Medicine for treating fibrotic diseases related to melanin metabolism and application thereof

Abstract

The invention relates to a medicine for treating fibrotic diseases related to melanin metabolism and application of the medicine. Specifically, a target MITF for inhibiting melanogenesis is found, and keloids can be effectively relieved by inhibiting melanogenesis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a drug for treating fibrotic diseases associated with melanin metabolism and an application thereof. Background Art

[0002] Keloids (KD) exhibit characteristics of tumorous growth, manifesting as persistent scarring that extends beyond the wound margin. Keloids are pathological scars formed during skin wound repair by excessive fibroblast proliferation and extracellular matrix deposition. They are disfiguring and often accompanied by symptoms of itching and pain, severely impacting patients' quality of life. During normal wound healing, collagen anabolism and catabolism maintain a balance. However, this balance is disrupted in keloids. Although the specific triggers of this change are unknown, the rate of collagen synthesis in keloids significantly exceeds its degradation, ultimately leading to a massive accumulation of collagen. The pathogenesis of this disease is complex, and no treatment has been approved by the FDA or CFDA. Keloid formation involves multiple factors, including abnormal wound healing, excessive tension, immune disorders, and hormonal imbalances. Cellular abnormalities related to melanin metabolism may contribute to the development and progression of scarring.

[0003] Melanogenesis is the biological pathway by which melanocytes synthesize melanin, involving a complex series of enzymatic and chemical reactions. Its regulation involves five signaling pathways, of which microphthalmia-associated transcription factor (MITF) is a key target shared by each pathway. Melanosomes are the specific sites of melanin synthesis, located within melanocytes in the basal layer of the epidermis. Their cell surface features numerous protruding structures called dendrites. When stimulated by ultraviolet light, melanocytes synthesize melanin within melanosomes and transfer it to keratinocytes via dendrites. The melanin synthesis process is highly complex, primarily involving tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). Pmel17 (gp100), a structural protein of melanosomes, is crucial for the formation of the internal fibrous matrix characteristic of stage II melanosomes. Melanosome formation is a key step in melanogenesis, but melanosomes must be transferred from melanocytes to keratinocytes to promote melanin production. The proteinase-activating receptor (PAR-2) induces melanosome transfer by enhancing keratinocyte phagocytosis of melanosomes. Studies have shown that melanin content is elevated in skin tissue from patients with KD and positively correlates with collagen expression. Furthermore, skin color is associated with keloid formation. Studies have also reported elevated tyrosinase activity and expression of the transcription factor MITF in KD melanocytes. More significantly, melanocytes can activate collagen synthesis in fibroblasts by secreting exosomal microRNAs. Although existing evidence suggests that melanocytes may be involved in the development and progression of KD, the specific role and mechanisms of melanocytes in KD, particularly the mechanism of action of melanin in KD, remain largely unknown.

[0004] Iron metabolism plays a crucial role in maintaining skin homeostasis. Iron production, transport, absorption, and excretion in the body are in a dynamic equilibrium, supporting a variety of biochemical catalytic reactions, hemoglobin synthesis, and various physiological functions. Iron deficiency can cause anemia, while iron overload leads to hemochromatosis. Iron can also trigger polyunsaturated phospholipid peroxidation through the Fenton reaction, producing excessive lipid peroxides (LipidROS), ultimately inducing ferroptosis. Disturbances in iron metabolism and the resulting dysregulation of ferroptosis contribute to the development and progression of various skin diseases. Zhao Ming et al. found that iron-dependent epigenetic regulation can promote the differentiation of pathogenic T cells in lupus. Iron overload and ferroptosis are also involved in the development of skin lesions such as melanoma, scleroderma, and psoriasis. Furthermore, iron overload and ferroptosis are widely involved in skin physiological processes such as wound healing, skin damage, inflammatory immunity, skin aging, and the skin barrier. Targeting iron metabolism and ferroptosis has been recognized as a promising therapeutic strategy for skin-related diseases. However, the existence of abnormalities in iron metabolism and ferroptosis in KD, their regulation, and their role in KD remain unclear. Further elucidation of the mechanisms of action of iron metabolism and ferroptosis in KD and the development of intervention strategies based on these mechanisms will significantly advance the diagnosis and treatment of KD. Summary of the Invention

[0005] The present invention discovered a target point MITF for inhibiting melanin production, and inhibiting melanin production can effectively relieve keloids.

[0006] The present invention provides a use of an agent for regulating the expression or activity of microphthalmia-associated transcription factor (MITF) or its downstream effector molecules in the preparation of a drug for one or more of the following effects: regulating (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barrier, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis in animals.

[0007] In one or more embodiments, the regulation is:

[0008] (1) downregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) inhibiting intracellular melanin production, (ii) inhibiting cell hyperproliferation and non-immortalization characteristics, (iii) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibiting abnormal extracellular matrix deposition, (v) inhibiting inflammatory response and destruction of basement membrane barrier, (vi) inhibiting iron overload and ferroptosis resistance, restoring ferroptosis sensitivity, (vi) inhibiting fibrosis, (viii) downregulating the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) inhibiting keratinocyte uptake of melanin, (x) reducing scar area, and / or (xi) treating or preventing skin fibrosis;

[0009] (2) Upregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) promoting intracellular melanin production, (ii) promoting cell hyperproliferation and immortalization characteristics, (iii) promoting cell proliferation, migration and collagen synthesis, (iv) promoting abnormal extracellular matrix deposition, (v) promoting inflammatory response and destruction of basement membrane barrier, (vi) promoting iron overload and iron death resistance, (vii) promoting fibrosis, (viii) upregulating the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) promoting keratinocyte uptake of melanin, and / or (x) increasing scar area and promoting darkening of skin color.

[0010] In one or more embodiments, the cell of item (ii) is a melanocyte.

[0011] In one or more embodiments, the cell of item (iii) is a fibroblast.

[0012] In one or more embodiments, the inflammatory response of item (v) is an inflammatory response triggered by melanin.

[0013] In one or more embodiments, the fibrogenesis described in item (vii) is melanin-induced fibrogenesis.

[0014] In one or more embodiments, upregulating the expression or activity of MITF protein or its downstream effector molecule in an animal comprises: transferring the coding sequence of MITF protein or its downstream effector molecule into the animal.

[0015] In one or more embodiments, downregulating the expression or activity of MITF protein or its downstream effector molecules comprises:

[0016] (a) specifically interfering with the transcription and / or expression of the MITF gene or its downstream effector molecule genes,

[0017] (b) downregulating the activity of MITF protein or its downstream effector molecules, or

[0018] (c) Expression of MITF protein or its downstream effector molecules with reduced activity in cells.

[0019] In one or more embodiments, the interference in (a) is interference with the transcription of the MITF gene or a gene of its downstream effector molecule or the translation of its transcript.

[0020] In one or more embodiments, the nucleic acid that specifically interferes with the transcription and / or expression of the MITF gene or its downstream effector molecule gene is selected from the following group: (i) dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that targets the MITF gene or its downstream effector molecule gene, or their transcripts for inhibition or silencing, or (ii) a construct that can express or form (i).

[0021] In one or more embodiments, the animal is a human or a mouse, preferably a human or a mouse.

[0022] In one or more embodiments, the MITF downstream effector molecule is a MITF downstream effector molecule associated with melanin production.

[0023] In one or more embodiments, the MITF downstream effector molecules include but are not limited to: tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome conjugate (DCT, also known as TYRP2), and premelanosome protein (PMEL).

[0024] The present invention also provides a method for regulating (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barrier, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis diseases in animals, the method comprising: regulating the expression or activity of MITF or its downstream effector molecules in animals.

[0025] In one or more embodiments, the regulation is:

[0026] (1) downregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) inhibiting intracellular melanin production, (ii) inhibiting cell hyperproliferation and non-immortalization characteristics, (iii) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibiting abnormal extracellular matrix deposition, (v) inhibiting inflammatory response and destruction of basement membrane barrier, (vi) inhibiting iron overload and ferroptosis resistance, restoring ferroptosis sensitivity, (vi) inhibiting fibrosis, (viii) downregulating the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) inhibiting keratinocyte uptake of melanin, (x) reducing scar area, and / or (xi) treating or preventing skin fibrosis;

[0027] (2) Upregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) promoting intracellular melanin production, (ii) promoting cell hyperproliferation and immortalization characteristics, (iii) promoting cell proliferation, migration and collagen synthesis, (iv) promoting abnormal extracellular matrix deposition, (v) promoting inflammatory response and destruction of basement membrane barrier, (vi) promoting iron overload and iron death resistance, (vii) promoting fibrosis, (viii) upregulating the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) promoting keratinocyte uptake of melanin, and / or (x) increasing scar area or promoting darkening of skin color.

[0028] In one or more embodiments, the cell of item (ii) is a melanocyte.

[0029] In one or more embodiments, the cell of item (iii) is a fibroblast.

