Use of l-kynurenine for the preparation of a medicament for the prevention and / or treatment of ultraviolet light-induced skin photodamage

Abstract

The application discloses application of L- kynurenine in preparation of a medicine for preventing and / or treating ultraviolet light induced skin photo-damage. The application proves that L- kynurenine has a significant anti-photo-damage effect by a UVA irradiation HaCaT cell model, can enhance mitochondrial function and promote mitochondrial biogenesis, thereby improving the antioxidant capacity of cells and reducing inflammatory reactions. The application also verifies that a hydrogel containing L- kynurenine can effectively repair mouse back skin damage caused by ultraviolet light through a UVA combined with UVB irradiation back depilation mouse model, improve the water content and antioxidant capacity of the skin, and reduce inflammatory reactions, edema, roughness, erythema or eschar and other phenomena. Therefore, the application provides a reference direction for development, utilization and popularization of L- kynurenine for preparing a medicine or a medical cosmetic product for repairing skin photo-damage.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of L-kynurenine in the preparation of drugs for the prevention and / or treatment of ultraviolet light-induced photodamage to the skin. Background Technology

[0002] Skin is the largest organ in the human body, accounting for approximately 10%-15% of body weight. As the body's first line of defense against the external environment, skin plays a vital role in protection, temperature regulation, sensation, secretion, excretion, and immunity. In recent years, skin health has faced greater threats due to increased exposure to ultraviolet radiation and chemical pollutants. Epidemiological surveys show that approximately 80% of exogenous skin aging is related to ultraviolet (UV) radiation, also known as photoaging. Mitochondria are unique organelles with their own genome. Ultraviolet radiation can induce oxidative stress, leading to the release of excessive reactive oxygen species (ROS). ROS primarily target mitochondrial DNA (mtDNA), making mitochondria particularly vulnerable to oxidative damage. Therefore, maintaining mitochondrial health is crucial for normal cellular function.

[0003] Kynurenine is an important product of tryptophan catabolism, participating in various biological processes and possessing a wide range of biological activities. Kynurenine helps stabilize the immune system and exerts neuroprotective effects. In existing technologies, kynurenine is primarily used as an immunomodulator and biomarker; however, there are currently no studies linking kynurenine to mitochondrial protection against UV damage to the skin. Summary of the Invention

[0004] The purpose of this invention is to provide the application of L-kynurenine in the preparation of drugs for the prevention and / or treatment of UV-induced photodamage to the skin. The L-kynurenine described in this invention not only alleviates oxidative stress damage and regulates inflammation levels, but also reduces mitophagy and promotes mitochondrial regeneration, further maintaining the structure and function of mitochondria, thereby playing a protective role in reducing photodamage to the skin. This multi-target protective mechanism has a more comprehensive advantage.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0006] This invention provides the use of L-kynurenine in the preparation of medicaments for the prevention and / or treatment of ultraviolet light-induced photodamage to the skin.

[0007] Furthermore, the structural formula of the L-kynurenine is as follows: .

[0008] Furthermore, the skin photodamage is skin photodamage caused by UVA and / or UVB ultraviolet light irradiation; the effective concentration of L-kynurenine is 50μM-200μM.

[0009] Furthermore, the photodamage is at least one of the following: skin erythema, desquamation, wrinkles, thickening of the stratum corneum, and collagen destruction.

[0010] Furthermore, the L-kynurenine can significantly increase the content of IL-37 and decrease the content of intracellular IL-6, thereby reducing the inflammation level of UVA-radiated cells.

[0011] Furthermore, L-kynurenine inhibits the increase of ROS and reduces oxidative stress; L-kynurenine lowers LDH levels, increases GSH levels, and enhances T-AOC levels, thereby improving the antioxidant capacity of skin keratinocytes exposed to UVA radiation.

[0012] Furthermore, the L-kynurenine enhances mitochondrial function and reduces the formation of autophagosomes; the L-kynurenine can promote mitochondrial regeneration by activating the PGC-1α / SIRT3 signaling pathway.

[0013] Furthermore, the functions of the mitochondria include effective restoration of mitochondrial membrane potential, restoration of the activity of mitochondrial complexes I, II and V, increased ATP synthesis, and improved mitochondrial morphology.

