Use of slc1a4 in the preparation of a medicament for treating a dyschromic skin disease

Abstract

The application provides application of SLC1A4 in preparation of a medicament for treating a pigmentation abnormality skin disease, specifically, application of a reagent for detecting SLC1A4 as a detection reagent or a detection reagent kit for detecting a pigmentation abnormality skin disease, and application of an enhancer or inhibitor targeting SLC1A4 in preparation of a medicament for treating a pigmentation abnormality skin disease. The enhancer targeting SLC1A4 is a SLC1A4 overexpression plasmid, and the inhibitor targeting SLC1A4 is an SLC1A4 siRNA or shRNA. The pigmentation abnormality skin disease includes a hyperpigmentation skin disease and a hypopigmentation skin disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of SLC1A4 in the preparation of drugs for treating pigmentation disorders of the skin. Background Technology

[0002] Pigmented skin diseases such as nevi and vitiligo are very common in clinical practice, affecting patients' appearance and mental and physical health. Currently, there are many methods for treating pigmented skin diseases, but the overall treatment effect is unsatisfactory and the treatment cycle is long. Therefore, developing new therapeutic targets is essential.

[0003] Melanogenesis primarily occurs in melanosomes within melanocytes. Melanogenesis begins with L-tyrosine or L-phenylalanine. L-phenylalanine can be converted to tyrosine by phenylalanine hydroxylase. Tyrosine is then catalyzed by tyrosinase (TYR) to produce L-DOPA, which is further converted to dopaquinone. Dopaquinone can be catalyzed by tyrosinase-related protein-2 (TYRP2, or DCT) and tyrosinase-related protein-1 (TYRP1) to produce two intermediates: 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid. These intermediates then undergo polymerization to form eumelanin. In the presence of cysteine ​​or glutathione, dopaquinone can be converted to cysteine-dopa, which then undergoes a series of oxidation and polymerization reactions to ultimately form pheomelanin. TYR, TYRP1, and DCT are key enzymes regulating melanogenesis, and the expression of these enzymes is regulated by the transcription factor MITF.

[0004] The amino acid transporter SLC1A4 (ASCT1) gene belongs to the solute carrier transporter 1 family, contains eight exons, and is located on chromosome 2p14.8. SLC1A4 protein is widely distributed throughout the human body, with high levels found in skeletal muscle, lungs, kidneys, ovaries, heart, and the entire digestive tract. The physiological functions of SLC1A4 have been studied since its initial discovery, but current understanding of its function is limited. The most accepted function of SLC1A4 appears to be related to brain homeostasis.

[0005] A search revealed no literature on the regulatory role of SLC1A4 in pigmentary skin diseases. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to study the regulatory effect of SLC1A4 on pigmentary skin diseases and to study the application of SLC1A4-targeting reagents in the preparation of drugs for treating pigmentary skin diseases.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] This invention provides the use of SLC1A4 enhancers or inhibitors in the preparation of drugs for treating pigmentation disorders of the skin.

[0009] Preferably, the pigmentary disorders include hyperpigmentation and hypopigmentation.

[0010] More preferably, the hyperpigmentation skin disease includes pigmented nevi, etc. The hypopigmentation skin disease includes vitiligo, etc.

[0011] More preferably, the SLC1A4-targeting enhancer is an SLC1A4 overexpression plasmid. The SLC1A4-targeting inhibitor is an SLC1A4 siRNA or shRNA.

[0012] The inhibitors targeting SLC1A4 are shown in SEQ ID NO.1 to SEQ ID NO.3.

[0013] This invention also provides the application of reagents for detecting SLC1A4 as diagnostic reagents or kits for detecting pigmentation disorders of the skin.

[0014] Compared with the prior art, the advantages of the present invention are:

[0015] This invention discovers that SLC1A4 is specifically highly expressed in melanocytes, and experimental verification confirms that SLC1A4 promotes melanin production. This invention also verifies that enhancers or inhibitors targeting SLC1A4 can influence melanin production in melanocytes by regulating SLC1A4 expression levels. SLC1A4 is a potential diagnostic marker and novel therapeutic target for pigmentary skin diseases. Therefore, reagents for detecting SLC1A4 can be used in diagnostic reagents or kits for detecting pigmentary abnormalities, and enhancers or inhibitors targeting SLC1A4 can be used in the preparation of drugs for treating pigmentary abnormalities.