[0030] In one or more embodiments, the inflammatory response of item (v) is an inflammatory response triggered by melanin.

[0031] In one or more embodiments, the fibrogenesis described in item (vii) is melanin-induced fibrogenesis.

[0032] In one or more embodiments, upregulating the expression or activity of MITF protein or its downstream effector molecule in an animal comprises: transferring the coding sequence of MITF protein or its downstream effector molecule into the animal.

[0033] In one or more embodiments, downregulating the expression or activity of MITF protein or its downstream effector molecules comprises:

[0034] (a) specifically interfere with the transcription and / or expression of the MITF gene or its downstream effector molecules,

[0035] (b) downregulating the activity of MITF protein or its downstream effector molecules, or

[0036] (c) Expression of MITF protein or its downstream effector molecules with reduced activity in cells.

[0037] In one or more embodiments, the interference in (a) is interference with the transcription of the MITF gene or a gene of its downstream effector molecule or the translation of its transcript.

[0038] In one or more embodiments, the nucleic acid that specifically interferes with the gene transcription and / or expression of the MITF gene or its downstream effector molecule is selected from the following group: (i) dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that targets the MITF gene or its downstream effector molecule, or their transcripts for inhibition or silencing, or (ii) a construct that can express or form (i).

[0039] In one or more embodiments, the animal is a human or a mouse, preferably a human or a mouse.

[0040] In one or more embodiments, the MITF downstream effector molecule is a MITF downstream effector molecule associated with melanin production.

[0041] In one or more embodiments, the MITF downstream effector molecules include but are not limited to: tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome conjugate (DCT, also known as TYRP2), and premelanosome protein (PMEL).

[0042] The present invention also provides a reagent targeting MITF, which can downregulate the expression or activity of MITF protein.

[0043] In one or more embodiments, downregulating the expression or activity of MITF protein comprises:

[0044] (a) specifically interfere with MITF gene transcription and / or expression,

[0045] (b) downregulating MITF protein activity, or

[0046] (c) MITF protein with reduced activity is expressed in cells.

[0047] In one or more embodiments, the interference in (a) is interference with the transcription of the MITF gene or the translation of its transcript.

[0048] In one or more embodiments, the nucleic acid that sexually interferes with the transcription and / or expression of the MITF gene is selected from the following group: (a) dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that targets the MITF gene or its transcript for inhibition or silencing, or (b) a construct that can express or form (a).

[0049] In one or more embodiments, the agent is selected from the group consisting of: a protein, a polypeptide, a nucleic acid, and / or a small molecule compound.

[0050] In one or more embodiments, the agent is a MITF inhibitor.

[0051] In one or more embodiments, the reagents include:

[0052] (1) Reagents that specifically interfere with MITF gene transcription and / or expression;

[0053] (2) Reagents that inhibit MITF protein expression or activity

[0054] (3) anti-MITF antibody or its expression vector;

[0055] (4) Small molecule inhibitors of MITF;

[0056] (5) A homologous recombination vector containing a nucleotide sequence encoding a mutated, inactive or activity-reduced MITF.

[0057] In one or more embodiments, the inhibitor is PLX4032, AZD8055, or ML329; preferably, the inhibitor is ML329.

[0058] The present invention also provides the use of an iron overload inhibitor and / or an iron death inducer in the preparation of a medicament for one or more of the following effects: (1) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (2) inhibiting abnormal deposition of extracellular matrix, (3) inhibiting fibrosis, and / or (4) reducing scar area.

[0059] In one or more embodiments, the iron overload inhibitors include iron chelators, iron absorption inhibitors, iron excretion promoters, iron metabolism regulatory protein inhibitors, and antioxidants.

[0060] In one or more embodiments, the iron overload inhibitor includes deferasirox, deferasirox, deferoxamine (DFO), HFE protein modulators, rotecept, captopril, Heme peptide analogs, FPN activating antibodies, and ruxolitinib; preferably, the iron overload inhibitor is DFO and / or deferasirox.

[0061] In one or more embodiments, the ferroptosis inducer includes a GPX4 inhibitor, a system Xc - Inhibitor, iron ion regulator, lipid peroxidation promoter.

[0062] In one or more embodiments, the ferroptosis inducer includes RSL-3, ML162, FIN56, DPI-1510, Erastin, Sulfasalazine, Sorafenib, Deferoxamine, Ferrostatin-3, A22, FINO2, and RSL-5; preferably, the ferroptosis inducer is RSL-3 and / or Erastin.

[0063] The present invention provides a method for screening candidate substances that can regulate (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barriers, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis diseases, comprising the steps of:

[0064] (a) contacting a substance with a system containing MITF protein or its downstream effector molecule, or their coding sequences; and

[0065] (b) Detecting the expression or activity of MITF or its downstream effector molecules in the system and comparing it with the control.

[0066] If the expression and activity of MITF or its downstream effector molecules are lower than the control, the candidate substance: (i) inhibits intracellular melanin production, (ii) inhibits cell hyperproliferation and non-immortalization characteristics, (iii) inhibits cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibits abnormal deposition of extracellular matrix, (v) inhibits inflammatory response and destruction of basement membrane barrier, (vi) inhibits iron overload and ferroptosis resistance, restores ferroptosis sensitivity, (vii) inhibits fibrosis, and (viii) downregulates F2R-like trypsin receptor 1

[0067] (ix) inhibiting melanin uptake by keratinocytes, (x) reducing scar area, and / or (xi) treating or preventing skin fibrosis;

[0068] If the expression and activity of MITF or its downstream effector molecules are higher than the control, the candidate substance: (i) promotes intracellular melanin production, (ii) promotes cell hyperproliferation and non-immortalization characteristics, (iii) promotes cell proliferation, migration and collagen synthesis, (iv) promotes abnormal deposition of extracellular matrix, (v) promotes inflammatory response and destroys basement membrane barrier, (vi) promotes iron overload and ferroptosis resistance, (vii) promotes fibrosis, (viii) upregulates the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) promotes keratinocyte uptake of melanin, and / or (x) increases scar area.

[0069] In one or more embodiments, the cell of item (ii) is a melanocyte.

[0070] In one or more embodiments, the cell of item (iii) is a fibroblast.

[0071] In one or more embodiments, the inflammatory response of item (v) is an inflammatory response triggered by melanin.

[0072] In one or more embodiments, the fibrogenesis described in item (vii) is melanin-induced fibrogenesis.

[0073] In one or more embodiments, the control is the same system without the substance.

[0074] In one or more embodiments, the system is a cell system, a tissue system, or an animal model.

[0075] The present invention also provides a pharmaceutical composition comprising the agent described in any embodiment herein, and other pharmaceutically acceptable excipients.

[0076] In one or more embodiments, the pharmaceutically acceptable excipients include diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants.

[0077] In one or more embodiments, the pharmaceutical composition is used to treat or prevent a fibrotic skin disease.

[0078] In one or more embodiments, the skin fibrotic disease is selected from any one or more of the following: scleroderma, morphea, keloids and hypertrophic scars; preferably, the skin fibrotic disease is keloids.

[0079] The present invention also provides a method for treating or preventing skin fibrosis, comprising administering a therapeutically effective amount of the agent or pharmaceutical composition described in any embodiment herein to a subject in need thereof.

[0080] In one or more embodiments, the skin fibrotic disease is selected from any one or more of the following: scleroderma, morphea, keloids and hypertrophic scars; preferably, the skin fibrotic disease is keloids.

[0081] The beneficial effects of the present invention are:

[0082] MITF inhibitors can inhibit melanin production. Inhibiting melanin production, iron overload, and ferroptosis resistance may become new targets for the treatment of keloids. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Hyperpigmentation of melanocytes in KD patients. (A) UMAP cell clustering diagram of single-cell RNA sequencing analysis of skin tissue; (B) re-clustering of melanocyte subsets based on PMEL, MITF, TYR, and MLANA marker genes; (C) scRNA-seq detection of PMEL, MITF, TYR, and MLANA mRNA expression levels in KD and KN skin tissues; (D-E) immunofluorescence staining (D) and counting analysis (E) of MITF-positive melanocytes in the epidermis of the skin. The white dotted line separates the upper epidermis from the lower dermis. n = 12, scale bar: 100 μm. ( F) Real-time fluorescence quantitative PCR (qPCR) analysis of PMEL, MITF, MLANA, TYRP1, and TYRP2 expression levels in keloid skin tissues, n = 12; (G) KEGG pathway analysis of differentially expressed genes (DEGs) in KD and KN melanocytes; (H) Changes in epidermal-dermal thickness in KD and KN skin tissues; (IJ) Masson-Fontana staining (I) and quantitative analysis (J) of epidermal melanin content; 40×, 100×, and 400× represent 40x, 100x, and 400x magnifications, respectively. Scale bars are 500 μm, 200 μm, and 50 μm, respectively. n = 12;

[0084] (K) Spectrophotometric determination of melanin content in skin tissue (490 nm absorbance). n = 12; (L) Correlation analysis between keloid area and severity index (KASI) and melanin content (n = 30);

[0085] (MN) are clinical photos of patients with keloid, *P<0.05; **P<0.01; ***P<0.001.