[0014] Furthermore, the L-kynurenine can reduce the level of mitochondrial autophagy in cells to an appropriate level, maintain the number and function of mitochondria, reduce the expression of mitochondrial autophagy proteins in keratinocytes of UVA-irradiated skin, thereby regulating excessive autophagy in cells.

[0015] The present invention also provides a topical preparation for preventing and / or repairing photodamage to the skin, wherein the active ingredient of the topical preparation is L-kynurenine, and the topical preparation is a cream, hydrogel, patch, ointment, or lotion.

[0016] Furthermore, the content of L-kynurenine in the hydrogel is 1-3%.

[0017] Furthermore, the hydrogel also contains carbomer 940, triethanolamine, glycerol, a 1% sodium benzoate aqueous solution and distilled water, wherein the mass ratio of carbomer 940 to triethanolamine is 1:1.

[0018] Furthermore, the topical preparation can improve at least one of the following phenomena in photodamaged skin: erythema, desquamation, wrinkles, thickening of the stratum corneum, and collagen destruction.

[0019] Furthermore, the topical formulation can increase the moisture content and antioxidant capacity of photodamaged skin.

[0020] Furthermore, the topical formulation can reduce the expression of autophagy proteins Parkin and LC3-II in photodamaged skin and increase the expression of SIRT3 protein.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] 1. This invention is the first to experimentally demonstrate the therapeutic effect of L-kynurenine on UV-induced skin damage. In a UVA-irradiated HaCaT cell model, L-kynurenine can alleviate oxidative stress, regulate inflammation levels, reduce mitophagy, and promote mitochondrial regeneration, thereby maintaining mitochondrial structure and function.

[0023] 2. The L-kynurenine hydrogel described in this invention can maintain skin moisture and collagen fiber content, improve antioxidant capacity, and regulate the expression of key proteins in the mitophagy and neonatal pathways in a mouse skin photodamage model induced by UVA+UVB radiation, further confirming the protective effect of L-kynurenine on UV skin photodamage.

[0024] 3. The L-kynurenine provided by this invention is a potential therapeutic agent that can effectively address skin damage caused by ultraviolet radiation and has broad application prospects, especially in the field of skin photodamage drugs and medical aesthetic products development. Attached Figure Description

[0025] Figure 1 The figure shows the results of measuring the proliferation activity of HaCaT cells under UVA radiation by L-kynurenine.

[0026] Figure 2 Figure 1 shows the results of measuring the antioxidant effect of L-kynurenine on HaCaT cells under UVA radiation; A: Effect of L-kynurenine on reactive oxygen species in UVA-radiated cells; B: Effect of L-kynurenine on lactate dehydrogenase release in UVA-radiated cells; C: Effect of L-kynurenine on glutathione content in UVA-radiated cells; D: Effect of L-kynurenine on total antioxidant capacity in UVA-radiated cells.

[0027] Figure 3 Figure 1 shows the results of measuring the release of inflammatory factors in HaCaT cells under UVA radiation by L-kynurenine; A: The promoting effect of L-kynurenine on the anti-inflammatory factor IL-37; B: The inhibitory effect of L-kynurenine on the pro-inflammatory factor IL-6.

[0028] Figure 4Figure 1 shows the results of L-kynurenine's effect on mitochondrial function in HaCaT cells under UVA radiation; A: L-kynurenine increases mitochondrial membrane potential; B: L-kynurenine increases the activity of mitochondrial complex I; C: L-kynurenine increases the activity of mitochondrial complex II; D: L-kynurenine increases the activity of mitochondrial complex V; E: L-kynurenine promotes ATP synthesis.

[0029] Figure 5 Electron microscopy results showing that L-kynurenine can improve mitochondrial structure and reduce autophagosomes in HaCaT cells exposed to UVA radiation.

[0030] Figure 6 A schematic diagram showing the bands (A) and relative content (BF) of L-kynurenine-regulated autophagy proteins PINK1, Parkin, LC3-II, Beclin-1 and p62 in HaCaT cells under UVA radiation.

[0031] Figure 7 A schematic diagram showing the bands (A) and relative content (BC) of L-kynurenine-regulated mitochondrial neoproteins PGC-1α and SIRT3 in HaCaT cells exposed to UVA radiation.

[0032] Figure 8 Figure (A), statistical results of the damage to the back skin of mice irradiated with UVA+UVB ultraviolet light are shown in the following figures: L-kynurenine hydrogel with three different formulations (B) and pathological section (C).

