Construction method and application of ultraviolet damage model of silkworms

Abstract

The invention discloses a construction method and application of a silkworm ultraviolet damage model, and belongs to the technical field of biomedical model construction. The method comprises the steps that bombyx mori with a dominant P site (+ P) reserved in a genome is placed in an ultraviolet irradiation box, UVC irradiation is used for 10-15 min or UVB irradiation is used for 6-8 h, quantifiable melanin deposition specifically appears within 6-24 h after irradiation, and the reliability of the model is evaluated by measuring the epidermal melanin content and the expression level of tyrosine hydroxylase or dopa pigment decarboxylase. The model can be used for evaluating the ultraviolet radiation resistance of sunscreen products and screening ultraviolet radiation resistant products. The method has the remarkable advantages of short period, low cost and no ethical dispute.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical model construction, and particularly relates to a construction method of a silkworm ultraviolet injury model and application thereof. BACKGROUND

[0002] UV rays are divided into UVA (315-400 nm), UVB (280-315 nm) and UVC (200-280 nm) according to wavelength, among which UVA and UVB penetrate the atmosphere and have a significant impact on organisms. Studies have shown that UVB mainly acts on the epidermis, induces the generation of reactive oxygen species (ROS), activates the MAPK signaling pathway, and up-regulates the expression of matrix metalloproteinases (MMPs), leading to collagen degradation and photoaging; and UVA penetrates the dermis, and through oxidative stress and inflammatory response, it synergistically accelerates skin damage. In recent years, the mechanism of ultraviolet-induced melanin production has become a research hotspot, which not only involves skin protective pigment deposition, but also is closely related to the risk of photocarcinogenesis. Therefore, the development of an efficient sunscreen agent has become an urgent need. Traditional chemical sunscreens have problems such as poor light stability and penetration toxicity, and physical sunscreens (such as titanium dioxide) are controversial due to the potential risks of nanoparticles. Therefore, researching a safe sunscreen based on natural biological mechanisms has become a new research direction.

[0003] At present, the animal models applied to ultraviolet irradiation models include mouse models, guinea pig models, nude mouse models, rabbit models, Xenopus models, zebrafish models, etc. Although certain research results have been achieved in various ultraviolet irradiation models, each animal model has certain shortcomings, such as the large difference between the skin structure of the mouse model and that of humans (such as the thin dermis), and the difficulty in completely simulating the pathological characteristics of UV response (such as photoaging wrinkles). In addition, the immune system is different from that of humans, which may affect the study of inflammation and immune suppression mechanisms. The guinea pig model has the problems of long experimental period (weeks to months are needed to induce chronic damage) and high cost. The nude mouse model also has differences in the immune system. In addition, long-term UV irradiation (such as 8 weeks) is needed to stabilize the phenotype, and the operation is complex. The lens damage mechanism of the rabbit model is different from that of humans, and the ocular UV exposure dose is difficult to accurately control. The skin structure of zebrafish and Xenopus models is quite different from that of mammals, lacking epidermal stratification and accessory organs (such as hair follicles and sweat glands), and cannot completely simulate the human skin photoaging phenotype. The sensitivity to UVB and the damage mechanism may have species-specific differences from mammals, which need to be verified in combination with mammalian models. In summary, the common shortcomings of the current ultraviolet light irradiation animal models are long experimental period, high cost, and complex operation. Therefore, it is urgent to establish a new, efficient, and stable ultraviolet light irradiation model for the screening of new sunscreen products and the testing of sunscreen effect and safety.

[0004] Silkworm as an important economic insect also has wide application value in biomedical research. CN107114561B discloses a method for preventing and treating silkworm BmNPV disease by irradiating mulberry leaves with ultraviolet light, which mentions that ultraviolet light can significantly change the content of secondary metabolites of mulberry leaves. CN116762767A introduces a silkworm feeding method for improving silk performance by spraying a melanin suspension on mulberry leaves. CN114680085A discloses a method for cultivating colored silkworms in a greenhouse, in which mulberry leaves are treated with purple light irradiation to improve vitamin content and antibacterial ability.

[0005] As a kind of insect with well-developed integumentary system, silkworm has certain similarity with human skin in terms of integument structure, and has the potential to be used as a model for ultraviolet radiation damage due to its low feeding cost, short growth cycle and simple experimental operation. However, there is no report on the use of silkworm to construct an ultraviolet radiation damage model for screening sunscreen products and evaluating the efficacy and safety of sunscreen products. Therefore, it is of great scientific significance and application value to develop an ultraviolet radiation damage model based on silkworm for the research and evaluation of sunscreen products. SUMMARY

[0006] In order to solve the technical problems of high feeding cost, long experimental period, unstable effect, ethical controversy and other shortcomings of traditional ultraviolet damage animal models, as well as the uneven quality of sunscreen products on the market, false propaganda and safety problems, and to achieve the technical effect of establishing a low-cost, high-efficiency and ethical controversy-free ultraviolet damage model for screening of new sunscreen products and sunscreen effect, the present application provides a method for constructing an ultraviolet damage model based on silkworm and its application.

