Application of L-kynurenine in preparation of medicine for preventing and / or treating skin light injury induced by ultraviolet light

Abstract

The invention discloses an application of L-kynurenine in preparation of a medicine for preventing and / or treating skin light injury induced by ultraviolet light. Through a UVA radiation HaCaT cell model, it is proved that L-kynurenine has a remarkable light damage resisting effect and can enhance the mitochondrial function and promote mitochondrial neogenesis, so that the oxidation resistance of cells is improved, and inflammatory response is relieved. Through a UVA combined UVB radiation back unhairing mouse model, it is verified that the hydrogel containing L-kynurenine can effectively repair mouse back skin injury caused by ultraviolet light, the water content and oxidation resistance of skin are improved, and the phenomena of inflammatory response, edema, roughness, erythema or eschar and the like are relieved. Therefore, the invention provides a reference direction for the development, utilization and popularization of the L-kynurenine for preparing the medicine for repairing the skin light injury or the medical beauty product.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to an application of L-kynurenine in preparing a medicine for preventing and / or treating ultraviolet light-induced skin photodamage. Background Art

[0002] Skin is the largest organ in the human body, accounting for about 10%-15% of body weight. The skin is the first barrier between the body and the external environment, and plays an important role in protection, temperature regulation, sensation, secretion, excretion and immunity. In recent years, skin health has also been under greater threat due to increased exposure to ultraviolet radiation and chemical pollutants. Epidemiological surveys show that about 80% of exogenous skin aging is related to ultraviolet (UV) radiation, also known as skin photoaging. Mitochondria are unique organelles with their own genome. UV radiation can induce oxidative stress, leading to the release of excessive ROS, which mainly targets mitochondrial DNA (mtDNA), which makes mitochondria particularly susceptible to oxidative damage. Therefore, maintaining mitochondrial health is essential for normal cell function.

[0003] Kynurenine is an important product in the process of tryptophan catabolism, participates in a variety of biological processes, and has a wide range of biologically active functions. Kynurenine can help stabilize the state of the immune system and exert a neuroprotective effect. In the existing technologies that have been disclosed, kynurenine is mostly used as an immunomodulator and biomarker, but there is currently no research linking kynurenine with mitochondrial protection against UV-damaged skin. Summary of the invention

[0004] The purpose of the present invention is to provide the use of L-kynurenine in the preparation of a drug for preventing and / or treating ultraviolet light-induced skin photodamage. The L-kynurenine of the present invention can not only alleviate oxidative stress damage and regulate inflammation levels, but also reduce mitochondrial autophagy and promote mitochondrial neogenesis, further maintain the structure and function of mitochondria, thereby playing a protective role in reducing skin photodamage. This multi-target protection mechanism has a more comprehensive advantage.

[0005] In order to achieve the above-mentioned invention object, the present invention adopts the following technical solutions: The present invention provides application of L-kynurenine in preparing a medicine for preventing and / or treating ultraviolet light-induced skin photodamage.

[0006] Furthermore, the structural formula of the L-kynurenine is: .

[0007] Furthermore, the skin photodamage is skin photodamage caused by UVA and / or UVB ultraviolet light irradiation; and the effective concentration of L-kynurenine is 50 μM-200 μM.

[0008] Furthermore, the photodamage is at least one of skin erythema, desquamation, wrinkles, stratum corneum thickening and collagen destruction.

[0009] Furthermore, the L-kynurenine can significantly increase the content of IL-37 and reduce the content of IL-6 in cells, thereby reducing the inflammatory level of UVA-irradiated cells.

[0010] Furthermore, the L-kynurenine inhibits the increase of ROS and reduces oxidative stress response; the L-kynurenine reduces the level of LDH, increases the level of GSH, increases the level of T-AOC, and improves the antioxidant capacity of skin keratinocytes irradiated by UVA.

[0011] Furthermore, the L-kynurenine improves mitochondrial function and reduces the formation of autophagosomes; the L-kynurenine can promote the regeneration of mitochondria by activating the PGC-1α / SIRT3 signaling pathway.

[0012] Furthermore, the functions of the mitochondria include effective recovery of mitochondrial membrane potential, recovery of the activities of mitochondrial complexes I, II and V, increased ATP synthesis and improved mitochondrial morphology.

