Construction method of animal model of chloasma in plateau area based on microbial interaction characteristics

Abstract

The present application relates to the technical field of animal model construction, in particular to a highland chloasma animal model construction method based on microbial interaction characteristics, 6-8 week old female C57BL / 6J mice are selected, and the mice are adaptively bred at an altitude of 3650m for 7 days; Aspergillus uncinatus is activated twice, and a 1x10 7 ~5x10 7 CFU / mL spore suspension is prepared; the compound mode of intramuscular injection of progesterone, middle wave ultraviolet irradiation and back fungal smearing is used, and the modeling is continuously performed for 42-49 days. The model is verified from multiple dimensions of apparent score, skin pathology, skin physiological parameters, oxidative stress biochemistry and neuroendocrine-immune factors. The present application firstly introduces a highland chloasma signature fungus, reenacts the pathogenesis pathway of pigment deposition induced by microbial flora disorder, and compared with the traditional hormone plus ultraviolet model, the skin lesion pathological characteristics are more typical, which is suitable for the mechanism research of highland chloasma and the screening of targeted microecological drugs.

Description

Technical Field

[0001] This invention relates to the field of animal model construction technology, specifically a method for constructing a plateau-based animal model of chloasma based on microbial interaction characteristics. Background Technology

[0002] Melasma is a common acquired facial pigmentation disorder, clinically manifested as symmetrically distributed light brown to dark brown patches, most commonly seen in women of childbearing age. It not only severely affects the patient's appearance but also leads to psychological problems such as anxiety and depression, significantly reducing the patient's quality of life. The pathogenesis of this disease is complex and is currently believed to be closely related to multiple factors, including genetic susceptibility, ultraviolet radiation, hormonal fluctuations, skin inflammation, angiogenesis, and neuroendocrine-immune (NEI) network disorders.

[0003] Due to the unique environmental characteristics of high altitude, strong ultraviolet radiation, long hours of sunshine, and low oxygen levels, the prevalence of melasma in high-altitude areas is significantly higher than in plains areas. Multiple epidemiological surveys show that the prevalence of melasma in the general population of Lhasa, Tibet (altitude approximately 3650 meters) in my country is as high as 14.94%, and the prevalence in women aged 31-40 reaches 37.50%; the prevalence of melasma in the native population of Ladakh, India, is even higher than the average level in lower-altitude areas, reaching 49.4%. The high-altitude environment has become a significant contributing factor to the development and progression of melasma, making research on the pathogenesis of melasma in high-altitude areas and the development of preventative and therapeutic drugs of great clinical and social value.

[0004] Animal models are crucial experimental tools for studying disease pathogenesis and screening and evaluating preventive and therapeutic drugs. Currently, commonly used methods for constructing animal models of melasma in plain areas mainly include simple ultraviolet irradiation, simple progesterone injection, emotional stimulation, and ultraviolet irradiation combined with progesterone injection, with the latter being the most widely used. However, these traditional modeling methods are all designed based on the pathogenic factors in plain areas and do not consider the unique skin microecological characteristics of high-altitude regions.

[0005] Recent studies have shown a close relationship between skin microbiota and the development of melasma. Compared with healthy individuals, the abundance of Aspergillus and Brachymycetes fungi on the skin surface of patients with high-altitude melasma was significantly increased. Among them, Aspergillus fusiformis was the most significant fungal marker, with a relative abundance of 0.9838±1.1082% in the skin of patients, significantly higher than 0.1893±0.2070% in healthy individuals (P=0.010), demonstrating disease risk prediction ability. This suggests that the excessive proliferation of specific fungi may directly participate in the pathogenesis of high-altitude melasma. Skin microbiota can influence skin physiological functions and pathological states through the NEI network regulatory pathway. However, traditional animal models of melasma do not incorporate this key regulatory factor of skin microbiota, and cannot simulate the core pathological process of "skin microbiota dysbiosis-skin inflammation-pigmentation." This results in significant differences between the constructed models and the actual pathological environment of melasma in high-altitude areas, making it difficult to meet the needs of research on the mechanism of melasma in high-altitude areas and drug development.

[0006] Furthermore, current explorations of animal models of melasma in high-altitude areas remain at the stage of optimizing combinations of traditional factors, such as progesterone injection combined with natural light exposure or natural light exposure alone. While these methods can induce a certain degree of skin pigmentation, they suffer from drawbacks such as poor model stability, atypical pathological features, and inability to reflect the microbiota-host interaction mechanism. Therefore, there is an urgent need to develop a novel animal model of melasma that can integrate the characteristics of the high-altitude environment with skin microbiome interactions, providing a more reliable experimental platform for the study of melasma in high-altitude regions. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing animal models of melasma in high-altitude areas, such as the lack of inclusion of skin microbial regulatory factors, inability to realistically simulate the pathological process of the disease, and low similarity between the model and the real pathological environment. This invention provides a method for constructing an animal model of melasma in high-altitude areas based on microbial interaction characteristics. For the first time, this invention introduces the key differentially abundant fungus *Aspergillus fusiforme*, enriched in the skin of patients with melasma in high-altitude areas, into the modeling system to construct an animal model induced by a combination of ultraviolet light, progesterone, and fungi. A multi-dimensional validation system ensures the effectiveness and stability of the model, making it closer to the real pathological environment of melasma in high-altitude areas and reflecting the role of microbiota-host interactions in the disease development.

[0008] The technical solution adopted by this invention to solve its technical problem is: a method for constructing a plateau region chrysoberylosis animal model based on microbial interaction characteristics, comprising the following steps:

[0009] S1. Selection and Adaptation of Experimental Animals: 6-8 week old female C57BL / 6J mice, weighing 18-22g, were selected and transported to a high-altitude experimental environment at 3650m. They were then acclimatized for 7 days. The high-altitude experimental environment was either the actual high-altitude environment or an artificial high-altitude environment that simulated the atmospheric pressure and oxygen concentration at the corresponding altitude through a hypobaric chamber. The feeding conditions were a temperature of 18-23℃ and a humidity of 19-41%, with free access to food and water.

