Application of substance for inhibiting TRPV3 in preparation of product for preventing or treating ultraviolet-induced / mediated diseases

Abstract

The invention discloses application of a substance for inhibiting TRPV3 in preparation of a product for preventing or treating ultraviolet-induced / mediated diseases, and belongs to the technical field of medicines. The invention describes the effect of a skin transient receptor potential cation channel, subgroup V and member 3 (TRPV3) in ultraviolet skin injury, and the inhibition of the TRPV3 can be used for developing medicines and skin care products for preventing or treating ultraviolet (UVB) injury. The invention also discloses evaluation of the anti-inflammatory activity of the natural selective TRPV3 inhibitor osthole by adopting a mouse skin ultraviolet injury model, and application prospects of the osthole in preparation of medicines and skin care products for preventing or treating ultraviolet injury are prompted. When a drug containing the TRPV3 inhibitor found according to the present invention or the TRPV3 inhibitor provided by the present invention is used in the prevention or treatment of ultraviolet damage, a good prevention or treatment effect can be obtained.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to the use of a substance that inhibits TRPV3 in the preparation of a product for preventing or treating ultraviolet-induced / mediated diseases. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] UV rays are an important component of sunlight. Studies have shown that although UV rays are indispensable, they cause many adverse effects on the skin, such as erythema, photoaging, inflammation, and even tumors (Bernard et al., 2019; Gordon and Brieva, 2012; Holick, 2020; Stern and Study, 2001). Compared with UVA, UVB has higher energy and is mainly absorbed by the epidermis. Therefore, UVB is the main component that causes skin sunburn and can cause strong dose-dependent hypersensitivity reactions (Abtahi et al., 2022; Byrne et al., 2002; Gu et al., 2021).

[0004] Previous studies have shown that transient receptor potential (TRP) is a type of ion channel protein present on the cell membrane or intracellular organelle membrane, consisting of seven subfamilies including TRPC, TRPV, TRPM, TRPML, TRPP, TRPA, and TRPN. The mammalian transient receptor potential vanilloid receptor subfamily (TRPV) is composed of TRPV1-6 (Nilius and Owsianik, 2011). The transient receptor potential vanilloid receptor 3 channel is a temperature-sensitive, calcium-permeable, non-selective cation channel (Xue et al., 2002). Transient receptor potential vanilloid receptor 3 is mainly expressed in human skin keratinocytes and is associated with the generation and development of inflammation. Function-enhancing transient receptor potential vanilloid receptor 3 mutations can cause skin inflammation in rats, mice, and humans. These mutations induce atopic dermatitis (AD)-like symptoms, such as infiltration of inflammatory cells, thickening of keratin, and upregulation of immunoglobulin E expression (Asakawa et al., 2006; Yoshioka et al., 2009). In addition to the described inflammatory skin symptoms, patients with Olmsted syndrome (OS) carrying a TRPV3 function-enhancing mutation (G573A) also show a variety of immune dysfunctions, including increased follicular T cells, persistent eosinophilia, and elevated immunoglobulin E (Lin et al., 2012), suggesting that TRPV3 is involved in inflammatory skin diseases.

[0005] At present, there are no reports on the role of transient receptor potential vanilloid receptor 3 channels in UV damage and the anti-UV damage effect of osthole on inhibiting transient receptor potential vanilloid receptor 3 channels. Summary of the invention

[0006] In order to address the deficiencies of the prior art, the present invention provides the use of a substance that inhibits TRPV3 in the preparation of a product for preventing or treating ultraviolet-induced / mediated diseases.

[0007] The present invention proves for the first time through pharmacological experiments that the TRPV3 inhibitor osthole can effectively inhibit skin thickening, keratinization of the stratum corneum, and inflammatory cell infiltration caused by ultraviolet radiation, so it has high clinical application value and development prospects. Through the mouse skin ultraviolet damage model, the TRPV3 inhibitor osthole can significantly reduce the swelling of the mouse ears, the keratinization of the stratum corneum, and the inflammatory cell infiltration. The above results show that the TRPV3 inhibitor osthole has a significant protective effect on skin damage caused by ultraviolet radiation.

[0008] Based on the above results, the first aspect of the present invention provides the use of a substance that inhibits transient receptor potential cation channel TRPV3 in the preparation of a product for preventing or treating ultraviolet-induced / mediated diseases.

