Application of miR-24 and analogue thereof in prevention and treatment of skin inflammation diseases

Abstract

The invention provides application of miR-24 and analogues thereof in prevention and treatment of skin inflammation diseases. The application of the miR-24 and the analogue thereof in inhibiting the skin inflammation and treating or preventing the skin inflammatory diseases is provided for the first time, and the internal mechanism of the miR-24 in inhibiting the skin inflammation is deeply studied. Based on the effect of miR-24 in inhibition of skin inflammation, the invention further provides a miR-24 transdermal delivery compound based on a sponge microneedle, and a transdermal delivery technology of the miR-24 transdermal delivery compound is optimized, so that a safer and more effective method and medicine for preventing and treating the skin inflammation are provided.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of miR-24 and its analogues in the prevention and treatment of inflammatory skin diseases. Background Technology

[0002] Skin inflammation refers to the immune response and related non-cancerous pathological changes of the skin triggered by endogenous or exogenous stimuli. It is typically characterized by immune cell aggregation, epidermal hyperplasia, and skin redness, swelling, and itching. Common types of inflammatory skin diseases include psoriasis, allergic dermatitis, contact dermatitis, seborrheic dermatitis, neurodermatitis, photodermatitis (sunburn), drug eruptions, acne, papules, sensitive skin redness, and various autoimmune skin diseases such as lupus erythematosus and dermatomyositis. Related stimuli include infections (such as invasion by pathogens like bacteria, viruses, and fungi), physical damage (such as ultraviolet radiation and trauma), chemical irritation, allergic reactions, and autoimmune diseases. [1] .

[0003] Although their causes are diverse, various inflammatory skin diseases are considered complex genetic disorders involving gene-gene and gene-environment interactions. Most rely on the interaction between immune cells and epidermal cells. Specifically, when epidermal cells are damaged or stimulated, they release immune factors such as antimicrobial peptides, prompting dendritic cells (DCs) to release inflammatory factors such as IFNα, TNFα, IL-23, and IL-6. These inflammatory factors recruit neutrophils to migrate to the skin and simultaneously promote the differentiation of skin T cells into pro-inflammatory helper T cells. The inflammatory factors released by these immune cells, such as IL-17 and IL-22, further stimulate epidermal cells to activate NF-κB and JAK-STAT. [3,4] Inflammatory signaling pathways [5] This leads to epidermal cells releasing more inflammatory factors, which in turn recruit immune cells, creating a positive feedback loop of mutual stimulation between epidermal and immune cells, resulting in inflammatory skin diseases. Therefore, disrupting this epidermal-immune cell stimulation loop is key to inhibiting skin inflammation.

[0004] Currently, most treatments for epidermal inflammatory diseases primarily focus on suppressing immune cell activity. Examples include anti-inflammatory drugs like methotrexate and T-cell inhibitors such as cyclosporine. However, these non-targeted immunosuppressive drugs all have significant toxicity; for instance, methotrexate has teratogenic effects, and cyclosporine has nephrotoxicity. Targeted therapies mainly target immune factors. Taking psoriasis as an example, first-line targeted therapies currently include TNFα inhibitors, IL-12 / 23 inhibitors, IL-17 inhibitors, and IL-23 inhibitors. [6]However, these immune factors often have widespread importance in various tissues and organs throughout the body, limiting the safe dosage and efficacy of related inhibitors in clinical application. Furthermore, drugs that induce epidermal cell apoptosis, such as anthralin or corticosteroids, can also be used topically to treat inflammatory skin diseases. However, anthralin is somewhat destructive to the epidermis and is not suitable for use on the face or flexural surfaces, nor in areas at risk of ulceration. Its irritant properties to healthy skin also make it unsuitable for prophylactic use.

[0005] Therefore, there is an urgent need in this field to develop new, safe, and effective methods and drugs for the prevention and treatment of skin inflammation. Summary of the Invention

[0006] The purpose of this invention is to provide a safe and effective method and medicine for preventing and treating skin inflammation.

[0007] In a first aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of:

[0008] (a) miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof whose core sequence at positions 2-8 of its 5' end is 5'-GGCUCAG-3', is 16-28nt in length, and has the same or substantially the same function as miR-24.

[0009] (b) a precursor miRNA, said precursor miRNA being processed within the host into the miRNA described in (a);

[0010] (c) A polynucleotide that can be transcribed in the host to form the precursor miRNA described in (b) and processed to form the miRNA described in (a);

[0011] (d) An expression vector containing the miRNA described in (a), or the precursor miRNA described in (b), or the polynucleotide described in (c);

[0012] Agonists of the miRNAs described in (e)(a);

[0013] The active ingredient is used to prepare a pharmaceutical composition or formulation for one or more applications selected from the group consisting of: (i) inhibiting skin inflammation; and (ii) preventing and / or treating inflammatory skin diseases.

[0014] In another preferred embodiment, the skin inflammation includes skin inflammation caused by the following irritants: infection (such as invasion by pathogens such as bacteria, viruses, and fungi), physical damage (such as ultraviolet radiation or trauma), chemical irritation, and allergic reactions.

[0015] In another preferred embodiment, the inflammatory skin disease includes psoriasis, photodermatitis (sunburn), allergic dermatitis, contact dermatitis, seborrheic dermatitis, neurodermatitis, drug eruption, acne, papules, sensitive skin redness, and other inflammatory skin diseases.

[0016] In another preferred embodiment, the inflammatory skin disease is associated with epidermal cell inflammatory responses mediated by the NF-κB and JAK-STAT signaling pathways.

[0017] In another preferred embodiment, the core sequence described in (a) is located within the first 8 nt of the 5' end of the miRNA.

[0018] In another preferred embodiment, the miRNA described in (a) is 16-28 nt in length and its sequence characteristics satisfy the following formula: 5'-(N)GGCUCAGN…-3', where N represents any nucleotide and (N) represents 1 or 0 N.

[0019] In another preferred embodiment, the miRNA is 18-26 nt in length.

[0020] In another preferred embodiment, "functionally the same as or substantially the same as miR-24" means retaining ≥40% and ≤500% of the skin inflammation-inhibiting function of miR-24.

[0021] In another preferred embodiment, the function of inhibiting skin inflammation includes one or more functions selected from the group consisting of:

[0022] (1) Reduce the expression of inflammatory factors in epidermal cells;

[0023] (2) Reduce the infiltration of immune cells in the epidermis;

[0024] (3) Reduces epidermal hyperplasia and skin redness and swelling; and

[0025] (4) Inhibit the NF-κB and JAK-STAT signaling pathways that mediate inflammatory responses in epidermal cells.

[0026] In another preferred embodiment, the sequence of miR-24 is shown in SEQ ID NO:1.

[0027] In another preferred embodiment, the miR-24 is derived from mammals, preferably from humans, mice, or rats.

[0028] In another preferred embodiment, the sequence of the miRNA described in (a) is shown in SEQ ID NO:2.