[0010] S2 strain activation and suspension preparation: The standard strain *Aspergillus penicillioides* GDMCC 3.587 was selected, activated twice on Sabouraud dextrose agar plates, and the colonies were washed with sterile physiological saline. After centrifugation at 3000 rpm for 5 min and washing twice, the suspension was resuspended, and the spore concentration was adjusted to 1×10⁻⁶. 7 -5×10 7 CFU / mL, prepare and use immediately;

[0011] S3 Composite Factor Modeling: The backs of mice were shaved, covering an area of ​​5cm × 4cm, 2-3 times per week. Mice were randomly divided into a control group and a model group. The model group received alternating intramuscular injections of 20mg / kg progesterone solution into both hind limbs daily, and simultaneously irradiated the back skin with UVB (275-320nm) at a distance of 20cm for 60 minutes each time. After irradiation, 100μL of Aspergillus spore suspension prepared in step S2 was applied to the shaved area on the back, evenly covered, and allowed to air dry. The control group received an equal volume of physiological saline and did not receive UV irradiation or fungal application. Modeling continued for 42-49 days.

[0012] S4 Model Validation: The successful construction of the model was verified through epigenetic feature scoring, skin pathological histology examination, skin physiological parameter detection, biochemical index detection, and neuro-endocrine-immune network factor detection.

[0013] Specifically, the specific conditions for strain activation in step S2 are as follows: take the strain from the -80℃ cryopreservation tube and inoculate it onto Sabouraud dextrose agar plates, and incubate at 30℃ for 48 hours to complete the first generation activation; pick a single smooth colony and streak it again, and incubate at 30℃ for 24 hours to complete the second generation activation.

[0014] Specifically, before applying the bacterial suspension in step S3, wipe the skin of the hair removal area on the back with a 75% alcohol swab for disinfection, let it dry for 1 minute, and then apply the suspension.

[0015] Specifically, in step S4, the appearance feature score is divided into 1-5 points based on the degree of skin pigmentation, where 1 point indicates no pigmentation and 5 points indicates large-area uniform dark gray-brown pigmentation.

[0016] Specifically, in step S4, the skin pathological histological examination used HE staining to observe the number of melanin granules in the basal layer and immunohistochemical staining to detect the expression level of HMB45-labeled melanin-positive cells.

[0017] Specifically, the skin physiological parameters in step S4 include E value, M value, ITA° value, L value, a value, b value, transepidermal water loss rate, and stratum corneum moisture content.

[0018] Specifically, the biochemical indicators in step S4 include superoxide dismutase activity, malondialdehyde content, tyrosinase activity, and lipofuscin content, and the test samples are serum, liver, and skin tissue.

[0019] Specifically, in step S4, the neuro-endocrine-immune network factors include substance P, α-melanocyte stimulating factor, and tumor necrosis factor-α, and the test samples are serum, liver, and skin tissue.

[0020] Specifically, in step S3, the model was continuously created for 49 days. At this time, the skin pigmentation of the mice in the model group was the most significant, and the differences in various indicators were the most obvious.

[0021] Specifically, female SD rats can also be used as experimental animals, with the modeling parameters adjusted accordingly.

[0022] Specifically, the high-altitude experimental environment described in step S1 is simulated by a hypobaric oxygen chamber, which is set to atmospheric pressure and oxygen concentration parameters corresponding to an altitude of 3650m.

[0023] The beneficial effects of this invention are:

[0024] (1) The method for constructing an animal model of melasma in high-altitude areas based on microbial interaction characteristics described in this invention introduces Aspergillus fusiforme, a key differential fungus enriched in the skin of patients with melasma in high-altitude areas, into the modeling system to construct an animal model of melasma in high-altitude areas induced by a combination of ultraviolet light, progesterone, and fungi. This method breaks through the limitation of traditional models that do not consider skin microbial factors and can realistically simulate the core pathological process of "skin flora imbalance-inflammatory response-pigmentation" in high-altitude areas. The model has a significantly improved similarity to the real pathological environment of melasma in high-altitude areas.

[0025] (2) The method for constructing a plateau-area chloasma animal model based on microbial interaction characteristics described in this invention optimizes the induction parameters of composite factors, including progesterone injection dosage, ultraviolet irradiation wavelength and time, Aspergillus spore concentration, and application method, ensuring good stability and reproducibility of the model. At 49 days of modeling, the model group mice showed obvious dark gray-brown pigmentation on their skin, and all pathological and biochemical indicators differed significantly from the blank group, and were superior to the traditional ultraviolet combined with progesterone model.

[0026] (3) The method for constructing a plateau-area chloasma animal model based on microbial interaction characteristics described in this invention establishes a multi-dimensional model validation system, integrating epigenetic features, pathological histology, skin physiological parameters, biochemical indicators, NEI network factors, and correlation analysis, comprehensively validating the model's effectiveness. Correlation analysis results show that the abundance of Aspergillus dermatophytes is significantly correlated with tyrosinase activity, oxidative stress indicators, and NEI network factors, confirming that the model can reflect the microbiome-host interaction mechanism.

[0027] (4) The method for constructing an animal model of chloasma in plateau regions based on microbial interaction characteristics described in this invention provides an ideal experimental platform for the study of the pathogenesis of chloasma in plateau regions. It is especially suitable for exploring the role of skin microorganisms in the occurrence of diseases and screening new drugs for prevention and treatment targeting microecology. It has important scientific research value and application prospects. Attached Figure Description

[0028] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0029] Figure 1 The images show the apparent changes in the skin on the back of mice on days 35, 42, and 49 under different modeling methods. Control is the blank group, M is the traditional ultraviolet light + progesterone model group, and M+Asp is the ultraviolet light + progesterone + Aspergillus broom model group of this invention.