[0009] In some embodiments of the present invention, the substance that inhibits the transient receptor potential cation channel TRPV3 achieves its inhibitory effect by reducing the expression level of TRPV3, accelerating the degradation of TRPV3, or blocking the function of TRPV3.

[0010] In some embodiments of the present invention, the substance that inhibits the transient receptor potential cation channel TRPV3 includes reversible, irreversible, competitive, non-competitive, anti-competitive inhibitors or channel blockers.

[0011] In some embodiments of the present invention, the substance that inhibits the transient receptor potential cation channel TRPV3 is osthole, wherein the osthole can inhibit skin thickening, reduce the degree of keratinization of the stratum corneum, and reduce the degree of inflammatory cell infiltration.

[0012] In some embodiments of the present invention, the products include pharmaceutical agents, experimental reagents, and skin care products.

[0013] Among them, the experimental reagents are used for basic research.

[0014] Preferably, the skin care products include sunscreen, shower gel, and skin damage detection kit.

[0015] In some embodiments of the present invention, the disease includes but is not limited to atopic dermatitis and skin cancer; preferably, the skin cancer includes melanoma.

[0016] The second aspect of the present invention provides the use of transient receptor potential cation channel TRPV3 in screening drugs and skin care products for preventing or treating ultraviolet-induced / mediated diseases.

[0017] In some embodiments of the present invention, the application is: using TRPV3 as a drug target to investigate whether drugs reduce the expression and / or activity of TRPV3 in skin damage caused by ultraviolet radiation.

[0018] In some embodiments of the present invention, the disease includes but is not limited to atopic dermatitis and skin cancer; preferably, the skin cancer includes melanoma.

[0019] The third aspect of the present invention provides a drug for preventing or treating ultraviolet damage, comprising a TRPV3 inhibitor and a pharmaceutically acceptable carrier.

[0020] In some embodiments of the present invention, the TRPV3 inhibitor is osthole, and the structure is shown in Formula I;

[0021]

[0022] The fourth aspect of the present invention provides the use of transient receptor potential cation channel TRPV3 in the treatment of skin diseases by light radiation or drugs;

[0023] The light is ultraviolet light or infrared light;

[0024] The application is to use TRPV3 as a target, and after applying light radiation or drugs, obtain the expression and / or increase the activity of the transient receptor potential cation channel TRPV in the skin, thereby exerting the therapeutic effect of light radiation or drugs on skin diseases.

[0025] Clinically, various skin diseases, such as atopic dermatitis, eczema, psoriasis, etc., are treated by light radiation, such as ultraviolet radiation or infrared radiation. The inventors found that ultraviolet radiation is not effective for all skin diseases, and the inventors found that ultraviolet rays have the effect of promoting the expression of transient receptor potential cation channel TRPV3, thereby causing inflammation and worsening the condition. Therefore, the inventors provide the use of transient receptor potential cation channel TRPV3 in the treatment of skin diseases by ultraviolet radiation or drugs.

[0026] The beneficial effects of the present invention are:

[0027] The present invention proves for the first time through pharmacological experiments that the TRPV3 inhibitor osthole can effectively inhibit skin thickening, keratinization of the stratum corneum, and inflammatory cell infiltration caused by ultraviolet radiation, so it has high clinical application value and development prospects. Through the mouse skin ultraviolet damage model, the TRPV3 inhibitor osthole can significantly reduce the swelling of the mouse ears, the keratinization of the stratum corneum, and the inflammatory cell infiltration. The above results show that the TRPV3 inhibitor osthole has a significant protective effect on skin damage caused by ultraviolet radiation.

[0028] The present invention suggests that transient receptor potential vanilloid receptor 3 is a target for treating ultraviolet damage, and a compound that inhibits transient receptor potential vanilloid receptor 3 channels can be used to treat ultraviolet skin damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0030] Figure 1 The following are pictures of the mouse ultraviolet ear injury model in Example 1; A represents exposure to 125 mJ / cm 2 ,250mJ / cm 2 ,500mJ / cm 2 ,1000mJ / cm 2 The appearance of the skin lesions in the mouse ultraviolet ear injury model induced by UVB radiation; B is the ear swelling score of the mouse inflammation model induced by different doses of UVB radiation; C is the change of ear thickness in the mouse inflammation model induced by different doses of UVB radiation; D is the H&E staining result of frozen sections of ear tissue in the mouse inflammation model induced by different doses of UVB radiation on the fifth day;