[0013] Furthermore, the L-kynurenine can reduce the level of mitochondrial autophagy in cells to an appropriate state, maintain the number and function of mitochondria, and reduce the expression of mitochondrial autophagy proteins in UVA-irradiated skin keratinocytes, thereby regulating excessive autophagy of cells.

[0014] The present invention also provides an external preparation for preventing and / or repairing skin photodamage, wherein the active ingredient of the external preparation is L-kynurenine, and the external preparation is a cream, a hydrogel, a patch, an ointment or a lotion.

[0015] Furthermore, the content of L-kynurenine in the hydrogel is 1-3%.

[0016] Furthermore, the hydrogel also contains carbomer 940, triethanolamine, glycerol, a 1% sodium benzoate aqueous solution and distilled water in a mass ratio, wherein the mass ratio of carbomer 940 to triethanolamine is 1:1.

[0017] Furthermore, the topical preparation can improve at least one of erythema, desquamation, wrinkles, stratum corneum thickening and collagen destruction in photodamaged skin.

[0018] Furthermore, the topical preparation can improve the water content and antioxidant capacity of photodamaged skin.

[0019] Furthermore, the topical preparation can reduce the expression of autophagy proteins Parkin and LC3-II in photodamaged skin and increase the expression of SIRT3 protein.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention experimentally proves for the first time that L-kynurenine has a therapeutic effect on UV-induced skin damage. In the UVA irradiated HaCaT cell model, L-kynurenine can relieve oxidative stress, regulate inflammation levels, reduce mitochondrial autophagy and promote mitochondrial neogenesis, thereby maintaining the structure and function of mitochondria.

[0021] 2. The L-kynurenine hydrogel described in the present invention can maintain the moisture and collagen fiber content of mouse skin, improve antioxidant capacity, and regulate the expression of key proteins in mitochondrial autophagy and neogenesis-related pathways in a mouse skin photodamage model irradiated by UVA+UVB, further confirming the protective effect of L-kynurenine against UV skin photodamage.

[0022] 3. The L-kynurenine provided by the present invention is a potential therapeutic agent that can effectively deal with skin damage caused by ultraviolet rays and has broad application prospects, especially in the development of skin photodamage drugs and medical cosmetic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the effect of L-kynurenine on the proliferation activity of UVA-irradiated HaCaT cells; Figure 2 The results of the determination of the antioxidant effect of L-kynurenine on UVA-irradiated HaCaT cells; A: The effect of L-kynurenine on reactive oxygen species in UVA-irradiated cells; B: The effect of L-kynurenine on the release of lactate dehydrogenase in UVA-irradiated cells; C: The effect of L-kynurenine on the glutathione content in UVA-irradiated cells; D: The effect of L-kynurenine on the total antioxidant capacity in UVA-irradiated cells; Figure 3 The results of the measurement of L-kynurenine on the release of inflammatory factors in HaCaT cells irradiated by UVA; 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; Figure 4 The results of the measurement of the effect of L-kynurenine on the mitochondrial function of UVA-irradiated HaCaT cells; A: L-kynurenine increases the 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 the synthesis of ATP; Figure 5 This is the electron microscopic result that L-kynurenine can improve the mitochondrial structure and reduce autophagosomes in HaCaT cells irradiated by UVA; Figure 6 Schematic diagram of the bands (A) and relative contents (BF) of autophagy proteins PINK1, Parkin, LC3-II, Beclin-1 and p62 in HaCaT cells irradiated by UVA. Figure 7 Schematic diagram of the bands (A) and relative contents (BC) of L-kynurenine regulating the mitochondrial nascent proteins PGC-1α and SIRT3 in HaCaT cells irradiated by UVA; Figure 8 The results of the damage to the back skin of mice irradiated with UVA+UVB ultraviolet light by three different formulations of L-kynurenine hydrogels (A), the statistical results of the damage area (B), and the results of pathological sections (C); Fig. 9 The results of the damage to the back skin of mice irradiated with UVA+UVB light by three different hydrogels (A) and the statistical results of the damage area (B); Fig.10 This is a picture showing the results of the pathological sections of the mouse back skin irradiated by UVA+UVB ultraviolet light by L-kynurenine hydrogel; Fig.11 This is the result of the L-kynurenine hydrogel on the back skin water content of mice irradiated with UVA+UVB ultraviolet light; Fig.12 The results of L-kynurenine hydrogel's antioxidant effect on the back skin of mice irradiated with UVA+UVB ultraviolet light; A: L-kynurenine hydrogel reduces the content of malondialdehyde in the back skin of mice; B: L-kynurenine hydrogel increases the content of glutathione in the back skin of mice; C: L-kynurenine hydrogel enhances the total antioxidant capacity of the back skin of mice; Fig.13 Schematic diagram of the bands (A) and relative contents (BC) of the autophagy proteins Parkin and LC3-II in the back skin of mice irradiated with UVA+UVB by L-kynurenine hydrogel; Fig.14 Schematic diagram of the bands and relative content of SIRT3 protein, a mitochondrial protein produced in the back skin of mice irradiated with UVA+UVB light, regulated by L-kynurenine hydrogel. DETAILED DESCRIPTION