[0016] The detailed structure of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0017] Figure 1 SLC1A4 is specifically highly expressed in melanocytes. (a) RNA expression levels of SLC1A4 in various skin cells were analyzed in the single-cell dataset GSE150672; (b) Protein levels of SLC1A4 in various skin cells were analyzed in the Skin Science website.

[0018] Figure 2 SLC1A4 is positively correlated with the expression of genes regulating melanin production. (a) Correlation analysis of SLC1A4 with genes regulating melanin production was performed using a single-cell dataset of melanocytes downloaded from https: / / www.immunesinglecell.org; (b) Correlation analysis of SLC1A4 with genes regulating melanin production was performed using the human skin tissue dataset GSE56754 exposed to ultraviolet light.

[0019] Figure 3 Expression of SLC1A4 in different skin tissue samples. IHC was used to detect the expression of SLC1A4 in normal skin, pigmented nevus tissue, and vitiligo lesions.

[0020] Figure 4 SLC1A4 protein is highly expressed in tissues with relatively high melanin content. The melanin content and SLC1A4 protein expression levels in different skin tissues were detected by ammoniacal silver staining and immunohistochemistry, respectively.

[0021] Figure 5 SLC1A4 can promote melanin production in MNT1 cells. (a) SLC1A4 RNA expression level was detected by qRT-PCR after MNT1 cells were transfected with SLC1A4 overexpression plasmid; (b) SLC1A4 RNA expression level was detected by qRT-PCR after MNT1 cells were transfected with SLC1A4 siRNAs; (c) SLC1A4 RNA expression level was detected by qRT-PCR after MNT1 cells were infected with SLC1A4 overexpression virus; (d) SLC1A4 RNA expression level was detected by qRT-PCR after MNT1 cells were infected with SLC1A4 silenced expression virus; (e) Melanin content was detected by ammonia silver staining after MNT1 cells were infected with SLC1A4 overexpression virus; (f) Melanin content was detected by ammonia silver staining after MNT1 cells were infected with SLC1A4 silenced expression virus.

[0022] Figure 6 SLC1A4 can promote melanin production in MCs. (a) MCs were observed under a microscope after extraction; (b) MCs were transfected with SLC1A4 overexpression plasmid and the melanin content was detected by ammonia silver staining; (c) MCs were transfected with SLC1A4 siRNAs and the melanin content was detected by ammonia silver staining.

[0023] Figure 7 : Expression of SLC1A4 in various skin diseases. Box plots showing the expression of SLC1A4 mRNA in lesion and non-lesion groups in the specific dermatitis dataset GSE32924 (a), the scleroderma dataset GSE19617 (b), the lichen planus dataset GSE130403 (c), and the androgenic alopecia dataset GSE90594 (d). Detailed Implementation Experimental Method 1. Cell Culture

[0024] Melanin-rich human melanoma cells (MNT1) were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum (Meisen, China) and 1% penicillin-streptomycin antibiotics (Biosharp, China). Primary human melanoma cells (MCs) were extracted from foreskin tissue donated after circumcision in adolescents and cultured in 254 medium (Gibco, USA) supplemented with 2.5% FBS (Gibco, USA), 1% HMGS, and 1% penicillin / mycin / amphotericidal b. Cells were cultured in an incubator at 37°C with 5% CO2.