[0031] Figure 2 Figure 1 shows the protein levels of TRPV3 and inflammatory factors TNF-α and IL-6 in the mouse ultraviolet ear injury model; A represents exposure to 125 mJ / cm 2 ,250mJ / cm 2 ,500mJ / cm 2 ,1000mJ / cm 2 Expression of TRPV3 protein in ear tissue of mouse inflammation model induced by UVB radiation; B is the expression of TRPV3 mRNA in ear tissue of mouse inflammation model induced by different doses of UVB radiation on the third day; C is the expression of TRPV3 mRNA in ear tissue of mouse inflammation model induced by 250mJ / cm 2 ,500mJ / cm 2 The expression of TRPV3, TNF-α, IL-6 and TSLP mRNA in the ear tissue of the mouse inflammation model induced by UVB radiation; D is the third day after receiving 250mJ / cm 2 ,500mJ / cm 2 The expression results of TRPV3, TNF-α and IL-6 proteins in the ear tissue of the mouse inflammation model induced by UVB radiation; E, F and G are the statistical results of the expression levels of TRPV3, TNF-α and IL-6 proteins in D respectively;

[0032] Figure 3 These are pictures related to the UV ear damage model of wild-type and transgenic mice in Example 2; A represents the exposure or non-exposure to 250 mJ / cm for five consecutive days. 2 and 500mJ / cm 2Appearance of skin lesions in UVB-induced ear damage model of wild-type mice and TRPV3 knockout mice; B shows whether the mice received or did not receive 250 mJ / cm 2 and 500mJ / cm 2 Ear swelling scores of wild-type mice and TRPV3 knockout mice induced by UVB radiation in the ultraviolet ear injury model; C represents the ear ... 2 and 500mJ / cm 2 Changes in ear thickness in UVB radiation-induced UV ear damage models in wild-type mice and TRPV3 knockout mice;

[0033] Figure 4 The pictures are related to the prevention and treatment of UVB radiation-induced ear damage by osthole in Example 3; A represents the ear receiving or not receiving 250 mJ / cm for five consecutive days. 2 Appearance of skin lesions in UVB-induced ear damage model in mice and mice treated with or without 10 mM osthole, a selective inhibitor of TRPV3; B shows treatment with or without 250 mJ / cm 2 Ear swelling scores of UVB-induced ear injury models in mice and mice treated with or without the TRPV3 selective inhibitor osthole; C represents the score of mice treated with or without 250 mJ / cm for five consecutive days 2 Changes in ear thickness in mice induced by UVB radiation and in mice treated with or without the TRPV3 selective inhibitor osthole; D is the fifth day of treatment with or without 250 mJ / cm 2 H&E staining results of frozen sections of ear tissues in UVB radiation-induced mice and mice treated with or without the TRPV3 selective inhibitor osthole;

[0034] Figure 5 The pictures of Example 4 show that osthole alleviates UV skin damage in a dose- and time-dependent manner; A represents exposure or non-exposure to 500 mJ / cm for five consecutive days. 2 Appearance of skin lesions in UVB-induced ear damage model in mice and mice treated with or without different doses of TRPV3 selective inhibitor osthole (0.1mM / 1mM / 10mM); B shows whether mice were treated with or without 250mJ / cm 2 Ear swelling scores of UVB-induced mice and mice treated with or without different doses of TRPV3 selective inhibitor osthole; C represents the score of mice treated with or without 250 mJ / cm for five consecutive days 2Changes in ear thickness in mice induced by UVB radiation and in mice treated with or without different doses of TRPV3 selective inhibitor osthole; D represents the fifth day of treatment with or without 250 mJ / cm 2 H&E staining results of frozen sections of ear tissues in UVB radiation-induced UV ear damage models in mice and in mice treated with or without different doses of TRPV3 selective inhibitor osthole. DETAILED DESCRIPTION

[0035] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0036] Example 1

[0037] This example is used to illustrate the application of the present invention in screening for preventing or treating ultraviolet skin damage using TRPV3 as a target.

