min; negatively stained with 5% uranyl acetate solution (SPI-02624, Hyde Biotech, Beijing, China) for 5 min and observed by transmission electron microscopy; the grids were rinsed three times with PBS to remove excess uranyl acetate solution and kept semi-dry with filter paper; images were observed using a transmission electron microscope (#HT7800, Hitachi, Tokyo, Japan) at 80 kV.

[0030] 1.2) Western blotting After the total protein concentration of AT-EVs was determined, 30 μg of AT-EVs were loaded onto 10%-15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels for electrophoresis; the proteins in AT-EVs were transferred to polyvinylidene fluoride (PVDF) membranes and incubated with primary antibodies such as anti-CD63 (1:1,000, #ab134045), anti-CD81 (1:1,000, #ab109201), anti-TSG101 (1:1,000, #ab125011), and anti-GM130 (1:1,000, #ab52649) (all antibodies were from Abcam, Cambridge, UK); then incubated with secondary antibodies (1:5,000, #111-035-045, Jackson ImmunoResearch, West Grove, PA, United States); BeyoECL Plus Protein expression was assessed by using the ELISA kit (#MA0186, Meilun Biotechnology, Dalian, Liaoning, China).

[0031] 1.3) Cellular uptake of AT-EVs AT-EVs were labeled with CM-Dil red fluorescent membrane-linked dye (C7000, Invitrogen, Waltham, MA, USA). 200 μg of AT-EVs were suspended in 500 μL PBS and labeled with 5 μL CM-Dil stock solution (1 mg / mL); incubation was performed at 37 °C for 5 min and 4 °C for 15 min. After the incubation, unbound CM-Dil was removed by ultrafiltration centrifugation with PBS. FBs (human dermal fibroblasts) were incubated with CM-Dil-labeled EVs (100 μg / mL) for 12 h. FBs were rinsed three times with PBS, fixed with 4% paraformaldehyde, and stained with phosphatidylcholine and DAPI. Cells were observed under a Zeiss Confocal LSM710 microscope (Carl Zeiss, Jena, Germany) to determine the uptake of labeled EVs.

[0032] 1.4) Construction of UVB-induced skin photoaging model All animal experiments were in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental procedures were approved by the Animal Research Committee of the Ninth Affiliated Hospital of Shanghai Jiao Tong University. A total of 80 female BALB / c nude mice (5 weeks old) were used.

[0033] Shanghai Chuansha Experimental Animal Farm (housed in a specific pathogen-free environment). Mice were randomly assigned to 6 groups (n = 10): [1] Control: no UVB irradiation and treatment; [2] UVB group: UVB irradiation + subcutaneous injection of 1 mL PBS / week; [3] UVB+young AT-EVs: UVB irradiation + subcutaneous injection of young AT-EVs (360 ug / mL (week / mouse)); [4] UVB+old AT-EVs: UVB irradiation + subcutaneous injection of old AT-EVs (360 ug / mL (week / mouse)).

[0034] UVB irradiation: Nude mice were irradiated with UVB lamps (Philip, 311 nm, 20 W / 01, Germany) for 8 weeks, 6 times a week; the distance between the animal back and the lamp was 9 cm; the energy density was measured with a UVB energy detector (UV-DETECTOR 150). The irradiation dose in the first week was the minimum erythema dose, i.e., 160 mJ / cm 2 From the 2nd to the 4th week, the radiation dose was increased by 1 / 3 each week compared with the previous week, and the dose was 210 mJ / cm 2 , 280 mJ / cm 2 and 370mJ / cm 2 ; From the 5th week onwards, the radiation dose was maintained at 370mJ / cm 2 Until the end of the 8th week, irradiation was performed 6 days a week.

[0035] AT-EVs were suspended in PBS and injected into the same dorsal subcutaneous position (2 cm × 2 cm) at a dose of 360 ug / mL (week / mouse) for 8 weeks.

[0036] 1.5) Gross observation and dermoscopy experiment During the UVB irradiation modeling of nude mice, the changes in the back skin of each group of nude mice were observed and recorded regularly every day, such as erythema, desquamation, ulceration, etc. 24 hours after the last UVB irradiation, all nude mice were anesthetized with a small animal gas anesthesia machine; after anesthesia, the nude mice were fixed on the operating table with paper tape; the skin detector was connected to the computer, the key was inserted, the software was opened, the epidermis phase was set to observe and photograph the skin roughness and wrinkles, and the roughness module was made with the built-in 3D roughness analysis software; the lens was adjusted to the dermis phase, and the degree of transparency of the subdermal vascular network of the nude mice was observed and photographed to reflect the thickness of the dermis. The thinner the dermis, the easier it is to see the subdermal vascular network; the skin wrinkles were quantitatively scored using a visual scale; treatment was started after the model quality was evaluated, and the above test was repeated after 8 weeks.

[0037] 1.6) Histological staining observation experiment The lower limbs of the nude mice were unfolded, with the angle formed by the junction of the lower limbs and the trunk as the bottom surface and 2 cm upward as the top surface. The skin tissue was taken from the center of the skin on the back of the nude mice, with a size of about 1 cm × 1 cm. The skin tissue was flattened and immediately immersed in a 15 mL centrifuge tube containing 4% paraformaldehyde for fixation. The volume of paraformaldehyde should be more than 1 cm above the skin tissue to ensure that the skin tissue is completely soaked in the fixative; after fixation for 1-2 days, subsequent histological examinations were performed. HE staining, Masson staining, and immunofluorescence staining were performed on the tissue to explore the effect of adipose tissue-derived extracellular vesicles on promoting the recovery of the in vivo photoaging model.