[0007] The technical solution adopted by the present application to solve its technical problems is as follows: In a first aspect, the present application provides a method for constructing a silkworm ultraviolet damage model, comprising the following steps: placing silkworms with genomic dominant P site (+P) in an ultraviolet irradiation box, and irradiating the silkworms with UVC or UVB to induce melanin deposition; The UVC irradiation time is 10-15 min, and the irradiation energy density is 6-9 J / cm2; the UVB irradiation time is 6-8 h, and the irradiation energy density is 216-288 J / cm2; The P site refers to a classic genetic locus located at 0 cM of the second linkage group of silkworm, which controls the presence or absence of body surface pattern of larvae, and its dominant allele (+P) can induce melanin deposition under ultraviolet induction.

[0008] Preferably, the ultraviolet irradiation box comprises a light-tight box body and a box cover movably connected with the box body, the inside of the box body constitutes an irradiation chamber, the inside of the box cover is fixedly provided with an ultraviolet lamp holder, the ultraviolet lamp holder is located at the center of the box cover, and a lamp tube capable of emitting ultraviolet rays is arranged on the ultraviolet lamp holder. Figure 14 ; Preferably, the box body is made of light-tight material and has a size of 300 mm x 200 mm x 210 mm, Preferably, the bottom of the ultraviolet irradiation box is uniformly spread with mulberry leaf fragments with a size of 1 cm2, so as to facilitate the feeding of the silkworms, but the body surface of the silkworms should not be blocked by the mulberry leaves.

[0009] Preferably, the silkworm strain is selected from D9L, DZ, N4 or a functionally derived strain with a similar genetic background and a dominant P site (+P) retained in the genome. Preferably, the silkworm with the P site is a 5th instar silkworm larva with a body weight of 1.240 (±0.2) g and a body surface area of 5 (±0.1) x 1.3 (±0.1) cm2.

[0010] Preferably, the melanin deposition starts to appear 6-8 hours after irradiation and tends to be stable after 24 hours.

[0011] Preferably, the expression of tyrosine hydroxylase and dopachrome decarboxylase genes in the silkworm epidermis is up-regulated after UVB and UVC irradiation.

[0012] Preferably, the UVB and UVC irradiation up-regulates the silkworm folate production, phenylalanine metabolism and melanin production pathways and down-regulates the tyrosine degradation pathway.

[0013] Preferably, the construction method further comprises evaluating the reliability of the obtained ultraviolet damage model. The evaluation method comprises: setting a blank control group, wherein the blank control group is the same as the conditions for constructing the silkworm ultraviolet damage model except that the silkworms are not irradiated with ultraviolet rays. 6-24h after ultraviolet irradiation, comparing any one or several of the phenotype changes, epidermal melanin content, tyrosine hydroxylase or dopachrome decarboxylase expression level of the silkworm ultraviolet damage model and the blank control silkworms, when any one or several of the phenotype changes, melanin content, tyrosine hydroxylase or dopachrome decarboxylase expression level of the silkworm ultraviolet damage model and the blank control silkworms have significant differences, it indicates that the silkworm ultraviolet damage model is reliable, otherwise it is an unreliable ultraviolet damage model.

[0014] In the second aspect, the present application provides the application of the silkworm ultraviolet damage model in evaluating the anti-ultraviolet radiation ability of sunscreen products or screening anti-ultraviolet radiation products.

[0015] In a third aspect, the present application provides a method for detecting the effect of sunscreen using the above-mentioned silkworm UV irradiation model, comprising the following steps: Applying the sunscreen to be tested on the body surface of the silkworm with a genomic dominant P site (+P); Irradiating for 10-15 minutes using UVC or 6-8 hours using UVB; After 6-24 hours, observing the melanin deposition, detecting any one or several of the melanin content, the expression level of tyrosine hydroxylase or dopachrome decarboxylase gene, and evaluating the UV protection effect of the sunscreen.

[0016] Preferably, the UV protection effect of the sunscreen is evaluated by the ultraviolet absorption spectrum and the degree of reduction of silkworm melanin deposition.

