Use of casin in preparing formulation for promoting synthesis of collagen and elastin

Abstract

Disclosed in the present invention is use of CASIN in preparing a formulation for promoting the synthesis of collagen and elastin. The present invention finds for the first time that CASIN can significantly promote the synthesis of type I collagen, type III collagen, and elastin in skin fibroblasts. Therefore, the present invention has good application prospects in the fields of biomedicine and cosmetics.

Description

Application of CASIN in the preparation of formulations that promote collagen and elastin synthesis Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, and in particular to the application of CASIN in the preparation of formulations that promote the synthesis of collagen and elastin. Background Technology

[0002] Collagen is the oldest and most abundant extracellular matrix protein, widely found in connective tissues, tendons, skin, bone, and cartilage. On the one hand, collagen provides mechanical support in tissues; on the other hand, it plays a crucial role in controlling cell adhesion, cell migration, and tissue repair. In mammals, collagen accounts for approximately 30% of the total protein mass (Zhu S, et al. Self-assembly of collagen-based biomaterials: preparation, characterizations and biomedical applications. J Mater Chem B, 2018. 6(18): 2650–2676.). Based on structural characteristics, collagen is generally classified into types I, II, III, and IV. Type I collagen has a triple helix structure with two α1 (I) chains and one α2 (II) chain. Type I collagen is a common collagen component in many other interstitial tissues such as tendons, skin, ligaments, and cornea, accounting for 25% of protein mass and more than 90% of bone matrix (Sorushanova A, et al. The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development. Adv Mater, 2019. 31(1): e1801651.). Type III collagen is also a homotrimer with three α1(III) chains. In addition, Type III collagen has disulfide bonds connecting the three C-terminal chains of the triple helix domain. In tissues with a certain degree of expansibility, such as skin, blood vessel walls, and reticular fibers of the lungs, liver, and spleen, Type III collagen usually coexists with collagen in Type I collagen fibrils (Shoulders MD, Raines RT. Collagen structure and stability. Annu Rev Biochem, 2009. 78: 929–958.). Collagen, with its good biocompatibility, biodegradability and low antigenicity, has been widely used in many fields of production and daily life, such as biomedicine, cosmetics, and health products in recent years.

[0003] Elastin is a protein essential for the elasticity of the natural extracellular matrix, contributing up to 2-4% of the skin's dry weight and responsible for conferring organ elasticity (Wen Q, et al. Elastin Biomaterials in Dermal Repair. Trends Biotechnol, 2020, 38(3):280-291.). It has a half-life greater than 70 years, and the monomer can reversibly extend to eight times its resting length. The mechanical properties of elastin can modulate cellular properties, meaning that the physical incorporation of elastin has a positive impact on cell growth; furthermore, the arrangement of elastin fibers affects cell phenotype, adhesion, and proliferation (Wen Q, et al. Elastin Biomaterials in Dermal Repair. Trends Biotechnol, 2020, 38(3):280-291.).

[0004] The small molecule compound CASIN is a specific inhibitor of the small Rho GTPase Cdc42. In recent years, some studies have preliminarily explored the biological effects of CASIN. 75-week-old aged C57BL / 6 mice were intraperitoneally injected with CASIN at a concentration of 25 mg / kg every 24 hours for 4 consecutive days. It was found that CASIN can significantly prolong the lifespan of aged female C57BL / 6 mice (Florian MC, et al. Inhibition of Cdc42 activity extends lifespan and decreases circulating inflammatory cytokines in aged female C57BL / 6 mice. Aging Cell, 2020. 19(9):e13208.); CASIN can restore the cell viability of senescent hematopoietic stem cells (Amoah A., et al. Aging of human hematopoietic stem cells is linked to changes in Cdc42 activity. Haematologica, 2022. 107(2):393-402.). In vivo treatment of aged mice with CASIN induced hair growth (Tiwari RL, et al. A Wnt5a-Cdc42 axis controls aging and rejuvenation of hair-follicle stem cells. Aging (Albany NY), 2021.13(4):4778-4793.). However, the effects of CASIN on collagen and elastin synthesis in fibroblasts remain unclear. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of CASIN in the preparation of formulations that promote the synthesis of collagen and elastin.

[0006] The objective of this invention is achieved through the following technical solution: the application of CASIN in the preparation of formulations that promote the synthesis of collagen and elastin. This is based on the results obtained by treating skin fibroblasts with the small molecule compound CASIN, and discovering that CASIN can significantly promote the synthesis of type I collagen, type III collagen, and elastin in skin fibroblasts.