[0086] Figure 2 Melanin derived from melanocytes promotes fibroblast growth, migration, and collagen deposition. (A) HE and Masson staining of KN and KD skin tissues, n = 12; scale bar: 200 μm;

[0087] (BC) Quantitative analysis of dermal thickness and collagen content; (D) Immunofluorescence staining and counting of S100A4-positive fibroblasts in KD skin tissue, n = 6, scale bar: 50 μm; (EF) Effects of pHEMs medium treatment on the growth (E) and migration (F) of KD fibroblasts; (GI) Effects of pHEMs medium treatment on the expression of fibrosis-related proteins in KD fibroblasts detected by qPCR (G) and Western blot (HI); (JK) Effects of melanin treatment on the growth (J) and migration (K) of KD fibroblasts;

[0088] (LN) qPCR (L) and Western blot (MN) were used to detect the regulatory effect of melanin treatment on the expression of fibrosis-related proteins; (O) Western blot detection of MITF after pHEMs were transfected with NC siRNA or si-MITF; (P) spectrophotometric determination of melanin content in pHEMs culture medium (absorbance at 490 nm);

[0089] (Q) Effects of pHEMs Medium+NC and pHEMs Medium+si-MITF treatment on the growth of KD fibroblasts; (R-R1) Regulation of cell migration ability by pHEMs Medium+NC and pHEMs Medium+si-MITF treatment; (SU) Effects of pHEMs Medium+NC and pHEMs Medium+si-MITF treatment on the expression of fibrosis-related proteins detected by qPCR (S) and Western blot (TU); Scale bars in Figures F, K, and R are 50 μm; EU experiments were repeated three times independently; *P<0.05; **P<0.01; ***P<0.001.

[0090] Figure 3 Melanin transfers to the dermis by disrupting the basement membrane structure. (A) Masson-Fontana staining and quantification of melanin content in the dermis, n = 12, 40× scale bar = 500 μm, 200× scale bar = 100 μm; (B) Determination of melanin content in the isolated dermis, n = 12; (C) Volcano plot of differentially expressed genes (DEGs) in KN and KD basal cells; (D) GO and KEGG pathway analysis of basal cell DEGs;

[0091] (E) Violin plots showing the expression of inflammatory, tight junction, and basal marker genes in KN and KD basal cells; (F) qPCR detection of inflammatory and tight junction genes in KN and KD skin, n=12; (G) ELISA analysis of CCL2 and S100A8 / A9 levels in KN and KD skin, n=12; (H) qPCR detection of COL17A1 and KRT15 expression, n=12; (I) Immunohistochemical staining of COL17A1 in KN and KD skin tissue, n=12; (J) Western blot detection of COL17A1 protein expression; (K) Schematic diagram of in vitro skin tissue culture and intraepidermal melanin injection; (L) qPCR analysis of inflammatory genes in skin tissue after melanin treatment; (M) ELISA detection of CCL2 and S100A8 / A9 after melanin treatment; (NO) Effect of melanin treatment on COL17A1 mRNA and protein levels; (P) Expression of CCL2 and S100A8 / A9 proteins in 354.T basal cells; (Q) Schematic diagram of keratinocyte-basal cell-fibroblast co-culture; (R) Cell permeability experiment; (S) Detection of melanin content in culture medium and fibroblasts before and after melanin treatment; PS experiment was repeated three times independently, *P<0.05; **P<0.01; ***P<0.001.

[0092] Figure 4 Melanin activates fibroblasts by inducing iron overload. (A) and (B) are the heatmap (A) and volcano plot (B) of differentially expressed genes in fibroblasts of the control group and the 100 μg / ml melanin-treated group.

[0093] (CD) GO and KEGG pathway analysis of up / down-regulated DEGs; (E) Heat map of up-regulated DEGs related to mineral absorption and ferroptosis; (F) mRNA expression of FTH1, FTL, TFRC, and FPN1 in primary KD fibroblasts; (GH) protein expression of FTH1, FTL, TFRC, and FPN1 in primary KD fibroblasts; (I) total iron, Fe 2+ and Fe 3+ level;

[0094] (JK) shows the expression of α-SMA and type I collagen in primary KD fibroblasts; (LM) shows the expression of α-SMA and type I collagen in primary KD fibroblasts; (N) shows Perl's iron staining of skin tissue, scale bars: 500 μm and 50 μm; (O) shows the expression of total iron and Fe in KN and KD fibroblasts. 2+ and Fe 3+Levels; (PQ) FTH1, FTL and TFRC protein expression in primary KD fibroblasts, n = 6; (RS) Immunofluorescence staining (R) and counting analysis (S) of FTH1+S100A4+ double-positive fibroblasts in KD skin tissue;

[0095] n=6, scale bar: 200 μm, AM experiments were repeated three times independently, *P<0.05; **P<0.01; ***P<0.001.

[0096] Figure 5 Melanin inhibits ferroptosis in KD fibroblasts. (AC) qPCR and Western blot analysis of the effects of melanin stimulation on the expression of ferroptosis-inhibiting proteins (SLC3A2 / SLC7A11 / GPX4) and ferroptosis-promoting proteins in KD fibroblasts; (D) scRNA-seq analysis of the mRNA levels of ACSL4, SLC7A11, GPX4, and SLC3A2 in KD and KN skin tissues.

[0097] (E) Evaluation of the effect of melanin on RSL-3-induced LipidROS in KD fibroblasts, scale bar: 200 μm; (F) CCK8 assay for the viability of fibroblasts treated with RSL-3 and melanin; (GH) Relative levels of 4-HNE and MDA in fibroblasts after treatment with RSL-3 and melanin; (IJ) Western blot analysis of SLC3A2, SLC7A11, GPX4, and ACSL4 protein levels in KN and KD primary fibroblasts, n=6; (KL) Immunofluorescence staining (K) and counting analysis (L) of GPX4+S100A4+ double-positive fibroblasts in KD skin tissue, n=6; scale bar: 200 μm, experiments AH were repeated three times independently; *P<0.05; **P<0.01; ***P<0.001.

[0098] Figure 6 Melanin induces melanocyte iron overload and ferroptosis resistance and promotes melanin uptake by keratinocytes. (AC) represents the expression of iron metabolism-related genes in pHEMs after incubation with 100 μg / ml melanin;

[0099] (D) Total iron and Fe in melanocytes treated with melanin 2+ and Fe 3+Levels; (E) CCK8 assay for the viability of pHEMs cells treated with RSL-3 and melanin; (FH) qPCR and Western blot analysis of the effects of melanin stimulation on the expression of ferroptosis inhibitory proteins (SLC3A2 / SLC7A11 / GPX4) and ferroptosis-promoting proteins in pHEMs; (I) Perl's staining of iron content in epidermal tissue, scale: 50 μm; (J) Evaluation of the effect of melanin on LipidROS in pHEMs induced by RSL-3, scale: 200 μm; (K) Melanin content in HaCaT cells; (LN) qPCR (L) and Western blot (MN) analysis of PAR-2 ​​protein levels in HaCaT cells. All experiments were repeated three times independently. *P<0.05; **P<0.01; ***P<0.001.

[0100] Figure 7 ML329 improves skin fibrosis in vivo. (A) Schematic diagram of the construction of the keloid transplantation mouse model and the subcutaneous injection of ML329; (B) Transplanted tissues obtained on day 21 (normal saline group and ML329 group); (CD) Transplanted tissue volume and weight measurements; (E) HE and Masson staining of transplanted tissues, scale: 200 μm; (F) Sircol collagen assay showing a decrease in extracellular matrix in the ML329 group; (GI) Melanin and total iron (Fe 2+ / Fe 3+ ) and MDA levels; (JM) Immunofluorescence staining of FTH1+S100A4+ and GPX4+S100A4+ double-positive fibroblasts, scale bar: 200 μm, n=6, *P<0.05; **P<0.01; ***P<0.001.

[0101] Figure 8 The expression of melanin-related genes in the skin was analyzed by scRNA-seq. *P<0.05;

[0102] **P<0.001; ***P<0.001.

[0103] Figure 9 Expression in KD fibroblasts. A is a Western blot analysis of MITF protein in KD fibroblasts treated with or without pHEMs medium; B is a Western blot analysis of MITF protein in KD fibroblasts treated with or without melanin; C is a Western blot analysis of MITF protein in KD fibroblasts treated with pHEMs medium + NC or pHEMs medium + si-MITF. *p < 0.05; **p < 0.001; ***p < 0.001.

[0104] Figure 10 This image shows the expression levels of the melanin receptor F2RL1 in fibroblasts analyzed by scRNA-seq. A shows F2RL1 levels in KN and KD fibroblasts; B shows F2RL1 levels in different subtypes of KN and KD skin fibroblasts. *p < 0.05; **p < 0.001; ***p < 0.001.