[0017] The present application has the following advantages: 1. Compared with traditional UV irradiation animal models (such as mice and guinea pigs), the silkworm UV irradiation model established in the present application has the advantages of clear genetic background, no ethical controversy, low breeding cost, and short experimental period; 2. The present application found that irradiating the D9L silkworm strain with a genomic dominant P site (+P) for 10 minutes using UVC and measuring the melanin content after 24 hours is the best experimental condition, and a stable and reliable silkworm UV irradiation model has been successfully established; 3. The present application found that the expression levels of key genes in the melanin production pathway such as tyrosine hydroxylase (TH) and dopachrome decarboxylase (DDC) in the silkworm epidermis increase after UVB and UVC irradiation.

[0018] 4. The present application elucidated the mechanism of the effect of UV on the gene expression in the silkworm epidermis by performing transcriptome sequencing and analysis on the silkworm epidermis after different UV irradiation. UVB and UVC irradiation up-regulate the folic acid production, phenylalanine metabolism, and melanin production pathways, and down-regulate the tyrosine degradation pathway. These changes in the pathways are closely related to melanin production.

[0019] 5. The application successfully applies the silkworm UV irradiation model to the effect test of sunscreen, and tests the physical sunscreen TiO2, the chemical sunscreen oxybenzone and the biological sunscreen silk fibroin by preparing the basic sunscreen cream containing the physical sunscreen, the chemical sunscreen and the biological sunscreen, and purchasing the commercial sunscreen. The UV absorption spectrum test shows that different sunscreens have different UV absorption capacity. After the sunscreen is applied to the body surface of the silkworm and UV irradiation, the melanin deposition is observed after 24 hours. The results show that, except the uncoated group and the base liquid group, the other groups all show the phenomenon of reduced melanin deposition, which is consistent with the result of UV absorption capacity shown by the UV absorption spectrum, indicating that the silkworm model successfully detects the effectiveness of the sunscreen. A new experimental platform is provided for the research and detection of sunscreen; 6. The application not only enriches the content of silkworm body color research, but also provides a scientific basis for the development of the silk industry and the sunscreen industry. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Homology analysis of silkworm TH gene and common mammalian TH gene sequence and structure; Figure 2 Homology analysis of silkworm DDC gene and common mammalian DDC gene sequence and structure; Figure 3 Phenotypic changes of different silkworm strains after UVB ultraviolet irradiation and feeding for 24 hours; Figure 4 Phenotypic changes of silkworm after irradiation under different ultraviolet irradiation conditions and feeding for 24 hours; Figure 5 Phenotypic changes of silkworm after different feeding time after UVB irradiation for 6 hours and UVC irradiation for 15 minutes; Figure 6 Epidermal melanin content of silkworm after feeding for 24 hours after irradiation under different ultraviolet irradiation conditions; Figure 7 Relative content changes of epidermal melanin of silkworm after different feeding time after UVC-10 min irradiation; Figure 8 Expression amount of TH (A) and DDC (B) genes under different ultraviolet irradiation conditions; Figure 9 KEGG pathway gene heat map analysis 1, wherein A is tyrosine pathway analysis, and B is Toll and IMD signal pathway analysis; Figure 10 KEGG pathway gene heat map analysis 2, wherein A is folate metabolism pathway analysis, and B is drug metabolism pathway analysis; Figure 11UV absorption spectra of various sunscreen creams, A: MISTINE; B: ANESSA; C: SUNCUT: D: base; E: TiO2+ base; F: oxybenzone+ base; G: oxybenzone+ TiO2+ base; H: silk fibroin+ base; I: silk fibroin+ TiO2+ base; J: silk fibroin+ oxybenzone+ TiO2+ base; Figure 12 Difference in phenotype of silkworm after UV irradiation after applying various sunscreen creams, A: MISTINE; B: ANESSA; C: SUNCUT: D: base; E: TiO2+ base; F: oxybenzone+ base; G: oxybenzone+ TiO2+ base; H: silk fibroin+ base; I: silk fibroin+ TiO2+ base; J: silk fibroin+ oxybenzone+ TiO2+ base; Figure 13 Relative melanin content in the epidermis of silkworm after UV irradiation after applying various sunscreen creams, taking UVC-10 min as control, n=3, **** (p<0.0001); A: MISTINE; B: ANESSA; C: SUNCUT: D: base; E: TiO2+ base; F: oxybenzone+ base; G: oxybenzone+ TiO2+ base; H: silk fibroin+ base; I: silk fibroin+ TiO2+ base; J: silk fibroin+ oxybenzone+ TiO2+ base; Figure 14 Structure diagram of UV irradiation box. DETAILED DESCRIPTION

[0021] The application provides a method for constructing a silkworm UV damage model, comprising the following steps: Step 1: placing silkworms containing a genomic retention dominant P site (+P) in a UV irradiation box, irradiating the silkworms with UVC or UVB to induce melanin deposition, and obtaining the silkworms after UV irradiation; Specifically, 5th instar silkworms of different strains are selected and placed in a UV irradiation box. 1cm 2 sized mulberry leaf fragments are uniformly spread on the bottom of the UV irradiation box, so that the silkworms can eat during the irradiation process, and at the same time, the body surface of the silkworms is not blocked by the mulberry leaves, so as to ensure that the UV can fully irradiate the body surface of the silkworms.