[0033] Figure 9 Figure (A) shows the skin damage on the back of mice irradiated with UVA+UVB light by three different hydrogels, and the statistical results of the damage area (B).

[0034] Figure 10 Image of a pathological section of mouse dorsal skin irradiated with UVA+UVB ultraviolet light using L-kynurenine hydrogel.

[0035] Figure 11 The image shows the results of L-kynurenine hydrogel on the water content of mouse back skin irradiated with UVA+UVB ultraviolet light.

[0036] Figure 12 Figure 1 shows the antioxidant effects of L-kynurenine hydrogel on mouse back skin irradiated with UVA+UVB light; A: L-kynurenine hydrogel reduces malondialdehyde content in mouse back skin; B: L-kynurenine hydrogel increases glutathione content in mouse back skin; C: L-kynurenine hydrogel enhances total antioxidant capacity of mouse back skin.

[0037] Figure 13A schematic diagram showing the bands (A) and relative content (BC) of Parkin and LC3-II autophagy proteins in mouse dorsal skin irradiated by L-kynurenine hydrogel under UVA+UVB ultraviolet light irradiation.

[0038] Figure 14 A schematic diagram showing the bands and relative content of SIRT3 protein, a mitochondrial protein produced by L-kynurenine hydrogels in mouse dorsal skin irradiated with UVA and UVB ultraviolet light. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below with reference to specific examples. It should be noted that the following descriptions are preferred embodiments of the present invention, not all embodiments. For those skilled in the art, all other embodiments obtained without making innovative contributions are within the scope of protection of the present invention. In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0040] The use of L-kynurenine, as shown in Formula I, in the preparation of medicaments for the prevention and / or treatment of UV-induced photodamage to the skin:

[0041] Formula I.

[0042] In this invention, the molecular formula of L-kynurenine is C 10 H 12 N2O3, with a molecular weight of 208.22. In this embodiment of the invention, the L-kynurenine was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., catalog number L864410.

[0043] Example 1: L-kynurenine significantly protects HaCaT cells from UVA radiation damage.

[0044] The specific implementation steps are as follows: Culture HaCaT cells and seed them in 96-well plates, 1.2 × 10⁶ cells per well. 4 Each well was incubated at 37°C for 48 hours. The culture medium was then discarded, and different concentrations of L-kynurenine (50 μM, 100 μM, and 200 μM) were prepared using phenol red-free culture medium. 200 μL was added to each well, the cap of the 96-well plate was removed, and the plate was irradiated under UVA at 0.5 J / cm². 2 The control group received no UVA irradiation. After culturing the 96-well plates back in the incubator for another 24 hours, cell viability was assessed using the MTT assay.

[0045] Experimental results: such as Figure 1 As shown, 0.5 J / cm 2UVA can reduce cell viability, while L-kynurenine (50-200 μM) shows a protective effect, significantly improving cell viability, and its protective effect is dose-dependent.

[0046] Example 2: L-kynurenine can scavenge intracellular ROS from UVA radiation.

[0047] The specific implementation steps are as follows: Culture HaCaT cells and seed them in 96-well plates, 1.2 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (50 μM, 100 μM, 200 μM) in phenol red-free medium. Add 200 μL to each well, remove the cap of the 96-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After returning the 96-well plate to the incubator and culturing for another 24 hours, the culture medium was discarded, and the prepared DCFH-DA fluorescent probe was added and incubated in the incubator for 20 minutes before detecting ROS at wavelengths of 488 / 525 nm.

[0048] Experimental results: such as Figure 2 As shown in Figure A, UVA increased intracellular ROS levels, while L-kynurenine in all groups inhibited the increase in ROS, thereby reducing oxidative stress.

[0049] Example 3: L-kynurenine can reduce the level of LDH in cells exposed to UVA radiation, and increase the levels of GSH and T-AOC.

[0050] The specific implementation steps are as follows: Culture HaCaT cells and seed them in 6-well plates, 70 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (200 μM) in phenol red-free culture medium. Add 2 mL to each well, remove the cap of the 6-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After returning the 6-well plate to the incubator and culturing for another 24 hours, the culture medium was discarded, cell proteins were extracted, and the levels of LDH, GSH, and T-AOC in each group of cells were measured according to the kit instructions.

