Skin aging animal model constructed by destroying skin barrier and modeling method and application thereof

Abstract

The invention discloses a skin aging animal model constructed by destroying a skin barrier and a modeling method and application of the skin aging animal model, and relates to the field of animal model construction, and the skin aging model is constructed by a method of destroying the skin barrier and is used for researching aging caused by chronic skin barrier injury. The invention provides a novel animal model for studying skin aging, and by constructing a chronic skin barrier damage model, the influence of a PPAR pathway in barrier damage and aging is studied, so that the potential relationship between skin barrier damage and aging is disclosed.

Description

Technical Field

[0001] The invention belongs to the field of animal model construction, and specifically relates to a skin aging animal model constructed by destroying the skin barrier, and a modeling method and application thereof. Background Art

[0002] Skin is the largest organ in the human body. Its aging is not only accompanied by an increase in wrinkles and age spots, but also brings varying degrees of anxiety to people. There is evidence that skin aging can lead to loss of skin function, increase the risk of skin cancer, and can also lead to physical aging and related functional disorders, affecting the health of patients. How to prevent and delay skin aging has become a hot topic and focus of current medical research.

[0003] Some domestic and foreign studies have explored the causes of aging: skin aging is caused by both intrinsic and extrinsic factors; intrinsic skin aging (natural aging) refers to physiological changes that occur over time, and the influencing factors include the production of reactive oxygen species (ROS), hormonal changes, etc., which manifests as the skin becoming thin, transparent and dry, showing fine wrinkles and irregular hair growth, and the skin becomes loose, loses elasticity, and cannot sweat fully; exogenous skin aging is the cumulative effect of lifestyle and environmental factors. Typical external influencing factors include ultraviolet radiation (UV), smoking and environmental pollution, which manifests as deep wrinkles, decreased elasticity, dryness, sagging, roughness, capillary dilation and pigmentation disorders. Currently, the generally recognized indicators for confirming skin aging include P16, P21, P53, Lamin B1, γ-H2AX, Col-Ⅰ, Col-Ⅲ, MMPs, β-galactosidase (SAβ-gal), etc. At the same time, indicators of oxidative stress in the skin: reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), glutathione catalase (GSH-PX), etc. are also classic indicators of skin aging.

[0004] Reports have shown that chronic skin barrier damage manifests itself as chronic inflammation, epidermal thickening, roughness, and lichenification. Clinically, repeated mechanical stress on the skin area, such as scratching and friction, can damage the skin barrier, leading to thickening, roughness, dryness, increased dandruff, and other aging-like manifestations of the skin stratum corneum. The above phenomena indicate that chronic skin barrier damage may cause or accelerate the occurrence of skin aging. After reviewing relevant domestic and foreign literature, the relationship between chronic barrier damage and aging and its potential mechanism have not been reported.

[0005] The skin barrier generally refers to the skin barrier in a narrow sense, that is, the epidermal permeability barrier, which is closely related to the immune barrier, microecological barrier and chemical barrier, and together constitutes the overall defense system of the skin barrier. The skin barrier function is extremely important, which can not only prevent damage to the skin by external chemical, physical, mechanical, biological and other factors, but also prevent the loss of water. When the skin is exposed to irritating conditions such as ultraviolet rays and toxins, it is very easy to destroy the integrity of the skin's stratum corneum, causing damage to the skin barrier and inducing oxidative stress and inflammation. Skin barrier abnormalities are considered to be one of the key factors in the pathogenesis of atopic dermatitis, acne, cutaneous squamous cell carcinoma, etc., and can induce or aggravate skin-related diseases. Whether repeated friction that damages the barrier will aggravate skin aging is a new research direction.

[0006] To this end, the present invention creates a new skin aging animal model by destroying the skin barrier, which is used to explore the impact of long-term skin barrier destruction on aging, so as to reveal its potential pathways, thereby providing a new perspective and approach for the defense and treatment of aging from the perspective of skin barrier protection, in order to provide a new strategy for the clinical treatment of skin aging and even body aging and age-related diseases. Summary of the invention

[0007] To achieve the above objectives, the present invention provides a skin aging animal model constructed by destroying the skin barrier, and a modeling method and application thereof, with the specific technical features as follows:

[0008] An animal model of skin aging through disruption of the skin barrier architecture.

[0009] Furthermore, the model damages the skin barrier and exacerbates skin aging by stimulating EGF-EGFR-JNK / ERK-PPARγ-NLR P3.

[0010] Furthermore, EGF-EGFR-JNK / ERK inhibitors, PPARγ inhibitors, and NLR P3 agonists can be used to prepare skin barrier-damaging drugs for aggravating skin aging.

[0011] Furthermore, the animal used to construct the model is mouse.