[0022] When UVC is used for irradiation, the irradiation time is 10-15 min, the irradiation intensity is 10 mw / cm 2 , and the irradiation energy density is 6-9 J / cm 2 ; when UVB is used for irradiation, the irradiation time is 6-8 h, the irradiation intensity is 10 mw / cm 2 , and the irradiation energy density is 216-288 J / cm 2 .

[0023] The silkworm strain containing dominant P site (+P) will gradually produce melanin deposition reaction on the epidermis during ultraviolet irradiation. This melanin deposition begins to appear 6-8 hours after irradiation and tends to be stable after 24 hours. It can be determined that the silkworm strain containing dominant P site (+P) will produce melanin deposition after ultraviolet irradiation. Melanin deposition is a protective response of silkworm to ultraviolet damage, which increases the content of body surface melanin to absorb and scatter ultraviolet light, reducing the damage of ultraviolet light to deep tissues.

[0024] Step two: place the silkworms irradiated by ultraviolet rays in the silkworm breeding box and continue breeding for 24 hours to obtain the silkworm ultraviolet damage model.

[0025] Specifically, the silkworms irradiated by ultraviolet rays are taken out from the ultraviolet irradiation box and gently transferred to the silkworm breeding box. Fresh mulberry leaves are laid in the silkworm breeding box in advance, and the temperature (25±1℃) and humidity (75±5%) are maintained, and the breeding is continued for 24 hours. In this process, a series of physiological and molecular level changes will occur in the silkworm body, including gene expression regulation, metabolic pathway change, etc., and finally a stable ultraviolet damage model is formed.

[0026] After UVB and UVC irradiation, the expression of tyrosine hydroxylase and dopachrome decarboxylase genes in the epidermis of silkworms is significantly up-regulated. Tyrosine hydroxylase is a key enzyme in the melanin synthesis pathway, which catalyzes the conversion of tyrosine to dopa, and dopachrome decarboxylase further catalyzes the conversion of dopa to dopaquinone, and finally forms melanin. The tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) genes of silkworm have high conservation with human homologous genes: as shown in Figure 1 and Figure 2 Sequence alignment shows that the sequence homology of silkworm TH with human TH and other mammalian TH is up to 85%, and the similarity of DDC is as high as 75%-80%, which is significantly higher than that of Drosophila (TH 48%-52%, DDC 60%-65%) and nematode (TH 40%-45%, DDC 50%-55%); two-dimensional structure analysis further confirms that the catalytic domain (β-helix propeller folding) of silkworm TH has an overlap degree of 85% with human, the PLP coenzyme binding pocket hydrogen bond network of DDC is completely matched, and the conformation similarity of substrate recognition region is >90%. This high homology at the molecular level makes silkworm an ideal model for studying human melanin formation.

[0027] The up-regulation of the expression of the two enzymes directly promotes the synthesis of melanin, which is an important defense mechanism of the silkworm against ultraviolet radiation. The folate production, phenylalanine metabolism and melanin production pathways are also up-regulated after UVB and UVC irradiation, and the tyrosine degradation pathway is down-regulated. Folate is an important cofactor in the DNA repair process, and the up-regulation of its production pathway helps to repair DNA damage caused by ultraviolet radiation. The up-regulation of the phenylalanine metabolism pathway provides precursor substances for melanin synthesis. The up-regulation of the melanin production pathway directly increases the production of melanin, and the down-regulation of the tyrosine degradation pathway reduces the consumption of tyrosine, ensuring that more tyrosine can be used for the synthesis of melanin.

[0028] In addition, the construction method of the present application also includes evaluating the reliability of the obtained ultraviolet damage model. The evaluation method is as follows: A blank control group is set up, and the blank control group is the same as the conditions for constructing the silkworm ultraviolet damage model except that the silkworm is not irradiated with ultraviolet rays, including silkworm strain, age, feeding environment, feed and the like.

[0029] 6-24h after ultraviolet irradiation, the phenotype changes and epidermal melanin content of the silkworm ultraviolet damage model and the blank control silkworm are compared. The phenotype changes mainly observe the body color, activity, feeding condition and the like of the silkworm; the epidermal melanin content is determined by extracting the epidermal tissue of the silkworm, and the absorbance value is determined at 405 nm after extracting melanin using an enzyme marker.

[0030] When the phenotype changes and melanin content have significant differences (p<0.05) between the silkworm ultraviolet damage model and the blank control silkworm, it indicates that the constructed silkworm ultraviolet damage model is reliable; otherwise, it is an unreliable ultraviolet damage model.