[0105] Figure 11 Mitochondrial function assay in pHEM cells after erastin treatment. A represents mitochondrial ROS levels in pHEM cells; B represents MMP levels in pHEM cells. ***P < 0.001.

[0106] Figure 12 Figure 3. Effects of ML329 on melanin synthesis in melanocytes and mast cell inflammation. A: Relative melanin content in KN and KD skin explants; B: Relative protein levels of c-Kit, IL-4, and TNF-α in KD skin explants. N = 6; *p < 0.05; **p < 0.001; ***p < 0.001. DETAILED DESCRIPTION

[0107] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form a preferred technical solution. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. It should be understood that the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0108] The present invention relates to a target for inhibiting melanin production, MITF, and a drug-induced inhibitor of melanin production, ML329, which can effectively alleviate keloids. The present invention confirms that the use of ML329, a microphthalmia-related transcription factor (MITF) inhibitor, to drug-inhibit melanin production can effectively alleviate keloids, providing a potential breakthrough strategy for clinical treatment. Specifically, single-cell transcriptome sequencing found that skin pigment-related pathways were significantly activated in melanocytes in keloids. Clinical sample verification showed that melanin levels in patients with keloids were elevated and positively correlated with scar area and severity index. Melanin secreted by melanocytes significantly promotes fibroblast proliferation, migration and collagen synthesis. Mechanistically, melanin is transferred to the dermis by enhancing basal cell permeability and inflammatory response, and further activates fibroblasts by inducing iron overload and anti-ferroptosis ability. Iron overload and ferroptosis resistance have been confirmed in primary fibroblasts and tissues of keloids. Inhibiting the two can effectively block melanin-induced fiber formation. Melanin induces iron overload and ferroptosis resistance in melanocytes in an autocrine manner. It also stimulates melanin uptake by keratinocytes by upregulating the F2RL1 (also known as PAR-2) receptor, darkening skin tone. In animal studies, the MITF inhibitor ML329 inhibited melanin production and reduced keloid scarring in nude mice, while also reducing iron levels and restoring ferroptosis sensitivity.

[0109] In this article, the term microphthalmia-associated transcription factor (MITF) belongs to the bHLH-Zip protein family. It is regulated by multiple signaling pathways and post-translational modifications through alternative splicing, and plays a central role in the development and differentiation of melanocytes, osteoclasts, and other cells. Its abnormalities are associated with diseases such as melanoma (high expression promotes tumors), vitiligo, and oculocutaneous albinism. It is both an auxiliary indicator for melanoma diagnosis and a potential therapeutic target, and is of great significance to cell physiology, pathology, and disease research.

[0110] The present invention provides the use of MITF or its downstream effector molecules for regulating (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barrier, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis in animals.

[0111] The present invention correspondingly provides a method for regulating (i) intracellular melanin production, (ii) cell overproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barrier, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis diseases in animals, the method comprising: regulating the expression or activity of MITF or its downstream effector molecules in the system.

[0112] The "regulation" described herein includes "upregulation" and "downregulation". Specifically, the use is: (1) downregulating the expression or activity of MITF, thereby (i) inhibiting intracellular melanin production, (ii) inhibiting cell hyperproliferation and non-immortalization characteristics, (iii) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibiting abnormal deposition of extracellular matrix, (v) inhibiting inflammatory response and destruction of basement membrane barrier, (vi) inhibiting iron overload and ferroptosis resistance, restoring ferroptosis sensitivity, (vi) inhibiting fibrosis, (viii) downregulating the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) inhibiting keratinocyte uptake of melanin, (x) reducing scar area, and / or (xi) improve skin fibrosis; or (2) upregulate the expression or activity of MITF, thereby (i) promoting intracellular melanin production, (ii) promoting cell hyperproliferation and immortalization characteristics, (iii) promoting cell proliferation, migration and collagen synthesis, (iv) promoting abnormal deposition of extracellular matrix, (v) promoting inflammatory response and destruction of basement membrane barrier, (vi) promoting iron overload and iron death resistance, (vii) promoting fibrosis, (viii) upregulating the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) promoting keratinocyte uptake of melanin, and / or (x) increasing scar area and promoting darkening of skin color.

[0113] Wherein, the cells described in item (ii) are melanocytes; the cells described in item (iii) are fibroblasts; the inflammatory response described in item (v) is an inflammatory response triggered by melanin; and the fibrogenesis described in item (vii) is fibrogenesis induced by melanin.

[0114] Any substance that can increase the activity of MITF or its downstream effector molecules, improve its stability, promote its expression, prolong its effective duration, or promote its gene transcription and translation can be used in the present invention as a "promoter" of MITF protein or its downstream effector molecules, for regulating animal traits. For example, a vector that increases the expression or activity of MITF or its downstream effector molecules. Promoters of MITF or its downstream effector molecules include, but are not limited to, small molecule compounds, nucleic acid molecules, or a combination thereof. Preferably, the nucleic acid molecule is a nucleic acid construct containing the coding sequence of MITF or its downstream effector molecules. The nucleic acid construct is an expression vector or an integration vector. On the other hand, any substance that can reduce the activity of MITF or its downstream effector molecules, reduce its stability, inhibit its expression, shorten its effective duration, or reduce its transcription and translation can be used in the present invention as an inhibitor of MITF or its downstream effector molecules.

[0115] For example, in order to downregulate the expression or activity of MITF or its downstream effector molecules, an inhibitory molecule that specifically interferes with the transcription and / or expression of MITF or its downstream effector molecules, or downregulates the activity of MITF protein or its downstream effector molecules, can be introduced into a cell or animal model so that the cell or animal model does not express or reduces the expression of the MITF gene or its downstream effector molecules. The inhibitory molecule uses the gene of MITF or its downstream effector molecules or their transcripts or expressed proteins as the inhibition target. The inhibitory molecule can be a small molecule compound known to inhibit the activity of MITF or its downstream effector molecules, an antibody or ligand of MITF protein or its downstream effector molecules, or a binding fragment thereof, or an antisense nucleic acid, microRNA, siRNA, shRNA, dsRNA, or sgRNA that interferes with the gene expression of MITF or its downstream effector molecules.

[0116] In addition, in order to lower the gene expression or activity of MITF or its downstream effector molecules, a gene knockout vector can be transferred into the cell. Therefore, the inhibitor can be a reagent, such as sgRNA, that knocks out or knocks down the MITF gene using a technology selected from ZFN, TALEN and CRISPR. ZFN, TALEN and CRISPR / Cas9 technology suitable for the present invention are well known in the art. Each technology realizes the knockout of the target gene by the joint action of a DNA recognition domain and an endonuclease. In these embodiments, the inhibitor also includes a Cas enzyme (such as Cas9), its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

[0117] The present invention also provides an agent targeting MITF, which can downregulate the expression or activity of MITF protein, i.e., an inhibitor of MITF. In one or more embodiments, the inhibitor is selected from the following group: protein, polypeptide, nucleic acid, and / or small molecule compound. For example, the protein can be an anti-MITF antibody, preferably a monoclonal antibody. A variety of anti-MITF antibodies have been disclosed in the art, such as the anti-MITF antibodies of Sigma-Aldrich, and these antibodies can be used to implement the present invention. The polypeptide can be a short peptide that is well known in the art and can inhibit the enzymatic activity of MITF, such as the TT-012 inhibitor of MedChemExpress, which can be used to implement the present invention. In one or more embodiments, the inhibitor is PLX4032 (Vemurafenib), AZD8055 (CAS No. 1009298-09-2, chemical formula C 35 H 37 N9), ML329 (CAS No. 19992-50-8, chemical formula C 16 H 12 N2O4S); preferably, the inhibitor is ML329.

[0118] Accordingly, the present invention also provides a method for screening candidate substances that regulate (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barrier, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis diseases, comprising the steps of: (a) contacting the substance with a system containing MITF protein or its downstream effector molecules or their coding sequences; and (b) detecting the expression or activity of MITF or its downstream effector molecules in the system and comparing it with a control.

[0119] If the expression and activity of MITF or its downstream effector molecules are lower than the control, the candidate substance: (i) inhibits intracellular melanin production, (ii) inhibits cell hyperproliferation and non-immortalization characteristics, (iii) inhibits cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibits abnormal deposition of extracellular matrix, (v) inhibits inflammatory response and destruction of basement membrane barrier, (vi) inhibits iron overload and ferroptosis resistance, and restores ferroptosis sensitivity, (vii) inhibits fibrosis, (viii) downregulates the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) inhibits keratinocyte uptake of melanin, and (x) reduces scar area. , and / or (xi) improve skin fibrosis; or, if the expression and activity of MITF or its downstream effector molecules are higher than the control, then the candidate substance: (i) promotes intracellular melanin production, (ii) promotes cell hyperproliferation and non-immortalization characteristics, (iii) promotes cell proliferation, migration and collagen synthesis, (iv) promotes abnormal deposition of extracellular matrix, (v) promotes inflammatory response and damages basement membrane barrier, (vi) promotes iron overload and iron death resistance, (vii) promotes fiber generation, (viii) upregulates the expression or activity of F2R-like trypsin receptor 1 (F2RL1), (ix) promotes keratinocyte uptake of melanin, and / or (x) increases scar area. Wherein, the cells in item (ii) are melanocytes; the cells in item (iii) are fibroblasts; the inflammatory response in item (v) is an inflammatory response triggered by melanin; the fiber generation in item (vii) is a fiber induced by melanin. In one or more embodiments, the control is the same system without the substance. In one or more embodiments, the system is a cell system, a tissue system, or an animal model.