[0029] In another preferred embodiment, the modified miRNA derivative is modified in one or more forms selected from the group consisting of: glycosyl modification of nucleotides, modification of the linkage between nucleotides, cholesterol modification, locked nucleotide modification, peptide modification, lipid modification, halogen modification, hydrocarbon modification, and nucleic acid modification.

[0030] In another preferred embodiment, the glycosyl modification of the nucleotide includes glycosyl modification with 2-O-methyl, glycosyl modification with 2-O-methoxyethyl ester, glycosyl modification with 2-O-alkyl, glycosyl modification with 2-fluoro, glycocycle modification, and locked nucleotide modification.

[0031] In another preferred embodiment, the modification of the linkage between the nucleotides includes thiophosphate modification and phosphoalkylation modification.

[0032] In another preferred embodiment, the nucleic acid modification includes "TT" modification.

[0033] In another preferred embodiment, the modified miRNA derivative described in (a) is a monomeric compound or a polymer thereof having the structure shown in Formula I:

[0034] (X)n-(Y)m(I)

[0035] In equation I,

[0036] Each X is a miRNA as described in (a);

[0037] Each Y is an independent modifier that promotes the stability of miRNA administration;

[0038] Y connects to the left, right, or middle of X;

[0039] n is a positive integer from 1 to 100 (preferably 1 to 20) (preferably n is 1, 2, 3, 4 or 5);

[0040] m is a positive integer from 1 to 1000 (preferably from 1 to 200);

[0041] Each "-" indicates a linker, chemical bond, or covalent bond.

[0042] In another preferred embodiment, the adapter is a nucleic acid sequence of 1-10 bases in length.

[0043] In another preferred embodiment, Y includes (but is not limited to) cholesterol, steroids, sterols, alcohols, organic acids, fatty acids, esters, monosaccharides, polysaccharides, amino acids, polypeptides, mononucleotides, and polynucleotides.

[0044] In another preferred embodiment, the polynucleotide described in (c) has the structure shown in Formula II:

[0045] Seq 正向-X-Seq 反向 (II)

[0046] In formula II,

[0047] Seq positive indicates that the microRNA nucleotide sequence can be processed in the host;

[0048] The reverse Seq sequence is a nucleotide sequence that is substantially or completely complementary to the forward Seq sequence.

[0049] X is an interval sequence located between the forward and reverse directions of Seq, and the interval sequence is not complementary to the forward and reverse directions of Seq;

[0050] Furthermore, the structure shown in Formula II, after being transferred into the host cell, forms the secondary structure shown in Formula III:

[0051]

[0052] In Equation III, the definitions of Seq forward, Seq backward, and X are as described above.

[0053] || indicates the complementary base pairing relationship formed between the forward and reverse sides of Seq.

[0054] In another preferred embodiment, the expression vector described in (d) includes: viral vectors and non-viral vectors.

[0055] In another preferred embodiment, the miR-24 agonist described in (e) is selected from the group consisting of substances that promote miR-24 expression, substances that enhance miR-24 activity, or combinations thereof.

[0056] In another preferred embodiment, the pharmaceutical composition or formulation comprises the active ingredient and a pharmaceutically acceptable carrier.

[0057] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, or combinations thereof.

[0058] In another preferred embodiment, the pharmaceutical composition or formulation further comprises additional anti-inflammatory active ingredients.

[0059] In another preferred embodiment, the pharmaceutical composition or formulation is administered via transdermal, percutaneous, or injectable route.

[0060] In another preferred embodiment, the dosage form of the pharmaceutical composition or preparation is a topical dosage form.

[0061] In another preferred embodiment, the dosage form of the composition or preparation includes ointments, creams, patches, liniments, sprays, microneedles, etc.

[0062] In a second aspect of the invention, a sponge spicule complex is provided, the sponge spicule complex containing miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof with a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and functioning the same as or substantially the same as miR-24 as an active ingredient; and a pharmaceutically acceptable carrier.

[0063] In another preferred embodiment, the pharmaceutically acceptable carrier includes a buffer solution and sodium hyaluronate.

[0064] In another preferred embodiment, the buffer solution is a PBS buffer.

[0065] In another preferred embodiment, the concentration of the active ingredient in the sponge-bone spicule complex is 1-10 mg / mL, preferably 2-5 mg / mL.

[0066] In another preferred embodiment, the sequence of the active ingredient is as shown in SEQ ID NO:1 or 2.

[0067] In a third aspect of the invention, a method for preparing the sponge-bone spicule composite as described in the second aspect of the invention is provided, the method comprising the following steps:

[0068] (S1) Dissolve the sponge spicules in buffer solution to prepare a sponge spicule solution;

[0069] (S2) Add an active ingredient to the sponge spicule solution prepared in step (S1). The active ingredient is selected from miR-24 or a modified miR-24 derivative, or miRNA with a core sequence of 5'-GGCUCAG-3', a length of 16-28nt, and a function that is the same as or substantially the same as miR-24, or a modified miRNA derivative thereof.

[0070] (S3) Sodium hyaluronate is added to the solution obtained in step (S2) to obtain the sponge spicule complex.

[0071] In another preferred embodiment, the buffer solution is a PBS buffer.

[0072] In another preferred embodiment, the concentration of sponge spicules in the sponge spicule solution prepared in step (S1) is 50-500 mg / mL, preferably 100-200 mg / mL.

[0073] In another preferred embodiment, the concentration of the active ingredient added in step (S2) is 1-10 mg / mL, preferably 2-5 mg / mL.

[0074] In another preferred embodiment, the final concentration of sodium hyaluronate in the complex after adding sodium hyaluronate in step (S3) is 0.5%-2%, preferably 1%-1.5%.

[0075] In a fourth aspect of the invention, a pharmaceutical composition is provided comprising the sponge spicule complex as described in the second aspect of the invention, and a pharmaceutically acceptable carrier.

[0076] In another preferred embodiment, the pharmaceutical composition further comprises additional anti-inflammatory active ingredients.

[0077] In a fifth aspect of the invention, a cosmetic composition is provided, the cosmetic composition comprising miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof having a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function identical or substantially identical to miR-24 as an active ingredient, or a sponge spicule complex as described in the second aspect of the invention; and a cosmetically acceptable carrier.

[0078] In a sixth aspect of the invention, a medicine box is provided, the medicine box comprising: (Z1) a medicine disposed in a container, the medicine being selected from: a sponge spur complex as described in the second aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention; and (Z2) an adhesive surgical film.

[0079] In another preferred embodiment, the medicine box also includes an instruction manual that describes how to use the medicine box:

[0080] The medication was applied to the intervention site on the subject's skin and the area was gently massaged for 2-5 minutes; then the intervention site was covered with an adhesive surgical film; the surgical film was removed approximately 5 minutes to 24 hours later.

[0081] In a seventh aspect of the invention, a microneedle array is provided, the microneedles containing an active ingredient selected from the group consisting of miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof with a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function that is the same as or substantially the same as miR-24.