[0012] A method for constructing a skin aging animal model comprises the following steps:

[0013] S1: After intraperitoneal injection of anesthesia, the hair on the back of the mouse was removed with a shaver without damaging the skin;

[0014] S2: Repeatedly apply adhesive peeling, knife scratching or sandpaper abrasion treatment to the hairless area after shaving, once a day for three months;

[0015] S3: Observe the aging degree of mice and take photos for record;

[0016] S4: Measure TEWL. When the TEWL value increases by 6-8 times, the model is successfully made and the pasting is stopped.

[0017] Furthermore, the tool for sticking and tearing off the hair-free area after shaving in S2 includes one or more of tape, a hair remover, a scraper or sandpaper.

[0018] A method for constructing an animal model of skin aging and the application of the constructed animal model as research material in the study of aging caused by chronic skin barrier damage.

[0019] Furthermore, the animal model is used in studying the effect of PPARγ on aging caused by chronic skin barrier damage.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) It provides a new skin aging model that can be used as a research material for aging caused by chronic skin barrier damage;

[0022] (2) To provide new perspectives and approaches for the prevention and treatment of aging from the perspective of skin barrier protection, in order to provide new strategies for the clinical treatment of skin aging and even body aging and age-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Skin appearance of Balb / c mice after chronic barrier disruption;

[0024] (A) Appearance of the back skin of mice in the two groups;

[0025] (B) Detection results of water content in the back skin tissue of two groups of mice;

[0026] (C) TEWL value test results of back skin tissue of two groups of mice; *P<0.05, **P<0.01,

[0027] ***P<0.001.

[0028] Figure 2 Detection of skin barrier-related indicators on the back of Balb / c mice;

[0029] (AE) Immunofluorescence images of epidermal barrier indicators of the two groups of mice: FLG, IVL, ZO-1, E-cadherin, and β-catenin, images were collected at 200× magnification;

[0030] (F) Quantitative analysis results of epidermal barrier markers in two groups of mice; *P<0.05, **P<0.01,

[0031] ***P<0.001.

[0032] Figure 3 Results of histopathological staining of the back skin of Balb / c mice.

[0033] Figure 4 Detection of aging indicators on the back skin of mice

[0034] (A, B) Immunohistochemical staining images of skin aging indicators of the two groups of mice at P16 and P21 were taken at 200× magnification, and the quantitative results of immunohistochemical expression.

[0035] (CD) Immunofluorescence staining images of skin aging indicators Col-Ⅰ, Col-Ⅲ, LaminB 1 and γ-H2AX in the two groups of mice, images taken at 200× magnification, and the quantitative results of immunofluorescence expression of Col-Ⅰ and Col-Ⅲ.

[0036] (F) Expression of ROS, SOD, MDA, CAT and GSH-Px in the dorsal skin of the two groups of mice.

[0037] (G, H) Western Blot analysis of mouse skin tissues using the indicated antibodies and quantitative results of protein expression. *P<0.05, **P<0.01, ***P<0.001.

[0038] Figure 5 PPARγ is enriched in the skin of aging mice with impaired barrier function

[0039] (A) Bar chart and bubble chart of indifferent gene enrichment analysis of transcriptome of dorsal skin tissue of control group and tape group mice combined with KEGG signaling pathway. Indifferent gene enrichment analysis of transcriptome of dorsal skin tissue of control group and tape group by GSEA-KEGG showed enrichment of PPAR pathway. Volcano plot analysis of differentially expressed genes in transcriptome of dorsal skin tissue of two groups of mice.

[0040] (B) Immunofluorescence images of PPARγ in the skin tissues of the two groups of mice, images were taken at 200× magnification, and PPARγ immunofluorescence quantitative analysis.

[0041] (C) Western Blot of PPARγ in skin tissues of the two groups of mice and quantitative analysis of PPARγ protein. *P<0.05, **P<0.01, ***P<0.001.

[0042] Figure 6 Down-regulation of PPARγ expression through EGF-EGFR-JNK / ERK for skin barrier disruption;

[0043] (A) Western Blot validation of the skin tissues of the two groups of mice using the indicated antibodies;

[0044] (B) Protein quantitative analysis of skin tissues of the two groups of mice using the indicated antibodies. *P<0.05,

[0045] **P<0.01, ***P<0.001.

[0046] Figure 7 The therapeutic effect of PPARγ agonist on the appearance of the back skin of tape-applied mice;

[0047] Figure 8 PPARγ agonist for the detection of FLG immunofluorescence and TEWL in the back skin of tape-applied mice;

[0048] (A) Detection of FLG indexes in the back skin of four groups of mice;

[0049] (B) Quantitative immunofluorescence analysis of FLG in the back skin of four groups of mice;

[0050] (C) TEWL index detection of the back skin of four groups of mice. "*" indicates a significant difference compared with the control group (***P < 0.001), and "#" indicates a significant difference compared with the negative control group (###P < 0.001);

[0051] Fig. 9 Effects of administration of PPARγ agonists on aging in tape-applied mice;

[0052] (A) HE and Masson staining results of the back skin of the four groups of mice, showing the histological changes of the four groups of mice and the statistical chart of the epidermal thickness of the four groups of mice. The images were collected at 200× magnification.