[0051] Experimental results: such as Figure 2 As shown in the middle BD, UVA irradiation increases intracellular LDH release, decreases GSH content, and further reduces T-AOC. In contrast, 200 μM L-kynurenine reduces LDH level, increases GSH level, and increases T-AOC, indicating that L-kynurenine can enhance the antioxidant capacity of UVA-damaged cells, thereby protecting cells from oxidative damage.

[0052] Example 4: L-kynurenine can regulate the release of inflammatory factors in cells exposed to UVA radiation.

[0053] The specific steps are as follows: Culture HaCaT cells and seed them in 96-well plates, 1.2 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (200 μM) in phenol red-free medium. Add 200 μL to each well, remove the cap of the 96-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After returning the 96-well plate to the incubator and culturing for another 24 hours, the supernatant was collected and the levels of IL-37 and IL-6 were detected by ELISA.

[0054] Experimental results: such as Figure 3 As shown, after UVA radiation, the content of the anti-inflammatory factor IL-37 in cells decreased significantly, while the content of the pro-inflammatory factor IL-6 increased significantly. L-kynurenine (200 μM) could significantly increase the content of IL-37 and decrease the content of IL-6 in cells, indicating that L-kynurenine can reduce the inflammation level of UVA-radiated cells.

[0055] Example 5: L-kynurenine increases mitochondrial membrane potential in UVA-irradiated cells

[0056] The specific implementation steps are as follows: Culture HaCaT cells and seed them in 96-well plates, 1.2 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (50 μM, 100 μM, 200 μM) in phenol red-free medium. Add 200 μL to each well, remove the cap of the 96-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After returning the 96-well plate to the incubator and culturing for another 24 hours, the culture medium was discarded. The prepared JC-1 fluorescent probe was added and incubated in the incubator for 20 minutes before detection. The detection wavelengths were 490 / 530 nm for monomers and 525 / 590 nm for polymers. The result was the ratio of polymer to monomer.

[0057] Experimental results: such as Figure 4 As shown in Figure A, the mitochondrial membrane potential of cells decreased after UVA radiation, indicating mitochondrial damage. However, 50-200 μM L-kynurenine effectively restored the mitochondrial membrane potential of the cells.

[0058] Example 6: L-kynurenine can enhance the activity of mitochondrial complexes I, II, and V in UVA-irradiated cells.

[0059] The specific implementation steps are as follows: Culture HaCaT cells and seed them in 6-well plates, 70 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (200 μM) in phenol red-free culture medium. Add 2 mL to each well, remove the cap of the 6-well plate, and irradiate it under UVA at 0.5 J / cm². 2After returning the 6-well plate to the incubator and culturing for another 24 hours, discard the culture medium and extract cell samples according to the kit instructions for testing.

[0060] Experimental results: such as Figure 4 As shown in Figure BD, the activities of mitochondrial complexes I, II, and V were significantly reduced after UVA radiation, indicating that UVA radiation disrupts the electron respiratory chain and fails to provide energy for cellular physiological activities. However, 200 μM L-kynurenine restored the activities of mitochondrial complexes I, II, and V, thereby repairing the damaged electron respiratory chain.

[0061] Example 7: L-kynurenine can promote ATP synthesis in UVA-irradiated cells.

[0062] The specific implementation steps are as follows: Culture HaCaT cells and seed them in 6-well plates, 70 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (200 μM) in phenol red-free culture medium. Add 2 mL to each well, remove the cap of the 6-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After returning the 6-well plate to the incubator and culturing for another 24 hours, discard the culture medium and extract cell samples according to the kit instructions for testing.

[0063] Experimental results: such as Figure 4 As shown in Figure E, the ATP content in cells decreased significantly after UVA radiation, while 200 μM L-kynurenine increased the ATP content, indicating that it can improve the energy state of cells.

[0064] Example 8: L-kynurenine can improve the mitochondrial morphology and structure of UVA-irradiated cells and reduce the formation of autophagosomes.

[0065] The specific steps are as follows: Culture HaCaT cells and seed them in 6-well plates, 70 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (200 μM) in phenol red-free culture medium. Add 2 mL to each well, remove the cap of the 6-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After returning the 6-well plate to the incubator and culturing for another 24 hours, discard the culture medium, add PBS, scrape off the cells using a scraper, and centrifuge at low speed for 10 minutes in a 1.5 mL centrifuge tube. Discard the supernatant; the precipitate should be at least half the size of a grain of rice. Carefully add 2.5% glutaraldehyde along the tube wall, being careful not to dislodge the precipitate. Fix overnight at 4°C. Prepare ultrathin sections as required, and observe the mitochondrial morphological changes of cells after different treatments using transmission electron microscopy.