[0120] The candidate substances can be selected from the group consisting of peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, organic small molecules, inorganic small molecules, and nucleic acid sequences. Depending on the type of substance to be screened, those skilled in the art will appreciate how to select an appropriate screening method.

[0121] The present invention also provides the use of an iron overload inhibitor and / or an iron death inducer in the preparation of a medicament for one or more of the following effects: (1) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (2) inhibiting abnormal deposition of extracellular matrix, (3) inhibiting fibrosis, and / or (4) reducing scar area.

[0122] In one or more embodiments, the iron overload inhibitors include iron chelators, iron absorption inhibitors, iron excretion promoters, iron metabolism regulatory protein inhibitors, and antioxidants. Among them, iron chelators include hydroxypyridine chelators (e.g., deferasirox, deferiprone), hydroxamic acid chelators (deferoxamine (DFO)); iron absorption inhibitors include targeting divalent metal transporter 1 (e.g., zinc preparations, HFE protein regulators), Luspatercept, ACE inhibitors (captopril); iron excretion promoters include ferroportin agonists (e.g., Heme peptide analogs, FPN activation antibodies), agents that promote iron autophagy or lysosomal degradation (e.g., chloroquine, hydroxychloroquine); iron metabolism regulatory protein inhibitors include inhibitors of iron response element binding proteins (e.g., small interfering RNA, antisense oligonucleotides), agents that block the JAK-STAT pathway (e.g., ruxolitinib). Preferably, the iron overload inhibitor is the iron chelator DFO and / or deferasirox.

[0123] In one or more embodiments, the ferroptosis inducer includes a GPX4 inhibitor, a system Xc - Inhibitors, iron ion regulators, lipid peroxidation promoters. Among them, GPX4 inhibitors include RSL-3 (chemical formula: C 17 H 17 ClF5N3O, CAS No.: 1260847-72-2), ML162 (chemical formula: C 15 H 12 F3N5, CAS No.: 1260847-74-4), FIN56 (chemical formula: C 22 H 25 FO4, CAS No.: 1416447-13-7), DPI-1510 (C 21 H 23 N5O2); system Xc - Inhibitors include Erastin (chemical formula: C 29 H 45 NO5, CAS No.: 571202-12-9), Sulfasalazine (chemical formula: C 18 H 14 N4O5S, CAS No.: 599-79-1), Sorafenib (chemical formula: C 21 H 16ClF3N4O3, CAS No.: 284461-73-0); iron ion regulators include iron ion carriers (such as Deferoxamine), iron excretion inhibitors (such as Ferrostatin-3), ferritin autophagy inducers (such as Autophagy inducers); lipid peroxidation promoters include A22 (chemical formula: C 14 H 12 ClF3N4O, CAS No.: 866405-64-3), FINO2 (chemical formula: C 22 H 24 ClFO4, CAS No. 1638847-25-1), RSL-5 (C 17 H 17 BrF5N3O). Preferably, the ferroptosis inducer is RSL-3 and / or Erastin.

[0124] In the present invention, the iron overload inhibitor and / or ferroptosis inducer can also block the MITF downstream effect, which can block the increase in melanin mediated by the MITF protein, and its further activation of iron overload and ferroptosis resistance. MITF downstream effector molecules are MITF downstream effector molecules related to melanin production, including but not limited to: tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), dopachrome interconvertase (DCT, also known as TYRP2), and premelanosome protein (PMEL). Cell experiments also found that melanin can promote fibroblast proliferation, migration, and activation, ultimately leading to excessive deposition of cellular collagen; mechanistic studies found that melanin causes iron overload in fibroblasts, and the use of the iron chelator DFO to inhibit iron overload can significantly reduce the above-mentioned fibroblast proliferation, migration, activation, and collagen deposition mediated by melanin; simultaneously, the phenomenon of ferroptosis resistance activated by melanin is an important factor that gives fibroblasts excessive proliferation in keloids; in one or more embodiments, the means of downregulating iron overload include: iron chelators (DFO), deferasirox; in one or more embodiments, the means of downregulating iron overload include: RSL-3 and Erastin.

[0125] In this article, the GeneIDs of MITF and its downstream effector molecules are: (1) MITF: 4286; (2) TYR: 7299; (3) TYRP1: 7306; (4) DCT: 1638; (5) PMEL: 6490.

[0126] Herein, skin fibrosis refers to persistent damage to skin tissue, which results in an increase in fibrous tissue, a gradual decrease in active cells in the parenchyma, localized sclerosis, functional impairment, etc., and in severe cases, can lead to organ dysfunction. In some embodiments herein, skin fibrosis is a keloid.

[0127] The pharmaceutical compositions herein contain the reagents described in any embodiment herein, and pharmaceutically acceptable excipients, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants. Excipients are preferably non-toxic to the recipient at the dosage and concentration employed. Such excipients include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, the pH, osmotic properties, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. These substances are known in the prior art. The optimal pharmaceutical composition can be determined based on the intended route of administration, mode of delivery, and desired dosage.

[0128] Pharmaceutical compositions for in vivo administration are typically provided as sterile formulations. Sterilization is achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, this method can be used for sterilization before or after lyophilization and rehydration. The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Compositions for parenteral administration can be stored in lyophilized form or in solution. For example, they can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution strip or vial with a stopper pierceable by a hypodermic needle. Alternatively, the composition can be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery methods (such as liposomal carriers, bioerodible microparticles or porous beads, and depot injections) are also known to those skilled in the art.

[0129] Once the pharmaceutical composition is formulated, it is stored in a sterile vial in the form of a solution, suspension, gel, emulsion, solid, crystal or in the form of a dehydrated or lyophilized powder. The formulation can be stored in a ready-to-use form or in the form of rehydration before administration (e.g., lyophilization). The present invention also provides a test kit for producing a single-dose administration unit. The test kit of the present invention can each contain a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, a test kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilizing syringe) is provided.

[0130] The present invention also provides a method for treating a patient (especially a keloid) by administering a binding molecule or pharmaceutical composition thereof according to any embodiment of the present invention. As used herein, the terms "patient," "subject," "individual," and "object" are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rats, mice, dogs, cats, rabbits, etc.), and most preferably a human. "Treatment" refers to the use of a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., inhibiting intracellular melanin production, inhibiting melanocyte hyperproliferation and non-immortalization characteristics, inhibiting fibroblast proliferation, migration, and collagen synthesis, inhibiting abnormal extracellular matrix deposition, inhibiting inflammatory responses triggered by melanin and destroying basement membrane barriers, inhibiting iron overload and ferroptosis resistance, restoring ferroptosis sensitivity, inhibiting melanin-induced fibrosis, downregulating the expression or activity of F2R-like trypsin receptor 1 (F2RL1), inhibiting keratinocyte uptake of melanin, and reducing scar area). The treatment regimen that effectively treats a patient can vary according to a variety of factors (e.g., the patient's disease state, age, weight, and the ability of the therapy to stimulate the subject's anti-disease response).

[0131] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are intended only to describe the specific embodiments and are not intended to limit the present invention. Although the numerical ranges and parameter approximations shown in the broad scope of the present invention are provided, the numerical values ​​shown in the specific embodiments are described as accurately as possible. However, any numerical value inherently contains a certain error, which is due to the standard deviation present in their respective measurements. In addition, all ranges disclosed herein are understood to encompass any and all subranges contained therein.

[0132] The present invention will be described below in the form of specific examples; it should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention; the methods and materials used in the examples are conventional materials and methods in the art unless otherwise specified.

[0133] Example

[0134] Example 1: Single-cell RNA sequencing (scRNA-seq) analysis reveals enhanced melanocyte pigmentation in keloid skin tissue, which is positively correlated with disease severity and prognosis.