[0053] (B) Western Blot analysis of mouse skin tissues using the indicated antibodies.

[0054] (C) Statistical graph of the expression of the proteins in the back skin of the four groups of mice, "*" indicates a significant difference compared with the control group (*P < 0.05, **P < 0.01), and "#" indicates a significant difference compared with the negative control group (#P < 0.05);

[0055] Fig.10 NLRP3 was significantly different and enriched in the tape + PPAR agonist group and the tape + DMSO solvent group; (A) The transcriptome of the back skin tissue of mice in the tape + PPARγ agonist group and the tape + DMSO solvent group was combined with the KEGG signaling pathway for indiscriminate gene enrichment analysis, the volcano plot analysis of the transcriptome differential genes of the back skin tissue, and the statistical bar chart of the number of differential genes; (B) Western Blot analysis of NLRP3 indicators in the skin tissue of mice in the tape + PPARγ agonist group and the tape + DMSO solvent group. *P < 0.05, **P < 0.01, ***P < 0.001. DETAILED DESCRIPTION

[0056] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. In addition, in the following invention, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.

[0057] Example 1: Animal modeling method

[0058] S1: 1-2 days before the experiment, the mouse back hair was removed with a shaver after intraperitoneal injection of anesthesia, and the skin was not damaged during the removal process; the injection dose of anesthesia was 0.15mL / 10g;

[0059] S2: Continue to apply tape to the shaved hairless area once a day for three months;

[0060] S3: Observe the aging degree of mice and take photos for record;

[0061] S4: Measure TEWL. When the TEWL value increases by 6-8 times, the model is successfully made and the pasting is stopped.

[0062] A control experiment was set up, and the hair on the back of the mice was removed with a shaver. The hair was removed gently to avoid damage to the skin by human factors. No depilatory cream was used to remove hair to avoid chemical reagents affecting the experimental results. The mice were shaved every day afterwards. The treatment methods for the two animal groups were:

[0063] ① Control group: The mice were raised normally and shaved after anesthesia, once a day for three months. The repeated shaving process did not damage the skin.

[0064] ② Tape stripping group: The mice were raised normally, shaved after anesthesia, and continued to be treated with tape stripping (knife scratch damage / sandpaper abrasion). The indicator for stopping tape stripping was: TEWL value increased by 6-8 times. This was done once a day for three months, and then the mouse skin was collected for experiments.

[0065] After the experiment was completed three months later, the aging degree of the mice was observed and photographed. The TEWL and water content values ​​were measured the next day. The specific operation was as follows:

[0066] (1) Measurement of TEWL value: The TEWL value was measured on the second day after the mouse model was established. The TEWL value was measured three times on the dorsal skin of the same fixed area of ​​the mouse using a CK measuring instrument to ensure that the probe was in complete contact with the mouse's dorsal skin. The measurement should be kept as unaffected by external air convection as possible and the temperature should be kept consistent.

[0067] (2) Measurement of water content: The water content was measured on the second day after the mouse model was established. The CK measuring instrument was used to measure the water content three times on the same fixed area of ​​the mouse's dorsal skin to ensure that the probe had consistent strength on the mouse's back skin. The measurement should be kept as unaffected by external air convection as possible and the temperature should be kept consistent.

[0068] The results are shown in the figure. The back skin of the mice in the control group was flesh-colored, smooth and elastic, with normal skin appearance without barrier damage. The back skin of the mice in the tape group showed barrier damage such as slight exudation and capillary dilation, as well as deeper wrinkles, roughness, dry epidermis, desquamation and other aging phenomena ( Figure 1 A). Non-invasive testing of the skin of the two groups of mice showed that the water content of the skin of the mice in the tape-sticking modeling group was lower than that in the control group (P < 0.001) ( Figure 1 B), TEWL value was higher than that of the control group (P<0.001) ( Figure 1 C) The results showed that tape sticking can aggravate the water loss of stratum corneum and damage the skin barrier of mice.

[0069] Example 2: Mouse skin collection, preservation and sectioning

[0070] In order to further detect the damage of the skin barrier after tape sticking (knife scratch injury / sandpaper abrasion), the mice in Example 1 were killed by spinal dislocation, and the mouse limbs were fixed on a special sterile operating table. After the mouse skin was disinfected with alcohol, the skin was cleaned with physiological saline, and finally the sample was taken. Take the skin of the same part, take a piece of skin of 1cm×1cm in size, put it into a centrifuge tube pre-filled with 4% paraformaldehyde, and store it at room temperature for subsequent tissue section staining. Continue to take tissues from the same part and put them in centrifuge tubes or cryopreservation tubes marked with the order and group in advance, put them into liquid nitrogen and then put them into a -80℃ refrigerator for subsequent experiments.