[0066] Experimental results: such as Figure 5As shown, UVA radiation caused mitochondria to exhibit abnormal morphology, structural damage, cristae breakage or disappearance, and increased autophagosome formation. Treatment with L-kynurenine significantly improved mitochondrial morphology and reduced autophagosome formation.

[0067] Example 9: L-kynurenine regulates mitophagy via the PINK1 / Parkin signaling pathway

[0068] The specific steps are as follows: Culture HaCaT cells and seed them in 6-well plates, 70 × 10⁶ cells per well. 4 After 48 hours, discard the culture medium and prepare L-kynurenine (200 μM) in phenol red-free culture medium. Add 2 mL to each well, remove the cap of the 6-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After culturing for another 24 hours, the culture medium was discarded, and pre-chilled lysis buffer was added. Lysis was performed on ice for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4°C. The supernatant was collected for Western blotting analysis. 20 μg of protein from the cell lysate was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed for 2 hours at room temperature using 5% BSA. The bands were incubated overnight at 4°C with specific primary antibodies (PINK1, Parkin, LC3A / B, Beclin-1, p62, GAPDH). After incubation, the bands were washed 6 times with TBST buffer and then incubated with the corresponding HRP-conjugated secondary antibodies. Chemiluminescence imaging was used for detection. The relative density of each protein band was analyzed using ImageJ, with GAPDH as an internal control. The optical density values ​​were normalized to obtain the corresponding protein expression levels.

[0069] Experimental results: such as Figure 6 As shown, compared with the control group, after UVA radiation, the expression levels of PINK1, Parkin, LC3-II, and Beclin-1 proteins increased, while the expression level of p62 protein decreased in HaCaT cells, indicating that mitochondrial stimulation led to excessive autophagy, which affects normal cell function. After treatment with L-kynurenine, the expression levels of PINK1, Parkin, LC3-II, and Beclin-1 proteins decreased, while the expression level of p62 protein increased, indicating that L-kynurenine can reduce the level of mitochondrial autophagy to an appropriate level, thereby maintaining the number and function of mitochondria and playing a protective role for cells.

[0070] Example 10: L-kynurenine regulates mitochondrial regeneration through the PGC-1α / SIRT3 signaling pathway

[0071] The specific steps are as follows: Culture HaCaT cells and seed them in 6-well plates, 70 × 10⁶ cells per well.4 After 48 hours, discard the culture medium and prepare L-kynurenine (200 μM) in phenol red-free culture medium. Add 2 mL to each well, remove the cap of the 6-well plate, and irradiate it under UVA at 0.5 J / cm². 2 After returning the cells to the incubator and culturing for another 24 hours, the culture medium was discarded, and pre-chilled lysis buffer was added. Lysis was performed on ice for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4°C. The supernatant was collected for Western blotting analysis. 20 μg of protein from the cell lysate was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed for 2 hours at room temperature using 5% BSA as the blocking buffer. The bands were incubated overnight at 4°C with specific primary antibodies (PGC-1α, SIRT3, GAPDH). After incubation, the bands were washed six times with TBST buffer and then incubated with the corresponding HRP-conjugated secondary antibodies. Chemiluminescence imaging was used for detection. The relative density of each protein band was analyzed using ImageJ, with GAPDH as an internal control. The optical density values ​​were normalized to obtain the corresponding protein expression levels.

[0072] Experimental results: such as Figure 7 As shown, compared with the control group, the expression of PGC-1α and SIRT3 proteins in cells was significantly reduced after UVA radiation, indicating that UVA inhibits mitochondrial regeneration. In cells treated with L-kynurenine, the expression levels of PGC-1α and SIRT3 proteins were significantly increased, indicating that L-kynurenine can promote mitochondrial regeneration by activating the PGC-1α / SIRT3 signaling pathway.