[0082] In another preferred embodiment, the microneedle array delivers the active ingredient transdermally to inhibit skin inflammation and prevent and / or treat inflammatory skin diseases.

[0083] In an eighth aspect of the invention, a method for inhibiting skin inflammation or preventing inflammatory skin diseases is provided, the method comprising administering to a subject in need miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof having a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function identical or substantially identical to miR-24, or a sponge-bone spicule complex as described in the second aspect of the invention, or a pharmaceutical composition as described in the fourth aspect of the invention, or a cassette as described in the sixth aspect of the invention, or a microneedle array as described in the seventh aspect of the invention.

[0084] In another preferred embodiment, the skin inflammation includes skin inflammation caused by the following irritants: infection (such as invasion by pathogens such as bacteria, viruses, and fungi), physical damage (such as ultraviolet radiation or trauma), chemical irritation, and allergic reactions.

[0085] In another preferred embodiment, the inflammatory skin disease includes psoriasis, photodermatitis (sunburn), allergic dermatitis, contact dermatitis, seborrheic dermatitis, neurodermatitis, drug eruption, acne, papules, sensitive skin redness, and other inflammatory skin diseases.

[0086] In another preferred embodiment, the inflammatory skin disease is associated with epidermal cell inflammatory responses mediated by the NF-κB and JAK-STAT signaling pathways.

[0087] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0088] Figure 1This demonstrates that miR-24 is an effective approach for preventing and treating inflammatory skin diseases. A. Mature miR-24 sequences in humans and mice, with the seed sequence highlighted in red. B. Schematic diagram of the construction of mice conditionally overexpressing miR-24 using K14. C. Overexpression efficiency of mice conditionally overexpressing miR-24 using K14. D. Schematic diagram of the experimental procedure for miR-24 intervention in an IMQ-induced mouse model of dorsal skin psoriasis / UVB irradiation sunburn. 7wo represents 7-week-old mice. IMQ / UVB intervention began 7 days after DOX induction and continued until the mice were sacrificed. IMQ and VAS: Topical application of imiquimod or Vaseline as controls. WT and DTG: WT mice and DTG (K14 conditionally overexpressing miR-24) mice. Sampling time point was day 10 of the procedure. E. Photograph of the dorsal skin surface taken under a dermatoscope. Scale bar is 0.5cm. F. Left: H&E image of a dorsal skin section. The middle image shows K6 immunofluorescence staining, and the right image shows F4 / 80 immunofluorescence staining. G. Statistical results of back skin sampling points from different treatment groups. Epidermal thickness refers to the distance from the basement membrane to the upper surface of the skin. n=3, two-tailed t-test. H. Statistical results of K6 immunofluorescence at back skin sampling points from different treatment groups. Ki67 is statistically analyzed as the proportion of K6-positive cells on a fixed-length basement membrane, standardized with the WT IMQ / UVB group as 1. n=3, two-tailed t-test. I. Statistical results of F4 / 80 immunofluorescence at back skin sampling points from different treatment groups. F4 / 80 is statistically analyzed as the proportion of F4 / 80-positive cells on a fixed-length basement membrane, standardized with the WT IMQ / UVB group as 1. n=3, two-tailed t-test. J. qPCR results of cytokines in WT and DTG epidermis under steady-state conditions. EPI refers to the epidermis. K. MK immunoblotting results of primary keratinocytes from WT and DTG. The left side is a schematic diagram. Keratinocytes from newborn mice were induced with DOX for 2 days, and then protein analysis was performed. DOX is doxycycline, and p65 is a classic marker of the NF-κB signaling pathway. pP65 is the phosphorylated form, STAT3 is a marker of the JAK-STAT signaling pathway, and pSTAT3 is the phosphorylated form. L represents the statistical results of the K-plot. n=3, two-tailed t-test.

[0089] Figure 2The images show the specific effects of miR-24 on the recruitment of epithelial inflammatory cells. A. Flow cytometry analysis of dorsal skin from WT and DTG mice after IMQ treatment. CD45 is a marker of immune cells. B. Flow cytometry analysis of dorsal skin from WT and DTG mice after IMQ treatment. Gr1 and CD11b are markers of granulocytes. C. Statistical results of Figure A. n=5, two-tailed t-test. D. Statistical results of Figure B. n=5, two-tailed t-test. E. Intestinal images of a mouse enteritis model. Scale bar: 1 cm. F. Weight statistics of a mouse enteritis model. G. qPCR results of cytokines in WT and DTG keratinocytes under basal culture conditions. n=3, two-tailed t-test.

[0090] Figure 3This image demonstrates that sponge spicules delivering miR-24 can prevent and treat inflammatory skin diseases. A. Schematic diagram of sponge spicule delivery of small molecules. B. Microscopic image of the sponge spicule. C. Microscopic image of the complete M24-SMN mixture prepared by mixing sponge spicules with miR-24 mimic molecules and hyaluronic acid; microscopic image of the complete M24-SMN mixture prepared by mixing sponge spicules with red hyaluronic acid. D. Top image of mouse back skin after M24-SMN intervention; bottom image of the skin after the surgical film is applied. E. Top microscopic image of mouse back skin after M24-SMN intervention; bottom microscopic image after the surgical film is removed. F. Microscopic photograph of the back skin surface taken with a skin microscope. Scale bar: 0.5 cm. G. The mice were divided into five groups according to different treatment methods: a sponge microneedle PBS group (nc), a group where FAM-siRNA dissolved in hyaluronic acid (HA) was applied to the back skin of mice after sponge microneedle treatment for 10 min, 30 min, and 60 min respectively, and a group where FAM-siRNA was applied to the sponge microneedle group for 60 min. The positive proportion of FAM in the epidermal cells (CD49f+) of mice in each group was detected by flow cytometry. H. The positive proportion of FAM in the epidermal cells of each group ((Q2 / Q2+Q3)*100%) was calculated and statistically analyzed. n=3, two-tailed t-test, P value <0.05 was considered statistically significant, and *, P<0.05; **, P<0.01; ***, P<0.001 and ****, P<0.0001. I. Schematic diagram of the experimental procedure for the M24-SMN intervention effect in an IMQ-induced mouse dorsal skin psoriasis / UVB irradiation model. 7wo represents 7-week-old mice, with M24-SMN or SCR-SMN intervention performed on day 0. IMQ and UVB stimulation were performed respectively. Sampling time point was day 4 of the procedure. J. Left: H&E image of dorsal skin sections. Middle: K6 immunofluorescence staining; Right: F4 / 80 immunofluorescence staining. K. Statistical results of the characteristics of the dorsal skin experimental sampling points in different treatment groups. Epidermal thickness refers to the distance from the basement membrane to the upper surface of the skin. n=3, two-tailed t-test. L. Statistical results of K6 immunofluorescence at the dorsal skin experimental sampling points in different treatment groups. The K6 statistical method is the proportion of K6-positive cells on a fixed-length basement membrane, standardized with the MSCR-SMN IMQ / UVB group as 1. n=3, two-tailed t-test. M. Statistical results of F4 / 80 immunofluorescence at the dorsal skin experimental sampling points in different treatment groups. The F4 / 80 statistical method refers to the proportion of F4 / 80 positive cells on a fixed-length basement membrane, standardized with the MSCR-SMN IMQ / UVB group as 1. n=3, two-tailed t-test. N represents the qPCR results of inflammatory factors in dorsal skin tissue from different treatment groups. Detailed Implementation