[0071] 1. Paraffin-embedded specimens

[0072] Fixed specimens: The fresh skin tissue was placed in a 4% paraformaldehyde solution and immersed at room temperature. The amount of paraformaldehyde should at least completely immerse the specimen. Then, it was continuously rinsed with tap water overnight to remove the paraformaldehyde.

[0073] Specimen dehydration: Soak the specimen in 70% ethanol solution for 24 hours, soak in 80% ethanol solution for 12 hours, soak in 90% ethanol solution for 12 hours, soak in 95% ethanol solution for 12 hours, soak in 95% ethanol II solution for 1 hour, soak in anhydrous ethanol I solution for 1 hour, and soak in anhydrous ethanol II solution for 1 hour. Specimen transparency: Soak in xylene I solution for 40 minutes; soak in xylene II solution for 40 minutes. Wax immersion: Immerse the tissue in paraffin liquid, keep at 62-64℃ for 1 hour, and repeat 3 times. Embedding: Place the tissue in an embedding device, place it on a cooling plate for 3 hours, and then store it in a 4℃ refrigerator for subsequent experimental operations.

[0074] 2. Slice production

[0075] Use a paraffin slicer to slice, and keep the thickness at about 4um. Then spread the slices in a 37℃ water bath, pick up the specimens with a slide (mark them in advance), and place the specimens in the center of the slide. Slice baking: Keep the paraffin slices at 60℃ for 60min. Dewaxing: Soak the sample in xylene I for 10min, then immerse the sample in xylene II for 10min, anhydrous ethanol for 15min, anhydrous ethanol II for 5min, 95% ethanol for 5min, 85% ethanol for 5min, 80% ethanol for 5min, and 75% ethanol for 5min. Complete the above steps and then proceed to the subsequent operations.

[0076] 3. Pathological staining and sectioning

[0077] (1) HE staining

[0078] Paraffin sections were routinely dewaxed and hydrated, and washed with distilled water for 5 minutes. Hematoxylin staining lasted for 16 minutes, and then rinsed with tap water to remove floating color, and differentiated with 1% hydrochloric acid alcohol for 2 seconds. The sections were rinsed with tap water for 1 minute, then rinsed with 1% dilute ammonia water for 30 seconds, and then rinsed with tap water for 1 minute. Use eosin staining at room temperature for 15 seconds. Use tap water to terminate color development. Dehydrate with gradient alcohol, in order of 75%, 80%, 85%, 90%, 100%, 100% (each stage lasts 10 minutes), and use xylene to transparentize the sample for 30 minutes. Use neutral gum to seal the slides.

[0079] (2) Masson staining

[0080] The sections were routinely dewaxed and hydrated. Stained with iron hematoxylin staining solution for 10 minutes. Differentiate with acidic ethanol differentiation solution for 5 seconds, rinse slowly and briefly with water (be careful not to go too fast). Masson blueing solution returns to blue for 3 minutes, rinse slowly and briefly with water again, and then wash with distilled water for 1 minute. Stain with Ponceau fuchsin staining solution for 10 minutes. (Weak acid working solution is configured with distilled water: weak acid solution = 2:1 ratio), wash with weak acid solution for 1 minute, and wash with phosphomolybdic acid solution for 2 minutes. Wash with the above-configured weak acid working solution for 1 minute. Quickly dehydrate with 95% ethanol and anhydrous ethanol, each time for 10 seconds. Use xylene to permeabilize the sections 3 times, 2 minutes each time. Use neutral gum to seal the slides.

[0081] (3)Weigert staining

[0082] After routine dewaxing, rinse the sections with tap water for 3 minutes. Stain with resorcinol fuchsin staining solution at room temperature for 3 hours, and rinse lightly with water (be careful not to rinse too quickly). Add 95% ethanol solution until no staining solution is removed. Rinse with running water for 10 minutes. Stain with VanGieson staining solution for 1 minute, then remove the staining solution. Seal the sections with neutral gum.

[0083] (4) VG staining

[0084] The sections were routinely dewaxed and hydrated. Weigert iron hematoxylin A solution was added dropwise, and then Weigert iron hematoxylin B solution was added dropwise and mixed (A solution: B solution = 1:1). The staining time was 5-10 minutes, and the sections were rinsed gently and briefly with water (be careful not to be too fast). The sections were quickly treated with 1% hydrochloric acid alcohol, washed with a trickle of water for several minutes, and then stained with VG stain solution for 1-2 minutes. After the stain solution was poured off, the sections were directly treated with 95% alcohol for a few seconds, dehydrated with anhydrous alcohol, and transparentized with xylene. The sections were sealed with neutral gum.