[0073] Example 11: Preparation of a topical formulation containing L-kynurenine

[0074] Based on the uses of L-kynurenine in the foregoing embodiments, this embodiment provides a topical preparation containing L-kynurenine. An experimental group will be set up below, and in conjunction with Examples 11-18, multiple tests will be performed on the mouse skin tissues to be tested in the experimental group to verify the effects of L-kynurenine in various aspects. In Examples 12-18 below, the mouse skin tissues used can be obtained according to the following steps 1-3:

[0075] Step 1: Preparation of L-kynurenine hydrogel

[0076] While stirring, slowly add carbomer 940 to distilled water and stir until completely dispersed; add glycerol and stir evenly; then mix in L-kynurenine and stir evenly; add triethanolamine, heat until uniform to remove air bubbles, and cool to room temperature; add 1% sodium benzoate aqueous solution and stir evenly to obtain a hydrogel containing L-kynurenine.

[0077] Three types of L-kynurenine hydrogels with a mass ratio of 1% were prepared by setting the ratio of carbomer 940 to triethanolamine to 1:4, 1:1, and 2:1, as detailed in Table 1.

[0078] Table 1: Three formulations of 1% L-kynurenine hydrogel

[0079]

[0080] Using the same preparation method and formula as KYN2, except for the absence of L-kynurenine, 1% ergothioneine gel (MJLY group) and blank matrix gel (blank matrix group) were prepared. See Table 2 for details.

[0081] Table 2: Hydrogel Formulation

[0082]

[0083] Step 2: Pre-experimental treatment

[0084] Balb / c female mice, 6-8 weeks old, SPF, weighing 20-22g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The fur on the backs of the mice was removed using a small animal razor, with gentle movements to avoid damaging the skin.

[0085] (a) The animals were randomly divided into 5 experimental groups, with 8 animals in each group. The 5 experimental groups were: blank group, model group, KYN1, KYN2, and KYN3.

[0086] (ii) The animals were randomly divided into 5 experimental groups, with 8 animals in each group. The 5 experimental groups were: blank group, model group, positive control group (MJLY group), L-kynurenine group (KYN group), and blank matrix gel group (blank matrix group).

[0087] Step 3: Obtaining mouse skin tissue

[0088] Before irradiation, each group applied the corresponding hydrogel to the back skin. LED UVA and UVB lamps were placed approximately 40 cm vertically above the mice's backs. After preheating for 5 minutes, mice were irradiated for 1 hour daily, 6 days a week, for 7 consecutive weeks. Within 24 hours of the final irradiation, mice were euthanized by cervical dislocation. Back skin tissue was collected, connective tissue and subcutaneous fat were removed, and the tissue was flash-frozen in liquid nitrogen and stored at -80°C for subsequent experimental studies.

[0089] Example 12: This example uses three different formulations of 1% L-kynurenine hydrogel prepared in Example 11 to verify the protective effect of the three different formulations of 1% L-kynurenine hydrogel on UVA+UVB irradiation-induced skin damage on the back of mice.

[0090] Experimental Results: The effects of three different formulations of 1% L-kynurenine hydrogel on the skin on the backs of mice after UV irradiation were as follows: Figure 8 As shown, the KYN2 formula (carbomer 940 to triethanolamine in a 1:1 ratio) is more effective than the other two formulas in improving skin redness and scabbing and reducing epidermal thickness. Therefore, the formula with a 1:1 ratio of carbomer 940 to triethanolamine was selected for subsequent experiments.

[0091] Example 13: This example uses the MJLY group hydrogel, KYN group hydrogel, and blank matrix group gel prepared in Example 11 to verify that L-kynurenine hydrogel (KYN group hydrogel) can improve the skin damage on the back of mice caused by UVA+UVB irradiation.

[0092] Experimental results: such as Figure 9 As shown, compared with the control group, after the first week of UV irradiation, the model group mice began to show obvious dryness, peeling, and epidermal thickening; after the third week of UV irradiation, the model group mice showed large areas of rough wrinkles, erythema, eschar, and sunburn. The control group did not improve the skin condition, indicating that the control group had no significant protective effect. Compared with the model group, the L-kynurenine group and the ergothioneine group showed significant improvement in skin roughness and sunburn symptoms such as redness and swelling. The effect of L-kynurenine in resisting UV damage was even better than that of ergothioneine. This indicates that L-kynurenine can significantly improve skin roughness and sunburn in mice after UV irradiation.