[0030] Figure 2 HE staining images of mouse dorsal skin tissue on days 35, 42, and 49, with different modeling methods, magnified 200 times, showing the distribution and number of melanin granules in the basal layer;

[0031] Figure 3 Immunohistochemical staining images of HMB45 in the dorsal skin tissue of mice under different modeling methods on days 35, 42 and 49, magnified 200 times, showing the expression level of melanin-positive cells;

[0032] Figure 4 Bar charts showing the changes in biochemical indicators in mouse serum, liver, and skin tissues on days 35, 42, and 49 under different modeling methods, including SOD activity, MDA content, TYR activity, and LF content;

[0033] Figure 5 Bar charts showing the changes in NEI network factors in mouse serum, liver, and skin tissues on days 35, 42, and 49 under different modeling methods, including the levels of SP, α-MSH, and TNF-α.

[0034] Figure 6Spearman correlation heatmaps are generated for the relationships between skin structural parameters, biochemical indicators, and NEI network factors, where * indicates P < 0.05 and ** indicates P < 0.01.

[0035] Figure 7 Principal component analysis (PCA) biplots for each index of different modeling methods are used to show the clustering characteristics of the blank group, the traditional model group, and the model group of this invention. Detailed Implementation

[0036] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0037] like Figures 1-7 As shown, the method for constructing a plateau-area yellow-brown animal model based on microbial interaction characteristics according to the present invention includes the following steps:

[0038] S1. Selection and Adaptation of Experimental Animals: Female C57BL / 6J mice aged 6-8 weeks, weighing 18-22g, were selected. All mice were transported from plains areas to a high-altitude experimental environment (3650m) and acclimatized for 7 days. The rearing conditions were controlled at a temperature of 18-23℃ and humidity of 19-41%, maintaining a 12-hour light / 12-hour dark cycle. Mice had free access to food and water. Female mice were chosen because melasma is highly prevalent in women of reproductive age and is closely related to fluctuations in estrogen levels. C57BL / 6J mice have a clear genetic background and stable skin pigmentation response, making them a commonly used experimental animal for constructing melasma models.

[0039] S2 strain activation and suspension preparation: The standard strain *Aspergillus penicillioides* GDMCC 3.587 was selected as the model strain. This strain is a key differentially expressed fungus obtained through skin microbiome sequencing of patients with melasma at high altitudes, and its relative abundance on the skin surface of patients is significantly higher than that of healthy individuals. Strain activation process: The frozen strain was removed from a -80℃ ultra-low temperature freezer, thawed with a small amount of sterile liquid culture medium, and then streaked onto Sabouraud dextrose agar (SDA) plates. It was then incubated at 30℃ for 48 hours to complete the first generation activation. Single colonies with regular morphology and smooth surfaces were picked from the plates and streaked again onto fresh SDA plates, incubated at 30℃ for 24 hours to complete the second generation activation, ensuring the strain was in the logarithmic growth phase and had optimal viability. Preparation of bacterial suspension: Wash the colonies on the second-generation activated agar plates with sterile physiological saline, collect the bacterial suspension into sterile centrifuge tubes, centrifuge at 3000 rpm for 5 min, discard the supernatant, resuspend the precipitate in sterile physiological saline, and repeat the washing twice to remove impurities from the culture medium; finally, count the spores using a hemocytometer and adjust the spore concentration to 1×10⁻⁶. 7 -5×107 CFU / mL, the bacterial suspension should be prepared and used immediately to avoid a decrease in spore viability.

[0040] S3 composite factor modeling: After the adaptation period, the fur on the backs of the mice was shaved, with a shaving area of ​​5cm × 4cm. Shaving was done 2-3 times per week. If hair regrowth was rapid, depilatory cream could be used to assist in hair removal. The depilatory cream was left on the skin for 3-5 minutes and then washed off with warm water to avoid skin damage. The mice were randomly divided into a control group and a model group, with at least 9 mice in each group to ensure statistical power. Model group procedures: Mice in the model group received intramuscular injections of progesterone solution every morning at a dose of 20 mg / kg, with an injection volume of 5 mL / kg. Injections were alternated between the hind limbs to minimize local tissue damage. In the afternoon, mice underwent local UVB irradiation (275-320 nm wavelength), maintaining a 20 cm distance between the mouse's back skin and the light source, with each irradiation lasting 60 minutes. After UV irradiation, the skin of the hair-removed area on the back was gently wiped with a 75% alcohol swab for disinfection. After air-drying for 1 minute, 100 μL of the Aspergillus spore suspension prepared in step S2 was added to the center of the hair-removed area using a 100 μL micropipette. The suspension was then gently spread evenly with a sterile cotton swab, ensuring the entire hair-removed area was covered. After air-drying, the mice were returned to their cages. The control group mice received the same volume of physiological saline as the model group daily, but did not undergo UV irradiation or fungal application. The mice were modeled continuously for 42-49 days, during which time their general condition was closely observed, including their diet, water intake, activity, fur luster, and bowel and bladder function.

[0041] S4 Model Validation: A multi-dimensional indicator system was used to verify the successful construction of the model, specifically including:

[0042] (1) Observation and scoring of appearance characteristics: On days 0, 7, 21, 35, 42 and 49 after modeling, the pigmentation of the skin on the back of the mice was observed, including the area of ​​the spots, the color depth, the gloss of the fur, etc. The pigmentation was scored according to the degree of pigmentation: 1 point (no pigmentation, the skin is normal pink), 2 points (a few light yellow spots, area <10%), 3 points (a lot of yellow spots, area 10%-30%), 4 points (large area of ​​light brown patches, area 30%-60%), 5 points (large area of ​​uniform dark gray brown patches, area >60%).