[0085] The results are shown below. HE staining showed that the epidermis of the mice in the tape group was significantly thickened (P<0.001). The epidermis thickness of normal mice is generally around 20 μm, while the epidermis thickness of the mice in the tape group reached about 60 μm ( Figure 3 ). At the same time, Masson staining, Van-Gieson (VG) staining, and Weigert resorcinol fuchsin staining were used to detect the structure of collagen fibers and elastic fibers in the mouse skin. The staining results showed that the collagen fibers and elastic fibers in the skin of the mice in the tape group were broken and disordered ( Figure 3 ). The phenomenon of epidermal thickening and destruction of collagen fibers and elastic fibers indicates from a pathological point of view that tape adhesion can cause skin aging in mice. The positive rate of β-gal staining near the hair follicles in the tape group was higher than that in the control group ( Figure 3 ). The above staining results indicate that chronic skin barrier damage aggravates skin aging.

[0086] Example 3: Immunofluorescence experiment of skin tissue

[0087] After paraffin embedding and sectioning as described above, proceed to the next step. Antigen repair, expose antigenic determinants, and serum blocking. Take out the previous slices, use filter paper to absorb the surrounding water, add serum, and then keep at 37℃ for 30min. Wipe the serum on the slide with absorbent paper, add primary antibody, and put it in a 4℃ refrigerator overnight. Take out the slide, rinse with PBS 3 times, 5min each time, wipe off PBS, add secondary antibody, and then keep at 37℃ for 30min. Take out the slide, rinse with PBS 3 times, 5min each time, wipe off PBS, add SABC, and then keep at 37℃ for 30min. Take out the slide, wash it in PBS 3 times, 5min each time, and then rinse the slide with clean water and stain it in hematoxylin for half a minute. Put the slide in PBS (PH7.4) solution and shake it on a decolorizing shaker for 3 times, 5min each time. Add autofluorescence quencher for 5min and rinse with running water for 10min. After the slices were slightly dried, they were sealed with an anti-fluorescence quencher and stored at 4°C in the dark. They were then photographed and recorded using a fluorescence microscope.

[0088] The results showed that compared with the control group, the expression of barrier-related proteins in the skin of mice in the tape group was significantly decreased, including ZO-1 (P < 0.01), FLG (P < 0.001), IVL (P < 0.01), E-cadherin (P < 0.001) and β-catenin (P < 0.001) ( Figure 2 ), the above results indicate that tape adhesion causes damage to the skin barrier of Balb / c mice.

[0089] Immunofluorescence was used to verify the expression of LaminB1, Col-Ⅰ, Col-Ⅲ, γ-H2AX( Figure 4 Compared with the control group, the content of Col-Ⅰ (P<0.001) and Col-Ⅲ (P<0.05) in the skin of mice in the tape group was reduced and the expression was disordered, the expression of γ-H2AX in the nucleus was increased, and the expression of Lamin B1 in the nuclear envelope was decreased. These results were consistent with the results of Western Blot. The above results confirmed that the expression of aging-related indicators in the skin of mice in the tape group was increased. In summary, the results show that chronic skin barrier damage accelerates skin aging.

[0090] Example 4: Detection of aging indicators of mouse back skin

[0091] In order to further verify that chronic barrier damage leads to skin aging, a more comprehensive test of aging-related indicators was performed on the back skin of the two groups of mice. The study found that the speed of aging is related to increased levels of oxidative stress. The skin oxidative stress indicators: ROS, SOD, MDA, CAT, and GSH-PX were tested. The results showed that compared with the control group, the skin oxidative stress indicators of mice in the tape group: ROS (P < 0.001) and MDA (P < 0.001) were significantly increased, and SOD (P < 0.001), CAT (P < 0.001), and GSH-PX (P < 0.01) were significantly decreased ( Figure 4 F), this experimental result confirmed that chronic barrier damage increased the oxidative stress indicators of mouse skin. At the same time, other indicators of skin aging were detected: changes in P16, P21, P53, LaminB1, γ-H2AX, MMP1, Col-Ⅰ, and Col-Ⅲ. First, the results of Western Blot showed that the levels of P53 (P < 0.01), P21 (P < 0.01), P16 (P < 0.01), γ-H2AX (P < 0.01), and MMP1 (P < 0.01) in the skin of mice in the tape group increased, and the expression of LaminB1 (P < 0.001), Col-Ⅰ (P < 0.001), and Col-Ⅲ (P < 0.001) decreased ( Figure 4 G, H). Immunohistochemistry verified the changes of P21 and P16 proteins, biomarkers of cell aging, in mouse skin tissue. The results showed that the positive rates of P21 (P < 0.01) and P16 (P < 0.001) proteins in the tape group mice were significantly increased compared with the control group mice ( Figure 4 A, B), which is the same as the results of Western Blot. We used immunofluorescence to verify LaminB1, Col-Ⅰ, Col-Ⅲ, γ-H2AX ( Figure 4 Compared with the control group, the content of Col-Ⅰ (P<0.001) and Col-Ⅲ (P<0.05) in the skin of mice in the tape group was reduced and the expression was disordered, the expression of γ-H2AX in the nucleus was increased, and the expression of Lamin B1 in the nuclear envelope was decreased. These results were consistent with the results of Western Blot. The above results confirmed that the expression of aging-related indicators in the skin of mice in the tape group was increased. In summary, the results show that chronic skin barrier damage accelerates skin aging.