[0093] Example 14: Effects of L-kynurenine hydrogel on mouse skin histopathology

[0094] The specific steps are as follows: Skin tissue from each group of mice was collected, fixed with 4% paraformaldehyde, and then embedded in paraffin for sectioning. The sections were mounted on glass slides, and the content of collagen in the epidermis and skin tissue was observed by eosin (HE) and Masson's trichrome staining. The obtained stained section images were analyzed using an inverted microscope.

[0095] Experimental results: such as Figure 10 As shown, HE and Masson staining results revealed that the epidermis of mice without UV exposure had normal structure and tightly packed collagen fibers. In mice exposed to UV radiation, the epidermis was significantly thickened with inflammatory cell aggregation, and the amount of collagen fibers in the green areas of the skin was greatly reduced, with some collagen fibers showing disorder and breakage, indicating that UV radiation damaged collagen fibers in the skin. After treatment with L-kynurenine hydrogel, the epidermis of mice was thinner than that of the UV group, and the collagen fibers in the dermis were more tightly packed and abundant. Therefore, for skin exposed to UV radiation, compositions containing L-kynurenine can reduce epidermal thickness and protect collagen fiber structure, thus playing a protective role for the skin.

[0096] Example 15: L-kynurenine hydrogel can increase the water content of mouse skin after UVA+UVB irradiation.

[0097] The specific steps are as follows: Measure skin moisture content using the direct drying method. Quickly cut off skin tissue, rinse rapidly with physiological saline, blot dry with absorbent paper, and weigh and record the weight; this is the wet weight. Set the oven temperature to approximately 105℃ and place the skin tissue inside for drying. After drying, remove the skin, weigh it, and record the weight. Place the sample back in the oven and continue drying for approximately 1 hour, then weigh it again. Repeat this process until the difference between two consecutive weighings does not exceed 0.5 mg; this weight is the dry weight. Skin moisture content (%) = (Wet weight - Dry weight) / Wet weight × 100%.

[0098] Experimental results: such as Figure 11 As shown, the skin moisture content of mice exposed to ultraviolet light decreased, while L-kynurenine hydrogel significantly increased the skin moisture content of mice, indicating that L-kynurenine has a significant effect on maintaining skin moisture content.

[0099] Example 16: L-kynurenine hydrogel can enhance the antioxidant capacity of mouse skin tissue after UVA+UVB irradiation.

[0100] The specific steps are as follows: Skin tissue is cut and ground on ice. The ground skin samples are processed according to the instructions of the corresponding kit, and the contents of MDA, GSH, and T-AOC in each group are detected.

[0101] Experimental results: such as Figure 12 As shown, compared with the control group, the levels of MDA increased and the levels of GSH and T-AOC decreased after UV irradiation. Treatment with L-kynurenine hydrogel effectively reduced MDA levels and restored GSH and T-AOC levels. This indicates that L-kynurenine can enhance the skin's total antioxidant capacity by reducing lipid peroxidation and promoting glutathione expression, demonstrating that L-kynurenine can enhance the antioxidant capacity of skin exposed to UV radiation.

[0102] Example 17: L-kynurenine hydrogel can promote the expression of autophagy-related proteins in mouse skin tissue after UVA+UVB irradiation.

[0103] The specific steps are as follows: Approximately 100 mg of frozen mouse skin tissue was placed in a 2 mL centrifuge tube, 1 mL of lysis buffer was added, and the tube was homogenized on ice using a tissue homogenizer. The homogenate was then transferred to a centrifuge at 4°C and 12000 rpm for 10 min. The supernatant was collected for Western blotting analysis. 30 μg of protein from the tissue lysis buffer was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed for 2 hours at room temperature using 5% BSA as the blocking buffer. The bands were incubated overnight at 4°C with specific primary antibodies (Parkin, LC3A / B, GAPDH). After incubation, the bands were washed 6 times with TBST buffer and then incubated with the corresponding HRP-conjugated secondary antibody. Detection was performed using a chemiluminescence imaging system. The relative density of each protein band was analyzed using ImageJ, with GAPDH as an internal control. The optical density values ​​were normalized to obtain the corresponding protein expression levels.

[0104] Experimental results: such as Figure 13 As shown, the expression levels of Parkin and LC3-II proteins were increased in mouse skin tissue irradiated with ultraviolet light, and the expression of these proteins was reduced by treatment with L-kynurenine hydrogel.