[0091] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time that miR-24 can inhibit epidermal inflammation, thus making it suitable for the prevention and treatment of inflammatory skin diseases. Experiments confirmed that specifically upregulating miR-24 in epidermal tissue can significantly inhibit skin inflammation caused by psoriasis and UV sunburn. Specifically, this manifests as reduced expression of inflammatory factors in the epidermis, reduced immune cell infiltration, and reduced epidermal hyperplasia and skin redness, with no visible side effects. Mechanistically, miR-24 significantly inhibited the NF-κB and JAK-STAT signaling pathways mediating inflammatory responses in epidermal cells and reduced the expression of inflammatory factors in epidermal cells. This indicates that miR-24 has a broad function of inhibiting epidermal inflammatory responses. Based on this, the inventors also developed a transdermal delivery complex M24-SMN based on sponge microneedles (miR-24 mimic) and optimized its transdermal delivery technology. Experiments confirmed that surface delivery of M24-SMN can achieve the therapeutic effect of inhibiting skin inflammation and has high practical value. Based on this, the present invention was completed.

[0092] miRNA and its precursors

[0093] MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA molecules, approximately 22 nucleotides in length, encoded by endogenous genes. They are widely involved in the regulation of most life activities and disease processes, and are highly promising therapeutic targets. [7,8] .

[0094] As used herein, “miRNA” refers to a class of RNA molecules derived from transcripts that can form miRNA precursors. Mature miRNAs typically have 18–26 nucleotides (nt) (more specifically, about 19–22 nt), but miRNA molecules with other numbers of nucleotides are also possible. miRNAs can usually be detected by Northern blotting.

[0095] Human-derived miRNAs can be isolated from human cells. As used herein, "isolated" means that the substance has been isolated from its native environment (or, in the case of a native substance, the native environment). Polynucleotides and polypeptides in their native state within living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in their native state.

[0096] miRNAs can be processed from precursor miRNAs (pre-miRNAs), which fold into a stable stem-loop (hairpin) structure. The stem-loop structure is typically 50-100 bp in length or longer. The precursor miRNA folds into a stable stem-loop structure, with two substantially complementary sequences on either side of the stem. The precursor miRNA can be natural or synthetically produced.

[0097] Precursor miRNAs can be cleaved to generate miRNAs that are substantially complementary to at least a portion of the sequence of the mRNA encoding the gene. As used herein, “substantially complementary” means that the nucleotide sequences are sufficiently complementary to interact in a predictable manner, such as forming secondary structures (e.g., stem-loop structures). Typically, two “substantially complementary” nucleotide sequences have at least 70% complementary nucleotides to each other; preferably, at least 80%; more preferably, at least 90%; and even more preferably, at least 95%; such as 98%, 99%, or 100%. Generally, two sufficiently complementary molecules may have up to 40 mismatched nucleotides; preferably, up to 30; more preferably, up to 20; and even more preferably, up to 10, such as 1, 2, 3, 4, 5, 8, or 11 mismatched nucleotides.

[0098] As used herein, a "stem-loop" structure, also known as a "hairpin" structure, refers to a nucleotide molecule that can form a secondary structure including a double-stranded region (stem) formed by two regions of the nucleotide molecule (located on the same molecule), positioned on either side of the double-stranded portion; it also includes at least one "loop" structure, comprising a non-complementary nucleotide molecule, i.e., a single-stranded region. Even if the two regions of the nucleotide molecule are not perfectly complementary, the double-stranded portion of the nucleotide can remain double-stranded. For example, insertions, deletions, substitutions, etc., can lead to a small region becoming non-complementary or that small region itself forming a stem-loop structure or other forms of secondary structure; however, the two regions can still be substantially complementary and interact in a predictable manner to form a double-stranded region of a stem-loop structure. Stem-loop structures are well known to those skilled in the art, and typically, after obtaining a nucleic acid with a nucleotide sequence having a primary structure, those skilled in the art can determine whether the nucleic acid can form a stem-loop structure.

[0099] In this invention, a specific miRNA, miR-24, was discovered that can specifically inhibit the inflammatory response of epidermal cells, thereby exerting an inhibitory effect on skin inflammation. Here, miR-24 specifically refers to the human hsa-miR-24-3p (sequence: 5'-UGGCUCAGUUCAGCAGGAACAG-3', miRbase number MIMAT0000080, SEQ ID NO:1) and the mouse miRNA mmu-miR-24-3p (sequence: 5'-UGGCUCAGUUCAGCAGGAACAG-3', miRbase number MIMAT0000219), whose core seed sequence of nucleotides 2-8 at the 5' end is GGCUCAG.

[0100] The miRNA mentioned in this invention refers to microRNA-24 (miR-24), which includes miR-24 or modified miR-24 derivatives, and whose functions are the same as or substantially the same as miR-24.

[0101] In another preferred embodiment, the microRNA is derived from humans or non-human mammals; preferably, the non-human mammals are rats or mice, and the miR-24 sequences of mice and humans are completely identical. The phrase "functionally the same as or substantially the same as miR-24" means retaining ≥40%, ≥50%, ≥60%, ≥70%, ≥80%, or ≥90% of the skin inflammation-inhibiting function of miR-24-3p.

[0102] This invention also includes miRNA variants and derivatives. Furthermore, miRNA derivatives in a broader sense may also include miRNA variants. Those skilled in the art can modify miR-24 using common methods, including (but not limited to): methylation, hydrocarbon modification, glycosylation (such as 2-methoxy-glycosylation, hydrocarbon-glycosylation, glycan ring modification, etc.), nucleic acid modification, peptide modification, lipid modification, halogen modification, nucleic acid modification (such as "TT" modification), etc.

[0103] Polynucleotide constructs

[0104] Based on the miRNA sequence provided by this invention, polynucleotide constructs that, upon introduction, can be designed to process miRNAs into miRNAs that can affect the expression of the corresponding mRNAs, i.e., the polynucleotide constructs can upregulate the amount of the corresponding miRNAs in vivo. Therefore, this invention provides an isolated polynucleotide (construct), which can be transcribed into a precursor miRNA by human cells, and the precursor miRNA can be cleaved and expressed into the miRNA by human cells.