[0092] The above tests show that the skin aging animal model constructed by destroying the skin barrier has:

[0093] (1) Appearance of mice: Compared with the control group, the mice in the tape group had skin erythema, increased wrinkles, aggravated desquamation, and a significantly thickened stratum corneum.

[0094] (2) Results of mouse skin barrier indexes: Compared with the control group, the TEWL value of the skin of the mice in the tape group increased and the water content decreased. Immunofluorescence staining results showed that the expressions of the core proteins of the skin barrier in the tape group: ZO-1, FLG, IVL, E-cadherin, and β-catenin were significantly reduced, indicating that the skin barrier was damaged.

[0095] (3) Results of mouse skin aging indicators: Compared with the control group mice, the skin pathological staining results of the mice in the tape group showed thickening of the stratum corneum, and rupture and disorder of collagen fibers and elastic fibers. In addition, the oxidative stress indicators of the mice in the tape group were also manifested as increased expression of ROS, MDA, and CAT, decreased expression of SOD and GSH-PX, significantly increased expression levels of aging markers P53, P21, P16, γ-H2AX, and MMP1, and significantly decreased protein expression levels of Col-Ⅰ, Col-Ⅲ, and LaminB1. (4) KEGG enrichment analysis was performed after RNA-seq detection, and the PPAR signaling pathway was significantly enriched.

[0096] From the above results, it can be seen that the skin aging model constructed by this method was successfully constructed, and the above animal model can be used as a research material to study skin aging caused by chronic skin barrier damage; it is of great significance for further in-depth understanding of the disease pathophysiology and histological changes, and is conducive to providing new insights for the subsequent prevention and treatment of clinical aging skin diseases.

[0097] Example 5: Application of the animal model constructed in Example 1

[0098] A skin barrier damage model was successfully constructed using Balb / c mice. In order to further explore the mechanism of skin aging caused by barrier damage, RNA-seq was used to detect the transcriptome of the back skin tissue of barrier-damaged mice and control mice, and the transcriptome of the back skin tissue of barrier-damaged mice was combined with KEGG signaling pathway for indiscriminate gene enrichment analysis. The results of GSEA-KEGG signaling pathway gene enrichment analysis showed that the PPAR signaling pathway was enriched ( Figure 5 A). These enrichment results suggest that the PPAR signaling pathway regulates the occurrence of barrier damage and skin aging. PPAR is divided into three factors, among which PPARγ is strongly enriched, and EGF is also strongly enriched. In order to further evaluate the difference in the expression of PPARγ in this experiment, Western Blot and immunofluorescence detection were performed. The results of Western Blot and immunofluorescence detection showed that the expression of PPARγ in the tape group decreased ( Figure 5 B, C).

[0099] In summary, long-term skin barrier damage will accelerate the aging process and degree. By studying the relationship between skin barrier and aging, the importance of barrier protection to aging is revealed, which provides a new direction for anti-aging from the perspective of barrier protection and a new perspective for the treatment and prevention of aging. The transcriptome of the back skin tissue of barrier-damaged mice combined with the KEGG signaling pathway was analyzed in an indiscriminate manner. The results showed that the PPAR signaling pathway was highly enriched. PPARγ is closely related to the skin and is involved in a variety of pathological and physiological processes of the skin. The PPAR signaling pathway may regulate the occurrence of aging caused by barrier damage.

[0100] Example 6: Skin barrier disruption downregulates PPARγ expression via EGF-EGFR-JNK / ERK

[0101] EGF in keratinocytes plays a key role in maintaining skin homeostasis. The destruction of the barrier is closely related to epidermal growth factor, and existing literature has shown that EGF-EGFR affects the expression of PPARγ through JNK / ERK. EGF was significantly enriched in the RNA-seq results of the back skin of mice in the tape group and the control group ( Figure 5 A). After the skin barrier is damaged, it is not clear whether the downregulation of PPARγ is related to EGF expression. In order to verify this pathway, the protein expression of EGFR, JNK, and ERK in the skin of mice in the tape group and the control group was detected by Western Blot. The results showed that compared with the control group, the expression of EGFR (P < 0.001), JNK (P < 0.001), and ERK (P < 0.001) in the tape group was downregulated ( Figure 6 ), indicating that chronic barrier destruction is mediated by EGF-EGFR-JNK / ERK, which in turn regulates PPARγ expression.