[0024] In order to make those skilled in the art better understand the present invention, the technical scheme of the present invention is further described in detail below in conjunction with specific examples. It should be pointed out that the following is a preferred embodiment of the present invention, rather than all embodiments. For those skilled in the art, all other embodiments obtained without making innovative premises belong to the scope of protection of the present invention. In the following examples, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used can be purchased from biological or chemical reagent companies.

[0025] Use of L-kynurenine as shown in Formula I in the preparation of a drug for preventing and / or treating ultraviolet-induced skin photodamage: Formula Ⅰ.

[0026] In the present invention, the molecular formula of L-kynurenine is C 10 H 12 N2O3, molecular weight is 208.22. In the embodiment of the present invention, the L-kynurenine is purchased from Shanghai MacLean Biochemical Technology Co., Ltd., with the product number L864410.

[0027] Example 1: L-kynurenine significantly protects HaCaT cells from damage by UVA radiation The specific implementation steps are as follows: HaCaT cells were cultured and seeded in 96-well plates, with 1.2×10 4 After incubation for 48 hours in a 37°C constant temperature cell culture incubator, the culture medium was discarded, and different concentrations of L-kynurenine (50 μM, 100 μM, and 200 μM) were prepared with phenol red-free culture medium. 200 μL was added to each well, and the cover of the 96-well plate was removed and placed under UVA irradiation at 0.5 J / cm 2 The blank group was not irradiated with UVA. After the 96-well plate was returned to the incubator and cultured for 24 hours, the cell viability was detected using the MTT method.

[0028] Experimental results: Figure 1 As shown, 0.5J / cm 2 UVA can reduce cell viability, while L-kynurenine (50-200 μM) shows a protective effect and can significantly increase cell viability, and its protective effect is dose-dependent.

[0029] Example 2: L-kynurenine can remove ROS in UVA-irradiated cells The specific implementation steps are as follows: HaCaT cells were cultured and seeded in 96-well plates, with 1.2×10 4After 48 hours, the culture medium was discarded, and L-kynurenine (50 μM, 100 μM, 200 μM) was prepared with phenol red-free culture medium, and 200 μL was added to each well. The cover of the 96-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2 After the 96-well plate was returned to the incubator and cultured for 24 hours, the culture medium was discarded, and the prepared DCFH-DA fluorescent probe was added to the incubator for 20 minutes to detect ROS at a detection wavelength of 488 / 525.

[0030] Experimental results: Figure 2 As shown in A, UVA increased the level of ROS in cells, and L-kynurenine in each group inhibited the increase of ROS, thereby reducing the oxidative stress response.

[0031] Example 3: L-kynurenine can reduce the level of LDH in UVA-irradiated cells and increase the level of GSH and T-AOC The specific implementation steps are as follows: HaCaT cells were cultured and seeded in 6-well plates, with 70×10 4 After 48 hours, the culture medium was discarded, phenol red-free culture medium was used to prepare L-kynurenine (200 μM), 2 mL was added to each well, the cover of the 6-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2 After the 6-well plate was returned to the incubator and cultured for 24 hours, the culture medium was discarded, the cell protein was extracted, and the levels of LDH, GSH and T-AOC in each group of cells were detected according to the instructions of the kit.