[0135] To elucidate the pathological mechanisms of skin fibrosis, we performed single-cell transcriptome sequencing on six pairs of keloid (KD) and adjacent normal control skin (KN) biopsies. After strict quality control, a total of 60,732 high-quality cells were obtained for subsequent analysis. After data normalization, uniform manifold approximation and projection (UMAP) dimensionality reduction was used for visualization. Based on known cell lineage-specific marker genes, these cells were divided into 21 subpopulations, mainly including T lymphocytes, B lymphocytes, fibroblasts, NK cells, mast cells, myeloid cells, epithelial cells, endothelial cells, oligodendrocytes, melanocytes, and unidentified cells ( Figure 1 A);

[0136] Given that melanocytes are the main functional cells for pigmentation, we focused on analyzing the melanocyte subpopulation in the scRNA-seq dataset; Figure 1 As shown in BC, the expression of key molecules of the pigment signaling pathway, PMEL, MITF, melan-A (MLANA), tyrosinase-related protein 1 (TYRP1), and dopachrome isomerase (DCT, also known as TYRP2), was significantly increased in keloid melanocytes. To confirm whether melanin is specifically produced by KD melanocytes, we evaluated the expression levels of melanin-related genes in other cell types. The results showed that PMEL, MITF, MLANA, TYR, TYRP1, and TYRP2 were mainly enriched in melanocytes ( Figure 8 );

[0137] Double immunofluorescence experiments further confirmed that the number of MITF-positive melanocytes in KD tissues was significantly higher than that in KN normal skin ( Figure 1 DE), indicating that melanocyte activity in keloids is enhanced; these findings were confirmed at the tissue level: PMEL, MITF, MLANA, TYRP1, and TYRP2 protein expressions were significantly increased in KD skin ( Figure 1 F);

[0138] Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the pigment granule formation, melanosome signaling, and pigmentation pathways were significantly enriched in KD melanocytes compared with adjacent normal skin ( Figure 1 G); Masson-Fontana staining showed thickening of the KD epidermis ( Figure 1 H), and quantitative analysis of the positive staining area confirmed increased epidermal melanin deposition ( Figure 1 IJ); Spectrophotometric detection (490nm absorbance) also showed that the melanin content in KD skin homogenate increased ( Figure 1 K); Clinical correlation analysis found that melanin content was significantly positively correlated with keloid area and severity index (KASI) ( Figure 1 L); In addition, the skin color of the KD lesion site is significantly darker than that of the KN normal skin ( Figure 1 M); It is worth noting that after surgical excision, the skin color of the lesion gradually became lighter as the scar recovered ( Figure 1 N);

[0139] Taken together, these results suggest that melanin metabolism is disturbed in keloids and may be associated with disease occurrence, severity, and prognosis;

[0140] Example 2: Melanin secreted by KD melanocytes is an important factor in activating melanocytes to induce fibroblasts

[0141] The histopathological characteristics of keloids were evaluated by hematoxylin-eosin (HE) and Masson's trichrome staining. We found that the dermis of KD skin tissue was thickened and the collagen content was significantly increased compared with normal skin tissue ( Figure 2 AC); S100A4 (also known as fibroblast-specific protein-1, FSP1) is a typical marker of skin fibroblasts. Its expression level increases significantly when fibroblasts proliferate or activate, and is associated with more severe fibrotic lesions; Immunofluorescence staining shows that S100A4-positive areas are significantly accumulated in KD skin tissue ( Figure 2 D) shows that fibroblast overactivation and collagen deposition are the prominent features of KD;

[0142] We collected the supernatant (pHEMs medium) of primary human epidermal melanocytes (pHEMs) cultured for 48 hours and added it to the fibroblast culture system (the control group was fresh medium without pHEMs culture); cell counting and wound healing assay results showed that pHEMs medium significantly promoted fibroblast proliferation and migration ( Figure 2 EF); qPCR analysis showed that the expression of fibrosis-related genes α-SMA, COL1A1, COL1A2, and COL3A1 was significantly upregulated ( Figure 2 G); Given that the TGF-β / p-SMAD3 signaling pathway is a key pathway regulating fibroblast activation, proliferation, and migration, Western blot further confirmed that melanocyte culture supernatant can significantly increase the expression levels of fibrosis-related proteins and p-SMAD3 in fibroblasts ( Figure 2 HI);

[0143] Based on the previous finding that the pigment signaling pathway is significantly activated in KD melanocytes, we further explored the activation effect of melanin on fibroblasts; exogenous addition of melanin can significantly promote fibroblast proliferation and migration ( Figure 2JK), qPCR and Western blot analysis showed that melanin could significantly upregulate the expression of fibrosis-related proteins such as α-SMA, COL1A1, COL1A2 and COL3A1 ( Figure 2 LN); by knocking down MITF, a key regulatory factor in melanin synthesis in pHEMs, pHEMs Medium + NC and pHEMs Medium + si-MITF culture media were prepared. It was found that the melanin level in the supernatant was significantly reduced after si-MITF treatment, and the effects of promoting fibroblast proliferation, migration and fibrosis were significantly weakened ( Figure 2 OU);

[0144] Since MITF is an essential factor for melanin synthesis, we detected the expression level of MITF in fibroblasts treated with pHEMs medium or melanin. The results showed that endogenous MITF expression in fibroblasts was weak, and exogenous addition of melanin or pHEMs medium did not significantly change its expression ( Figure 9 ), suggesting that melanin in fibroblasts comes from the transfer of melanocytes rather than autonomous synthesis;

[0145] Regarding how fibroblasts take up extracellular melanin, single-cell sequencing analysis found that the expression of F2RL1 (PAR-2, a key receptor for skin pigmentation, mediating melanosome uptake) was elevated in KD fibroblasts (especially CCL19+ and CTHRC1+ subpopulations) ( Figure 10 , AB);

[0146] In conclusion, melanocytes in KD promote fibroblast proliferation, migration, and fibrosis at least by oversynthesizing melanin;

[0147] Example 3: Melanin destroys the basement membrane structure and promotes its migration to the dermis of KD

[0148] A key scientific question is whether melanin can be transferred to the dermis of KD. The mechanism and specific compartment of action remain to be elucidated. By using Masson-Fontana staining to detect the distribution of melanin in the skin, we found that melanin deposition was present in the papillary dermis of KD, but not in the dermis of KN ( Figure 3 A). The increase in melanin content in KD dermal separation was also verified ( Figure 3 B). Considering that the basement membrane is the barrier between the epidermis and dermis, we first analyzed the differentially expressed genes (DEGs) of basal cells and found that IL-17, TNF-α, cytokine-receptor interaction, chemokine and tight junction pathways were widely altered in KD basal cells ( Figure 3CD). Pro-inflammatory genes CCL2, JUN, FOS, TNFSF10, S100A9, and S100A8 were significantly upregulated, while the expression of NFKBIA, which has anti-inflammatory effects, was decreased; tight junction-related genes CLDN1, ACTG1, and ACTN4 were significantly downregulated ( Figure 3 E). qPCR confirmed that inflammatory factors increased and tight junction proteins decreased in KD ( Figure 3 F), ELISA assay showed that the levels of CCL2 and S100A8 / A9 proteins were higher in KD skin ( Figure 3 G).

[0149] Single-cell sequencing showed that the expression of KRT15, ​​a marker of KD basal cells, and COL17A1, a component of hemidesmosomes, were downregulated ( Figure 3 E), and qPCR confirmed this finding ( Figure 3 H). Immunohistochemistry showed that COL17A1 protein was reduced in KD epidermis ( Figure 3 I), Western blot confirmed that COL17A1 protein was significantly decreased in KD tissues ( Figure 3 J). To explore whether basement membrane damage is related to excessive melanin, we conducted an in vitro skin tissue experiment: exogenous addition of melanin can significantly induce the expression of inflammatory factors in skin tissue ( Figure 3 LM), while reducing the mRNA and protein levels of COL17A1 ( Figure 3 NO). In vitro experiments using human skin basal cells TE 354.T further confirmed that melanin promotes the secretion of inflammatory factors such as CCL2 and S100A8 / A9 ( Figure 3 P), and 100 μg / ml melanin treatment increased cell permeability ( Figure 3 QR). Chamber experiments showed that melanin can penetrate the basal cell layer and be detected in fibroblasts ( Figure 3 S).

[0150] In summary, the abnormally increased melanin in KD can induce inflammatory response in basal cells and increase their permeability, thereby breaking through the basement membrane barrier and transferring to the dermis.

[0151] Example 4: Melanin activates fibroblast fibrosis by inducing iron overload

[0152] To further explore the mechanism of melanin-induced fibrosis in skin fibroblasts, we performed an integrated analysis of differentially expressed genes (DEGs) (|fold change|>1 and q value<0.05). RNA-seq analysis showed that there were significant differences in the gene expression heatmaps between the negative control group (NC) and the melanin-treated group ( Figure 4 A total of 2,928 DEGs were identified, including 1,461 down-regulated genes and 1,467 up-regulated genes ( Figure 4B). GO and KEGG analysis showed that the upregulated DEGs were mainly enriched in melanogenesis, cytokine-receptor interaction, focal adhesion, PI3K / AKT and ECM-receptor pathways ( Figure 4 C), suggesting that the profibrotic effect of melanin is dependent on multiple pathways and multiple biological processes. The down-regulated genes mainly involve apoptosis, cell cycle and P53 signaling pathways ( Figure 4 D). It is worth noting that the mineral absorption pathway (especially the iron metabolism-related pathway) was significantly enriched in the melanin group ( Figure 4 E), indicating that abnormal iron metabolism is involved in melanin-mediated ECM deposition.