[0038] (1) Ultraviolet ear damage model in mice induced by different doses of UVB radiation

[0039] 7-8 week old male C57BL / 6 mice weighing 20±2 g were purchased from Beijing Weitong Lihua and randomly divided into blank control group, model group with different ultraviolet radiation B (UVB) doses (125 mJ / cm 2 ,250mJ / cm 2 ,500mJ / cm 2 ,1000mJ / cm 2 ), 5-10 mice per group. Before the experiment, the mice were placed in a traditional laboratory environment for at least 3 days to adapt to the environment, with a normal 12-hour alternating circadian rhythm, and the mice had free access to food and water. Before the experiment, the radiation intensity of the ultraviolet lamp was measured using an ultraviolet radiation meter, and the radiation time was calculated based on the pre-set radiation dose. The ultraviolet radiation intensity measured by the ultraviolet radiation meter was about 0.28mW / cm 2 According to the radiation dose = radiation intensity * radiation time, the irradiation time is about 7.5 minutes, 15 minutes, 30 minutes and 60 minutes respectively. The mice were taken out immediately after a single dose of ultraviolet irradiation, and then anesthetized with isoflurane. The mouse characteristics were recorded, the thickness of the mouse ears was measured, and finally the score was given according to the degree of inflammation of the ear swelling. The ear appearance was as follows for 5 consecutive days during the modeling process. Figure 1 As shown in A.

[0040] (2) Evaluation of inflammatory symptoms and ear thickness changes in the mouse ultraviolet ear injury model

[0041] The inflammatory symptoms of the mouse UV ear injury model were scored using the following criteria: 0 points for no swelling, 1 point for mild swelling, 2 points for moderate swelling, 3 points for severe swelling, and 4 points for extremely severe swelling. Ear swelling was evaluated at 0 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours after receiving a single dose of UV radiation.

[0042] The results are as follows Figure 1 As shown in B, mice irradiated with a single dose of ultraviolet radiation will have ear swelling, which is significantly different from the control group ( Figure 1 B). The thickness of mouse ears was measured in vivo using a vernier caliper. The statistical results showed that the thickness of the ears of mice exposed to UVB radiation increased significantly ( Figure 1 C). Note: The measurement data were expressed as mean ± SD and tested by two-way ANOVA. In the analysis results, **P<0.01, ***P<0.001, compared with the control group.

[0043] (3) Histopathological examination of the mouse ultraviolet ear injury model

[0044] After the mice were killed by cervical dislocation, ear tissues were obtained, fixed with 4% paraformaldehyde solution, embedded in optimal cutting temperature compound (OCT), sliced ​​at 6 μm, and stained with H&E. The Eclipse Ti inverted microscope (Nikon Corporation, Tokyo, Japan) was used to observe and photograph, and the tissues were photographed and recorded using NIS-element software (Nikon Corporation, Tokyo, Japan).

[0045] The results are as follows Figure 1 As shown in D, H&E stained ear tissue sections show that in the ear injury induced by ultraviolet radiation, the skin is significantly thickened, the stratum corneum is hyperkeratotic, and there is inflammatory cell infiltration. The skin layers of the control group showed normal physiological morphology, and the thickness of the epidermis was significantly lower than that of the modeling group receiving UVB radiation.

[0046] (4) TRPV3 and inflammatory factors TNF-α and IL-6 protein levels in the mouse ultraviolet ear injury model