[0007] The aforementioned CASIN, also known as 2-[(2,3,4,9-tetrahydro-6-phenyl-1H-carbazol-1-yl)amino]ethanol, is a specific inhibitor of Cdc42 activity, with the molecular formula C 20 H 22 N2O, CAS No.: 425399-05-9, molecular weight 306.40, its structural formula is shown in Formula I. Purified CASIN (HPLC purity > 98%) is a white powder, readily soluble in organic solvents such as ethanol and DMSO.

[0008]

[0009] The collagen mentioned is type I collagen and type III collagen.

[0010] The concentration of CASIN in the formulation for cell experiments is preferably 0.125–0.5 μmol / L, and most preferably 0.5 μmol / L.

[0011] The concentration of CASIN in the formulation in animal experiments is preferably 8.15–81.5 μmol / L, and most preferably 16.3 μmol / L.

[0012] The preparation is preferably a topical liniment or an injectable reagent.

[0013] The formulation described above has the following functions: improving skin elasticity and promoting skin firmness. Therefore, the formulation has strong application value in the fields of biomedicine and cosmetics.

[0014] The present invention has the following advantages and effects compared with the prior art:

[0015] This invention utilizes the CCK8 assay to determine the optimal concentration of CASIN for promoting fibroblast proliferation; and employs Western blotting (WB) to detect the regulatory effects of CASIN on the synthesis of type I, type III, and elastin in skin fibroblasts. Through this experimental model, this invention screens for active compounds that significantly upregulate the levels of type I, type III, and elastin at the cellular level. This invention is the first to publicly report that CASIN has a significant activity in promoting the synthesis of type I, type III, and elastin in mouse skin fibroblasts, and both in vivo and in vitro experiments demonstrate that CASIN significantly increases skin collagen content and significantly improves skin elasticity. This invention has strong application value in the biomedical and cosmetic fields. Attached Figure Description

[0016] Figure 1 shows the CCK8 experimental results of the effect of CASIN on fibroblast proliferation; where * indicates P < 0.05, **P < 0.01, and ***P < 0.001.

[0017] Figure 2 shows the results of the Western blot experiment on the effect of CASIN on collagen and elastin synthesis in fibroblasts; where A is a photograph of the Western blot experiment results; B is the statistical result of A, *P<0.05, **P<0.01, ***P<0.001.

[0018] Figure 3 shows the results of MASSON staining to detect the effects of different concentrations of CASIN injection on collagen and elastin synthesis in mouse skin after 14 days; where A is the MASSON staining result and B is the statistical graph of collagen area and density; * indicates P < 0.05, **P < 0.01, ***P < 0.001.

[0019] Figure 4 shows the results of aldehyde fuchsin staining to detect the effect of CASIN injection on elastin synthesis in mouse skin 14 days later; where A is the aldehyde fuchsin staining result of elastic fibers, and B is the statistical graph of elastic fiber area; * indicates P < 0.05.

[0020] Figure 5 shows the results of the skin physiological analyzer detecting the effect of injection on the skin elasticity of mice 14 days later; where ** indicates P<0.01.

[0021] Figure 6 shows the results of MASSON staining to detect the effect of CASIN application for 21 days on collagen synthesis in mouse skin; where A is the MASSON staining result and B is a statistical graph of collagen area and density; ** indicates P < 0.01.

[0022] Figure 7 shows the results of aldehyde fuchsin staining to detect the effect of CASIN 21 on elastin synthesis in mouse skin; where A is the aldehyde fuchsin staining result of elastic fibers, and B is the statistical graph of elastic fiber area; ** indicates P < 0.01.

[0023] Figure 8 shows the results of the effect of CASIN application on the skin elasticity of mice after 21 days, as detected by a skin physiology analyzer; where ** indicates P < 0.01. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0025] Example 1. CCK8 assay to detect the effect of different concentrations of CASIN on the proliferation of mouse skin fibroblasts.

[0026] Fifteen-month-old female mice were selected. After euthanasia, the hair on their backs was removed, and the skin was thoroughly disinfected. The full-thickness skin was then cut off and placed in a culture dish with the epidermis facing upwards and the dermis downwards. The cells were trypsinized at 4°C for 18 hours to separate the epidermis and dermis. The dermal tissue was completely minced, digested with type I collagenase, filtered, centrifuged at 1000 rpm for 5 minutes, and resuspended in DMEM low-glucose medium containing 10% fetal bovine serum. The cells were then seeded into 6 cm cell culture dishes to obtain primary mouse fibroblasts. The medium was changed according to the cell condition, and the cells were passaged.