[0043] (2) Skin pathological histological examination: After the modeling was completed, the mice were sacrificed, and about 1 cm of skin tissue from the lesion area on the back was taken. 2 The tissue was fixed in 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) and immunohistochemically. HE staining was used to observe the skin tissue structure and the number and distribution of melanin granules in the basal layer; immunohistochemical staining was performed by labeling melanocytes with mouse anti-HMB45 monoclonal antibody to observe the number and expression intensity of melanin-positive cells.

[0044] (3) Detection of skin physiological parameters: On days 0, 7, 21, 35, 42, and 49 of modeling, a skin detector is used to measure the E value, M value, ITA° value, L value, a value, b value, transepidermal water loss rate (TEWL) and stratum corneum moisture content (CM) of the back skin of mice, so as to assess the degree of skin pigmentation and barrier function.

[0045] (4) Detection of biochemical indicators: After modeling, mouse blood is collected and serum is separated; liver and back skin tissues are taken to prepare tissue homogenates. Enzyme-linked immunosorbent assay (ELISA) is used to detect superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, tyrosinase (TYR) activity and lipofuscin (LF) content in serum, liver and skin tissues, so as to assess the oxidative stress level and melanin synthesis capacity.

[0046] (5) Detection of NEI network factors: The ELISA method is used to detect the contents of substance P (SP), α-melanocyte-stimulating hormone (α-MSH) and tumor necrosis factor-α (TNF-α) in serum, liver and skin tissues, so as to assess the functional state of the neuro-endocrine-immune network.

[0047] (6) Correlation analysis: The Spearman correlation coefficient is used to analyze the correlation between the abundance of Aspergillus penicillioides in the skin and various model indicators, so as to verify the role of flora-host interaction in the model.

[0048] Example 1: Construction of a chloasma mouse model in plateau areas based on microbial interaction characteristics (49 days of modeling)

[0049] Experimental materials:

[0050] 1.1 Experimental animals: 27 female C57BL / 6J mice aged 6-8 weeks, weighing 18-22 g, are selected and purchased from Chengdu Ensiweier Biotechnology Co., Ltd., with the experimental animal production license number: SCXK (Xiang) 2021-0002. The mice are transported from Chengdu, Sichuan to the Animal Experiment Center of Xizang University (at an altitude of 3650 m) and acclimatized for 7 days. The feeding conditions are: temperature 18-23°C, humidity 19-41%, 12 h light / 12 h dark cycle, and free access to food and water. All animal experimental operations are approved by the Institutional Animal Care and Use Committee of Xizang University. This example is carried out in a real plateau environment. Under the same conditions, a hypobaric oxygen chamber can also be used to simulate the corresponding altitude environment in plain areas to complete the entire modeling process.

[0051] 1.2 Strain: Aspergillus penicillioides GDMCC 3.587, purchased from Guangdong Microbial Culture Collection Center.

[0052] 1.3. Main Reagents and Instruments: Progesterone injection solution (Zhejiang Xianju Pharmaceutical Co., Ltd.); SOD, MDA, TYR, LF, SP, α-MSH, TNF-α ELISA kits (Shanghai Enzyme-Linked Biotechnology Co., Ltd.); Sabouraud dextrose agar medium (Qingdao Haibo Biotechnology Co., Ltd.); Skin color measuring instrument, skin moisture loss tester, skin stratum corneum moisture content measuring instrument (Delfin Technologies Ltd., Finland); Three-in-one constant temperature water bath (Shanghai Jinghong Instrument Manufacturing Co., Ltd.); Benchtop large-capacity low-speed centrifuge (Shanghai Anting Scientific Instrument Factory); 0.01% electronic balance (METTLERTOLEDO, Switzerland); Ultraviolet lamp (UVB, wavelength 275-320nm, medical reference); Optical microscope (Olympus, Japan).

[0053] Experimental methods

[0054] 2.1. Activation of the bacterial strain and preparation of bacterial suspension: The *Aspergillus oryzae* frozen tube was removed from the -80℃ ultra-low temperature freezer, thawed at room temperature, and resuspended in 1 mL of sterile physiological saline. 100 μL of the bacterial suspension was streaked onto an SDA plate and incubated at 30℃ for 48 h to complete the first generation activation. Regularly shaped, white, fluffy colonies (approximately 2-3 mm in diameter) were picked from the plate and streaked again onto fresh SDA plates. The plates were incubated at 30℃ for 24 h to complete the second generation activation. The colonies on the second-generation plate were washed with 5 mL of sterile physiological saline. The bacterial suspension was collected into a 15 mL sterile centrifuge tube, centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the precipitate was resuspended in 5 mL of sterile physiological saline, washed twice. Finally, the number of spores was counted under a microscope using a hemocytometer, and the spore concentration was adjusted to 3 × 10⁻⁶. 7 CFU / mL, bacterial suspension should be prepared and used immediately.

[0055] 2.2 Animal Grouping and Model Establishment: After the acclimatization period, 27 mice were randomly divided into 3 groups of 9 mice each: a blank control group (Control group), a traditional model group (M group, ultraviolet light + progesterone), and the model group of this invention (M+Asp group, ultraviolet light + progesterone + Aspergillus fulvicina). All mice had their backs shaved, covering an area of ​​5cm × 4cm, twice a week. If hair regrowth was significant, depilatory cream was used to assist in hair removal. The depilatory cream was left on for 3 minutes and then rinsed off with warm water.

[0056] Modeling procedures: M mice were injected intramuscularly with progesterone solution 20 mg / kg (5 mL / kg) daily in the morning, alternating between the hind limbs; their backs were irradiated with UVB every afternoon at a distance of 20 cm for 60 minutes each time, for 49 consecutive days. M+Asp mice, in addition to the M group modeling, had their backs disinfected with 75% alcohol swabs after daily UV irradiation, allowed to air dry for 1 minute, and then 100 μL of the prepared Aspergillus spore suspension was applied evenly to the entire hairless area, allowing it to air dry naturally. Control group mice were injected with an equal volume of physiological saline daily, without UV irradiation or fungal application. During the modeling period, the mice's diet, water intake, activity, fur condition, and bowel movements were observed daily, and their weight was measured weekly.