[0038] HE staining steps: (1) Dewaxing and rehydration of paraffin sections: Place the sections in xylene I for 10 min; xylene II for 10 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; 95% alcohol for 5 min; 90% alcohol for 5 min; 80% alcohol for 5 min; 70% alcohol for 5 min; wash with distilled water to remove residual paraffin in the tissue and rehydrate the tissue for subsequent staining.

[0039] (2) Hematoxylin staining of cell nuclei: Place the slices in Harris hematoxylin staining for 3-8 minutes, rinse gently with tap water, then differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, then use 0.6% ammonia water to turn blue, and rinse with running water.

[0040] (3) Place the slices in eosin staining solution and stain for 1-3 minutes.

[0041] (4) Dehydration and sealing: Place the slices in 95% alcohol I for 5 min; 95% alcohol II for 5 min; anhydrous ethanol I for 5 min; anhydrous ethanol II for 5 min; xylene I for 5 min; and xylene II for 5 min to dehydrate and make them transparent. Take the slices out of xylene, let them dry slightly, and then seal them with neutral gum.

[0042] Use a slide scanner to observe, scan images and collect analysis.

[0043] Masson staining: (1) Dewax and rehydrate the sections as usual; then stain with the prepared Weigert iron hematoxylin staining solution for 5 min-10 min.

[0044] (2) Wash thoroughly with water. If over-stained, use hydrochloric acid and alcohol to differentiate.

[0045] (3) Incubate in Masson bluing solution for 3-5 minutes, then rinse with water; then rinse with distilled water for 1 minute.

[0046] (4) Stain with Ponceau fuchsin solution for 5-10 minutes.

[0047] (5) During the above operation, prepare weak acid working solution in the ratio of distilled water to weak acid solution = 2:1, and wash with weak acid working solution for 1 minute.

[0048] (6) Wash with 1% phosphomolybdic acid solution for 1-2 minutes; wash with the prepared weak acid working solution for 1 minute.

[0049] (7) Stain the sample directly in aniline blue staining solution for 1-2 minutes without washing; then wash the sample with the prepared weak acid working solution for 1 minute.

[0050] (8) Rapid dehydration with 95% ethanol; dehydration with anhydrous ethanol three times, each time for 5-10 seconds.

[0051] Transparent with xylene three times, 1-2 min each time; seal with neutral gum.

[0052] Immunofluorescence staining: (1) Dewaxing of paraffin sections: Place the sections in xylene I for 8 min; xylene II for 8 min; xylene III for 8 min; anhydrous ethanol I for 6 min; anhydrous ethanol II for 6 min; 95% alcohol I for 3 min; 95% alcohol II for 3 min; 80% alcohol for 3 min; 75% alcohol for 3 min; and wash with distilled water.

[0053] (2) Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (PH 9.0) in a microwave oven for antigen repair; heat on high for 3 minutes until boiling and then lower the heat for 9 minutes. During this process, prevent the buffer from evaporating excessively and do not let the slides dry; after natural cooling, wash the slides once in PBS (PH 7.4) for 5 minutes.

[0054] (3) Serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), add 10% rabbit serum in the circle to evenly cover the tissue, and incubate in a 37°C oven for 30 minutes.

[0055] (4) Add primary antibody: Gently shake off the blocking solution, and add anti-CK14 (1:100), anti-ki67 (1:100), anti-CD31 (1:100) and anti-CD68 (1:100) prepared in a certain ratio in PBS on the slices; lay the slices flat in a humidified box and incubate overnight at 4°C. Add a small amount of water to the humidified box to prevent the evaporation of the antibody.

[0056] (5) Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash three times, 5 min each time; after the sections are slightly dried, add secondary antibody of the same species as the primary antibody in the circle to cover the tissue (1:400) and incubate at 37°C for 45 min in the dark.

[0057] (6) DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash 4 times, 5 min each time. After slightly drying, add DAPI staining solution in the circle and incubate at room temperature for 10 min in the dark.

[0058] (7) Sealing: Wash the slides three times in distilled water, 5 min each time. Shake the sections dry and seal with anti-fluorescence quenching sealing medium.

[0059] (8) Microscopic examination and photography: The sections were observed and images were collected under an inverted fluorescence microscope (ultraviolet excitation wavelength 330-380 nm, emission wavelength 420 nm; FITC green light excitation wavelength 465-495 nm, emission wavelength 515-555 nm; red light excitation wavelength 510-560 nm, emission wavelength 590 nm).

[0060] 1.7) In vitro ultraviolet irradiation experiment on human dermal fibroblasts Use 96-well plates (2000 cells / well) or 6-well plates (1 × 10 5 Human fibroblasts were cultured in 4% paraformaldehyde (100 cells / well).

[0061] After removing the supernatant, the cells were rinsed twice with PBS and covered with a thin layer of PBS; the cells were exposed to UVB (Philip, 311 nm, 20 W / 01, Germany) with a total dose of 100 mJ / cm 2 After irradiation, PBS was removed and replaced with culture medium, and the cells were cultured for 24 or 72 hours before further experiments.