[0105] In a preferred embodiment of the present invention, the polynucleotide construct contains the structure shown in Formula II:

[0106] Seq 正向 -X-Seq 反向 (II)

[0107] In formula II,

[0108] Seq 正向 To obtain the nucleotide sequence that can be expressed in cells as miRNA-27b, Seq 反向 To be with Seq 正向 Essentially complementary nucleotide sequences; or, Seq 反向 Seq provides the nucleotide sequence that can be expressed as the miRNA in cells. 正向 To be with Seq 正向 Essentially complementary nucleotide sequences; X is located in Seq 正向 and Seq 反向 The interval sequence between, and the interval sequence with Seq 正向 and Seq 反向 Not complementary;

[0109] The structure shown in Formula I, after being transfected into cells, forms the secondary structure shown in Formula III:

[0110]

[0111] In Equation III, Seq 正向 Seq 反向 The definitions of X and X are as described above;

[0112] || indicates that in Seq 正向 and Seq 反向 The complementary base pairing relationship formed between them.

[0113] Typically, the polynucleotide construct is located on an expression vector. Therefore, the present invention also includes a vector containing the miRNA or the polynucleotide construct. The expression vector typically also contains a promoter, a replication origin, and / or a marker gene. Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells, such as resistance to kanamycin, gentamicin, hygromycin, and ampicillin.

[0114] In this invention, the promoter may be constitutive, inductive, or a combination thereof.

[0115] Sponge-bone spicule complex

[0116] This invention also provides a sponge spicule complex containing miR-24 or a modified miR-24 derivative, or a miRNA with a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function identical or substantially identical to miR-24, or a modified miRNA derivative thereof, as the active ingredient, and a pharmaceutically acceptable carrier (buffer solution for dissolving the sponge spicules and the active ingredient, and sodium hyaluronate). The sponge spicules are commercially available products, such as sponge microneedle powder made from deep-sea microcrystalline silica sponges from pristine waters, wherein the spicule content is 99%, and their size is 20-200 micrometers. Sponges rely on filtering and absorbing nutrients from seawater to sustain life; long-term evolutionary selection has enabled them to retain a strong metabolic capacity for various substances in seawater. Sponges degrade and transform various bioactive substances, such as β-sitosterol and astaxanthin. The main structure of the sponge microneedle, formed by silica, has a bone needle size of only 20 to 200 micrometers. When used, it can penetrate into the epidermis with a slight massage, forming microchannels in the epidermis, activating the epidermal microcirculation, and helping active ingredients to penetrate the stratum corneum.

[0117] The sponge spicule complex of the present invention is a viscous solution prepared by dissolving the active ingredient miR-24 or its derivatives, sponge spicules, and sodium hyaluronate in a buffer solution (e.g., PBS). In use, it is applied to the intervention site of the subject and gently massaged in.

[0118] Furthermore, the inventors have optimized the method of using the sponge-bone needle composite, namely, after application and massage, covering the intervention site with an adhesive surgical film, and removing the film after the intervention. On the one hand, covering the intervention site with the surgical film can protect the delivery part for a long time, preventing microneedle detachment, liquid drying, external infection, etc., and promoting microneedle transdermal penetration through continuous pressure; on the other hand, the adhesive surgical film removes most of the microneedles when it is peeled off, significantly reducing microneedle residue on the skin and minimizing the adverse effects of microneedles on the skin.

[0119] medicine box

[0120] Based on optimized transdermal delivery technology using sponge spurs, this application also provides a kit for transdermal delivery of the miR-24 active ingredient. In one embodiment of the invention, the kit contains a drug disposed in a container, comprising the sponge spur complex as described herein, and an adhesive surgical film. Further, the kit also includes an instruction manual describing the method of use: applying the drug to the intervention site on the subject's skin and gently massaging the site for 2-5 minutes; subsequently covering the intervention site with the adhesive surgical film; removing the surgical film after approximately 24 hours.

[0121] microneedle array

[0122] This invention also provides a microneedle array for transdermal drug delivery of the miR-24 active ingredient of this invention. The microneedle array can be any existing microneedle array, wherein the cross-sectional shape of the microneedles can be conical or multifaceted conical, the height of the microneedles can be between 50 and 400 micrometers, and the tip diameter can be between 100 nanometers and 10 micrometers. The microneedle array can be made of various materials, such as stainless steel microneedles, metal microneedles, glass microneedles, silicon needles, or biodegradable microneedles integrally molded with a polymer material. The microneedle array of this invention can improve the transdermal permeability of the transdermal drug composition or formulation of this invention, thereby enabling the drug to effectively enter the body and achieve superior efficacy.

[0123] Pharmaceutical Composition

[0124] This invention provides the use of an active ingredient for preparing pharmaceutical compositions that inhibit skin inflammation and treat and / or prevent inflammatory skin diseases.

[0125] As used herein, the term "active ingredient" or "miR-24 active ingredient" refers to miR-24, miR-24 derivatives or their precursor sequences, or expression vectors containing them, which can be used in this invention. Preferably, the active ingredient is selected from the group consisting of:

[0126] (a) miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof with a core sequence of 5'-GGCUCAG-3', a length of 16-28nt, and a function that is the same as or substantially the same as miR-24.

[0127] (b) a precursor miRNA, said precursor miRNA being processed within the host into the miRNA described in (a);

[0128] (c) A polynucleotide that can be transcribed in the host to form the precursor miRNA described in (b) and processed to form the miRNA described in (a);

[0129] (d) An expression vector containing the miRNA described in (a), or the precursor miRNA described in (b), or the polynucleotide described in (c);

[0130] Agonists of miRNAs as described in (e)(a).

[0131] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0132] As used herein, the term "pharmaceuticalally acceptable" refers to a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents.

[0133] The pharmaceutical compositions of the present invention contain a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage forms of the pharmaceutical compositions of the present invention can be injections, transdermal formulations, oral formulations (tablets, capsules, oral liquids), and sustained-release formulations. For example, they can be prepared using physiological saline or aqueous solutions containing glucose and other excipients by conventional methods. The pharmaceutical compositions are preferably manufactured under aseptic conditions.

[0134] The effective amount of the active ingredient described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. Generally, satisfactory results are obtained when the active ingredient of this invention is administered daily at a dose of approximately 0.00001 mg to 50 mg / kg animal body weight (preferably 0.0001 mg to 10 mg / kg animal body weight). For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0135] The pharmaceutically acceptable carriers described in this invention include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0136] cosmetic compositions

[0137] In this invention, another type of composition or product is provided, which is a cosmetic or daily chemical product containing the active ingredient of this invention.

[0138] In this invention, the dosage form of the cosmetic composition or formulation includes (but is not limited to): solid dosage forms, liquid dosage forms, gel dosage forms, and semi-solid dosage forms. Some preferred dosage forms include ointments, creams, patches, lotions, sprays, microneedles, etc. In a preferred embodiment of this invention, the cosmetic composition contains the sponge-bone nebula complex described in this invention.

[0139] In this invention, representative products (or daily chemical products) include (but are not limited to): skin care products, facial masks, body lotions, shower gels, etc.

[0140] The main advantages of this invention include:

[0141] (1) This invention unexpectedly confirmed for the first time that miR-24 is a miRNA closely related to skin inflammation. Overexpression of miR-24 in the epidermis can significantly inhibit skin inflammation, and therefore it can be used to prevent and treat inflammatory skin diseases.