[0032] Experimental results: Figure 2 As shown in BD, UVA irradiation increased the release of intracellular LDH, decreased the content of GSH, and further decreased T-AOC, while 200 μM L-kynurenine reduced the level of LDH, increased the level of GSH, and increased T-AOC, indicating that L-kynurenine can enhance the antioxidant capacity of UVA-damaged cells, thereby protecting cells from oxidative damage.

[0033] Example 4: L-kynurenine can regulate the release of inflammatory factors in UVA-irradiated cells The specific steps are as follows: HaCaT cells were cultured and seeded in 96-well plates, with 1.2 × 10 4 After 48 hours, the culture medium was discarded, L-kynurenine (200 μM) was prepared with phenol red-free culture medium, and 200 μL was added to each well. The cover of the 96-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2 After the 96-well plate was returned to the incubator and cultured for 24 hours, the supernatant was taken and the contents of IL-37 and IL-6 were detected by ELISA.

[0034] Experimental results: Figure 3 As shown in the data, after UVA irradiation, the content of anti-inflammatory factor IL-37 in cells was significantly decreased, and the content of pro-inflammatory factor IL-6 was significantly increased, while L-kynurenine (200 μM) could significantly increase the content of IL-37 and reduce the content of IL-6 in cells, which indicates that L-kynurenine can reduce the inflammatory level of UVA irradiated cells.

[0035] Example 5: L-kynurenine increases mitochondrial membrane potential in UVA-irradiated cells The specific implementation steps are as follows: HaCaT cells were cultured and seeded in 96-well plates, with 1.2×10 4 After 48 hours, the culture medium was discarded, and L-kynurenine (50 μM, 100 μM, 200 μM) was prepared with phenol red-free culture medium, and 200 μL was added to each well. The cover of the 96-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2 After the 96-well plate was returned to the incubator and cultured for 24 hours, the culture medium was discarded, and the prepared JC-1 fluorescent probe was added and placed in the incubator for incubation for 20 minutes before detection. The detection wavelength was 490 / 530 for monomers and 525 / 590 for polymers. The result was the ratio of polymer to monomer.

[0036] Experimental results: Figure 4 As shown in A, the mitochondrial membrane potential of cells decreased after UVA irradiation, indicating that the cells had mitochondrial damage, while 50-200 μM L-kynurenine effectively restored the mitochondrial membrane potential of the cells.

[0037] Example 6: L-kynurenine can enhance the activity of mitochondrial complexes I, II and V in UVA-irradiated cells The specific implementation steps are as follows: HaCaT cells were cultured and seeded in 6-well plates, with 70×10 4 After 48 hours, the culture medium was discarded, phenol red-free culture medium was used to prepare L-kynurenine (200 μM), 2 mL was added to each well, the cover of the 6-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2 After the 6-well plate was returned to the incubator and cultured for 24 hours, the culture medium was discarded, and the cell samples were extracted and tested according to the instructions of the kit.

[0038] Experimental results: Figure 4 As shown in BD, the activity of mitochondrial complexes I, II and V in cells irradiated by UVA was significantly reduced, indicating that UVA radiation destroyed the electron respiratory chain and could not provide energy for the physiological activities of cells. However, 200 μM L-kynurenine restored the activity of mitochondrial complexes I, II and V in cells, thereby repairing the damaged electron respiratory chain.

[0039] Example 7: L-kynurenine can promote the synthesis of ATP in UVA-irradiated cells The specific implementation steps are as follows: HaCaT cells were cultured and seeded in 6-well plates, with 70×10 4 After 48 hours, the culture medium was discarded, phenol red-free culture medium was used to prepare L-kynurenine (200 μM), 2 mL was added to each well, the cover of the 6-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2 After the 6-well plate was returned to the incubator and cultured for 24 hours, the culture medium was discarded, and the cell samples were extracted and tested according to the instructions of the kit.

[0040] Experimental results: Figure 4 As shown in E, the ATP content of cells was significantly reduced after UVA irradiation, while 200 μM L-kynurenine could increase the ATP content, indicating that it could improve the energy status of cells.