[0062] 1.8) Cell viability assay The cell viability of HDFs (human dermal fibroblasts) was analyzed using a cell counting kit (CCK-8, #CK04-01). After culturing with CCK-8 for 2 h, the optical density (OD) value was measured using a microplate reader (#CK04-01); the optical density (OD value) was measured at a wavelength of 450 nm using a microplate reader (Thermo Electron Corporation, Waltham, MA, United States).

[0063] 1.9) Cell β-galactosidase (SA-β-Gal) staining experiment Cell SA-β-Gal staining was performed using the Senescence β-galactosidase Staining Kit (C0602, Beyotime, C0602, Beyotime, China); SA-β-Gal-positive cells were observed using an inverted microscope (Carl Zeiss, Oberkochen, Germany).

[0064] Images were taken with an inverted microscope (Carl Zeiss, Oberkochen, Germany) and counts were performed using Image-Pro Plus 6.0 software (Rockville, MD, USA); five fields were randomly selected from each sample (n = 3 samples / group) to determine the cell senescence rate.

[0065] 1.10) Detection of reactive oxygen species (ROS) The intracellular ROS level was detected using a ROS detection kit (S0033, Biotime, China).

[0066] HDFs cells were incubated with DCFH2-DA (2',7'-dichlorodihydrofluorescein diacetate, 10 μM) at 37 °C for 20 min; washed three times with DMEM, and the HDFs cells were exposed to UVB irradiation; half of the FBs were observed.

[0067] The cells were observed and imaged under a fluorescence microscope (Olympus, Tokyo, Japan) at a wavelength of 488 nm, and the other half of the HDFs were collected and quantitatively analyzed using a flow cytometer (BD FACS Calibur, Beckman Coulter).

[0068] 1.11) Cell cycle detection experiment Cell cycle detection was performed 24 h after UVB irradiation using a cell cycle and apoptosis analysis kit (C1052, Bio-Time, China). 5 × 10 5 cells and fixed with 70% ethanol at 4°C overnight; after washing twice with PBS, the cells were incubated with staining solution (a mixture containing 25 μL 20× propidium iodide, 10 μL 50× RNase A, and 0.5 mL staining buffer) at 37°C in the dark for 30 min; the DNA content was determined by flow cytometry (BD FACS Calibur, Beckman Coulter).

[0069] 1.12) Protein spectrum detection experiment Total protein was extracted from young HDFs, HDFs irradiated with UVB at the same time, and HDFs treated with young AT-EVs after irradiation; a portion was taken out for protein concentration determination and SDS-PAGE detection, and another portion was hydrolyzed with trypsin; after desalting the hydrolyzed peptides, the samples were identified by LC-MS / MS; LC-MS / MS identification used DIA technology to collect mass spectrometry data of each sample, perform spectrum matching, extract quantitative information, and perform subsequent statistical analysis.

[0070] 1.13) DIA detection experiment Before mass spectrometry injection, each sample was mixed at a volume ratio of iRT: sample to be tested = 1:20 as an internal standard; equal amounts of peptides were taken from all enzymatically digested samples and separated using U3000 liquid phase, mobile phase A was 0.1% FA aqueous solution, mobile phase B was 0.1% FA in ACN; gradient elution conditions: 0-48 min, 5-22% B; 48-53 min, 22-35% B; 53-56 min, 35-90% B; 56-57 min, 90% B; 57-60 min, 3% B; the peptides were separated by an ultra-high performance liquid phase system and injected into a timsTOF Pro mass spectrometer (Bruker) for analysis. The mass spectrometry conditions were as follows: capillary voltage of 1.5 KV, drying gas temperature of 180 °C, drying gas flow rate of 3.0 L / min, mass spectrometry scanning range of 100-1700 m / z, and ion mobility range of 0.7-1.3 Vs / cm2, and the collision energy range is 20-59 eV.

[0071] 1.14) Protein qualitative, quantitative and functional analysis experiments Spectronaut Pulsar™ 18.4 (Biognosys, Swiss) software was used to process the DIA raw data. The mass spectrometry search parameters were precursor mass value threshold of 0.01, protein mass value threshold of 0.01, fixed modification of carbamidomethyl (C), variable modification of oxidation (M) and acetyl (N-term), and maximum missed cleavage sites of 2.

[0072] Differential proteins must meet the conditions of P<0.05 and FC>1.2 or FC<1 / 1.2. When P<0.05 and FC>1.2, they are significantly up-regulated proteins, and when P<0.05 and FC<1 / 1.2, they are significantly down-regulated proteins. The Gene Ontology (GO) database was used to analyze the biological process (BP), cellular component (CC), and molecular function (MF) of differentially expressed proteins according to their biological functions and classifications. The Kyoto encyclopedia of genes and genomes (KEGG) database was used to analyze the main pathways involved in differentially expressed proteins. The protein protein interaction (PPl) analysis of differentially expressed proteins was performed based on the string database, and a differential protein interaction network was constructed.

[0073] 1.15) RNA extraction and library construction experiments Total RNA was extracted using TRIzol reagent. RNA purity and quantification were determined using a NanoDrop 2000 spectrophotometer (ThermoScientific, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (AgilentTechnologies, Santa Clara, CA, USA). Transcriptome libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit. Transcriptome sequencing and analysis were performed by Shanghai Ouyi Biotechnology Co., Ltd. (Shanghai, China).