[0142] (2) This invention also reveals the intrinsic mechanism by which miR-24 inhibits skin inflammation and confirms that the effect of overexpressing miR-24 in the epidermis to inhibit skin inflammation is skin-specific and does not affect the overall immune system.

[0143] (3) This invention is the first to develop a sponge bone needle complex based on miR-24 active ingredient and verify the transdermal therapeutic effect of the sponge bone needle complex.

[0144] (4) The present invention further optimizes the transdermal treatment technology of sponge bone needle composite. When applying the composite, the application area is covered with an adhesive surgical film and removed after application. This not only improves the transdermal efficiency (nearly 3 times higher than the general method), but also reduces bone needle residue and greatly reduces the adverse effects of applying sponge bone needle to the skin.

[0145] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0146] Materials and methods

[0147] 1) Animal model

[0148] 1.1 Wild-type mice: C57BL / 6 strain, purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0149] 1.2K14-rtTA-TRE-miR-24 mice: miR-24 can be overexpressed under doxycycline-driven conditions in the presence of rtTA, and were produced by Shanghai Southern Model Biotechnology Co., Ltd.

[0150] 1.3K14-Cre-miR-24 Mice: These mice, with a K14 promoter-driven overexpression of Cre protein and a genetic background of C57BL / 6, are tool mice for conditional gene knockout in the skin epithelium. Southern Model Biotechnology Co., Ltd. was commissioned to use CRISPR-Cas9 technology to precisely knock in LoxP sites approximately 300 bp upstream and downstream of the Mir24-1 gene region (Chr13: 63301208-63301275) in C57BL / 6 wild-type mice. This resulted in Mir24-1LoxP mice (FloxP for short), with a theoretical knockout region of chr13: 63300966-63301497, completely covering the mouse Mir24-1 gene region without affecting any known surrounding genes. Using CRISPR-Cas9 technology, 35 base pairs containing the mature miR-24 sequence were knocked out at specific sites in the Mir24-2 gene region (Chr8:84208815-84208921) of C57BL / 6 wild-type mice. This resulted in Mir24-2KO mice (KO for short), with the theoretical knockout region being Chr8:84208873-84208907, completely covering the mature mouse miR-24 sequence region without affecting any known surrounding genes.

[0151] 2) Imiquimod cream-induced psoriasis model

[0152] Seven-week-old female C57BL / 6 mice were selected. On the first day, the mice were shaved and treated with Veet depilatory cream for localized hair removal. Two points were taken on the upper and lower back skin, for a total of four points, for the experiment. On the second day, 5% imiquimod cream and petrolatum were applied to the four hair-removed sites as controls. The creams were applied daily for three days, after which the samples were collected. Imiquimod cream was purchased from Aldara, and petrolatum was purchased from Sangon Biotech (Shanghai) Co., Ltd. (A510146-0500).

[0153] 3) Ultraviolet radiation-induced skin inflammatory hyperplasia model

[0154] Seven-week-old female C57BL / 6 mice were selected. On the first day, the mice were shaved and treated with Veet hair removal cream, with two points taken on the upper and lower back skin, for a total of four points. On the second day, 125 mJ / cm² was applied to each of the four treated areas. 2 UVB irradiation was performed on samples, along with a control treatment (no intervention). Samples were collected three days after irradiation. UVB irradiation was achieved using a UV crosslinker (Beijing Saizhi Venture Technology Co., Ltd. #SGLinker II).

[0155] 4) Primary isolation and culture of MK

[0156] The dermis and epidermis of mice within 3 days of birth were separated by digestion with 2.5 mg / mL Dispase II (Roche #4942078001) overnight at 4°C. The epidermis was then digested with Trypsin-Versene (Lonza #17-161F) at room temperature for 15 minutes. The mice were cultured in CNT-Prime (cellntec #CnT-PR) medium.

[0157] 5) Induction of miR-24 overexpressing mice and miR-24 overexpressing cells

[0158] Six-week-old (6wo) female C57BL / 6 miR-24 overexpressing mice and WT control mice were fed a diet supplemented with DOX for one week. Subsequent experiments were then initiated, with mice also fed DOX-supplemented diet (Research Diets#C11300-2000) during this period. MK was isolated from miR-24 overexpressing mice within 3 days of birth. Once the mice reached 80% kinase, DOX (MCE#HY-15142) was added to a final concentration of 2 μg / ml. Subsequent experiments began 48 hours later, with continued DOX supplementation.

[0159] 5) Preparation and application of miR-24-sponge spicule complex (M24-SMN)

[0160] Both miR-24 mimic (B02001) and the scramble control mimic, abbreviated as SCR mimic (B04002), were purchased from Shanghai Genomics. The miR-24 mimic sequence is: 5'-UGGCUCAGUUCAGCAGGAACA-3' (SEQ ID NO:2, only lacking a G at the 3' end compared to miR-24). The SCR mimic sequence is: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO:3).

[0161] The preparation steps for M24-SMN or control SCR-SMN are as follows: Sponge spicules (Hebei Gongchuang Biotechnology Co., Ltd.) were dissolved in PBS to prepare a 100 mg / mL solution, and then miR-24mimic or SCR mimic was added to a final concentration of 0.2 ug / ul. Sodium hyaluronate powder was then added to a final concentration of 1%. After mixing, a skin-friendly viscous solution of M24-SMN or SCR-SMN was prepared.

[0162] The usage method for M24-SMN or SCR-SMN is as follows: Remove the hair from the back of the mouse using depilatory cream and clean it thoroughly. Per 1cm 2Apply 100ul of M24-SMN or SCR-SMN solution to the skin and gently massage the intervention area for 2 minutes. Then cover the intervention area with a surgical film (3L adhesive surgical drape #SP1107). Remove the film after 30 minutes to 24 hours. This additional step serves several purposes. First, it provides prolonged protection for the delivery site, preventing microneedle dislodgement, fluid drying, and external infection, and promotes microneedle transdermal absorption through continuous pressure. Second, the adhesive film removes most of the microneedles upon removal, minimizing any adverse effects on the skin.

[0163] 6) M24-SMN skin inflammation intervention in mice

[0164] Six female mice were used in each experiment and randomly divided into two groups: three as the control group (treated with SCR-SMN) and three as the experimental group (treated with M24-SMN). On the first day, the mice were shaved and treated with either SCR-SMN or M24-SMN, followed by a surgical film covering the treatment area. The film was removed 30 minutes to 24 hours later. Subsequently, mice were treated with IMQ (imiquimod cream) and VAS (petroleum jelly) as a control, and then irradiated with UVB. Imiquimod and VAS were applied daily, while UVB irradiation was performed only once. Samples were collected for analysis on the fifth day.