[0031] The silkworm ultraviolet damage model constructed by the present application can be applied to evaluate the anti-ultraviolet radiation ability of sunscreen products. The specific application method is: evenly applying the sunscreen product to be tested on the body surface of the silkworm, and then irradiating with ultraviolet rays after the product is fully absorbed, then observing the melanin deposition on the body surface of the silkworm and determining the melanin content, and comparing with the control group without applying the sunscreen product to evaluate the protection effect of the sunscreen product. The better the protection effect of the product, the less the melanin deposition on the body surface of the silkworm, and the lower the melanin content.

[0032] In addition, the silkworm ultraviolet damage model constructed by the present application can also be applied to screen anti-ultraviolet radiation products. The specific application method is: applying a plurality of candidate products to the silkworm ultraviolet damage model respectively, and screening out the best anti-ultraviolet radiation product by comparing the protection effects of each product against ultraviolet radiation. The screening indexes include the degree of melanin deposition on the body surface of the silkworm, the melanin content, the expression levels of tyrosine hydroxylase and dopachrome decarboxylase genes and the like.

[0033] The technical solutions of the present application will be further illustrated below by combining with specific examples. Those skilled in the art should understand that the examples are only for helping understanding the present application, and should not be regarded as specific limitation to the present application.

[0034] The process, conditions, reagents, experimental methods, etc. for implementing the present application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the present application has no specific limitation. The experimental methods not specifically mentioned in the examples are usually carried out according to the conventional conditions or the conditions suggested by the manufacturers.

[0035] Unless otherwise specified, the meanings of all professional terms and scientific terms used in the present specification are the same as those generally understood by the skilled person in the technical field to which the present application belongs. However, if there is a conflict, the present specification including the definition shall prevail.

[0036] The silkworm strains DZ (systematic number: 19-200), DZ derived species (D9L), and N4 were provided by the silkworm resource library of Southwest University, and Gui5 was provided by the Guangxi Sericultural Science Research Institute. After the silkworm eggs were hatched, they were fed with fresh mulberry leaves, and the incubator feeding conditions were temperature 25℃, relative humidity (75±5)%, and fresh mulberry leaves were fed 3 times a day until 5th instar 3 days. The 300*200*210mm opaque logistics box body was used as the main body of the ultraviolet irradiation box, the ultraviolet lamp holder was fixed on the box cover of the logistics box, and the ultraviolet lamp holder was ensured to be in the center of the box cover, and then different wavelength ultraviolet lamp tubes were placed on the lamp holder.

[0037] Example 1 Construction of a silkworm ultraviolet injury model The silkworm strains D9L, DZ and N4 containing the gene locus P site, and the silkworm strain Gui5 not containing the gene locus P site (control) were taken, the silkworm eggs were hatched, and they were fed with fresh mulberry leaves, and the incubator feeding conditions were temperature 25℃, relative humidity (75±5)%, and light period 12L:12D, and fresh mulberry leaves were fed 3 times a day until 5th instar 3 days. Each group of 20 silkworms was placed in the ultraviolet irradiation box to give UVC (256nm) 10min and 5min and UVB (302nm) irradiation 6h and 8h (UVC irradiation intensity was 10 mw / cm 2 , and the irradiation energy density was 6J / cm²; the UVB irradiation intensity was 10 mw / cm 2 , and the irradiation energy density was 216J / cm²). 1cm 2 size of mulberry leaf fragments were uniformly spread on the bottom of the box to facilitate the silkworms to eat, but it was ensured that the silkworm body surface would not be blocked by the mulberry leaves. After irradiation, they were taken out and placed in the silkworm breeding box for further feeding for 48h.

[0038] Another 20 silkworms were placed in the silkworm rearing box and fed with mulberry leaves as the control group.

[0039] After irradiation, the phenotypic changes of silkworms irradiated for different times were observed, and 8 silkworms fed for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h and 48 h after irradiation were collected and placed in a-80℃ refrigerator for standby.

[0040] (1) Phenotypic observation after irradiation The silkworms were observed and photographed 24 h after irradiation. The results of UVB irradiation of different strains of silkworms are as follows Figure 3 As can be seen from the figure, only silkworm strains containing P sites showed obvious melanin deposition bands after UVB irradiation, among which D9L and DZ silkworms showed the most obvious phenotype after irradiation; D9L silkworms were used as experimental objects, and the results of UVB and UVC ultraviolet irradiation are as follows Figure 4 As can be seen from the figure, compared with the control group, UVC (254 nm) irradiation for 10 min and 15 min, UVB (302 nm) irradiation for 6 h and 8 h, D9L silkworms showed obvious melanin deposition bands on the intersegmental epidermis.