[0027] Primary mouse fibroblasts were seeded at a rate of 2000 cells per well in 96-well plates. When the cells reached 50%–60% confluence, they were starved for 8 hours in serum-free medium (DMEM low-glucose medium, Gibco, 11885084). The cells were then divided into a control group and an experimental group treated with CASIN. The original medium was discarded. The CASIN-treated groups were added 100 μL of serum-free medium containing 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, and 0.125 μM CASIN (three replicates per concentration). The control group received only serum-free medium. The 96-well plates were incubated at 37°C with 5% CO2 for 12 h, 24 h, 48 h, and 72 h for CCK8 assay. Absorbance at 450 nm was measured using a microplate reader. Data analysis was performed using SPSS 25 statistical software. Paired t-tests were used to compare measurements at different time points. For statistically significant differences, * indicates P < 0.05, **P < 0.01, and ***P < 0.001. The experimental results are shown in Figure 1. Figure 1 shows that CASIN at concentrations greater than or equal to 2 μM can induce cell death; a CASIN concentration of 0.5 μM can significantly promote cell proliferation.

[0028] Example 2. Western blot analysis of the effects of CASIN on the expression levels of type I collagen, type III collagen, and elastin in mouse skin fibroblasts.

[0029] Primary fibroblasts from 15-month-old mice were divided into groups of 1×10⁻⁶ cells per well. 5Cells were seeded in 6-well plates and starved for 8 hours in DMEM serum-free low-glucose medium. Cells were then divided into a control group and a CASIN treatment group. The original medium was discarded. The control group was treated with DMEM serum-free low-glucose medium, while the CASIN treatment group was treated with CASIN medium at a final concentration of 0.5 μM. After 48 hours of culture, cell proteins were collected. Western blot was used to detect the levels of type I collagen, type III collagen, and elastin synthesized in fibroblasts, with each group repeated three times. ImageJ software was used to identify the gray values ​​of the bands and analyze the relative expression levels. SPSS 25 statistical software was used for data analysis, and the results are shown in Figure 2. Figure 2 shows that CASIN can promote the synthesis of type I collagen, type III collagen, and elastin in fibroblasts (*** indicates P < 0.001).

[0030] Example 3. MASSON staining to detect the effect of CASIN injection 14 days on collagen synthesis in mouse skin.

[0031] 15-month-old C57BL / 6 mice (Laboratory Animal Center of Southern Medical University, SCXK(Guangdong)2021-0041) were purchased and fed adaptively for one week. After the adaptation period ended, experimental treatments were carried out. To exclude the influence of individual differences, autologous control was adopted in this experiment. Taking the midline axis of the mouse back as the symmetry, treatments were performed on both sides of the posterior back. Control group: Inject 200 μL of PBS once every 3 days, for a total of 4 injections, and sample on the 14th day; The CASIN treatment groups were divided into 8.15 μM, 16.3 μM and 81.5 μM treatment groups. The 0.5 μg CASIN treatment group injected 200 μL of a CASIN solution with a concentration of 8.15 μmol / L (total amount of 0.5 μg, solvent is PBS) once every 3 days, for a total of 4 injections, and sampled on the 14th day. The 1 μg CASIN treatment group injected 200 μL of a CASIN solution with a concentration of 16.3 μmol / L (total amount of 1 μg, solvent is PBS) once every 3 days, for a total of 4 injections, and sampled on the 14th day. The 5 μg CASIN treatment group injected 200 μL of a CASIN solution with a concentration of 81.5 μmol / L (total amount of 5 μg, solvent is PBS) once every 3 days, for a total of 4 injections, and sampled on the 14th day. Carefully excise 1 cm of intact skin at the treatment site, fix it in 4% paraformaldehyde solution (PFA), make 5-μm continuous paraffin sections, and then perform Masson staining on the paraffin sections. The results of MASSON staining can stain collagen fibers bright blue, and the results are shown in Figure 3: Compared with the control group, the collagen area increased in the 8.15 μM, 16.3 μM and 81.5 μM treatment groups, and the difference was statistically significant (P < 0.01); The collagen density increased in the 8.15 μM and 16.3 μM treatment groups, and the difference was statistically significant (P < 0.05), but there was no statistically significant difference in the collagen density between the 81.5 μM treatment group and the control group. The 16.3 μM treatment group could significantly promote the increase in collagen area and density, and the difference was highly statistically significant (P < 0.001). The results in Figure 3 show that the results of MASSON staining show that CASIN injection can promote an increase in collagen synthesis in the dermis of mouse skin, and the collagen arrangement is denser and more orderly. The 16.3 μM treatment group is the most suitable concentration; * indicates P < 0.05, **P < 0.01, ***P < 0.001.