[0047] Weigh each ear tissue in turn and place it in a 1.5mL centrifuge tube. Prepare the lysis solution containing protease inhibitors in advance according to the ratio of adding 100μL lysis solution and 1μL protease inhibitor to 10mg, and add it to each tissue according to the weight of the ear tissue. Then add the cleaned grinding beads to the centrifuge tube, use SCIENTZ-48 high-throughput tissue grinder (Xinzhi, Zhejiang, China) to grind the tissue at 60HZ. After the tissue is ground, shake it up and down at 4℃ for 1 hour. Then centrifuge at 4℃, 12000rpm for 15 minutes, transfer the supernatant to a new 1.5mL centrifuge tube and place it on ice, and determine the protein concentration by BCA method. Take a part and heat it with 5× loading buffer at 95℃ for 10 minutes for denaturation, and store it at -20℃ for standby use after denaturation. Prepare standard protein and sample protein solutions according to the instructions of the BCA protein concentration determination kit, and determine the protein standard curve and sample protein content at a wavelength of 562nm on the microplate reader. The specific operation process will not be repeated. Prepare 10% separation gel and concentrated gel. The denatured protein samples were vortexed and centrifuged, and then the pre-stained protein marker (3 μL) and protein samples (the volume converted to 20 μg based on the concentration measured by BCA) were loaded into the sample wells. The loading volume of each sample well must be consistent (if inconsistent, 1× loading buffer must be used to fill it up). The remaining sample wells on both sides can also be added with an equal volume of 1× loading buffer. Electrophoresis was performed at a constant voltage of 80V for about 30 minutes, and then the voltage was adjusted to 120V for electrophoresis to the bottom. The protein was transferred from the gel to the nitrocellulose membrane using a wet method, and the constant current was 150mA for 90 minutes. After the transfer, the nitrocellulose membrane was removed and blocked with 5% skim milk powder blocking solution at room temperature for 1.5 hours; after the blocking, TRPV3 antibody (Abcam, catalog number: ab85022) was diluted with fresh blocking solution at a ratio of 1:1000, and incubated at 4°C overnight with shaking; after the primary antibody incubation, the membrane was washed 3 times with 1×TBST solution at room temperature, 5 minutes each time. Use 5% skim milk powder to prepare the secondary antibody at a ratio of 1:10000 and incubate at room temperature for 1 hour. After the secondary antibody incubation, wash the membrane 4 times with 1×TBST at room temperature for 6 minutes each time. Add ECL luminescent solution to the washed nitrocellulose membrane with the protein side facing up for reaction and place it in a developer (Bio-Rad, California, USA). Set appropriate parameters after preview to avoid overexposure and develop, such as Figure 2As shown in A, compared with the control group, the expression level of TRPV3 protein in the ear tissue of mice exposed to UVB radiation was upregulated. Tissues of mice in each group exposed to different doses of ultraviolet radiation were selected on the third day to extract proteins. TRPV3 and inflammatory factors TNF-α (Abcam, Catalog No.: ab1793) and IL-6 (Abcam, Catalog No.: ab208113) were used as primary antibodies for overnight incubation according to the above method. Appropriate secondary antibodies were selected according to the source of the primary antibodies, and ECL luminescent solution was still used for development, as shown in Figure 2. Figure 2 As shown in D, the OD value of the target protein was calculated using Image Lab software, and the statistical results were obtained using Graph Prism 5. Compared with the control group, the protein expression levels of TRPV3 and inflammatory factors TNF-α and IL-6 in the ear tissue of mice exposed to UVB radiation were significantly upregulated. The quantitative data were expressed as mean ± standard deviation (mean ± SD) and tested using one-way ANOVA. * indicates P < 0.05, and *** indicates P < 0.001.

[0048] (5) TRPV3 and inflammatory factors TNF-α, IL-6, and TSLP mRNA levels in the mouse ultraviolet ear injury model

[0049] Weigh the control group, 250mJ / cm 2 UV radiation group, 500mJ / cm 2 The ear tissues of mice in the UV radiation group were placed in 1.5mL centrifuge tubes, and 1mL TRI reagent RNA isolation reagent (Sigma-Aldrich, catalog number: T9424) was added. The tubes were repeatedly blown several times, and grinding beads were added to grind. After grinding, they were placed at room temperature for 10 minutes. 200μL chloroform was added and shaken vigorously for 15 seconds, and then allowed to stand for 15 minutes. Centrifuge at 4°C, 12000rpm for 15 minutes. The upper aqueous phase was aspirated into a new 1.5mL RNase-free centrifuge tube, and an equal amount of isopropanol was added. The tubes were shaken vigorously for 20 seconds and allowed to stand for 20 minutes to precipitate RNA. The supernatant was discarded, and 1mL of 75% ethanol prepared with DEPC water was added to wash the RNA precipitate. Centrifuge at 4°C, 7500rpm for 15 minutes, and the washing was repeated twice. The supernatant was discarded, the RNA precipitate was dried in a clean bench, and the RNA was dissolved with 20μL DEPC water. 1 μL was taken for concentration detection, and the detection instrument was NanoDrop ONE ultra-micro spectrophotometer (Thermo Scientific, Waltham, USA). RNA was then taken for reverse transcription reaction, and the operation was performed according to the instructions of FastKing RT Kit (With gDNase) (Tiangen, Beijing, China, Cat. No.: KR116). The reverse transcription product was stored at -20°C for future use.