[0105] Example 18: L-kynurenine hydrogel promotes the expression of SIRT3 protein in mouse skin tissue after UVA+UVB irradiation.

[0106] The specific steps are as follows: Approximately 100 mg of frozen mouse skin tissue was placed in a 2 mL centrifuge tube, 1 mL of lysis buffer was added, and the tube was homogenized on ice using a tissue homogenizer. The homogenate was then transferred to a centrifuge at 12,000 rpm for 10 min at 4°C. The supernatant was collected for Western blotting analysis. 30 μg of protein from the tissue lysate was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Blocking was performed for 2 hours at room temperature using 5% BSA as the blocking buffer. The bands were incubated overnight at 4°C with specific primary antibodies (SIRT3, GAPDH). After incubation, the bands were washed 6 times with TBST buffer and then incubated with the corresponding HRP-conjugated secondary antibodies. Detection was performed using a chemiluminescence imaging system. The relative density of each protein band was analyzed using ImageJ, with GAPDH as an internal control. The optical density values ​​were normalized to obtain the corresponding protein expression levels.

[0107] Experimental results: such as Figure 14 As shown, compared with the control group, the expression of SIRT3 protein in mouse skin tissue exposed to ultraviolet radiation was reduced, while the expression of SIRT3 protein in skin tissue treated with L-kynurenine hydrogel was increased.

[0108] In summary, this invention verifies the multiple protective effects of L-kynurenine in treating UV-induced photodamage to the skin. L-kynurenine can alleviate oxidative stress damage, reduce inflammation levels, and maintain mitochondrial structure and function. This multi-target protective mechanism provides a more comprehensive therapeutic effect. In a UVA-irradiated HaCaT cell model, L-kynurenine alleviates oxidative stress damage, reduces inflammation levels, decreases mitophagy, and promotes mitochondrial regeneration, thereby improving cell viability. In a mouse skin photodamage model induced by UVA+UVB radiation, L-kynurenine hydrogel increased skin moisture and collagen fiber content, enhanced antioxidant capacity, and regulated the expression of key proteins in mitophagy and regeneration pathways, verifying the protective effect of L-kynurenine on UV-damaged skin. This invention fills a gap in the existing technology regarding the treatment of photodamage to the skin and has broad application prospects.

[0109] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. The use of L-kynurenine in the preparation of hydrogel formulations for the prevention and / or treatment of UV-induced photodamage to the skin, characterized in that, The effective concentration of L-kynurenine is 50 μM-200 μM; the skin photodamage is skin photodamage caused by UVA and / or UVB ultraviolet light irradiation; the formulation can improve at least one of the following phenomena in photodamaged skin: erythema, desquamation, wrinkles, thickening of the stratum corneum, and collagen destruction; the hydrogel also contains carbomer 940, triethanolamine, glycerol, 1% sodium benzoate aqueous solution and distilled water by mass ratio, wherein the mass ratio of carbomer 940 to triethanolamine is 1:1; the hydrogel formulation can reduce epidermal thickness, protect collagen fiber structure, reduce the expression of Parkin and LC3-II proteins, and increase the expression of SIRT3 protein.

2. The application according to claim 1, characterized in that, The structural formula of L-kynurenine is: .

3. The application according to claim 1, characterized in that, The L-kynurenine significantly increases the content of IL-37 and decreases the content of intracellular IL-6, thereby reducing the inflammation level of UVA-irradiated cells. The L-kynurenine also inhibits the increase of ROS, reduces oxidative stress, lowers the level of LDH, increases the levels of GSH and T-AOC, and enhances the antioxidant capacity of keratinocytes in UVA-irradiated skin.

4. The application according to claim 1, characterized in that, The L-kynurenine enhances mitochondrial function and reduces the formation of autophagosomes. The L-kynurenine can promote mitochondrial regeneration by activating the PGC-1α / SIRT3 signaling pathway. The mitochondrial function includes effective restoration of mitochondrial membrane potential, restoration of the activity of mitochondrial complexes I, II and V, increased ATP synthesis, and improved mitochondrial morphology.

5. The application according to claim 1, characterized in that, The L-kynurenine can reduce the level of mitochondrial autophagy in cells to an appropriate level, maintain the number and function of mitochondria, reduce the expression of mitochondrial autophagy proteins in keratinocytes of skin exposed to UVA radiation, thereby regulating excessive autophagy in cells.

Images