[0074] 1.16) RNA sequencing and differentially expressed gene analysis experiments The library was sequenced using the Illumina Novaseq 6000 sequencing platform, and 150 bp double-end reads were generated, with a total of 81.46G CleanData. Fastp software was used to process the raw reads in fastq format, and clean reads were obtained after removing low-quality reads for subsequent data analysis; HISAT2 software was used for reference genome alignment and gene expression (FPKM) calculation; PCA analysis and drawing of genes (counts) were performed using R (v3.2.0) to evaluate sample biological repeatability.

[0075] DESeq2 software was used for differentially expressed gene analysis, where genes that met the thresholds of P < 0.05 and FC > 1.5 were defined as differentially expressed genes (DEGs). GO and KEGG Pathway enrichment analysis was performed on differentially expressed genes based on the hypergeometric distribution algorithm to screen for significantly enriched functional items. GSEA software was used for gene set enrichment analysis. Using predefined gene sets, genes were ranked according to the degree of differential expression in the two types of samples, and then it was tested whether the pre-set gene set was enriched at the top or bottom of this ranking table.

[0076] 1.17) Small RNA library construction experiment Total RNA was extracted using a kit (Qiagen miRNeasy Serum / Plasma Kit); the total amount and integrity of RNA were assessed using Nanodrop2000 (Thermo Fisher Scientific Inc., USA) and Agilent 2100 Bioanalyzer (Agilent Technology, USA), respectively; a small RNA library was constructed using a kit (NEBNext Small RNA Library Prep Set for Illumina, Cat. No. NEB#E7330S, NEB, USA). After the library quality was qualified by Agilent 2100 Bioanalyzer, it was sequenced using the Illumina Novaseq 6000 platform to generate 150 bp double-end sequences. Small RNA sequencing and analysis were performed by Ouyi Biotechnology Co., Ltd. (Shanghai, China).

[0077] 1.18) Small RNA sequencing analysis The raw image data files obtained by high-throughput sequencing were converted into raw sequencing sequences through base calling analysis, and low-quality reads were further filtered to obtain clean reads. First, the length distribution of clean reads in the reference genome was compared with the Rfam v10.1 database (http: / / www.sanger.ac.uk / software / Rfam) using Bowtie software, and rRNA, scRNA, Cis-reg, snRNA, tRNA and other sequences were annotated and filtered; Bowtie software was then used to compare and annotate with cDNA sequences, species repeat sequence library Repbase database, and miRBase database (http: / / www.mirbase.org / ) in turn to remove degraded transcript sequences and repeat sequences, and to identify and annotate known miRNAs, and analyze the expression patterns of known miRNAs in different samples.

[0078] When calculating differentially expressed miRNAs, the default thresholds for filtering were P < 0.05 and FC > 1.5.

[0079] When predicting target genes, the target genes of differential miRNAs were predicted using miranda software with the following parameters: S ≥ 150, ΔG ≤ −20 kcal / mol, and Demand strict 5' seed pairing.

[0080] The GO enrichment and KEGG pathway enrichment analyses were performed on the target genes of differentially expressed miRNAs based on the hypergeometric distribution using the R package.

[0081] 1.19) Statistical analysis Numerical data are expressed as mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) and Tukey post-test. Statistical analysis was performed using SAS (SAS Institute Inc.). P < 0.05 indicated significant differences.

[0082] Results and Analysis 2.1) AT-EVs were purified from young (20 ± 2.3 years) and old (55 ± 2.9 years) human fat and characterized using TEM, NTA, Western blotting, and cellular uptake of AT-EVs. Figure 1 As shown. (youngAT-EVs: young AT-EVs; old AT-EVs: old AT-EVs) AT-EVs were purified from young (20±2.3 years) and old (55±2.9 years) human fat. Transmission electron microscopy (TEM) showed that most AT-EVs purified from young and old human fat showed a characteristic round vesicle morphology and varied in size, but purified young AT-EVs had a more complete characteristic morphology and size ( Figure 1 a). Nanoparticle tracking analysis (NTA) showed that the particle size of EVs in young fat and old fat was similar, with a peak of about 115 nanometers and the same order of magnitude. There were 1.8×10 EVs per milliliter in young fat. 10 There are 1.5×10 particles per milliliter in old age fat. 10 Particles ( Figure 1 b, Figure 1 c, Nanoparticle tracer analysis (NTA): The collected AT-EVs samples were detected and reported by the Zeta View PMX 110 instrument); Immunoblotting analysis showed that the purified young and old AT-EVs were highly enriched with classic EVs markers (CD63, CD81, and TSG101), but without GM130, indicating that the isolated AT-EVs were not contaminated by plasma and intracellular components (Figure 1d); After the purified young and old AT-EVs were cultured with HDFs for 12 hours, AT-EVs labeled with red fluorescence (CM-Dil) were observed to be internalized into HDFs (Figure 1e).

[0083] 2.2) Effects of young AT-EVs and old AT-EVs on cell senescence phenotypes. Figure 2 As shown, young AT-EVs can improve the activity and function of HDFs and improve ultraviolet B (UVB)-induced senescence. Control: HDFs without UVB irradiation; UVB: HDFs irradiated with UVB; UVB+young AT-EVs: HDFs treated with young AT-EVs after irradiation; UVB+old AT-EVs: HDFs treated with old AT-EVs after irradiation.