[0057] 2.3 Indicator Testing

[0058] 2.3.1 Observation and scoring of appearance characteristics: On days 0, 7, 21, 35, 42 and 49 after modeling, the pigmentation of the skin on the back of the mice was observed under the same light conditions. The scores were given according to the scoring standard of 1-5 points, and the average value of each group was taken.

[0059] 2.3.2 Skin physiological parameter detection: On days 0, 7, 21, 35, 42 and 49 after modeling, the E, M, ITA°, L, a, b values, TEWL and CM values ​​of the dorsal skin of each group of mice were measured using a skin analyzer. Each mouse was measured 3 times and the average value was taken.

[0060] 2.3.3 Pathological and histological examination: On day 49 of modeling, after the mice were fasted for 12 hours but allowed free access to water, they were euthanized by cervical dislocation, and approximately 1 cm of skin tissue from the lesion area on their backs was immediately harvested. 2 The tissue was fixed in 4% paraformaldehyde for 24 hours, then routinely dehydrated, cleared, and embedded in paraffin to prepare 4 μm thick serial sections. HE staining and HMB45 immunohistochemical staining were performed, and the skin tissue structure, the number of melanin granules in the basal layer, and the expression of melanin-positive cells were observed under a light microscope.

[0061] 2.3.4 Biochemical Indicators and NEI Network Factor Detection: After euthanizing mice, blood was collected from the abdominal aorta, allowed to stand at room temperature for 30 min, centrifuged at 3000 rpm for 10 min, and the serum was separated and stored at -80℃. Liver and dorsal skin tissue were collected, rinsed with pre-cooled physiological saline, blotted dry with filter paper, and 10% tissue homogenate was prepared by adding pre-cooled physiological saline at a weight-to-volume ratio of 1:9. The homogenate was centrifuged at 3000 rpm for 10 min, and the supernatant was collected and stored at -80℃. Following the ELISA kit instructions, the SOD activity, MDA content, TYR activity, LF content, and the contents of SP, α-MSH, and TNF-α in serum, liver, and skin tissue were measured.

[0062] 2.3.5 Correlation analysis: SPSS 26.0 software was used for data analysis. One-way ANOVA was used for comparisons among multiple groups. Spearman correlation coefficient was used for correlation analysis. P < 0.05 was considered statistically significant.

[0063] Experimental results

[0064] 3.1 Skin Microbiome Differentiation Analysis: The relative abundance of skin fungal species in patients with high-altitude melasma and healthy individuals was analyzed, and the results are shown in Table 1. The relative abundance of *Aspergillus penicillioides* in the skin of melasma patients was 0.9838±1.1082%, significantly higher than that in healthy individuals (0.1893±0.2070%) (P=0.010). Other differentially abundant fungi showed no statistical significance, confirming that *Aspergillus penicillioides* is the key differentially abundant fungus in the skin of patients with high-altitude melasma.

[0065] Table 1. Differences in the relative abundance of dermatofungal flora at the species level between the melasma group and the control group (X±S):

[0066] Alternaria_japonica 2.1153±6.9926 0.0659±0.1978 0.428 Aspergillus penicillioides (broom-shaped Aspergillus) 0.9838±1.1082 0.1893±0.207 0.010 Capnocheirides_sp 0.8117±1.2255 2.0697±2.4668 0.656 Aspergillus_elegans 0.7621±2.5276 0±0 0.766 Knufia_sp 0.3399±1.1085 0.0004±0.0011 0.766 Preussia_africana 0.2309±0.5979 0.0023±0.0069 0.766 Aspergillus_ochraceus 0.174±0.4976 0.002±0.006 0.503 Aspergillus_restrictus 0.0973±0.1222 0.0152±0.0309 0.095 Neocelosporium_eucalypti 0.0741±0.1228 0.1314±0.226 0.552 Aspergillus_baarnensis 0.0711±0.1221 0.0837±0.1422 0.824 Cladosporium_aggregatocicatricatum 0.0662±0.0987 0.0837±0.1403 0.882 Exophiala_dermatitidis 0.066±0.1802 0.0479±0.061 0.503 Sydowia_polyspora 0.0608±0.2018 0±0 0.766 Epicoccum_proteae 0.0575±0.1087 0.0572±0.1716 0.656 Cladosporium_limoniforme 0.0537±0.1576 0.0377±0.0481 0.331 Neocucurbitaria_unguishominis 0.049±0.1314 0.0027±0.0082 0.175 Endosporium_sp 0.0476±0.1299 0.0086±0.0238 0.449 Cladosporium_halotolerans 0.047±0.0599 0.0623±0.0986 0.817 Phaeococcomyces_mexicanus 0.0464±0.1361 0.1636±0.4908 0.656 Aspergillus_candidus 0.0396±0.1183 - 0.503 Italica_achilleae 0.0372±0.1189 0.0008±0.0025 0.766

[0067] 3.2 Changes in Phenotypic Characteristics: During the modeling period, mice in all groups had normal diet and water intake, good activity, and no deaths. On day 35 after modeling, a small number of yellow spots began to appear on the back skin of mice in the M group and the M+Asp group; on day 42, the spots in the M+Asp group became darker and larger; on day 49, obvious grayish-brown pigmentation was visible on the back skin of both groups of model mice, with the M+Asp group showing more uniform and darker pigmentation covering more than 60% of the skin, while the Control group's skin remained a normal pink without pigmentation (see...). Figure 1 The apparent scoring results are shown in Table 2. The score of the Control group was 1.7±0.67, the score of the M group was 3.3±0.67 (compared with the Control group, P<0.001), and the score of the M+Asp group was 4.2±0.79 (compared with the Control group, P<0.001; compared with the M group, P<0.05), indicating that the pigmentation degree of the model group of the present invention was significantly higher than that of the traditional model group.