[0032] Example 4: Detection of the effect of CASIN injection for 14 days on elastin synthesis in mouse skin by aldehyde fuchsin staining.

[0033] 15-month-old C57BL / 6 mice (Experimental Animal Center of Southern Medical University, SCXK(Guangdong) 2021-0041) were purchased and fed adaptively for one week. After the adaptation period ended, experimental treatments were carried out. To exclude the influence of individual differences, self-control was adopted in this experiment, and the treatments were carried out on both sides of the posterior back symmetrically with the mid-axis of the mouse back as the symmetry axis. Control group: Inject 200 μL of PBS once every 3 days, for a total of 4 injections, and sample on the 14th day; CASIN treatment group: Inject 200 μL of CASIN solution with a concentration of 16.3 μM (the solvent is PBS) once every 3 days, for a total of 4 injections, and sample on the 14th day. Carefully excise 1 cm of intact skin at the treatment site, fix it in 4% paraformaldehyde solution (PFA), make 5-μm continuous paraffin sections, and then perform aldehyde fuchsin staining on the paraffin sections. Aldehyde fuchsin staining can stain elastic fibers dark purple. The results are shown in Figure 4: The aldehyde fuchsin staining results show that CASIN injection can promote the increase in the number and thickness of elastic fibers in the dermis of mouse skin, and the differences are all statistically significant (P < 0.05); * indicates P < 0.05.

[0034] Example 5. Detection of the effect of CASIN injection for 14 days on the skin elasticity of mice by a skin physiological detector.

[0035] 15-month-old C57BL / 6 mice (Experimental Animal Center of Southern Medical University, SCXK(Guangdong) 2021-0041) were purchased and fed adaptively for one week. After the adaptation period ended, experimental treatments were carried out. To exclude the influence of individual differences, self-control was adopted in this experiment, and the treatments were carried out on both sides of the posterior back symmetrically with the mid-axis of the mouse back as the symmetry axis. Control group: Inject 200 μL of PBS once every 3 days, for a total of 4 injections, and sample on the 14th day; CASIN treatment group: Inject 200 μL of CASIN solution with a concentration of 16.3 μM (the solvent is PBS) once every 3 days, for a total of 4 injections. Before sampling on the 14th day, measure the elasticity of the skin on the left and right sides of the mouse back respectively using a skin physiological instrument (Multi Skin Test Center MC 750, Germany). SPSS 25 statistical software was used for data analysis. For the measured data, paired t-tests were used to compare the measured values at different time points. For data with significant differences after statistics, P < 0.01 is marked with ** in the figure. The experimental results are shown in Figure  5. Figure 5 shows that compared with the control group, the elastic content of the skin of mice in the CASIN injection treatment group has a significant increase, with a significant difference (P < 0.01); ** indicates P <  0.01.

[0036] Example 6. Detection of the effect of CASIN application for 21 days on the collagen synthesis of mouse skin by MASSON staining.

[0037] 15-month-old C57BL / 6 mice (Experimental Animal Center of Southern Medical University, SCXK(Guangdong)2021-0041) were purchased and fed adaptively for one week. After the adaptation period ended, experimental treatments were carried out. To exclude the influence of individual differences, autologous control was adopted in this experiment. Symmetrical treatments were carried out at symmetrical positions on both sides of the posterior back with the mid-axis of the mouse back as the symmetry axis. Control group: 200 μL of PBS was applied every day for a total of 21 days; CASIN treatment group: 200 μL of CASIN solution with a concentration of 16.3 μM (the solvent was PBS) was applied every day for a total of 21 days. The intact skin with a diameter of 1 cm at the treatment site was carefully excised, fixed in 4% paraformaldehyde solution (PFA), and 5-μm continuous paraffin sections were made. Then, MASSON staining was performed on the paraffin sections. The MASSON staining results can stain collagen fibers bright blue. The results showed that CASIN application could promote the increase of collagen synthesis in the dermis of mouse skin, with an increase in collagen area and density, and the arrangement was denser and more orderly. The difference was statistically significant (P < 0.01). As shown in Figure 6, CASIN application for 21 days promoted the synthesis of collagen in mouse skin; ** indicates P < 0.01.