[0050] TRPV3 Primers:

[0051] Front citation: 5'-CGCTGGCCTCACTGATTGAGAA-3'(SEQ ID No.01)

[0052] Post-introduction: 5'-CCCATGCGGAATCTGCTTCTCA-3', Yingweijieji (SEQ ID No.02)

[0053] TNF-α Primers:

[0054] Front citation: 5'-ACTCCAGGCGGTGCCTATAT-3'(SEQ ID No.03)

[0055] Post-induction: 5'-GTGAGGGTCTGGGCCATAGAA-3', Invitrogen (SEQ ID No. 04)

[0056] IL-6 Primers:

[0057] Front citation: 5'-CCACTTCACAAGTCGGAGGCTTA-3' (SEQ ID No.05)

[0058] Post-induction: 5'-GCAAGTGCATCGTTGTTCATAC-3', Invitrogen (SEQ ID No. 06)

[0059] TSLP Primers:

[0060] Front citation: 5'-CCAGGCTACCCTGAAACTGA-3' (SEQ ID No.07)

[0061] Post-induction: 5'-5'-TCTGGAGATTGCATGAAGGA-3', Invitrogen (SEQ ID No. 08)

[0062] Real-time quantitative PCR reaction was carried out in a real-time fluorescence quantitative PCR system (QuantStudio3, Thermo Fisher Scientific, USA) according to the instructions of SuperReal PreMix Plus (SYBR Green) (Tiangen, Beijing, China). The reaction system was 2 μL of reverse transcription product, 0.6 μL of forward primer, 0.6 μL of backward primer, 6.4 μL of DEPC water and 10 μL of dye premix; the reaction conditions were: 95°C for 3 minutes, 94°C for 15 seconds, 60°C for 40 cycles for 30 seconds, 72°C for 30 seconds, 95°C for 10 seconds, 65°C for 60 seconds, and 97°C for 1 second. The data of real-time quantitative PCR were processed by the 2-ΔΔCt method to reflect the effect of ultraviolet radiation on TRPV3 in ear tissue relative to the mRNA transcription level of the control group. The results are shown in the figure. Figure 2Compared with the control group, the 2 、500mJ / cm 2 UVB and 1000mJ / cm 2 The TRPV3 mRNA expression level in the ear tissue of mice irradiated with UVB was significantly increased on the third day.

[0063] On the third day, the mRNA levels of inflammatory factors TNF-α, IL-6, and TSLP in the mouse ear tissue were detected by the same method as above. Figure 2 Compared with the control group, the 2 and 500mJ / cm 2 The expression levels of TNF-α, IL-6, and TSLP mRNA in the ear tissue of mice irradiated with UVB were significantly increased. Note: The quantitative data are expressed as mean ± standard deviation (mean ± SD). In the analysis results, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, relative to the control group, # indicates P < 0.05, ## indicates P < 0.01, relative to the UVB250 group.

[0064] Example 2

[0065] This example is used to further illustrate the application of the present invention in preventing or treating ultraviolet skin damage by targeting TRPV3.

[0066] (1) Different doses 250mJ / cm 2 and 500mJ / cm 2 UVB radiation-induced ear damage in wild-type and transgenic mice

[0067] 7-8 week old male C57BL / 6 mice purchased from Beijing Weitonglihua and systemic TRPV3 gene knockout male C57BL / 6 mice purchased from Saiye Biotechnology were used, weighing 20±2g. Wild-type C57BL / 6 mice were randomly divided into blank control group, different UVB radiation dose model groups (250mJ / cm 2 ,500mJ / cm 2 ), 5-10 mice per group. Transgenic C57BL / 6 mice were randomly divided into different UVB radiation dose model groups (250mJ / cm 2 ,500mJ / cm 2 ), 5 mice per group. Before the experiment, the mice were placed in a traditional laboratory environment for at least 3 days to adapt to the environment, with a normal 12-hour alternating circadian rhythm, and the mice had free access to food and water. After receiving a single dose of UVB radiation, the mice were immediately taken out and anesthetized with isoflurane. The mouse characteristics were recorded by photographing, the thickness of the mouse ears was measured, and finally the score was given according to the degree of inflammation of the ear swelling. During the experiment, the ear appearance was as follows for 5 consecutive days: Figure 3 As shown in A.

[0068] (2) Evaluation of inflammatory symptoms and ear thickness changes in wild-type and transgenic mouse models of UV ear damage

[0069] The inflammatory symptoms of the mouse UV ear injury model are consistent with the scoring criteria described above and will not be repeated here. Ear swelling was evaluated at 0 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours after receiving a single dose of UV radiation. Figure 3 As shown in B, wild-type mice exposed to UVB radiation developed more severe ear swelling, which was significantly different from the control group and the transgenic group ( Figure 3 B). Note: The quantitative data were expressed as mean ± standard deviation (mean ± SD) and tested by two-way ANOVA. In the analysis results, ** indicates P < 0.01, *** indicates P < 0.001, relative to the wild-type UVB500 group, and ### indicates P < 0.001, relative to the wild-type UVB250 group.