[0068] Table 2 Apparent scores for different modeling methods:

[0069] Blank control group 1.7±0.67 Traditional model group (ultraviolet light + progesterone) 3.3±0.67*** The model group of this invention (ultraviolet light + progesterone + Aspergillus oryzae) 4.2±0.79***，#

[0070] Note: * indicates comparison with Control, # indicates comparison between M+Asp group and M group, # indicates P < 0.05; ## indicates P < 0.01; ### indicates P < 0.001.

[0071] 3.3 Changes in Skin Physiological Parameters: The results of skin physiological parameter detection on day 49 after modeling are shown in Table 3-1. Compared with the Control group, the E, M, and b values ​​of the skin of mice in the M group and the M+Asp group increased, while the ITA°, L, and a values ​​decreased, with the M+Asp group showing the greater changes; TEWL increased to 92.2±110.6 g / (m 2 The CM level significantly decreased to 5.3±3.5 (P<0.05), indicating impaired skin barrier function in the model mice. Skin physiological parameters on day 42 and day 35 after modeling are shown in Tables 3-2 and 3-3, respectively. On day 35, the TEWL in group M significantly decreased to 12.0±7.7 g / (m²). 2 ·h) (P<0.05), the CM in the M+Asp group was significantly reduced to 1.7±0.8 (P<0.05), indicating that the skin barrier damage gradually worsened with the extension of the modeling time.

[0072] Table 3-1 Changes in skin structural parameters on day 49 under different modeling methods:

[0073] E-value (elasticity value) 358.3±12.7 372.3±30.6 382.7±21.5 0.487 M value (melanin value) 646.7±9.9 773.7±99.9 783.0±13.9 0.068 ITA° (Individual Type Angle) 75.7±17.7 -6.7±59.6 -24.7±27.0 0.066 L-value (brightness value) 56.7±4.9 46.7±11.0 47.0±2.6 0.206 a-value (redness index) 6.3±2.5 5.0±5.2 4.3±3.1 0.880 b-value (yellowness value) 0.0±1.0 4.0±1.7 6.3±4.2 0.061 <![CDATA[TEWL (transepidermal water loss rate, g / (m 2 ·h))]]> 78.0±94.4 72.3±41.0 92.2±110.6 0.791 CM (Clones Moisture Content) 26.8±8.6 3.3±0.3* 5.3±3.5* 0.022

[0074] Note: Compared with the Control group, *p < 0.05, **p < 0.01, ***p < 0.001, and the same applies below.

[0075] Table 3-2 Changes in skin structural parameters on day 42 under different modeling methods:

[0076] E-value (elasticity value) 351.7±23.8 369.7±30.3 359.3±33.8 0.825 M value (melanin value) 643.3±14.0 734.7±91.0 738.7±113.2 0.191 ITA° (Individual Type Angle) 85.7±4.5 9.3±82.9 17.0±91.4 0.487 L-value (brightness value) 60.0±1.0 51.7±7.6 50.0±11.3 0.127 a-value (redness index) 5.7±1.5 4.0±4.6 3.3±6.0 0.88 b-value (yellowness value) 0.3±1.2 2.7±2.5 1.7±1.5 0.463 <![CDATA[TEWL (transepidermal water loss rate, g / (m 2 ·h))]]> 133.3±82.5 33.1±18.2 112.1±153.3 0.463 CM (Clones Moisture Content) 27.2±7.7 11.4±6.1 19.8±17.8 0.146

[0077] Table 3-3 Changes in skin structural parameters on day 35 under different modeling methods:

[0078] E-value (elasticity value) 331.0±42.7 327.0±23.3 343.7±22.6 0.825 M value (melanin value) 825.0±138.6 877.0±59.8 832.7±59.2 0.825 ITA° (Individual Type Angle) -29.3±100.2 -86.0±6.2 -50.7±44.6 0.463 L-value (brightness value) 39.0±19.3 34.0±10.6 42.7±8.1 0.825 a-value (redness index) -0.7±5.5 -3.0±5.3 -1.0±1.7 0.88 b-value (yellowness value) 1.0±0.0 0.0±4.0 3.3±2.5 0.328 <![CDATA[TEWL (transepidermal water loss rate, g / (m 2 ·h))]]> 22.2±4.6 12.0±7.7* 17.5±2.1 0.038 CM (Clones Moisture Content) 29.4±35.0 5.5±2.5 1.7±0.8* 0.038

[0079] 3.4 Pathological and Histological Changes: HE staining results showed that the skin structure of mice in the Control group was intact, with few melanin granules in the basal layer; the basal layer melanin granules in the M group increased, and the epidermis was slightly thickened; the basal layer melanin granules in the M+Asp group were extensively aggregated, the epidermis was more significantly thickened, and a small amount of inflammatory cell infiltration was visible in the dermis (see...). Figure 2 Immunohistochemical staining results showed that the number of HMB45-positive cells was extremely low in the Control group; the number of positive cells increased in the M group, showing a weak to moderate positive reaction; and the number of positive cells increased significantly in the M+Asp group, showing a strong positive reaction (see...). Figure 3 This indicates that the model group of the present invention has a higher degree of melanocyte proliferation and activation.

[0080] 3.5 Changes in Biochemical Indicators: On day 49 of modeling, compared with the Control group, the liver SOD activity in the M+Asp group and the M group was significantly decreased (P<0.05), while the MDA content showed an increasing trend; liver and skin TYR activity was significantly increased (P<0.05); liver LF content in the M+Asp group was significantly increased (P<0.01), and serum LF content in the M group was significantly increased (P<0.01) (see...). Figure 4 On day 35 of modeling, serum SOD activity in both model groups decreased significantly (P<0.01), serum MDA content decreased extremely significantly (P<0.001), and serum TYR activity in the M+Asp group increased extremely significantly (P<0.01), indicating that oxidative stress was enhanced and melanin synthesis was active in the model mice.