[0025] 2. Cell infection / transfection

[0026] A shRNA sequence targeting SLC1A4 (ACCCUUCCCUCUAUGAUGATT), see SEQ ID NO.1, and siRNA sequences targeting SLC1A4 (si_2:ACCCUUCCCUCUAUGAUGATT (see SEQ ID NO.2); si_589:GCAUCGCUGUCGCCUACUUTT (see SEQ ID NO.3)) were used to downregulate SLC1A4 expression. An SLC1A4 overexpression plasmid (based on the NM_003038.5 transcript) was used to upregulate SLC1A4 expression. Cells were transfected using Lipofectamine 3000 (Invitrogen, USA). Furthermore, an SLC1A4 overexpression virus (based on the NM_003038.5 transcript) and an SLC1A4 knockdown virus (based on the si_2 sequence) were constructed to build SLC1A4 overexpression / low expression cell lines. All sequences were synthesized by Gemma Biosciences (Shanghai, China).

[0027] 3. RT-PCR

[0028] Total RNA was extracted from cells using the RNAfast200 reagent (Fastagen, China) and reverse transcribed into cDNA using a reverse transcription kit (Vazyme, China). qRT-PCR analysis was performed on a Roche LightCycler 480II real-time PCR system (Basel, Switzerland). GAPDH was used as an internal control for calculating the expression level of the target mRNA.

[0029] 4. Ammoniacal silver staining (Fontana-Masson staining)

[0030] Cells were fixed with 4% paraformaldehyde for 30 minutes, washed 4-5 times with double-distilled water, and then incubated with warmed ammoniacal silver solution (Sloarbio, China) in a 56 ℃ water bath for 5 minutes. Paraffin sections were dewaxed and hydrated, and ammoniacal silver solution was added to the tissue, which was then incubated in a 56 ℃ water bath for 30 minutes, followed by counterstaining with neutral red solution for 5 minutes. Melanin granules were observed using an inverted microscope (Olympus X71, Japan).

[0031] 5. Immunohistochemistry

[0032] Paraffin sections were dewaxed and hydrated, retrievald with sodium citrate antigen, blocked with endogenous peroxidase, and blocked with serum. They were then incubated overnight at 4°C with SLC1A4 antibody (1:400). Subsequently, they were incubated with reaction enhancement solution and secondary antibody for 30 minutes each, and stained with DAB. The staining results were observed using an inverted fluorescence microscope (Zeiss, China). Immunoassay kits were purchased from BIOSS (China).

[0033] 6. Statistical Analysis

[0034] Statistical analysis was performed using GraphPad Prism 8.0.2 software. When the sample distribution largely conforms to a normal distribution, the Student's t-test was used to compare the means between the two groups. When the distribution does not conform to a normal distribution, a nonparametric rank-sum test was used. p < 0.05 was considered statistically significant. ns indicates no statistically significant difference between groups; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001.

[0035] Experimental Results 1. SLC1A4 is specifically highly expressed in melanocytes

[0036] Analysis of SLC1A4 RNA expression levels in various skin cells using the single-cell dataset GSE150672 revealed relatively high expression in melanocytes. Figure 1 a). Meanwhile, analysis on the Skin Science website revealed that the protein level of SLC1A4 is also specifically highly expressed in melanocytes (a). Figure 1 b).

[0037] 2. SLC1A4 is positively correlated with the expression of genes regulating melanin production.

[0038] Correlation analysis of single-cell datasets of melanocytes downloaded from https: / / www.immunesinglecell.org revealed a significant positive correlation between SLC1A4 and the expression of genes regulating melanin production. Figure 2a). Similarly, in the UV-exposed human skin tissue sample dataset GSE56754, we also found that SLC1A4 is highly positively correlated with melanin production regulatory genes ( Figure 2 b).

[0039] 3. Expression and localization of SLC1A4 in skin tissue

[0040] Normal skin was collected, and immunohistochemistry (IHC) was used to preliminarily validate the expression of SLC1A4. Immunohistochemical staining results showed that SLC1A4 was mainly located in the basal layer of skin cells. In addition, pigmented nevus tissue and vitiligo lesions were collected, and the results showed that SLC1A4 was highly expressed in nevus tissue, while its expression was downregulated in vitiligo lesions, suggesting that SLC1A4 is mainly located in melanocytes. Figure 3 ).

[0041] 4. The expression levels of SLC1A4 showed good correlation with melanin content.