[0041] The D9L silkworms were observed and photographed after UVB irradiation for 6 h and UVC irradiation for 15 min, and the results are as follows Figure 5 Within the first 8 hours after UVB-6 h and UVC-15 min irradiation, the silkworm epidermis was only yellow all over the body, but after 8 h, the intersegmental epidermis of the silkworm began to darken, and the melanin deposited in the intersegmental epidermis became band-shaped; but with the increase of time, the melanin deposition was not limited to the intersegmental epidermis, and after 16 h of irradiation, the silkworm epidermis began to appear melanin deposition all over the body. After 48 h of irradiation, the silkworms also showed obvious bending phenotype.

[0042] (2) Determination of epidermal melanin content after 24 h feeding under different ultraviolet irradiation conditions After UVC (254 nm) irradiation for 10 min and 15 min, UVB (302 nm) irradiation for 6 h and 8 h, 3 D9L silkworms with similar body size were selected, dissected on ice, and the epidermis from the third segment to the ninth segment was directly torn off in the frozen state, and the torn epidermis was weighed in a centrifuge tube. After weighing, the epidermis was placed in a 60℃ oven and dried overnight until the second weighing was constant.

[0043] The epidermis after drying was ground into powder with a glass grinding rod in a centrifuge tube, the weight of the ground epidermis was recorded, and then 200 μL of 5 M NaOH melanin dissolving solution was added to each centrifuge tube, and the electric grinding rod was used to grind thoroughly until no obvious tissue was left; (2) Each sample was supplemented with 200 μL of 5 M NaOH solution, and boiled in a boiling water bath for 20 min; (4) 4°C high-speed refrigerated centrifuge 9200 g, 5 min; (5) After centrifugation, the melanin-containing supernatant was collected in a new 1.5 mL centrifuge tube and labeled; (6) Similarly, 200 μL of the collected melanin supernatant was added to each well of a 96-well enzyme-labeled plate, and the absorbance value was measured at 405 nm using an enzyme-labeled instrument.

[0044] The melanin content was calculated according to the melanin standard curve, and the experimental results are shown in Figure 6 Compared with the control group, the melanin content of the epidermis of the irradiation group increased significantly (p<0.05), and the amount of melanin produced by UVC irradiation was much higher than that by UVB irradiation.

[0045] (3) Changes in the relative content of melanin in the epidermis of silkworms of different feeding times after UVC-10 min irradiation The epidermis of silkworms of different feeding times after UVC irradiation for 10 min was selected, and the melanin content was determined. The results are shown in Figure 7 as shown, which is consistent with the melanin deposition phenotype, the melanin content is relatively stable in the first 8 h after irradiation, and there is no significant difference in the relative melanin content compared with the control group, the melanin synthesis amount increases suddenly after 8 h after irradiation, and the melanin content reaches a peak in 16-20 h after irradiation, and the melanin content decreases at 24 h.

[0046] Example 2 Expression detection of key genes in the melanin production pathway of silkworms irradiated by ultraviolet light The epidermis of D9L strain silkworms of the control group WT and the experimental groups UVB-8 h and UVC-10 min was selected as experimental material 4 h after irradiation, RNA extraction and reverse transcription analysis were performed, and the results are shown in Figure 8 As shown, the expression levels of TH and DDC genes in the UVB irradiation group increased significantly compared with the control group, and the expression levels of TH and DDC genes in the UVC irradiation group were the highest among the four groups, which increased several times compared with the control group.

[0047] Example 3 Transcriptome analysis of silkworm epidermis irradiated by ultraviolet light Transcriptome sequencing and analysis were performed on the epidermis of silkworms irradiated with WT (whole-wheat tadpoles), UVB-8 h, and UVC-10 min for 4 h. Differential gene analysis showed 1087 differentially expressed genes between UVB and WT, and 802 differentially expressed genes between UVC and WT. GO and KEGG annotation enrichment analyses identified several pathways highly associated with the silkworm melanin pathway, and heatmap analysis was performed on genes in each pathway. The results are shown below. Figure 9 and Figure 10 The results showed that, in the tyrosine synthesis pathway, the TH gene (which promotes the conversion of tyrosine into melanin) plays a crucial role. ple The DDC gene and other genes are significantly upregulated in UVB and UVC, while a series of genes that lead to tyrosine degradation ( TAT , HPD (etc.) significantly downregulated; in the Toll and Imd signaling pathway of the immune pathway, it regulates the melanin synthesis pathway by regulating phenol oxidase. Hayan Genes and SP7 The gene was significantly upregulated in the UVC group. It controls the synthesis of the cofactor BH4 in the folate-related pathway. SPR The gene was significantly upregulated in the UVC group, which promoted melanin synthesis. Analysis showed that UVB and UVC irradiation upregulated folate production, phenylalanine metabolism, and melanin production pathways, while downregulating tyrosine degradation pathways. These pathway changes are closely related to melanin production.