[0081] 3.6 Changes in NEI network factors: On day 49 of modeling, compared with the Control group, the liver SP content in the M+Asp group and the M group was significantly decreased (P<0.001), and the skin SP content in the M group was significantly decreased (P<0.05); serum α-MSH content was increased in both model groups, and skin α-MSH content was significantly increased in the M group (P<0.01); liver and skin TNF-α content was decreased in both model groups (see...). Figure 5 On day 35 of modeling, the serum α-MSH content in the M+Asp group was significantly higher than that in the M group (P<0.01) and the Control group (P<0.05), indicating that the model mice had obvious NEI network disorder.

[0082] 3.7 Changes in organ indices: The organ indices of mice in each group at different modeling times are shown in Table 4. There were no significant differences in organ indices between the M+Asp group and the M group at days 35 and 49 after modeling. On day 42, the liver index of the M+Asp group was higher than that of the control group (P < 0.05), and the skin index of the M group was higher than that of the control group (P < 0.05), indicating that the modeling process had some effect on the liver and skin of mice, but no obvious toxic reaction.

[0083] Table 4. Changes in organ indices at different modeling times using different modeling methods:

[0084] 49 liver 4.845±0.267 4.941±0.946 4.749±0.107 0.825 heart 0.676±0.037 0.386±0.164 0.659±0.193 0.098 spleen 0.356±0.102 0.331±0.115 0.292±0.096 0.88 lung 0.963±0.103 0.980±0.100 0.870±0.132 0.487 kidney 1.271±0.057 1.320±0.244 1.253±0.174 0.88 skin 4.597±0.654 5.567±0.292 5.098±0.395 0.098 42 liver 3.875±0.226 4.300±0.234 4.714±0.191* 0.021 heart 0.540±0.049 0.614±0.044 0.549±0.107 0.487 spleen 0.294±0.042 0.410±0.011 0.364±0.140 0.191 lung 0.909±0.134 0.957±0.191 1.255±0.543 0.487 kidney 1.183±0.078 1.160±0.094 1.180±0.072 0.88 skin 4.574±1.055 7.004±0.851* 6.274±0.837 0.021 35 liver 4.283±0.052 4.885±0.530 4.835±1.254 0.487 heart 0.699±0.137 0.592±0.074 0.481±0.049 0.066 spleen 0.377±0.032 0.271±0.078 0.340±0.137 0.487 lung 1.078±0.222 0.880±0.032 0.599±0.634 0.191 kidney 1.322±0.114 1.184±0.067 1.144±0.391 0.487 skin 5.860±1.450 6.853±0.176 5.310±0.357 0.127

[0085] 3.8 Correlation Analysis: The results of Spearman correlation analysis are as follows... Figure 6 As shown, the abundance of Aspergillus flavomarginata in skin tissue was significantly positively correlated with TYR activity, MDA level, SP and α-MSH levels (P < 0.05), and significantly negatively correlated with SOD activity, L value and ITA° value (P < 0.05), suggesting that Aspergillus flavomarginata promotes melanin synthesis by regulating oxidative stress and NEI network.

[0086] 3.9 Principal Component Analysis: The PCA analysis results of various indicators for different modeling methods are as follows: Figure 7 As shown, there are obvious clustering features among the blank group, the traditional model group and the model group of the present invention at the overall level. As the modeling time is extended, the classification of the model group and the blank group becomes more and more obvious. On the 49th day of modeling, the clustering features of the model group of the present invention and the traditional model group are highly consistent and both are significantly separated from the blank group, indicating that the model of the present invention is an optimization and upgrade of the traditional model and can better reflect the pathological features of plateau chloasma.

[0087] Example 2: Construction of a high-altitude mouse model of melasma based on microbial interaction characteristics (42 days of modeling).

[0088] The only difference between this embodiment and Embodiment 1 is that the modeling time is 42 days; all other experimental materials and methods are the same as in Embodiment 1.

[0089] The experimental results showed that on day 42 after modeling, mice in the M+Asp group developed obvious light brown pigmentation on their backs, with an apparent score of 3.8±0.63, significantly higher than the M group's 3.0±0.58 (P<0.05) and the Control group's 1.6±0.52 (P<0.001). Skin physiological parameters showed that the CM value in the M+Asp group was 19.8±17.8, lower than the Control group's 27.2±7.7, but the difference was not statistically significant (see Table 3-2). Histopathological examination showed an increase in melanin granules in the basal layer of the M+Asp group, and a higher number of HMB45-positive cells compared to the M group. Biochemical indicators showed a decreasing trend in TYR activity in the skin of the M+Asp group (P<0.05), and a significantly increased α-MSH content in the liver (P<0.01).

[0090] The results showed that a plateau chloasma animal model could be successfully established after 42 days of modeling, exhibiting typical pigmentation characteristics, but the degree of difference in various indicators was slightly lower than that of the model established after 49 days. Therefore, if a model with more significant pathological features is required, it is recommended to choose a model established after 49 days; if a shorter experimental period is required, a model established after 42 days can be chosen.

[0091] Example 1 and blank control group:

[0092] This control example is the Control group in Example 1. The experimental method is as follows: after the mice were acclimatized, their backs were shaved, and they were injected with an equal amount of physiological saline every day. They were not exposed to ultraviolet light or fungal smears. The treatment was carried out for 49 consecutive days. The detection methods for other indicators were the same as in Example 1.

[0093] The experimental results showed that the skin on the backs of mice in the control group remained a normal pink color without any pigmentation, and the apparent score was 1.7 ± 0.67. Skin physiological parameters were normal, and TEWL and CM values ​​were stable. HE staining showed intact skin structure with no obvious melanin granule aggregation in the basal layer. Immunohistochemical staining showed very few HMB45-positive cells. Biochemical indicators and NEI network factors were all at normal levels, showing significant differences compared to the model group.