[0084] HDFs irradiated with UVB were incubated with young AT-EVs at 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, and 300 μg / mL for 72 hours. The CCK-8 results showed that UVB significantly reduced cell activity. However, compared with the UVB group, cell activity also increased significantly in a dose-dependent manner within the range of 50 μg / mL to 200 μg / mL, but did not increase significantly at 250 μg / mL and 300 μg / mL ( Figure 2a). Young AT-EVs at a concentration of 200 μg / mL were selected for subsequent in vitro experiments ( Figure 2 b- Figure 2 g). Compared with the Control group, the expression of Col-1 in the UVB group was significantly reduced, and the expression of MMP-3 was significantly increased. The treatment of the UVB+young AT-EVs group significantly alleviated the photoaging effect observed in the UVB group, but the UVB+oldAT-EVs group showed higher expression of Col-1 and lower expression of MMP-3. Compared with the UVB group, the UVB+old AT-EVs group did not have a significant improvement effect ( Figure 2 b). Compared with the Control group, the expression of P53 and P16 in the UVB group increased significantly. P53 and P16 are important tumor suppressor proteins and transcription factors, playing a key role in cell cycle regulation, DNA repair, apoptosis, aging and genome stability maintenance. The UVB+young AT-EVs group showed lower expression of P53 and P16, indicating that the use of UVB+young AT-EVs treatment significantly reduced the cell aging effect observed in the UVB group, but the UVB+old AT-EVs group did not show significant improvement compared with the UVB group ( Figure 2 c). The scratch experiment showed that young AT-EVs could improve the slowing of HDF cell migration caused by UVB irradiation, but the use of old AT-EVs had no such effect ( Figure 2 d, Figure 2 e). Compared with the Control group, the UVB group showed strong SA-β-Gal staining, while the UVB+young AT-EVs group showed a significant decrease in SA-β-Gal staining, but the UVB+old AT-EVs group had no significant effect compared with the UVB group, indicating that the use of old AT-EVs to treat aged HDFs cells cannot improve photoaging and may further promote their aging ( Figure 2 f, Figure 2 g).

[0085] 2.3) Young AT-EVs improve cell aging by affecting cell cycle, oxidative stress, metabolic pathways, etc. Figure 3 Control: HDFs without UVB irradiation; UVB: HDFs irradiated with UVB; UVB+young AT-EVs: HDFs treated with young AT-EVs after irradiation; UVB+old AT-EVs: HDFs treated with old AT-EVs after irradiation.

[0086] Photoaging leads to significant changes in the expression of some genes and proteins. Figure 3As shown in a-3d, the volcano plot of genes significantly upregulated (red dots) and significantly downregulated (blue dots) in HDFs after UVB irradiation is shown in Figure 3 a; KEGG pathway analysis results of differentially expressed genes are shown in Figure 3 b. After UVB irradiation, the volcano map of proteins that are significantly upregulated (red dots) and significantly downregulated (blue dots) in HDFs is shown in Figure 3 c; KEGG pathway analysis of differentially expressed proteins is shown in Figure 3 d. These results indicate that these genes and proteins are significantly enriched in pathways related to the cell cycle, oxidative stress, and metabolism. In order to elucidate the mechanism by which young AT-EVs improve skin photoaging, the generated reactive oxygen species (ROS) were stained, observed under a fluorescence microscope, and detected using a flow cytometer. Compared with the control group, the average DCF fluorescence intensity (representing the number of ROS) in the UVB group was significantly increased. However, the average DCF fluorescence intensity in the UVB+youngAT-EVs group was significantly lower than that in the UVB group, while treatment with old AT-EVs had no such effect. The results indicate that young AT-EVs can improve the generation of reactive oxygen species in HDFs cells caused by UVB irradiation, but old AT-EVs do not have this function ( Figure 3 e- Figure 3 g). Flow cytometry results showed that the number of cells in the G1 phase increased significantly due to UVB irradiation, and the cell cycle arrest in the UVB+young AT-EVs group was improved and returned to a level close to that of the control group, while the cell cycle arrest in the UVB+oldAT-EVs group was still obvious and even aggravated ( Figure 3 h, Figure 3 i).

[0087] In summary, young AT-EVs can alleviate cell cycle arrest and cellular aging related to oxidative metabolism, but the use of old AT-EVs has no such effect. HDFs cells treated with old AT-EVs show accelerated aging.

[0088] 2.4) Young AT-EVs improve the proteome of aged HDFs, as shown in the following results Figure 4 shown.

[0089] Control: young HDFs cells; UVB: HDFs cells irradiated with UVB at the same time; UVB+young AT-EVs: HDFs cells treated with young AT-EVs after irradiation. In order to reveal the mechanism of young AT-EVs treatment at the proteomic level, data-independent acquisition (DIA)-based proteomics quantification and proteomic analysis were performed on a single sample. A total of 8028 proteins were identified, and 7937 proteins were quantified. The fuzzy clustering principle of the R package Mfuzz was used to correspond the proteins to several clusters. By calculating the protein membership value under each cluster, the degree of protein belonging to a certain cluster was intuitively felt, so as to understand the overview of the proteomic level changes induced by young AT-EVs to improve aging. Although the expression levels of most proteins did not differ significantly among the control group, UVB group, and UVB+youngAT-EVs group, some proteins showed significant upward or downward adjustments after treatment. Taking the level after treatment as close to that of the control group as possible as the standard, the proteins in cluster 4 and cluster 6 showed significant adjustments back to normal levels. There were 68 proteins in cluster 4 that adjusted upward, and 38 proteins in cluster 6 that adjusted downward ( Figure 4 a). Subsequently, KEGG ( Figure 4 b) and GO( Figure 4 c) The database performed enrichment analysis on the above 106 proteins (68 proteins in cluster4 and 38 proteins in cluster6) to further clarify the impact of proteomic changes induced by young AT-EVs on biological processes. The KEGG pathway enrichment results showed that there was significant enrichment in the AMPK signaling pathway, MAPK signaling pathway, PPAR signaling pathway, ferroptosis, NOD receptor signaling pathway, and Rap1 signaling pathway, indicating that young AT-EVs may improve cell aging by regulating energy metabolism and cell homeostasis. The 999 GO terms were clustered and enriched into 3 types of GO functions, as shown below: Group 1 was more specifically enriched in “biological-process” (typical terms: mitotic spindle organization, regulation of centriole duplication, glial migration, regulation of protein degradation, regulation of BMP signaling, mitochondrial fission) Group 2 is closely related to "cellular-component". (Typical terms: chromosome passenger complex, spindle pole centrosome, collagen-containing extracellular matrix, spindle midzone, Golgi cavity) Group 3 is closely related to “molealar-funcyion” (typical terms: rRNA binding, nuclear steroid receptor activity, glycosaminoglycan binding, NF-κB binding, ADP binding).