[0165] 7) Collection and analysis of mouse dorsal skin samples

[0166] Data were collected after mouse IMQ / UVB intervention modeling. The surface morphology of the dorsal skin was recorded using a dermatological microscope. Mice were then euthanized using a carbon dioxide lethal device, shaved, and the dorsal skin treated with IMQ or Vaseline, as well as the UVB-treated skin, were collected. Dermal adipose tissue was removed with a scalpel, and the tissue was washed with PBS and placed in OCT embedding medium to prepare embedding blocks. The embedding blocks were sectioned using a Leica cryostat and stained with K6 and F4 / 80. After staining, images were taken using a Zeiss Axio Imager A2 (Zeiss) fluorescence microscope, followed by semi-quantitative fluorescence analysis using ImageJ to calculate the relative K6 and F4 / 80 expression levels per unit skin length. OCT tissue tissue was purchased from Sakura (4583). K6 (from the inventor's laboratory) and F4 / 80 were purchased from BioLegend (123101).

[0167] 8) Immunofluorescence

[0168] Sections were removed from a -80°C freezer and dried at room temperature for 10 minutes. They were then fixed with 4% paraformaldehyde for 10 minutes, washed three times with PBS for 5 minutes each time, and blocked with blocking buffer (2.5% normal donkey serum, 2.5% normal goatserum, 1% BSA, 0.3% Triton X-100) for 1 hour. Afterward, they were incubated with primary antibody overnight at 4°C, washed with PBS, and then incubated with secondary antibody at room temperature for 1 hour. Excess secondary antibody was washed away with PBS, and the sections were mounted with Fluoromount-G mounting media (Invitrogen) containing DAPI. The sections were then observed and photographed. K6 (Rabbit, 1:1000), F4 / 80 (Rat, 1:200), and 4% paraformaldehyde were purchased from Sangon Biotech (WH1013). PBS was purchased from HYCLONE (SH30256.01). Normal Donkey Serum and Normal Goat Serum were purchased from Jackson, and BSA was purchased from Yisheng (36101ES25).

[0169] 9) Western Blots

[0170] Cells were lysed using cell lysis buffer, and proteins were extracted using a column-based animal tissue / cell total protein extraction kit (YAG). Protein precast gels (YAG) were used for electrophoresis until the bromophenol blue indicator reached the appropriate position. Transfer, blocking, primary antibody, secondary antibody, and color development were performed according to the manufacturer's instructions. The primary antibodies used were ACTB (mouse, Genscript, 1:1000), p65 (rabbit, CST, 1:1000, (D14E12)), Phospho-p65 (Ser536) (Rabbit, CST, 1:1000, 3033T), STAT3 (rabbit, CST, 1:1000, D3Z2G), and Phospho-Stat3 (Tyr705) (Rabbit, CST, 1:1000, 9145t). Western blot primary antibody dilution buffer, Western blot secondary antibody dilution buffer, protein-free rapid blocking buffer (5×), TBS / Tween buffer (10×), trichrome pre-stained protein markers 10kDa~250kDa, PVDF membrane (0.45μm), HRP-labeled secondary antibody-goat anti-mouse IgG, HRP-labeled secondary antibody-goat anti-rabbit IgG, Omni-ECL TM The ultrasensitive chemiluminescence detection kits were all purchased from Yageo. The Bio-rad imaging system was used for color development, and ImageJ was subsequently used for quantification of the target protein.

[0171] 10) qRT-PCR

[0172] The collected total RNA was transcribed using a reverse transcription kit. After transcription with V Reverse Transcriptase (YEASEN, 11300ES92), a qPCR reaction was performed using a qPCR kit. Green Master Mix (No Rox) (YEASEN, 11201ES08). The instrument used was a BIO-RADCFX Connect model.

[0173] 11) Flow cytometry

[0174] IMQ-induced mouse dorsal skin was digested into single-cell suspensions using collagenase (Sigma / flu / Ald) and Dispase II. The suspensions were then placed in PFE (PBS + 1% FBS + 1mM EDTA) staining solution on ice for CD45 (1:500, biolegend), Gr1 (1:500, biolegend), and Cd11b (1:500, biolegend) staining. After staining, the cells were washed and treated with the viable cell dye Helix NP NIR (biolegend, 425301). The stained cells were then analyzed using a flow cytometer (BD, CytoFLEX LX).

[0175] 12) DSS enteritis model

[0176] Seven-week-old mice were selected, and their water was replaced with water containing 3% sodium dextran sulfate (DSS). They were fed this diet for one week, and their weight was measured daily. The severity of enteritis was determined by the length of the colon and the degree of weight loss. The DSS was purchased from Meilun Biotechnology (MB5535).

[0177] 10) Image analysis and statistical methods

[0178] Western blotting (WB) quantification involved calculating the ratio of target protein signal to internal reference protein signal. ImageJ was used for quantification. Skin thickness was determined by measuring the length from the basement membrane vertically to the stratum corneum. Ki67 counts represented the number of Ki67-positive cells per 100µm length of skin, and F4 / 80 counts represented the F4 / 80 expression level per 100µm length of skin. ImageJ was used to analyze the area occupied by positive cells (n=3). A p-value < 0.05 was considered statistically significant (marked with 1 star); p < 0.01 (marked with 2 stars); p < 0.001 (marked with 3 stars); and p > 0.05 was considered not statistically significant.

[0179] Example 1: miR-24 overexpression inhibits skin inflammation

[0180] miR-24, sequence (5'-UGGCUCAGUUCAGCAGGAACAG-3')(SEQ ID NO:1, Figure 1 (A). In mice, it is named mmu-miR-24-3p (sequence 5'-UGGCUCAGUUCAGCAGGAACAG-3', miRBase number MIMAT0000219), and in humans, it is named hsa-miR-24-3p (sequence 5'-UGGCUCAGUUCAGCAGGAACAG-3', miRBase number MIMAT0000080). Its seed sequence is GGCUCAG. It can be found that the mature sequence of miR-24-3p in humans and mice is consistent, indicating that its expression is highly conserved in humans and mice.

[0181] In this embodiment, the epidermal-specific miR-24-induced overexpression mouse model DTG (K14-rtTA, Tre-miR-24) described in previous literature was used. Figure 1 (B) [9] This literature has demonstrated that DTG mice can specifically overexpress miR-24 in skin epithelial tissues, including the epidermis, under Dox-induced stimulation. Figure 1 (C) and will not have a visible effect on the normal development and homeostasis of the epidermis. [9] .

[0182] To investigate the effects of epidermal miR-24 overexpression on skin inflammation, an IMQ-induced psoriasis model was used in mice.

[10] and UV radiation-induced sunburn model

[11] ( Figure 1 (D). In these two typical models of skin inflammation, the results showed that DTG mice exhibited significantly reduced psoriasis symptoms compared to the WT group, and dermoscopy revealed a significant reduction in the degree of erythema and scaling on the skin surface. Figure 1 (Middle E); H&E images showed significantly reduced skin thickness and significantly reduced immune cell infiltration ( Figure 1 Immunofluorescence staining of sections containing the classic callus-associated factor K6 and the epidermal basement membrane marker CD104 showed a significant reduction in skin inflammation. Figure 1 Immunofluorescence staining of sections containing F4 / 80, a marker for dendritic cells and mature macrophages, showed a significant reduction in the infiltration of immune cells in the skin. Figure 1 (F). Statistical analysis of these immune signals showed that overexpression of miR-24 significantly reduced the production of K6 and F4 / 80. Figure 1 H in the middle Figure 1 This allows the skin to maintain a low-inflammatory environment and preserve the homeostasis of skin inflammation.