[0070] The thickness of mouse ears was measured in vivo using a vernier caliper. The statistical results showed that the thickness of the ears of wild-type mice exposed to UVB radiation increased significantly, with significant differences compared with the control group and the transgenic group ( Figure 3 C). Note: The quantitative data were expressed as mean ± standard deviation (mean ± SD) and tested by two-way ANOVA. In the analysis results, *** means P < 0.001, relative to the wild-type control group, ## means P < 0.01, ### means P < 0.001, relative to the wild-type UVB250 group, and &&& means P < 0.001, relative to the wild-type UVB500 group.

[0071] Example 3

[0072] This example is used to further illustrate the application of osthole, a natural inhibitor targeting TRPV3 provided by the present invention, in preventing or treating ultraviolet skin damage.

[0073] (1) Prevention and treatment with natural TRPV3 selective inhibitor osthole 250mJ / cm 2 UVB radiation-induced ear damage

[0074] 7-8 week old male C57BL / 6 mice weighing 20±2g were purchased from Beijing Weitonglihua and randomly divided into blank control group, 250mJ / cm 2UVB radiation dose model group, 10mM osthole preventive administration group, 10mM osthole therapeutic administration group, 5-10 mice in each group. The preventive administration method is: administer once 6 hours before receiving UVB radiation, administer once after receiving UVB radiation, and then administer once at intervals of 24 hours, 48 ​​hours, and 96 hours, for a total of 5 times. The therapeutic administration method is: administer after receiving UVB radiation, and then administer once at intervals of 24 hours, 48 ​​hours, and 96 hours, for a total of 4 times. 2 After UVB irradiation, the mice were immediately taken out and anesthetized with isoflurane. The mouse characteristics were recorded by photographing, the thickness of the mouse ears was measured, and finally the degree of inflammation of ear swelling was scored. Figure 4 As shown in A.

[0075] (2) Osthole inhibits TRPV3 channels to alleviate inflammation symptoms and ear thickness changes in the mouse ultraviolet ear injury model

[0076] The inflammatory symptoms of the mouse UV ear injury model are consistent with the scoring criteria described above and will not be repeated here. Ear swelling was evaluated at 0 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours after receiving a single dose of UV radiation. Figure 4 B, The ear swelling degree of mice in the osthole-treated group was compared with that in the mice that only received 250 mJ / cm 2 The swelling of mice irradiated with UVB was milder and significantly different. The thickness of the mouse ears was measured in vivo with a vernier caliper. The statistical results showed that the swelling of the ears of mice in the osthole-treated group was significantly greater than that of mice in the group that received only 250 mJ / cm 2 The increase in ear thickness in UVB-irradiated mice was lower and significantly different ( Figure 4 C). Note: The quantitative data were expressed as mean ± standard deviation (mean ± SD) and tested by two-way analysis of variance. In the analysis results, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, relative to the UVB250 group.

[0077] (3) Histopathological examination of the mouse model of ultraviolet ear damage by inhibiting TRPV3 channels with osthole

[0078] After the mice were killed by cervical dislocation, ear tissues were obtained and frozen sections and H&E staining were performed as described above. The results were observed and photographed. Figure 4As shown in D, from the H&E stained ear tissue sections, it can be seen that in the ear damage of mice only exposed to ultraviolet radiation, the skin was significantly thickened, the stratum corneum was hyperkeratinized, and inflammatory cell infiltration was occasionally seen in the epidermis, while the skin thickening, stratum corneum hyperkeratinization, and inflammatory cell infiltration of mice treated with osthole were less, and the thickness of the ear tissue was significantly lower than that of the group only exposed to UVB radiation.

[0079] Example 4

[0080] This example is used to further illustrate the application of the natural inhibitor osthole with TRPV3 as the target provided by the present invention in alleviating ultraviolet skin damage in a dose- and time-dependent manner.

[0081] (1) Prevention and treatment of different doses of natural TRPV3 selective inhibitor osthole (0.1mM, 1mM, 10mM) 500mJ / cm 2 UVB radiation-induced ear damage

[0082] 7-8 week old male C57BL / 6 mice weighing 20±2g were purchased from Beijing Weitonglihua and randomly divided into blank control group, 500mJ / cm 2 UVB radiation dose model group, 0.1mM osthole treatment group, 1mM osthole treatment group, 10mM osthole treatment group, 5-10 mice in each group. 2 After UVB irradiation, the mice were immediately taken out and anesthetized with isoflurane. The mouse characteristics were recorded by photographing, the thickness of the mouse ears was measured, and finally the degree of inflammation of the ear swelling was scored. Figure 5 As shown in A.