[0048] Example 4: Application of the silkworm UV damage model in sunscreen detection (1) Sunscreen formulation: Based on online research and the book "7 Steps to Cosmetic Formulation Design" by Dong Yinmao et al., following the principles of sunscreen design, the oil phase was determined to be No. 26 white oil, the emulsifier was a commercially available white oil emulsifier, and the aqueous phase was pure water. Different sunscreen formulas were constructed by adjusting and adding different sunscreen agents. To explore the response of the silkworm model to different types of sunscreen agents, seven homemade sunscreen formulas, including the base, were finally determined. As shown in Table 1, three popular sunscreens on the market—MISSTINE, ANESSA, and SUNCUT—were selected. In addition, there were self-formulated physical sunscreens containing 5% TiO2, chemical sunscreens containing oxybenzone, biological sunscreens containing silk fibroin, and sunscreens composed of three different types of sunscreen agents.

[0049] Table 1. Formulations or sources of various sunscreen agents

[0050] (2) Ultraviolet irradiation experiment Take 5 μL of the prepared sunscreen lotion and apply it evenly to the surface of the D9L strain silkworm to be irradiated (do not apply it to the pores), ensuring that the sunscreen is completely applied and forms a uniform film on the silkworm's body surface.

[0051] Twenty silkworms of the D9L strain, each coated with a different sunscreen, were placed in a UVC irradiation chamber. Additionally, another 20 silkworms were placed in a silkworm rearing box and fed normal mulberry leaf treatment, serving as a control group. The control group was placed under natural light; the experimental group silkworms coated with sunscreen were irradiated under UVC (254 nm) light for 10 minutes (UVC irradiation intensity was 10 mw / cm²). 2 The irradiation energy density is 9 J / cm³. 2 After irradiation, silkworms of three varieties were collected at 0 h, 4 h, and 24 h post-irradiation and placed in a -80℃ refrigerator for later use. Phenotypic changes in each group of irradiated silkworms were observed and photographed 24 h later.

[0052] (3) Determination of the effectiveness of sunscreen lotion Take appropriate amounts of the prepared base solution, various sunscreen lotions, and commercially available sunscreen lotions, and disperse them with white oil at a ratio of 1:100, ensuring that they contain equal amounts of sunscreen agents. Take an equal volume of solution from the white oil dispersion and place it in a UV-permeable ELISA plate. Use an ELISA reader to measure the absorbance at a wavelength of 200-400 nm and plot the absorption curve.

[0053] See results Figure 11 The MISSTINE and ANESSA sunscreens exhibit significantly higher UV absorption in the 250-350 nm wavelength range compared to other formula sunscreens. This indicates that these two sunscreens contain a large amount of chemical sunscreens with high absorption in this wavelength range, achieving better sun protection through the combination of these chemical sunscreens. The UV absorption spectrum of the SUNCUT brand sunscreen shows that while it also has a UV absorption peak in the 250-350 nm range, its peak value is lower compared to the other two brands. The homemade sunscreen's overall UV absorption spectrum only shows a slight increase in absorption compared to the base liquid; only the sunscreen with added chemical sunscreens shows a significant absorption peak.

[0054] (4) Phenotypic changes and melanin content analysis of silkworms after UV irradiation with sunscreen Various sunscreens were evenly applied to the surface of silkworms, and then the silkworms were exposed to UVC for 10 minutes. The phenotypic characteristics of the silkworms were observed and photographed 24 hours after the exposure. Results are as follows: Figure 12The comparison clearly shows that MISSTINE sunscreen has the best sun protection effect, followed by the remaining two commercially available sunscreens. Among the remaining homemade sunscreens, the base liquid, lacking any added sunscreen ingredients, offered virtually no sun protection, and its application to silkworms resulted in melanin deposition. The remaining homemade sunscreens, such as those containing the physical sunscreen TiO2, the chemical sunscreen oxybenzone, and silk fibroin, while offering some sun protection, still caused melanin production on the silkworms' epidermis after exposure to sunlight, showing a significant difference from commercially available products and indicating that the ingredients need further optimization.