[0153] qPCR and Western blot analysis confirmed that melanin significantly upregulated the expression of ferritin heavy chain 1 (FTH1), ferritin light chain (FTL), transferrin receptor (TFRC) and iron transporter (SLC40A1 / FPN1). Figure 4 FH). Iron content detection shows that melanin increases iron deposition in fibroblasts ( Figure 4 I). Further experiments found that iron supplementation (ferric citrate, FAC) promoted collagen synthesis and α-SMA expression ( Figure 4 JK), while the iron chelator deferoxamine (DFO) significantly inhibited the melanin-induced upregulation of collagen and α-SMA ( Figure 4 LM), demonstrating that the profibrotic effect of melanin is iron-dependent in nature.

[0154] To verify whether iron overload is involved in the pathogenesis of KD, we detected the expression of iron metabolism-related genes in KD patients. Perl's staining showed that there was obvious iron overload in the dermis of KD patients ( Figure 4 N). Compared with KN fibroblasts, the total iron and Fe 2+ and Fe 3+ The levels were significantly increased ( Figure 4 O). The expression of key iron metabolism genes FTH, FTL, and TFRC increased in KD fibroblasts ( Figure 4 PQ). Double immunofluorescence staining revealed that the proportion of S100A4+FTH1+ fibroblasts in KD skin tissue was significantly higher than that in KN group ( Figure 4 RS), further confirming that iron metabolism disorder and iron overload are involved in the process of KD.

[0155] In summary, this study found that melanin activated the fibrotic response of fibroblasts by inducing iron overload, ultimately exacerbating the development of KD.

[0156] Example 5: Melanin imparts ferroptosis resistance to fibroblasts

[0157] Iron overload is one of the key factors inducing ferroptosis. Figure 4 As shown in Figure E, melanin did significantly upregulate the expression of ferroptosis-related genes. However, surprisingly, in melanin-treated fibroblasts, the expression of three key ferroptosis inhibitors - glutathione peroxidase 4 (GPX4), solute carrier family 3 member 2 (SLC3A2) and solute carrier family 7 member 11 (SLC7A11) - was significantly upregulated, while the expression of the ferroptosis-promoting factor long-chain acyl-CoA synthetase 4 (ACSL4) was downregulated ( Figure 5 AC). Single-cell sequencing results also showed that ACSL4 was decreased, while SLC3A2 and GPX4 were increased in KD fibroblasts ( Figure 5 D). Lipid peroxide (LipidROS) detection found that melanin could reduce the production of LipidROS in KD fibroblasts, both under basal conditions and RSL-3 induction conditions ( Figure 5 E). Cell counting experiments showed that melanin significantly inhibited the decrease in fibroblast viability caused by the ferroptosis inducer RSL-3 (GPX4 inhibitor) ( Figure 5 F). At the same time, melanin also significantly reduced the content of 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) ( Figure 5 GH), suggesting that melanin acquires ferroptosis resistance by activating the antioxidant signaling pathway in fibroblasts.

[0158] To verify whether ferroptosis resistance is involved in the pathogenesis of KD, we detected the expression of ferroptosis-related genes in KD. The results showed that the expression of GPX4, SLC3A2, and SLC7A11 in KD fibroblasts was significantly increased compared with normal controls ( Figure 5 IJ). Immunofluorescence staining further revealed that GPX4 significantly accumulated in S100A4-positive fibroblasts in KD skin tissue ( Figure 5 These results consistently indicate that the ferroptosis-resistant properties of fibroblasts are involved in the pathological process of KD.

[0159] Example 6: Melanin induces melanocyte iron overload and ferroptosis resistance through autocrine pathway and promotes its transport to keratinocytes

[0160] We found that melanin secreted by melanocytes can activate fibroblasts by resisting iron overload and ferroptosis. So what effect does melanin have on melanocytes themselves? qPCR and Western blot assays showed that melanin can upregulate the expression of iron metabolism-related genes FTH / FTL / TFRC / SLC40A1 in primary human epidermal melanocytes (pHEMs). Figure 6 AC), iron content detection confirmed that intracellular iron ions increased significantly after melanin treatment ( Figure 6D), indicating that melanin induces iron overload in melanocytes. Further cell counting experiments found that melanin not only promotes pHEMs activity, but also effectively blocks RSL-3-induced ferroptosis ( Figure 6 E). Accordingly, melanin significantly increased the expression of ferroptosis resistance-related proteins GPX4 and System XC-(SLC3A2 / SLC7A11), while significantly reducing ACSL4 levels ( Figure 6 FH). Perl's staining showed increased iron deposition in KD keratinocytes, and LipidROS assays showed that melanin could reduce LipidROS production in keratinocytes under basal and RSL-3-induced conditions ( Figure 6 These results strongly suggest that melanin can also lead to melanocyte iron overload and ferroptosis resistance in an autocrine manner.

[0161] Melanin synthesized by melanocytes needs to be transported to keratinocytes, and skin color differences will only appear under stimulation such as ultraviolet rays. To this end, we constructed a melanocyte-keratinocyte co-culture system. Transwell experiments found that compared with the untreated group, pHEMs treated with ferric citrate (FAC) or ferroptosis inhibitor Ferrostatin-1 (Fer-1) could significantly increase the melanin content in HaCaT cells; while pHEMs treated with deferoxamine (DFO) and ferroptosis inducer Erastin significantly reduced the melanin content in HaCaT cells ( Figure 6 K). Erastin is known to exert its effects by binding to voltage-dependent anion channels 2 / 3 (VDAC2 / 3). We found that mitochondrial function in pHEMs treated with erastin was altered: mitochondrial membrane potential (MMP) and mitochondrial ROS levels increased ( Figure 11 , AB), suggesting that mitochondrial dysfunction may be involved in the erastin-mediated melanin transport process. qPCR and Western blot experiments showed that the supernatant of pHEMs incubated with melanin could significantly upregulate the expression of PAR-2 ​​in HaCaT cells compared with the control group ( Figure 6 LN). These results suggest that the ferroptosis-resistant properties of melanocytes facilitate melanin transport to keratinocytes.

[0162] Example 7: Inhibiting melanin synthesis can alleviate keloid formation in nude mice

[0163] Based on the effect of melanin on the proliferation, migration and collagen deposition of dermal fibroblasts, we explored the possibility of inhibiting melanin production in vivo to alleviate keloids. After establishing a keloid transplantation mouse model, the mice were given normal saline or 5 mg / kg of the melanin production inhibitor ML329 (injected once every 3 days), and samples were collected on the 21st day ( Figure 7 A). Compared with the control group, the volume of subcutaneously transplanted keloids in the ML329-treated group was ( Figure 7 B) and weight ( Figure 7 CD) were significantly reduced. Histological analysis showed that ML329 reduced the dermal tissue density ( Figure 7 E), Sircol collagen assay showed that collagen fiber bundles were loose and the content was downregulated ( Figure 7 F). Melanin content in transplanted tissue was detected and found to be significantly reduced after ML329 treatment ( Figure 7 G). In addition, ML329 also reduced the iron ion level ( Figure 7 H) while increasing MDA content ( Figure 7 I). Immunofluorescence staining confirmed that ML329 reduced the expression of ferritin and GPX4 in keloid fibroblasts, suggesting that iron overload and ferroptosis resistance were alleviated ( Figure 7 JM). These results suggest that inhibiting melanogenesis may attenuate keloid progression by reducing melanin activity, iron overload, and ferroptosis.

[0164] Given that MITF inhibitors may cause vitiligo, affect mast cells and bone regeneration, and other side effects in human applications, we further evaluated the safety of ML329 treatment. In vitro skin tissue explant experiments showed that ML329 specifically inhibited melanin synthesis in KD tissues only, without affecting the melanin level of normal skin (KN), and could restore KD melanin to the typical level of KN ( Figure 12 , A). It is worth noting that, as a key factor in immune regulation, ML329 treatment can restore the mast cell marker c-Kit (CD117) and its secreted cytokines such as IL-4 and TNF-α to a near physiological state ( Figure 12 , B). This suggests that ML329 is more likely to restore abnormally elevated melanin and inflammation to normal levels, thereby promoting skin homeostasis. Because keloid tissue grows outward, the dermis is distant from the bone. Potential effects on osteoblasts and osteoclasts can be circumvented by topical or epidermal administration. Therefore, selecting an appropriate concentration and dosing regimen to restore homeostasis in skin melanin metabolism and immune responses will effectively reduce the risk of side effects.

[0165] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the present invention and without unnecessary experiments. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the scope disclosed in this application and are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following claims.