[0083] (2) Evaluation of different doses of osthole inhibiting TRPV3 channels to alleviate inflammatory symptoms and ear thickness changes in the mouse ultraviolet ear injury model

[0084] The inflammatory symptoms of the mouse UV ear injury model are consistent with the scoring criteria described above and will not be repeated here. Ear swelling was evaluated at 0 hours, 24 hours, 48 ​​hours, 72 hours, and 96 hours after receiving a single dose of UV radiation. Figure 5 As shown in B, the ear swelling of mice receiving 10 mM osthole was higher than that of mice receiving only 250 mJ / cm 2 The swelling of mice irradiated with UVB was less severe and the difference was significant ( Figure 5 B). The thickness of mouse ears was measured in vivo using a vernier caliper. The statistical results showed that the ear thickness of mice in the 10mM and 1mM osthole groups was significantly thicker than that in the mice treated with 500mJ / cm 2 The increase in ear thickness in mice exposed to UVB radiation was lower and significantly different ( Figure 5 C). Note: The quantitative data were expressed as mean ± standard deviation (mean ± SD) and tested by two-way analysis of variance. In the analysis results, *P < 0.05, **P < 0.01, ***P < 0.001, relative to the UVB500 group.

[0085] (3) Histopathological examination of different doses of osthole to inhibit TRPV3 channel and alleviate the ultraviolet ear damage model in mice

[0086] After the mice were killed by cervical dislocation, ear tissues were obtained and frozen sections and H&E staining were performed as described above. The results were observed and photographed. Figure 1 As shown in D, H&E stained ear tissue sections show that in the ear lesions of mice that were only exposed to UV radiation and treated with 0.1 mM osthole, the skin was significantly thickened, the stratum corneum was hyperkeratinized, and inflammatory cell infiltration was occasionally seen in the epidermis, while the skin thickness of mice treated with 1 mM and 10 mM osthole was significantly lower than that of the UVB500 group.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of substances that inhibit transient receptor potential cation channel TRPV3 in the preparation of products for preventing or treating ultraviolet-induced / mediated diseases.

2. The use according to claim 1, characterized in that: Substances that inhibit the transient receptor potential cation channel TRPV3 achieve their inhibitory effects by reducing the expression level of TRPV3, accelerating TRPV3 degradation, or blocking TRPV3 function; Substances that inhibit the transient receptor potential cation channel TRPV3 include reversible, irreversible, competitive, non-competitive, uncompetitive inhibitors or channel blockers.

3. The use according to claim 1, characterized in that: The substance that inhibits the transient receptor potential cation channel TRPV3 is osthole; The osthole can inhibit skin thickening, reduce the degree of keratinization of the stratum corneum, and reduce the degree of inflammatory cell infiltration.

4. The use according to claim 1, characterized in that: The products include pharmaceuticals, experimental reagents, and skin care products; The experimental reagents are for basic research use; Preferably, the skin care products include sunscreen, shower gel, and skin damage detection kit.

5. The use according to claim 1, characterized in that: The diseases include dermatitis and skin cancer; preferably, the skin cancer includes melanoma.

6. Application of transient receptor potential cation channel TRPV3 in screening drugs and skin care products for preventing or treating ultraviolet-induced / mediated diseases.

7. The use according to claim 6, characterized in that: TRPV3 was used as a drug target to investigate whether drugs could reduce the expression and / or activity of TRPV3 in skin damage caused by ultraviolet radiation.

8. The use according to claim 6, characterized in that: The diseases include dermatitis but not limited to atopic dermatitis and skin cancer; preferably, the skin cancer includes melanoma.

9. A drug for preventing or treating ultraviolet damage, characterized in that: comprising a TRPV3 inhibitor and a pharmaceutically acceptable carrier; The TRPV3 inhibitor is osthole, and its structure is shown in Formula I; 10. Application of transient receptor potential cation channel TRPV3 in the treatment of skin diseases by light radiation or drugs; The light is ultraviolet light or infrared light; The application is to use TRPV3 as a target, and after applying light radiation or drugs, obtain the expression and / or increase the activity of the transient receptor potential cation channel TRPV in the skin, thereby exerting the therapeutic effect of light radiation or drugs on skin diseases.

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