[0094] Control Example 2, Traditional Ultraviolet Radiation Combined with Progesterone Model Group:

[0095] This control example is group M in Example 1. The experimental method is as follows: after the mice were acclimatized, their backs were shaved, and they were injected intramuscularly with 20 mg / kg of progesterone solution daily, while being irradiated with UVB for 60 minutes each time. The model was established for 49 consecutive days. No fungal smear was performed. The detection methods for other indicators were the same as in Example 1.

[0096] The experimental results showed that on day 49 of modeling, grayish-brown pigmentation appeared on the back skin of mice in group M, but the color was lighter and the distribution was uneven. The apparent score was 3.3±0.67 points, significantly lower than the 4.2±0.79 points of the M+Asp group of this invention (P<0.05). The changes in skin physiological parameters were smaller than those in the M+Asp group, with TEWL increasing to 72.3±41.0 g / (m 2 In the M+Asp group, the CM level decreased to 3.3±0.3, indicating less damage. Pathological examination showed that the number of melanin granules in the basal layer was lower than in the M+Asp group, and the expression intensity of HMB45 positive cells was weaker. Among biochemical indicators, the increase in TYR activity was lower than in the M+Asp group, and the level of oxidative stress was also relatively lower. The changes in NEI network factors were also less pronounced than in the M+Asp group.

[0097] The results showed that although the traditional ultraviolet light combined with progesterone model could induce skin pigmentation, the pathological features were not typical and the degree of pigmentation was relatively mild, which could not reflect the role of skin microorganisms in the pathogenesis of melasma. However, after introducing Aspergillus fulvicina in this invention, the pathological features of the model were more significant and closer to the real pathological environment of high-altitude melasma.

[0098] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a plateau-area yellow-brown animal model based on microbial interaction characteristics, characterized in that, Includes the following steps: S1. Selection and Adaptation of Experimental Animals: 6-8 week old female C57BL / 6J mice weighing 18-22g were selected and transported to a high-altitude experimental environment at 3650m. They were then adapted for 7 days under the following conditions: temperature 18-23℃, humidity 19-41%, and free access to food and water. S2 strain activation and suspension preparation: The standard strain *Aspergillus penicillioides* GDMCC 3.587 was selected, activated twice on Sabouraud dextrose agar plates, and the colonies were washed with sterile physiological saline. After centrifugation at 3000 rpm for 5 min and washing twice, the suspension was resuspended, and the spore concentration was adjusted to 1×10⁻⁶. 7 -5×10 7 CFU / mL, prepare and use immediately; S3 Composite Factor Modeling: The backs of mice were shaved, covering an area of ​​5cm × 4cm, 2-3 times per week. Mice were randomly divided into a control group and a model group. The model group received alternating intramuscular injections of 20mg / kg progesterone solution into both hind limbs daily, and simultaneously irradiated the back skin with UVB (275-320nm) at a distance of 20cm for 60 minutes each time. After irradiation, 100μL of Aspergillus spore suspension prepared in step S2 was applied to the shaved area on the back, evenly covered, and allowed to air dry. The control group received an equal volume of physiological saline and did not receive UV irradiation or fungal application. Modeling continued for 42-49 days. S4 Model Validation: The successful construction of the model was verified through epigenetic feature scoring, skin pathological histology examination, skin physiological parameter detection, biochemical index detection, and neuro-endocrine-immune network factor detection.

2. The method for constructing a plateau-area brown spot animal model based on microbial interaction characteristics according to claim 1, characterized in that: The specific conditions for strain activation in step S2 are as follows: take the strain from the -80℃ cryopreservation tube and inoculate it onto Sabouraud dextrose agar plates, and incubate at 30℃ for 48 hours to complete the first generation activation; pick a single smooth colony and streak it again, and incubate at 30℃ for 24 hours to complete the second generation activation.

3. The method for constructing a plateau-area brown spot animal model based on microbial interaction characteristics according to claim 1, characterized in that: Before applying the bacterial suspension in step S3, wipe the skin of the hair removal area on the back with a 75% alcohol swab for disinfection, let it dry for 1 minute, and then apply the suspension.

4. The method for constructing a plateau-area chrysoberylosis animal model based on microbial interaction characteristics according to claim 1, characterized in that: In step S4, the appearance feature score is divided into 1-5 points based on the degree of skin pigmentation, where 1 point is no pigmentation and 5 points is large area of ​​uniform dark gray-brown pigmentation.

5. The method for constructing a plateau-area brown spot animal model based on microbial interaction characteristics according to claim 1, characterized in that: In step S4, skin pathological histological examination used HE staining to observe the number of melanin granules in the basal layer and immunohistochemical staining to detect the expression level of HMB45-labeled melanin-positive cells.

6. The method for constructing a plateau-area brown spot animal model based on microbial interaction characteristics according to claim 1, characterized in that: In step S4, the skin physiological parameters include E value, M value, ITA° value, L value, a value, b value, transepidermal water loss rate, and stratum corneum moisture content.

7. The method for constructing a plateau-area brown spot animal model based on microbial interaction characteristics according to claim 1, characterized in that: The biochemical indicators in step S4 include superoxide dismutase activity, malondialdehyde content, tyrosinase activity, and lipofuscin content. The test samples are serum, liver, and skin tissue.

8. The method for constructing a plateau-area yellow-brown animal model based on microbial interaction characteristics according to claim 1, characterized in that: In step S4, the neuro-endocrine-immune network factors include substance P, α-melanocyte stimulating factor, and tumor necrosis factor-α. The test samples are serum, liver, and skin tissue.

9. The method for constructing a plateau-area brown spot animal model based on microbial interaction characteristics according to claim 1, characterized in that: The high-altitude experimental environment described in step S1 is simulated by a hypobaric oxygen chamber, which is set to atmospheric pressure and oxygen concentration parameters corresponding to an altitude of 3650m.

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