Claims

1. Use of an agent for regulating the expression or activity of MITF or its downstream effector molecules in the preparation of a medicament for one or more effects selected from the group consisting of: regulating (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barriers, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) expression or activity of F2RL1, (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis in animals. Preferably, the MITF downstream effector molecule is a MITF downstream effector molecule related to melanin production; Preferably, the cells in item (ii) are melanocytes; the cells in item (iii) are fibroblasts; the inflammatory response in item (v) is an inflammatory response triggered by melanin; and / or the fibrogenesis in item (vii) is fibrogenesis induced by melanin.

2. The use according to claim 1, characterized in that The regulation is: (1) downregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) inhibiting intracellular melanin production, (ii) inhibiting cell hyperproliferation and non-immortalization characteristics, (iii) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibiting abnormal extracellular matrix deposition, (v) inhibiting inflammatory response and destruction of basement membrane barrier, (vi) inhibiting iron overload and ferroptosis resistance, restoring ferroptosis sensitivity, (vi) inhibiting fibrosis, (viii) downregulating the expression or activity of F2RL1, (ix) inhibiting keratinocyte uptake of melanin, (x) reducing scar area, and / or (xi) treating or preventing skin fibrosis; (2) Upregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) promoting intracellular melanin production, (ii) promoting cell hyperproliferation and immortalization characteristics, (iii) promoting cell proliferation, migration and collagen synthesis, (iv) promoting abnormal extracellular matrix deposition, (v) promoting inflammatory response and destruction of basement membrane barrier, (vi) promoting iron overload and ferroptosis resistance, (vii) promoting fibrosis, (viii) upregulating the expression or activity of F2RL1, (ix) promoting keratinocyte uptake of melanin, and / or (x) increasing scar area or promoting darkening of skin color; Preferably, The upregulation of the expression or activity of the MITF protein or its downstream effector molecules includes: transferring the coding sequence of the MITF protein or its downstream effector molecules into animals; the downregulation of the expression or activity of the MITF protein or its downstream effector molecules includes: (a) specifically interfering with the transcription and / or expression of the MITF gene or its downstream effector molecules, (b) downregulating the activity of the MITF protein or its downstream effector molecules, or (c) expressing the MITF protein or its downstream effector molecules with reduced activity in cells.

3. The use according to claim 2, characterized in that The nucleic acid that specifically interferes with the transcription and / or expression of the MITF gene or its downstream effector molecule gene is selected from the following group: (i) dsRNA, antisense nucleic acid, small interfering RNA, microRNA, shRNA, reRNA, sgRNA that inhibits or silences the MITF gene or its downstream effector molecule gene, or their transcripts, or (ii) a construct that can express or form (i), The agent that downregulates the activity of MITF or its downstream effector molecules is selected from one or more of the following: PLX4032, AZD8055 and ML329.

4. The use according to any one of claims 1 to 3, characterized in that The MITF downstream effector molecules include: tyrosinase, tyrosinase-related protein 1, dopachrome interconvertase, or premelanosome protein.

5. A method for regulating (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barrier, (vi) iron overload and resistance to ferroptosis, (vii) fibrosis, (viii) (ix) reducing the expression or activity of F2RL1, (ix) melanin uptake by keratinocytes, (x) reducing scar area, and / or (xi) improving skin fibrosis, the method comprising regulating the expression or activity of MITF or its downstream effector molecules in an animal; Preferably, the MITF downstream effector molecule is a MITF downstream effector molecule related to melanin production; Preferably, the cells in item (ii) are melanocytes; the cells in item (iii) are fibroblasts; the inflammatory response in item (v) is an inflammatory response triggered by melanin; and / or the fibrogenesis in item (vii) is fibrogenesis induced by melanin.

6. The method according to claim 5, wherein The regulation is: (1) downregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) inhibiting intracellular melanin production, (ii) inhibiting cell hyperproliferation and non-immortalization characteristics, (iii) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibiting abnormal extracellular matrix deposition, (v) inhibiting inflammatory response and destruction of basement membrane barrier, (vi) inhibiting iron overload and ferroptosis resistance, restoring ferroptosis sensitivity, (vi) inhibiting fibrosis, (viii) downregulating the expression or activity of F2RL1, (ix) inhibiting keratinocyte uptake of melanin, (x) reducing scar area, and / or (xi) improving skin fibrosis; and / or (2) Upregulating the expression or activity of MITF or its downstream effector molecules, thereby (i) promoting intracellular melanin production, (ii) promoting cell hyperproliferation and immortalization characteristics, (iii) promoting cell proliferation, migration and collagen synthesis, (iv) promoting abnormal extracellular matrix deposition, (v) promoting inflammatory response and destruction of basement membrane barrier, (vi) promoting iron overload and ferroptosis resistance, (vii) promoting fibrosis, (viii) upregulating the expression or activity of F2RL1, (ix) promoting keratinocyte uptake of melanin, and / or (x) increasing scar area or promoting darkening of skin color; Preferably, The upregulation of the expression or activity of the MITF protein or its downstream effector molecules includes: transferring the coding sequence of the MITF protein or its downstream effector molecules into animals; the downregulation of the expression or activity of the MITF protein or its downstream effector molecules includes: (a) specifically interfering with the transcription and / or expression of the MITF gene or its downstream effector molecules, (b) downregulating the activity of the MITF protein or its downstream effector molecules, or (c) expressing the MITF protein or its downstream effector molecules with reduced activity in cells.

7. Use of an iron overload inhibitor and / or an iron death inducer in the preparation of a medicament for one or more of the following effects: (1) inhibiting cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (2) inhibiting abnormal deposition of extracellular matrix, (3) inhibiting fibrogenesis, (4) reducing scar area, and / or (5) treating or preventing skin fibrosis. Preferably, The iron overload inhibitors include iron chelators, iron absorption inhibitors, iron excretion promoters, iron metabolism regulatory protein inhibitors, and antioxidants. The ferroptosis inducers include GPX4 inhibitors, system Xc - Inhibitor, iron ion regulator, lipid peroxidation promoter.

8. The use according to claim 7, characterized in that The iron overload inhibitor includes one or more selected from the following: deferasirox, delazosane, deferoxamine, HFE protein modulators, rotecept, captopril, Heme peptide analogs, FPN activation antibodies, ruxolitinib, The ferroptosis inducer includes one or more selected from the following: RSL-3, ML162, FIN56, DPI-1510, Erastin, Sulfasalazine, Sorafenib, Deferoxamine, Ferrostatin-3, A22, FINO2, and RSL-5.

9. A method for screening candidate substances having one or more of the following effects: regulating (i) intracellular melanin production, (ii) cell hyperproliferation and non-immortalization characteristics, (iii) cell proliferation, migration and collagen synthesis, (iv) abnormal extracellular matrix deposition, (v) inflammatory response and destruction of basement membrane barriers, (vi) iron overload and resistance to ferroptosis, (vii) fibrogenesis, (viii) expression or activity of F2RL1, (ix) melanin uptake by keratinocytes, (x) scar area, and / or (xi) improvement of skin fibrosis, the method comprising the steps of: (a) contacting a substance with a system containing MITF protein or its downstream effector molecule, or their coding sequences, and (b) detecting the expression or activity of MITF or its downstream effector molecules in the system and comparing it with the control; If the expression and activity of MITF or its downstream effector molecules are lower than the control, the candidate substance: (i) inhibits intracellular melanin production, (ii) inhibits cell hyperproliferation and non-immortalization characteristics, (iii) inhibits cell proliferation, cell migration, cell activation, collagen synthesis and / or collagen deposition, (iv) inhibits abnormal extracellular matrix deposition, (v) inhibits inflammatory response and destruction of basement membrane barrier, (vi) inhibits iron overload and ferroptosis resistance, restores ferroptosis sensitivity, (vii) inhibits fibrosis, (viii) downregulates the expression or activity of F2RL1, (ix) inhibits keratinocyte uptake of melanin, (x) reduces scar area, and / or (xi) treats or prevents skin fibrosis; If the expression and activity of MITF or its downstream effector molecules are higher than the control, then the candidate substance: (i) promotes intracellular melanin production, (ii) promotes cell hyperproliferation and non-immortalization characteristics, (iii) promotes cell proliferation, migration and collagen synthesis, (iv) promotes abnormal deposition of extracellular matrix, (v) promotes inflammatory response and damages basement membrane barrier, (vi) promotes iron overload and ferroptosis resistance, (vii) promotes fibrosis, (viii) upregulates the expression or activity of F2RL1, (ix) promotes keratinocyte uptake of melanin, and / or (x) increases scar area or promotes darkening of skin color, Preferably, the system is a cell system, a tissue system or an animal model.

10. The method according to claim 9, wherein The cells described in item (ii) are melanocytes; the cells described in item (iii) are fibroblasts; the inflammatory response described in item (v) is an inflammatory response triggered by melanin; and / or the fibrosis described in item (vii) is fibrosis induced by melanin.

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