[0041] Example 8: L-kynurenine can improve the mitochondrial morphology and reduce the formation of autophagosomes in UVA-irradiated cells The specific steps are as follows: HaCaT cells were cultured and seeded in 6-well plates, with 70 × 10 4 After 48 hours, the culture medium was discarded, phenol red-free culture medium was used to prepare L-kynurenine (200 μM), 2 mL was added to each well, the cover of the 6-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2 . After the 6-well plate was returned to the incubator and continued to be cultured for 24 hours, the culture medium was discarded, PBS was added, and the cells were scraped off with a cell scraper. The cells were centrifuged at low speed for 10 minutes in a 1.5 mL centrifuge tube, and the supernatant was discarded. The volume of the precipitate was not less than half the size of a rice grain. 2.5% glutaraldehyde was carefully added along the tube wall. Do not wash away the precipitate, and fix it in a 4°C refrigerator overnight. Ultrathin sections were made as required, and the changes in mitochondrial morphology of cells after different treatments were observed by transmission electron microscopy.

[0042] Experimental results: Figure 5 As shown, after UVA radiation, the mitochondrial morphology was abnormal, the structure was damaged, the cristae were broken or disappeared, and the formation of autophagosomes increased. After L-kynurenine treatment, the mitochondrial morphology was significantly improved, and the formation of autophagosomes was also reduced.

[0043] Example 9: L-kynurenine regulates mitochondrial autophagy via the PINK1 / Parkin signaling pathway The specific steps are as follows: HaCaT cells were cultured and seeded in 6-well plates, with 70 × 10 4 After 48 hours, the culture medium was discarded, phenol red-free culture medium was used to prepare L-kynurenine (200 μM), 2 mL was added to each well, the cover of the 6-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2. After returning to the incubator and continuing to culture for 24 hours, the culture medium was discarded, and the pre-cooled lysis solution was added to lyse on ice for 30 minutes, and then transferred to a centrifuge at 4°C and 12000rpm for 10 minutes. The supernatant was collected for Western blotting analysis. 20ug of cell lysate protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. 5% BSA was used as a blocking solution and blocked at room temperature for 2 hours. The bands were incubated with specific primary antibodies (PINK1, Parkin, LC3A / B, Beclin-1, p62, GAPDH) at 4°C overnight. After incubation, they were washed 6 times with TBST buffer, incubated with the corresponding HRP-conjugated secondary antibody, and detected by chemiluminescence imaging. The relative density of each group of protein bands was analyzed by Image J, and the optical density value was normalized with GAPDH as an internal reference to obtain the corresponding protein expression level.

[0044] Experimental results: Figure 6 As shown in the figure, compared with the control group, after UVA irradiation, the expression levels of PINK1, Parkin, LC3-II and Beclin-1 proteins in HaCaT cells increased, and the expression level of p62 protein decreased, indicating that excessive autophagy was caused when mitochondria were stimulated, which would affect the normal function of cells. After L-kynurenine treatment, the expression levels of PINK1, Parkin, LC3-II and Beclin-1 proteins in cells decreased, and the expression level of p62 protein increased, indicating that L-kynurenine can reduce the level of mitochondrial autophagy in cells to an appropriate state, thereby maintaining the number and function of mitochondria and protecting cells.

[0045] Example 10: L-kynurenine regulates mitochondrial neogenesis via the PGC-1α / SIRT3 signaling pathway The specific steps are as follows: HaCaT cells were cultured and seeded in 6-well plates, with 70 × 10 4 After 48 hours, the culture medium was discarded, phenol red-free culture medium was used to prepare L-kynurenine (200 μM), 2 mL was added to each well, the cover of the 6-well plate was removed and the plate was placed under UVA irradiation at 0.5 J / cm 2. After returning to the incubator and continuing to culture for 24 hours, the culture medium was discarded, and pre-cooled lysis solution was added to lyse on ice for 30 minutes, transferred to a centrifuge and centrifuged at 12000rpm for 10 minutes at 4°C, and the supernatant was collected for Western blotting analysis. 20ug of cell lysate protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane. 5% BSA was used as a blocking solution and blocked at room temperature for 2 hours. The bands were incubated with specific primary antibodies (PGC-1α, SIRT3, GAPDH) at 4°C overnight. After incubation, they were washed 6 times with TBST buffer, incubated with the corresponding HRP-coupled secondary antibody, and detected by chemiluminescence imaging. The relative density of each group of protein bands was analyzed using Image J, and the optical density values ​​were normalized with GAPDH as an internal reference to obtain the corresponding protein expression level.