[0042] To further verify the relationship between SLC1A4 and melanocytes and melanin production, we collected pigmented nevus and halo nevus tissues with different melanin contents. Ammoniacal silver staining was used to analyze the melanin content and distribution in these tissues. Further immunohistochemical detection of SLC1A4 revealed that SLC1A4 expression was relatively increased in areas with high melanin content and relatively decreased in areas with low melanin content. This indicates that the expression level and location of SLC1A4 are well consistent with the level and location of melanin. Figure 4 ).

[0043] 5. SLC1A4 affects melanin production in MNT1.

[0044] Interference with SLC1A4 expression in MNT1 cells, a tool for studying melanin production, revealed by RT-PCR that the SLC1A4 overexpression plasmid (oe-SLC1A4) significantly promoted SLC1A4 expression. Figure 5 a), while SLC1A4 siRNAs can significantly inhibit SLC1A4 expression ( Figure 5 (b) In this embodiment, both groups of SLC1A4 siRNAs designed showed significant differences compared with the blank control group. Furthermore, we constructed an SLC1A4 overexpression virus using an SLC1A4 overexpression plasmid and an SLC1A4 knockdown virus (sh-SLC1A4) using siRNA-2 (si-2). qRT-PCR was used to detect SLC1A4 expression levels, revealing that the overexpression virus promoted SLC1A4 expression (Figure-c), while the knockdown virus inhibited SLC1A4 expression (…). Figure 5 d). Ammoniacal silver staining results showed that, compared to the NC group, the ammoniacal silver staining color was darker after MNT1 overexpression of SLC1A4 ( Figure 5 e), after downregulating SLC1A4 expression, the silver staining became lighter ( Figure 5 f). This suggests that upregulation of SLC1A4 expression can promote melanin production in MNT1 cells, while downregulation of SLC1A4 expression can reduce melanin production in MNT1 cells.

[0045] 6. SLC1A4 affects melanin production in primary melanocytes of human skin.

[0046] Furthermore, we validated this finding in human primary melanocytes (MCs) extracted from foreskin tissue donated after circumcision in children. In MCs, after intervening in SLC1A4 expression using overexpression plasmids or siRNAs, we further validated the effect of SLC1A4 on melanin production in melanocytes using ammoniacal silver staining. Compared to the NC group, upregulating SLC1A4 expression in MCs resulted in a deeper ammoniacal silver staining. Figure 6 a, b), Downregulating SLC1A4 expression can lighten the color of ammoniacal silver staining ( Figure 6 c). Therefore, upregulating SLC1A4 expression can promote melanin production in MCs, while downregulating SLC1A4 expression can reduce melanin production in MCs.

[0047] 7. Expression of SLC1A4 in various skin diseases

[0048] We analyzed the expression of SLC1A4 mRNA in the lesion group and the non-lesion group in the atopic dermatitis (AD) dataset GSE32924, the scleroderma (SSc) dataset GSE19617, the lichen planus (LP) dataset GSE130403, and the androgenetic alopecia (AGA) dataset GSE90594, and found no significant difference. Figure 7 The results suggest that SLC1A4 may not be associated with the development of atopic dermatitis, scleroderma, lichen planus, or androgenetic alopecia.

[0049] Experimental conclusion:

[0050] 1. SLC1A4 is specifically highly expressed in melanocytes.

[0051] 2. SLC1A4 promotes melanin production.

[0052] 3. Enhancers or inhibitors targeting SLC1A4 can affect melanin production in melanocytes by regulating the expression level of SLC1A4.

[0053] 4. SLC1A4 is a potential diagnostic marker and novel therapeutic target for pigmented skin diseases.

[0054] 5. Reagents for detecting SLC1A4 can be used in diagnostic reagents or kits for detecting pigmentary disorders of the skin, and enhancers or inhibitors targeting SLC1A4 can be used in the preparation of drugs for treating pigmentary disorders of the skin.

[0055] The above description is a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the claims of the present invention.

Claims

1. The application of an SLC1A4-targeting enhancer in the preparation of a drug for treating vitiligo, wherein the SLC1A4-targeting enhancer is an SLC1A4 overexpression plasmid.

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