[0102] Example 7: Administration of PPARγ agonists in the skin of barrier-disrupted mice

[0103] (1) The mechanism was explored by giving PPARγ agonists and DMSO solvent to mice with tape adhesion and setting up tape group and control group experiments. The details are as follows:

[0104] Forty mice were randomly divided into four groups, with 40 mice in each group.

[0105] ① Control group (Control), the mice were raised normally and shaved after anesthesia;

[0106] ② The tape stripping group: mice were raised normally, shaved after anesthesia, and continued to be treated with tape stripping once a day for three months;

[0107] ③ Adhesive tape + DMSO solvent group (Negative Control), the mice were raised normally, shaved after anesthesia, and continued to be treated with adhesive tape for three months. After the adhesive tape was applied, DMSO solvent was applied once a day. The method of using DMSO solvent was: 1% DMSO in normal saline, and oscillated with an ultrasonic device for 30 minutes.

[0108] ④ The tape + PPAR agonist group (pioglitazone, PGZ) was raised normally. After anesthesia, the mice were shaved and the tape was applied continuously for three months (the tape was stopped when the TEWL value increased by 6-8 times). After the tape was applied, the PPARγ agonist (pioglitazone) was applied once a day. The method of using pioglitazone was: 30μg / kg mouse, dissolved in 1% DMSO saline, and vibrated with an ultrasonic device for 30 minutes to fully dissolve the pioglitazone.

[0109] The experimental results showed that the PPARγ agonist administered after tape application reduced the degree of barrier damage such as erythema, roughness, and inflammation on the back skin of mice compared with the tape group and the tape + DMSO solvent group, and reduced the degree of aging such as wrinkles and epidermal thickness. However, there was no significant difference in appearance between the tape group and the tape + DMSO solvent group. The tape + PPARγ agonist group still had aging phenomena such as wrinkles and epidermal thickening compared with the control group ( Figure 7 ). From the appearance, it can be seen that PPARγ agonists have the effect of alleviating skin aging caused by barrier damage.

[0110] (2) The TEWL values ​​and FLG immunofluorescence indexes of the mouse skin in the control group, tape group, tape + DMSO solvent group, and tape + PPARγ agonist group were measured. The test results showed:

[0111] Compared with the tape + DMSO solvent group, the TEWL value of the tape + PPARγ agonist group was reduced (P < 0.001) ( Figure 8 C). The results of immunofluorescence staining showed that the expression of FLG in the tape + PPARγ agonist group was higher than that in the tape + DMSO solvent group ( Figure 8 A, B), these results suggest that PPARγ agonist has a certain repair effect on the damaged skin barrier of the back of mice.

[0112] (3) Pathological staining, oxidative stress index detection and Western Blot were used to measure the skin aging indicators of the four groups of mice.

[0113] ①HE staining results showed that the stratum corneum of the mice in the tape group and the tape+DMSO solvent group was significantly thickened, and the stratum corneum thickness of the mice in the tape+PPARγ agonist group was thinner than that in the tape+DMSO solvent group (P<0.001).

[0114] ②The results of Masson staining showed that the structure of the elastic fibers in the skin of mice in the tape + PPARγ agonist group was less broken and disordered than that in the tape + DMSO solvent group ( Fig. 9 A).

[0115] ③Western Blot detected the protein expression levels of aging markers P16, P21, and P53 in the skin tissues of the four groups of mice. The results showed that the administration of PPARγ agonists on the basis of skin barrier damage reduced the expression of aging markers P16, P21, and P53 in the tissues compared with the tape + DMSO solvent group ( Fig. 9 B, C). In conclusion, these results suggest that PPARγ agonists can improve and restore the degree of aging caused by barrier damage. They further indicate that PPARγ agonists can be used as drugs or skin care products for improving and restoring aging caused by barrier damage.

[0116] (4) Transcriptome analysis of the skin of barrier-damaged mice after administration of PPARγ agonists showed that inflammatory signaling pathways were significantly enriched ( Fig.10 A). These enrichment results suggest that inflammatory signals may be a strongly related pathway downstream of PPAR, which may regulate the occurrence of skin aging caused by barrier damage. At the same time, we further found the differential gene-inflammasome-NLRP3. In order to further evaluate the difference in the expression of NLRP3 in this experiment, we performed Western Blot detection. The test results showed that NLRP3 was inhibited in the tape + PPARγ agonist group ( Fig.10 B), consistent with the RNA-seq results.