[0046] Experimental results: Figure 7 As shown in the figure, compared with the control group, the expression of PGC-1α and SIRT3 proteins in cells was significantly reduced after UVA irradiation, indicating that UVA hindered the mitochondrial regeneration in cells. In cells treated with L-kynurenine, the expression levels of PGC-1α and SIRT3 proteins were significantly increased, indicating that L-kynurenine can promote the regeneration of mitochondria by activating the PGC-1α / SIRT3 signaling pathway.

[0047] Example 11: Preparation of topical preparations containing L-kynurenine According to the use of L-kynurenine in the above-mentioned embodiments, this embodiment provides an external preparation containing L-kynurenine. The following experimental groups will be set up, and the mouse skin tissue to be tested in the experimental groups will be tested in multiple aspects in combination with Experimental Examples 11-18 to verify the effects of L-kynurenine in various aspects. In the following Examples 12-18, the mouse skin tissue used can be obtained in the manner shown in the following steps 1-3: Step 1: Preparation of L-kynurenine hydrogel Under stirring, slowly add Carbomer 940 into distilled water and stir until it is completely dispersed; add glycerol and stir evenly; then mix L-kynurenine therein and stir evenly; add triethanolamine, heat until uniform to drive out air bubbles, and cool to room temperature; add 1% sodium benzoate aqueous solution and stir evenly to obtain a hydrogel containing L-kynurenine.

[0048] The ratio of carbomer 940 to triethanolamine was set to 1:4, 1:1, and 2:1 to prepare three L-kynurenine hydrogels with a mass ratio of 1%, as shown in Table 1.

[0049] Table 1: Three 1% L-kynurenine hydrogel formulations

[0050] According to the formula of KYN2, the same preparation method was used, except that L-kynurenine was not added, to prepare 1% ergothioneine gel (MJLY group) and blank matrix gel (blank matrix group). See Table 2 for details.

[0051] Table 2: Hydrogel formulation

[0052] Step 2: Pre-experimental treatment 6-8 weeks old Balb / c female mice, SPF, weighing 20-22 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. A small animal razor was used to remove the hair on the back of the mice. The hair was removed gently to avoid damage to the skin by human factors.

[0053] (I) The animals were randomly divided into 5 experimental groups, with 8 mice in each group. The 5 experimental groups were blank group, model group, KYN1, KYN2, and KYN3.

[0054] (II) The mice were randomly divided into 5 experimental groups, with 8 mice 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).

[0055] Step 3: Obtaining mouse skin tissue Before irradiation, each group applied the corresponding hydrogel to the back skin, and used LED UVA and UVB lamps to be placed at a vertical height of about 40 cm above the back of the mouse. After preheating for 5 minutes, irradiation was given for 1 hour every day, 6 days a week, and 7 consecutive weeks. Within 24 hours after the last irradiation, the mice were killed by cervical dislocation. The back skin tissue was collected, the connective tissue and subcutaneous fat were peeled off, and they were quickly frozen with liquid nitrogen and stored at -80°C for subsequent experimental studies.

[0056] Example 12: This example uses the three different formulations of 1% L-kynurenine hydrogels prepared in Example 11 to verify the protective effects of the three different formulations of 1% L-kynurenine hydrogels on the skin damage on the back of mice caused by UVA+UVB irradiation Experimental results: Effects of 1% L-kynurenine hydrogels with three different formulations on the back skin of mice after ultraviolet irradiation. Figure 8 As shown, the KYN2 formula (the ratio of carbomer 940 to triethanolamine is 1:1) is better than the other two formulas in improving skin redness, swelling and scab formation and reducing epidermal thickness. Therefore, the subsequent experiments selected the formula with a ratio of carbomer 940 to triethanolamine of 1:1.

[0057] 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 Experimental results: Fig. 9 As shown, compared with the blank group, after the first week of ultraviolet irradiation, the skin of the mice in the model group began to show obvious dryness and desquamation, and epidermal thickening; after the third week of ultraviolet irradiation, the skin of the mice in the model group showed large areas of rough wrinkles, erythema eschar, and sunburn; the blank matrix group did not improve the skin condition, indicating that the blank matrix had no obvious protective effect; compared with the model group, the L-kynurenine group and the ergothioneine group showed significantly improved sunburn phenomena such as rough skin and redness. The effect of L-kynurenine in resisting ultraviolet damage is even better than ergothioneine. This shows that L-kynurenine can significantly improve the rough skin and sunburn of mice after ultraviolet irradiation.