[0038] Example 7. Detection of the effect of CASIN application for 21 days on the synthesis of elastin in mouse skin by aldehyde fuchsin staining.

[0039] 15-month-old C57BL / 6 mice (Experimental Animal Center of Southern Medical University, SCXK(Guangdong)2021-0041) were purchased and fed adaptively for one week. After the adaptation period ended, experimental treatments were carried out. To exclude the influence of individual differences, autologous control was adopted in this experiment. Symmetrical treatments were carried out at symmetrical positions on both sides of the posterior back with the mid-axis of the mouse back as the symmetry axis. Control group: 200 μL of PBS was applied every day for a total of 21 days; CASIN treatment group: 200 μL of CASIN solution with a concentration of 16.3 μM (the solvent was PBS) was applied every day for a total of 21 days. The intact skin with a diameter of 1 cm at the treatment site was carefully excised, fixed in 4% paraformaldehyde solution (PFA), and 5-μm continuous paraffin sections were made. Then, aldehyde fuchsin staining was performed on the paraffin sections. Aldehyde fuchsin staining can stain elastic fibers dark purple. The staining results showed that CASIN application could promote the increase and thickening of elastic fibers in the dermis of mouse skin, and the differences were all statistically significant (P < 0.01). As shown in Figure 7, CASIN application for 21 days promoted the synthesis of elastin in mouse skin; ** indicates P < 0.01.

[0040] Example 8. Detection of the effect of CASIN application for 21 days on the elasticity of mouse skin by a skin physiological detector.

[0041] 15-month-old C57BL / 6 mice (Experimental Animal Center of Southern Medical University, SCXK(Guangdong) 2021-0041) were purchased and fed adaptively for one week. After the adaptation period ended, experimental treatments were carried out. To exclude the influence of individual differences, autologous control was adopted in this experiment. Symmetrical treatments were performed at symmetrical positions on both sides of the posterior back with the mid-axis of the mouse back as the symmetry axis. Control group: 200 μL of PBS was applied every day for a total of 21 days. CASIN treatment group: 200 μL of CASIN solution with a concentration of 16.3 μM (the solvent was PBS) was applied every day for a total of 21 days. The elasticity of the skin on the left and right sides of the mouse back was measured separately using a skin physiometer (Multi Skin Test Center MC 750, Germany). SPSS 25 statistical software was used for data analysis. For the measured data, paired t-tests were used to compare the measured values at different time points. For data with significant differences after statistics, P < 0.01 was marked with ** in the figure. The experimental results are shown in Figure 8. Figure 8 shows that compared with the control group, the elasticity of the skin of the mice in the CASIN treatment group was significantly increased, with a significant difference (P < 0.01); ** indicates P < 0.01.

[0042] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of CASIN in the preparation of preparations that improve skin elasticity.

2. The application of CASIN according to claim 1 in the preparation of skin elasticity-enhancing agents, characterized in that: The improvement in skin elasticity mentioned above is achieved by promoting the synthesis of collagen and elastin.

3. The application of CASIN according to claim 2 in the preparation of skin elasticity-enhancing agents, characterized in that: The collagen mentioned is type I collagen and type III collagen.

4. The application of CASIN according to claim 1 in the preparation of skin elasticity-enhancing agents, characterized in that: The concentration of CASIN in the formulation used in cell experiments is 0.125–0.5 μmol / L.

5. The application of CASIN according to claim 4 in the preparation of skin elasticity-enhancing agents, characterized in that: The concentration of CASIN in the formulation used in cell biology experiments is 0.5 μmol / L.

6. The application of CASIN according to claim 1 in the preparation of skin elasticity-enhancing agents, characterized in that: The concentration of CASIN in the formulation, as observed in animal experiments, was 8.15–81.5 μmol / L.

7. The application of CASIN according to claim 6 in the preparation of skin elasticity-enhancing agents, characterized in that: The concentration of CASIN in the formulation was 16.3 μmol / L in animal experiments.

8. The application of CASIN according to claim 1 in the preparation of skin elasticity-enhancing agents, characterized in that: The preparation is a topical liniment or an injectable reagent.

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