[0055] The relative melanin content of the treated silkworm epidermis was measured after irradiation, and the results are as follows: Figure 13 As shown in the figure, there was no significant difference in the relative melanin content of silkworm epidermis after applying MISSTINE sunscreen compared to the unexposed control group. However, the base liquid without sunscreen showed a large amount of melanin production, similar to the unexposed and exposed silkworm epidermis, indicating that the base liquid had virtually no sun protection effect. Other sunscreens, including SUNCUT and homemade sunscreens such as those containing TiO2 and oxybenzone, those containing silk fibroin, and those containing both silk fibroin and TiO2, while offering some sun protection, did not provide as much protection as MISSTINE sunscreen based on their relative melanin content. In terms of relative melanin content, the ability to prevent melanin production is ranked as follows: MISSTINE > ANESSA > oxybenzone sunscreen > silk fibroin sunscreen > silk fibroin > TiO2 > oxybenzone combination sunscreen > SUNCUT sunscreen > TiO2 sunscreen > silk fibroin and TiO2 combination sunscreen > base liquid. This indicates that by comparing the differences in the relative melanin content of silkworm epidermis after irradiation, the sun protection ability of different sunscreens can be determined to a certain extent.

[0056] The applicant declares that the above description is only a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

Claims

1. A method for constructing a model of ultraviolet damage in Bombyx mori, characterized by, The method comprises the following steps: The silkworm strain with dominant P locus is placed in the ultraviolet irradiation box, and the silkworm is irradiated by UVC or UVB to induce melanin deposition; the UVC irradiation time is 10-15 min, and the irradiation energy density is 6-9 J / cm2; the UVB irradiation time is 6-8 h, and the irradiation energy density is 216-288 J / cm2; the P locus refers to a classic genetic locus located at 0 cM of the second linkage group of the silkworm, which controls the presence or absence of larval body surface pattern, and the dominant allele thereof can initiate melanin deposition under ultraviolet induction.

2. The construction method of claim 1, wherein, The ultraviolet irradiation box comprises a light-tight box body and a box cover movably connected with the box body, the inside of the box body constitutes an irradiation chamber, an ultraviolet lamp holder is fixedly installed on the inner side of the box cover, and the ultraviolet lamp holder is located at the center of the box cover; the ultraviolet lamp holder is provided with a lamp tube capable of emitting ultraviolet rays; Preferably, the bottom of the ultraviolet irradiation box is uniformly spread with 1 cm2-sized mulberry leaf fragments, so as to facilitate the silkworm to eat, but to ensure that the body surface of the silkworm is not blocked by the mulberry leaves.

3. The construction method of claim 1, wherein, The silkworm strain is selected from D9L, DZ, N4 or a functionally derived strain with a similar genetic background and a genome retaining a dominant P locus.

4. The construction method according to claim 3, characterized in that, The silkworm with the P locus is a 5th instar 3-day-old silkworm larva, the body weight is 1.240 (±0.2) g, and the body surface area of the silkworm is 5 (±0.1) x 1.3 (±0.1) cm2.

5. The construction method of claim 1, wherein, The melanin deposition starts to appear 6-8 hours after irradiation, and tends to be stable after 24 hours.

6. The construction method of claim 1, wherein, The expression of tyrosine hydroxylase and dopachrome decarboxylase genes in the silkworm epidermis is up-regulated after UVB and UVC irradiation.

7. The construction method of claim 1, wherein, The silkworm folate generation, phenylalanine metabolism and melanin generation pathways are up-regulated, and the tyrosine degradation pathway is down-regulated after UVB and UVC irradiation.

8. The construction method of claim 1, wherein, The construction method further comprises evaluating the reliability of the obtained ultraviolet damage model; The evaluation method comprises: setting a blank control group, which is the same as the conditions for constructing the silkworm ultraviolet damage model except that the silkworm is not irradiated by ultraviolet rays; 6-24 hours after ultraviolet irradiation, any one or several of the phenotype changes, epidermal melanin content, tyrosine hydroxylase or dopachrome decarboxylase expression level of the silkworm ultraviolet damage model and the blank control silkworm are compared, and when any one or several of the phenotype changes, melanin content, tyrosine hydroxylase or dopachrome decarboxylase expression level have significant differences between the silkworm ultraviolet damage model and the blank control silkworm, it indicates that the silkworm ultraviolet damage model is reliable, otherwise it is an unreliable ultraviolet damage model.

9. The application of the silkworm ultraviolet damage model constructed by the construction method of any one of claims 1-8 in evaluating the anti-ultraviolet radiation ability of sunscreen products or screening anti-ultraviolet radiation products.

10. A method for detecting the effect of sunscreen agents by using the silkworm ultraviolet damage model constructed by the construction method of any one of claims 1-8, comprising the following steps: Applying the sunscreen agent to be tested on the body surface of the silkworm with a genome retaining a dominant P locus; Irradiating for 10-15 min by UVC or 6-8 h by UVB; After 6-24 hours, the melanin deposition is observed, and any one or several of the melanin content, the tyrosine hydroxylase or dopachrome decarboxylase gene expression level is detected to evaluate the UV protection effect of the sunscreen.

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