[0058] Example 14: Effect of L-kynurenine hydrogel on mouse skin pathological histology The specific steps are as follows: The skin tissue of each group of mice was fixed with 4% paraformaldehyde, and then embedded in paraffin and sliced. The slices were mounted and fixed on a slide, and the content of collagen in the epidermis and skin tissue was observed by HE and Masson trichrome staining. The stained slice images were analyzed by an inverted microscope.

[0059] Experimental results: Fig.10 As shown in the figure, HE and Masson staining results show that the epidermis structure of mice without UV irradiation is normal and the collagen fibers are tightly organized; the epidermis of mice irradiated with UV irradiation is significantly thickened and accompanied by the aggregation of inflammatory cells, the collagen fibers in the green part of the skin are greatly reduced, and some collagen fibers are disordered and broken, indicating that UV irradiation destroys the collagen fibers in the skin; after treatment with L-kynurenine hydrogel, the thickness of the mouse epidermis is thinner than that of the UV group, and the collagen fibers in the dermis are tightly organized and the content is increased. Therefore, for skin irradiated with UV irradiation, the composition containing L-kynurenine can reduce the thickness of the epidermis, protect the collagen fiber structure, and play a role in protecting the skin.

[0060] Example 15: L-kynurenine hydrogel can increase the water content of mouse skin after UVA+UVB irradiation The specific steps are as follows: Measure the skin moisture content by direct drying method. Quickly cut the skin tissue, quickly rinse with saline, absorb the moisture with absorbent paper, weigh and record the weight, which is the wet weight. Set the oven temperature to about 105°C and put the skin tissue in to dry. After drying, take out the skin, weigh and record the weight, put the sample back into the oven and continue to dry for about 1 hour, weigh it again, until the difference between two consecutive weighings does not exceed 0.5 mg, at which time the weight is the dry weight. Skin moisture content (%) = (wet weight-dry weight) / wet weight × 100%.

[0061] Experimental results: Fig.11 As shown, the water content of mouse skin irradiated with ultraviolet light decreased, while L-kynurenine hydrogel significantly increased the water content of mouse skin, indicating that L-kynurenine has a significant effect on maintaining the water content of the skin.

[0062] Example 16: L-kynurenine hydrogel can improve the antioxidant capacity of mouse skin tissue after UVA+UVB irradiation The specific steps are as follows: cut the skin tissue and grind it on ice. Process the ground skin samples according to the instructions of the corresponding kit, and detect the content of MDA, GSH and T-AOC in each group.

[0063] Experimental results: Fig.12 As shown in the figure, compared with the control group, the level of MDA increased and the levels of GSH and T-AOC decreased after ultraviolet irradiation. After treatment with L-kynurenine hydrogel, the level of MDA was effectively reduced and the levels of GSH and T-AOC were restored. This shows that L-kynurenine can enhance the total antioxidant capacity of the skin by reducing lipid peroxidation and promoting the expression of glutathione, indicating that L-kynurenine can enhance the antioxidant capacity of ultraviolet irradiated skin.

[0064] Example 17: L-kynurenine hydrogel can promote the expression of autophagy-related proteins in mouse skin tissue after UVA+UVB irradiation The specific steps are as follows: Take about 100 mg of frozen mouse skin tissue, place it in a 2 mL centrifuge tube, add 1 mL of lysis buffer, place it on ice and grind it with a tissue homogenizer, transfer it to a centrifuge and centrifuge it at 12000 rpm for 10 minutes at 4 ° C, and collect the supernatant for Western blotting analysis. Use SDS-PAGE to separate 30 ug of tissue lysate protein and transfer it to a nitrocellulose membrane. Use 5% BSA as a blocking solution and block it at room temperature for 2 hours. Incubate the bands with specific primary antibodies (Parkin, LC3A / B, GAPDH) at 4 ° C overnight, wash with TBST buffer 6 times after incubation, incubate the corresponding HRP-coupled secondary antibody, and detect it using a chemiluminescent imager. Image J was used to analyze the relative density of each group of protein bands, and GAPDH was used as an internal reference to normalize the optical density value to obtain the corresponding protein expression level.