[0090] The results showed that protein treatment with young AT-EVs resulted in similar protein change trends in multiple GO entries. These results reflect that young AT-EVs can reverse age-related damage through multiple mechanisms and exert a comprehensive rejuvenating effect.

[0091] 2.5) Changes in miRNA content and enrichment analysis in adipose tissue-derived extracellular vesicles during aging. The results are as follows Figure 5 shown.

[0092] The specific microRNAs in AT-EVs change with age, but it is still unclear which component of young AT-EVs is responsible for the rejuvenation effect. The inherent structure of AT-EVs enables efficient transfer of information molecules, including proteins, lipids, DNA, and RNAs, between cells. To date, a great deal of research has been devoted to understanding the RNA components of EVs, especially microRNAs (miRNAs). In fact, miRNAs encapsulated in AT-EVs can be transferred to recipient cells, where they can have important functional effects on target genes and cellular pathways.

[0093] Small RNA sequencing was used to detect the miRNA profiles in young AT-EVs and old AT-EVs. According to the criteria of P < 0.05 and FC > 1.5, 120 miRNAs were significantly differentially expressed in old AT-EVs compared with young AT-EVs, 71 of which were significantly upregulated and 49 were significantly downregulated ( Figure 5 a). Based on the AT-EVs miRNA profile, hierarchical clustering analysis can clearly distinguish old fat samples (old) from young fat samples (young) ( Figure 5 b), indicating that young AT-EVs and old AT-EVs contain significantly different miRNAs. The sequencing results of small RNA sequencing showed that ( Figure 5c) There were 12 miRNAs with large differences between the old group and the young group, among which the elderly AT-EVs had 9 higher miRNAs and 3 lower miRNAs than the young AT-EVs. The contents of hsa-miR-1-3p, hsa-miR-19b-3p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-328-3p, hsa-miR-34a-5p, and hsa-miR-423-5p were significantly increased in the elderly AT-EVs ( Figure 5 c), while the contents of hsa-let-7a-5p, hsa-let-7e-5p, and hsa-let-7f-5p were significantly increased in young AT-EVs ( Figure 5 c), therefore, hsa-let-7a-5p, hsa-let-7e-5p, and hsa-let-7f-5p are considered to be representative contents of young AT-EVs, while hsa-miR-1-3p, hsa-miR-19b-3p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-328-3p, hsa-miR-34a-5p, and hsa-miR-423-5p are the characteristics of elderly AT-EVs. The target genes of the above 12 miRNAs were enriched using the KEGG, GO, and Reactome databases ( Figure 5 d, Figure 5 e, Figure 5 f), the results showed that during the aging process, miRNAs in AT-EVs changed in AMPK signaling pathway, MAPK signaling pathway, collagen chain trimer formation, elastic fiber formation, O-glycosylation and metabolic process, immune system process, extracellular region, organelle part, and antioxidant activity function, all of which were crosstalked with aging and anti-aging. This discovery not only deepens the understanding of the aging mechanism, but also provides new directions and potential targets for anti-aging research and treatment.

[0094] 2.6) Trend analysis was performed in the order of Control group > UVB group > UVB+young AT-EVs group to explore the effects of young AT-EVs on proteins and genes. The results are as follows Figure 6 , Figure 7 , Figure 8 shown.

[0095] Young AT-EVs improve skin aging by affecting the AMPK pathway. There are 68 proteins that are first down-regulated and then up-regulated, and 38 proteins that are first up-regulated and then down-regulated ( Figure 6 a, Figure 6 b). In order to explore the possible mechanism by which young AT-EVs improve aging HDFs, we collected Control (young HDFs cells), UVB (HDFs cells irradiated with UVB at the same time), and UVB+young AT-EVs (HDFs cells treated with young AT-EVs after irradiation), and performed transcriptome sequencing on each sample to find genes with opposite expression trends in the UVB group compared to the Control group, and in the UVB+youngAT-EVs group compared to the UVB group. The samples were analyzed for trend analysis in the order of Control >UVB > UVB+young AT-EVs. All data were divided into 8 modules through mathematical models, and a total of 5 modules that were called back after processing were screened (modules 2, 3, 4, 6, and 7), and module trend graphs and heat maps were drawn. The results showed that ( Figure 6 c, Figure 6 d), there are 800 genes that are first down-regulated and then up-regulated, and 635 genes that are first up-regulated and then down-regulated. The intersection of these and the 12 miRNA target gene prediction results described in 2.5) was used to screen out 817 common genes ( Figure 7 b: Venn diagram of the overlap between target genes of characteristic miRNAs in old AT-EVs and young AT-EVs and trend genes in transcriptome sequencing).