[0117] In summary, PPARγ and NLRP3 have a direct protein interaction relationship. According to experimental results and literature reports, PPARγ-NLRP3 is directly related to skin aging. Repeated damage to the skin barrier is another important cause of skin aging, which works through the EGF-EGFR-JNK / ERK-PPARγ-NLRP3 pathway and exacerbates the phenomenon and degree of skin aging by stimulating EGF-EGFR-JNK / ERK-PPARγ-NLR P3.

[0118] From the above conclusions, it can be concluded that PPARγ agonists and / or NLRP3 inhibitors can be used to prepare anti-aging drugs or cosmetics caused by barrier damage.

[0119] Example 8: Another method for constructing a skin aging animal model, comprising the following steps:

[0120] S1: After intraperitoneal injection of anesthesia, the hair on the back of the mouse was removed with a shaver without damaging the skin;

[0121] S2: Repeatedly scrape the hairless area after shaving, once a day, for three months;

[0122] S3: Observe the aging degree of mice and take photos for record;

[0123] S4: To mouse skin Figure 1 As shown, TEWL was measured and found to have increased 8 times, proving that this method can successfully construct an animal model of skin aging.

[0124] Example 9: Another method for constructing a skin aging animal model, comprising the following steps:

[0125] S1: After intraperitoneal injection of anesthesia, the hair on the back of the mouse was removed with a shaver without damaging the skin;

[0126] S2: Repeated sandpaper abrasion treatment of the shaved hairless area once a day for three months;

[0127] S3: Observe the aging degree of mice and take photos for record;

[0128] S4: To mouse skin Figure 1 As shown, TEWL was measured and found to have increased by 7 times, proving that this method can successfully construct an animal model of skin aging.

[0129] Example 10: Another method for constructing a skin aging animal model, comprising the following steps:

[0130] S1: After intraperitoneal injection of anesthesia, the hair on the back of the mouse was removed with a shaver without damaging the skin;

[0131] S2: The hairless area after shaving was treated with a sticker containing PM2.5, which was applied once a day for 2 hours each time for three months to damage the mouse skin barrier;

[0132] S3: Observe the aging degree of mice every day and take photos and record them;

[0133] S4: To mouse skin Figure 1 As shown, TEWL was measured and found to have increased by 7 times, proving that this method can successfully construct an animal model of skin aging.

[0134] From the above methods for constructing the four skin aging animal models of Examples 1-10, it can be seen that PM2.5 and repeated friction can cause skin barrier damage, and the EGF-EGFR-JNK / ERK-PPARγ-NLRP3 pathway of the damaged skin is affected. Since PM2.5 and repeated friction can cause skin barrier damage, EGF-EGFR-JNK / ERK is downregulated, PPARγ is also downregulated, and NLRP3 is upregulated, it can be seen from this pathway that EGF-EGFR-JNK / ERK agonists, PPARγ agonists, and NLRP3 inhibitors can alleviate or repair skin aging problems caused by damaged skin barrier, and can be used to prepare anti-aging products.

[0135] It should be understood that the above specific embodiments of the present invention are only used for illustrative inventions or to explain the principles of the present invention, and do not constitute limitations on the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included in the protection scope of the present invention. In addition, the appended claims of the present invention are intended to cover all changes and modifications that fall within the scope and boundaries of the appended claims, or the equivalent forms of such scope and boundaries.

Claims

1. A skin aging animal model, characterized in that: The model was constructed by disrupting the animal skin barrier.

2. A skin aging animal model according to claim 1, characterized in that: The model exacerbates skin aging by stimulating EGF-EGFR-JNK / ERK-PPARγ-NLR P3 to damage the skin barrier.

3. A skin aging animal model according to claim 2, characterized in that: EGF-EGFR-JNK / ERK inhibitors, PPARγ inhibitors, and NLR P3 agonists can be used to prepare skin barrier-damaging drugs to aggravate skin aging.

4. A skin aging animal model according to claim 1, characterized in that: The animal is a mouse.

5. A method for constructing a skin aging animal model according to claim 1, characterized in that: The steps include: S1: After intraperitoneal injection of anesthesia, the hair on the back of the mouse was removed with a shaver without damaging the skin; S2: Repeatedly apply adhesive peeling, knife scratching or sandpaper abrasion treatment to the hairless area after shaving, once a day for three months; S3: Observe the aging degree of mice and take photos for record; S4: Measure TEWL. When the TEWL value increases by 6-8 times, the model is successfully made and the pasting is stopped.

6. The method for constructing a skin aging animal model according to claim 5, characterized in that: The tool for sticking and tearing off the hair-free area after shaving in S2 includes one or more of tape, a hair remover, a scraper or sandpaper.

7. Use of an animal model constructed according to the method for constructing an animal model of skin aging according to claim 5 as a research material in a study on aging caused by chronic skin barrier damage.

8. The use according to claim 7, characterized in that: The animal model is used in studying the effect of PPARγ on aging caused by chronic skin barrier damage.

Images