[0065] Experimental results: Fig.13 As shown, the expression levels of Parkin and LC3-II proteins were increased in the skin tissue of mice irradiated with UV light, and the treatment with L-kynurenine hydrogel reduced the expression of these proteins.

[0066] Example 18: L-kynurenine hydrogel can promote the expression of SIRT3 protein in mouse skin tissue after UVA+UVB irradiation The specific steps are as follows: Take about 100 mg of frozen mouse skin tissue, place it in a 2 mL centrifuge tube, add 1 mL of lysis buffer, place it on ice and grind it with a tissue homogenizer, transfer it to a centrifuge and centrifuge it at 12000 rpm for 10 min at 4 ° C, and collect the supernatant for Western blotting analysis. Use SDS-PAGE to separate 30 ug of tissue lysate protein and transfer it to a nitrocellulose membrane. Use 5% BSA as a blocking solution and block it at room temperature for 2 hours. Incubate the bands with specific primary antibodies (SIRT3, GAPDH) at 4 ° C overnight, wash them with TBST buffer 6 times after incubation, incubate the corresponding HRP-coupled secondary antibody, and detect it using a chemiluminescent imager. Image J was used to analyze the relative density of each group of protein bands, and GAPDH was used as an internal reference to normalize the optical density value to obtain the corresponding protein expression level.

[0067] Experimental results: Fig.14 As shown, compared with the control group, the expression of SIRT3 protein in the skin tissue of mice irradiated with ultraviolet light was reduced, and the expression of SIRT3 protein in the skin tissue treated with L-kynurenine hydrogel was increased.

[0068] In summary, the present invention verifies the multiple protective effects of L-kynurenine in the treatment of ultraviolet-induced skin photodamage. L-kynurenine can alleviate oxidative stress damage, reduce inflammation levels, and maintain the structure and function of mitochondria. This multi-target protection mechanism has a more comprehensive therapeutic effect. In the UVA irradiated HaCaT cell model, L-kynurenine alleviates oxidative stress damage, reduces inflammation levels, reduces mitochondrial autophagy, and promotes mitochondrial regeneration, thereby improving cell vitality; in the UVA+UVB irradiated mouse skin photodamage model, L-kynurenine hydrogel increases the moisture and collagen fiber content of mouse skin, improves antioxidant capacity, and regulates the expression of key proteins in mitochondrial autophagy and regeneration-related pathways, verifying the protective effect of L-kynurenine on UV light-damaged skin. The present invention fills the gap in the prior art regarding L-kynurenine in the field of skin photodamage treatment, and has broad application prospects.

[0069] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. Use of L-kynurenine in the preparation of a drug for preventing and / or treating ultraviolet-induced skin photodamage.

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

3. The use according to claim 1, characterized in that: The skin photodamage is skin photodamage caused by UVA and / or UVB ultraviolet light irradiation; the effective concentration of L-kynurenine is 50 μM-200 μM.

4. The use according to claim 1, characterized in that: The L-kynurenine can significantly increase the content of IL-37 and reduce the content of IL-6 in cells, thereby reducing the inflammation level of UVA-irradiated cells.

5. The use according to claim 1, characterized in that: The L-kynurenine inhibits the increase of ROS, reduces oxidative stress reaction, reduces the level of LDH, increases the levels of GSH and T-AOC, and improves the antioxidant capacity of skin keratinocytes irradiated by UVA.

6. The use according to claim 1, characterized in that: The L-kynurenine improves mitochondrial function and reduces the formation of autophagosomes; the L-kynurenine can promote the regeneration of mitochondria by activating the PGC-1α / SIRT3 signaling pathway.

7. The use according to claim 6, characterized in that: The mitochondrial function includes effective recovery of mitochondrial membrane potential, recovery of activity of mitochondrial complexes I, II and V, increased ATP synthesis and improved mitochondrial morphology.

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

9. An external preparation for preventing and / or repairing skin photodamage, characterized in that: The external preparation has L-kynurenine as an active ingredient and is a cream, a hydrogel, a patch, an ointment or a lotion.

10. The external preparation according to claim 9, characterized in that The content of L-kynurenine in the hydrogel is 1-3%.

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