[0096] The target relationship results of 12 characteristic miRNAs in old AT-EVs and young AT-EVs and trend genes in transcriptome sequencing are as follows Figure 7 As shown in a, these 12 miRNAs can affect and intervene in many target genes.

[0097] The relationship network diagram of trend genes and trend proteins in AMPK, MAPK, and autophagy signaling pathways is shown in the figure Figure 7 As shown in Figure c, AMPK, MAPK, and autophagy signaling pathways are closely related to skin repair and anti-aging, and the target genes of these 12 miRNAs play an important role in these three pathways, indicating that further research on the functions of these miRNAs and their target genes can bring new breakthroughs in the field of skin repair and anti-aging.

[0098] The third-level KEGG pathway analysis of DEGs showed that Figure 7 As shown in d, DEGs are the genes in the above cross-sets, showing the ten most enriched terms. These 12 miRNAs are involved in lifespan regulation, gene homologous recombination, mismatch repair and other pathways, indicating that they may play an important role in regulating these processes.

[0099] The KEGG and GO databases were used to perform enrichment analysis on each gene. The KEGG results showed that the AMPK signaling pathway, MAPK signaling pathway, autophagy, NOD receptor signaling pathway, ferroptosis, calcium signaling pathway, etc. were enriched, and there were associations and interactions between them ( Figure 7 e); and also enriched in functions such as nuclear-cytoplasmic transport, longevity regulation pathway, mismatch repair and developmental process, immune system process, metabolic process, antioxidant activity, and translation regulation activity. Focusing on the expression of proteins and genes in the above pathways, the results showed that in the AMPK signaling pathway, MAPK signaling pathway, autophagy, NOD receptor signaling pathway, RAP1 signaling pathway and other signaling pathways, young AT-EVs significantly improved the expression disorders of proteins and genes caused by aging ( Figure 7 f, Figure 8 g). It is worth noting that the AMPK signaling pathway is a recognized pathway for regulating aging, which has been reported in many previous studies. Among the factors related to the AMPK signaling pathway, the expression levels of SIRT1, AKT3, and PRKAA2 were all shown to be regulated by young AT-EVs, and this result was consistent with the characteristics of miRNA in AT-EVs. The AMPK signaling pathway delays aging through multiple mechanisms, including regulating energy metabolism, autophagy, NAD+ / SIRT1 signaling pathway, and miRNA expression.

[0100] Animal skin tissue sections were stained, and the results were as follows Figure 8 As shown in h, young AT-EVs improved the effects of SIRT1 and AKT3 in the skin caused by UVB. UVB reduced the expression of SIRT1 and AKT3 in the skin. After treatment with young AT-EVs, the expression of SIRT1 and AKT3 increased, which is consistent with the improvement of skin photoaging under the influence of the AMPK pathway, but the use of old AT-EVs had no such effect.

[0101] 2.7) Young AT-EVs can improve UVB-induced skin photoaging. Fig. 9 Control: nude mouse skin without UVB irradiation; UVB: nude mouse skin treated with PBS after UVB irradiation; UVB+youngAT-EVs: nude mouse skin treated with young AT-EVs+PBS after UVB irradiation; UVB+old AT-EVs: nude mouse skin treated with old AT-EVs+PBS after UVB irradiation.

[0102] In vivo models of skin photoaging Fig. 9As shown in a, the backs of nude mice were placed under UV light for 8 weeks to establish an in vivo model of skin photoaging. To simulate the natural concentration of AT-EVs in fat, purified AT-EVs were dissolved in PBS and adjusted to 360 μg total protein per ml, and then injected subcutaneously. The operation process is as follows Fig. 9 As shown in b; the nude mice were divided into four groups (Control, UVB, UVB+young AT-EVs, UVB+old AT-EVs) to create an in vivo model of skin photoaging, refer to Fig. 9 a, The backs of four groups of nude mice were irradiated with UV light for 8 weeks to establish an in vivo skin photoaging model. After 8 weeks, gross observation and clinical wrinkle scoring results were shown (Fig. 9c, Fig. 9 d, ), the UVB group, UVB+youngAT-EVs group, and UVB+old AT-EVs group all showed obvious skin keratinization and wrinkles compared with the Control group, with thinning skin and significantly increased roughness. The polarized light mode showed ultraviolet-induced skin thinning, and subcutaneous blood vessels were visible, indicating that the dermis was thinning. The above-mentioned UVB+young AT-EVs group and UVB+old AT-EVs group were injected with young AT-EVs+PBS and old AT-EVs+PBS to treat the back skin of nude mice, and the UVB group was injected with PBS to treat the back skin of nude mice. After 8 weeks of treatment, gross observation and clinical wrinkle scoring results showed that injection of young AT-EVs can significantly improve skin thinning; the three-dimensional roughness of the skin was consistent, indicating that injection of young AT-EVs can reduce skin roughness, but the UVB+old AT-EVs group and the UVB group had no obvious effect (Figure 9e, Fig. 9 f, The photoaging model in vivo at this time had undergone a total of 16 weeks of treatment, including 8 weeks of modeling and 8 weeks of injection treatment).

[0103] 2.8) Animal skin tissue sections were stained, and the results were as follows Fig.10 As shown, Control: nude mouse skin without UVB irradiation; UVB: nude mouse skin irradiated with UVB; UVB+young AT-EVs: nude mouse skin treated with young AT-EVs after irradiation; UVB+old AT-EVs: nude mouse skin treated with old AT-EVs after irradiation.

[0104] HE staining combined with Masson staining results showed ( Fig.10 a— Fig.10d) Compared with the Control group, the epidermis of the UVB group was thicker and the dermis was thinner. No obvious thickening of the epidermis and thinning of the dermis was observed in the UVB+young AT-EVs group, indicating that the injection of young AT-EVs can treat skin damage caused by UVB irradiation. However, there was no difference between the UVB+old AT-EVs group and the UVB group, indicating that the injection of old AT-EVs could not repair skin damage.

[0105] To further assess aging, skin tissue was stained with β-galactosidase, and the results showed that ( Fig.10 e. Fig.10 f) During skin photoaging, UVB can significantly induce the expression of β-galactosidase, while young AT-EVs can inhibit this effect, but old AT-EVs have no significant inhibitory effect on the expression of β-galactosidase. Therefore, histological evaluation showed that young AT-EVs can significantly improve UVB-induced photoaging of mouse skin, but old AT-EVs have no significant effect.

[0106] In order to observe the changes in peripheral nerves, the tissues were stained with PGP9.5, and the results are shown in Figures 10g, Fig.10 As shown in h, after 8 weeks of UVB irradiation, PGP9.5 expression decreased, and after treatment with young AT-EVs, PGP9.5 expression increased and peripheral nerve abundance improved, but treatment with old AT-EVs had no obvious effect. This indicates that young AT-EVs can repair aging and damaged peripheral nerves and promote their regeneration, but old AT-EVs treatment does not have this function.

[0107] Cell proliferation in the epidermis and dermis was measured by anti-PCNA immunohistochemical staining (Fig. Fig.10 j). Compared with the Control group, after UVB irradiation, the cell proliferation index in the epidermis and dermis of the UVB group decreased significantly, and the proliferation index of the epidermis and dermis of the UVB+youngAT-EVs group was significantly higher than that of the UVB group, but the UVB+old AT-EVs group did not show significant improvement. This indicates that young AT-EVs can repair cells in the aging and damaged epidermis and dermis and promote proliferation and regeneration, but the use of old AT-EVs does not have this function.

[0108] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. A drug for treating skin photoaging, characterized in that: The drug for treating skin photoaging comprises extracellular vesicles derived from adipose tissue.

2. The drug for treating skin photoaging according to claim 1, characterized in that: The extraction of the extracellular vesicles comprises the following steps: S1. Adipose tissue extraction The collected fat tissue was rinsed, allowed to stand, and centrifuged. After centrifugation, excess water at the bottom was removed and the fat tissue in the middle was retained. The fat tissue in the middle was treated with shock waves with an intensity of 2.5 par, a frequency of 4 Hz, and 30 rounds / cm 2 Then, the red light source was irradiated for 10 minutes, stirring during the process, and finally ultrasonic treatment was used with an ultrasonic intensity of 1.5w / cm 2 , ultrasound 1.5-2.0 min; S2. Adipose tissue processing Add an equal volume of sterile physiological saline to the adipose tissue in step S1, homogenize on ice at 20,000-25,000 rpm in a homogenizer to make the fat homogenous, discard the upper residual tissue after centrifugation, and retain the lower clear liquid; S3. Adipose tissue processing The lower clear liquid retained in step S2 is sequentially concentrated by microfiltration, ultrafiltration, and low-temperature centrifugation, and then transferred into a sterile cryopreservation tube and stored frozen.

3. The drug for treating skin photoaging according to claim 2, characterized in that: In step S1, the collected fat tissue is rinsed with sterile saline.

4. The drug for treating skin photoaging according to claim 2, characterized in that: In step S1, the adipose tissue is derived from the autologous adipose tissue of the liposuction patient.

5. The drug for treating skin photoaging according to claim 2, characterized in that: In step S1, during centrifugation, the rinsed adipose tissue is placed in a centrifuge and centrifuged at 500-2000 rpm, 37° C., for 5 min.

6. The drug for treating skin photoaging according to claim 2, characterized in that: In step S2, the adipose tissue was homogenized on ice for 1 min.

7. The drug for treating skin photoaging according to claim 2, characterized in that: In step S2, the fat is processed into a homogenous state, and then centrifuged at 1000-4000g, 4°C, for 5 min, the upper residual tissue is discarded, and the lower clear liquid is retained.

8. The drug for treating skin photoaging according to claim 2, characterized in that: In step S3, the lower clear liquid is filtered using a filter, placed in an ultrafiltration centrifuge tube with a 100KD membrane, concentrated by centrifugation at 3000g and 4°C to an inner tube volume of 500 μL, transferred into a sterile cryopreservation tube, and transferred to a -80°C refrigerator for cryopreservation.

9. The drug for treating skin photoaging according to claim 2, characterized in that: In step S3, the lower layer supernatant is filtered using a 0.22 μm filter.

10. The drug for treating skin photoaging according to claim 1, characterized in that: The drug for treating skin photoaging includes any one of tablets, capsules, granules, injections, ointments, hydrogels, water injections, powder injections or oral solutions.

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