[0183] Furthermore, under normal physiological conditions, the epidermis of 7-week-old mice was isolated and RNA was extracted for qPCR analysis. The analysis revealed a significant decrease in the expression of typical immune factors CXCL1, IL-6, IL-23, and the antimicrobial peptide LL37 in the epidermis of Dox-induced DTG mice. Figure 1 (J). This indicates that miR-24 can also inhibit the expression of epidermal inflammatory factors under normal physiological conditions.

[0184] In cultured MK (keratinocyte) cells, MK cells from DTG mice showed significant downregulation of phosphorylated P65 and phosphorylated STAT3 after induction of miR-24 expression, representing downregulation of NF-κB and JAK-STAT signaling pathways. This indicates that miR-24 can inhibit NF-κB...

[12] and JAK-STAT

[13] The two main inflammatory signaling pathways in epidermal cells ( Figure 1 Zhong K, Figure 1 (Middle L).

[0185] Example 2: The anti-inflammatory effect of skin miR-24 overexpression is skin-specific.

[0186] Locally induced skin inflammation may also affect systemic immunity. The inventors observed the spleen of mice treated with IMQ and found no significant difference between DTG and WT mice; however, skin tissue was collected, digested into single cells, and analyzed by flow cytometry. The results showed that, compared to WT mice, DTG mice treated with IMQ had a significantly lower number of CD45+ immune cells in their skin. Figure 2 China A, Figure 2 (C). Furthermore, neutrophils, which play an important role in the early stages of inflammatory diseases, also show a significant decrease (C). Figure 2 B, Figure 2 (D). This indicates that miR-24 can inhibit the recruitment and aggregation of immune cells.

[0187] Because DTG mice are epithelial-specific transgenic mice, to rule out leakage, the inventors constructed a DSS intestinal inflammation model. They found no significant difference in intestinal inflammation between DTG mice and WT mice, specifically, there were no significant differences in intestinal length and body weight changes between the two groups. Figure 2 E, Figure 2 This indicates that in DTG mice, the anti-inflammatory effect of skin miR-24 overexpression is skin-specific and does not affect the overall immune system.

[0188] Example 3: miR-24's ability to inhibit epidermal cell inflammatory response is unrelated to cell proliferation.

[0189] To clarify whether the function of miR-24 is related to its effect on proliferation, MK cells from Dox-induced DTG mice and control mice were cultured in a growth factor-deficient basal medium for 16 hours before analysis. The disappearance of proliferation differences was confirmed by Ki67 proliferation signal detection. Immune factors were also detected, revealing that even after the elimination of proliferation differences, the expression of typical immune factors CXCL1, IL-6, IL-23, and the antimicrobial peptide LL37 in DTG MK cells remained significantly lower than in control MK cells. Figure 2 (G), which indicates that miR-24's ability to inhibit epidermal cell inflammatory response is unrelated to cell proliferation.

[0190] Example 4: Preparation and verification of anti-inflammatory effects of sponge microneedles loaded with miR-24mimic (M24-SMN) on the skin.

[0191] Sponge microneedles (SMNs), also known as sponge spicules, are extremely tiny needle-like structures. Derived from sponges, they are composed of countless tiny silica spicules, appearing as minute needles. In recent years, sponge microneedles have been widely used in the cosmetics industry because they can create microchannels in the skin, allowing the active ingredients in skincare products to penetrate more easily into the deeper layers of the skin.

[14] miRNAs (mimics) are a class of chemically synthesized miRNA analogs that mimic the functions of endogenous miRNAs. They also possess enhanced stability through specific chemical modifications and are widely used for treating diseases by overexpressing miRNAs. [15,16] .

[0192] Based on the transdermal delivery principle of sponge microneedles and the mechanism of action of miRNA mimics, the inventors combined them with miR-24 mimics to invent sponge microneedles loaded with miR-24 mimics (M24-SMN), and detailed the method of use. Figure 3 (A): Insert sponge bone needles ( Figure 3 A suspension was prepared by mixing B, miR-24mimic, and hyaluronic acid. Figure 3 (C) Apply an appropriate amount to the skin of a hairless mouse and massage for 3-5 minutes. Then apply a suitable-sized transparent surgical film. After 24 hours, remove the film, taking with it most of the spongy spicules. Figure 3 (D, E). To verify whether hyaluronic acid promotes the delivery of FAM-siRNA-loaded sponge microneedles (FAM-SMN) into epidermal cells, the inventors, following the above method, applied FAM-siRNA with or without hyaluronic acid and attached appropriately sized transparent surgical films. After 10 min, 30 min, and 60 min of application, the proportion of FAM+ epidermal cells (CD49f+) was detected by flow cytometry. Figure 3(G). Statistical analysis revealed that hyaluronic acid (HA) enhanced the delivery efficiency of FAM-SMN to epidermal cells. At 60 min, the delivery efficiency with HA addition was 21.6%, while the delivery efficiency without HA addition was only 8.1%, and this efficiency was positively correlated with time. Figure 3 (H).

[0193] To verify the efficacy of M24-SMN in antagonizing skin inflammatory diseases, the above methods were applied to an IMQ-induced psoriasis model and an ultraviolet radiation-induced sunburn model in mice. Figure 3 Middle I).

[0194] The results showed that a single dose of M24-SMN significantly suppressed the inflammatory phenotype of the skin in both the psoriasis and sunburn models. Compared with control mice treated with SCR-SMN, the efficacy of M24-SMN was specifically manifested in reducing scaling and erythema on the skin surface, reducing epidermal thickening, reducing the expression of the inflammatory marker K6 in the epidermis and the infiltration of F4 / 80+ macrophages in the skin, and reducing the levels of inflammatory factors such as IL-6 and 1137 in the skin. Figure 3 (JN). This indicates that M24-SMN is an effective anti-inflammatory agent for the skin.

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[0212] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of an active ingredient selected from the group consisting of: (a) miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof having a core sequence of 5'-GGCUCAG-3' at the 5' end, a length of 16-28 nt, and a function identical or substantially identical to that of miR-24; (b) a precursor miRNA capable of being processed into the miRNA of (a) in a host; (c) a polynucleotide capable of being transcribed into the precursor miRNA of (b) and processed into the miRNA of (a) in a host; (d) an expression vector containing the miRNA of (a), or the precursor miRNA of (b), or the polynucleotide of (c); and (e) an agonist of the miRNA of (a); for the preparation of a pharmaceutical composition or preparation for one or more applications selected from the group consisting of: (i) inhibiting skin inflammation; and (ii) preventing and / or treating a skin inflammatory disease. The skin inflammatory disease includes psoriasis, sunburn, allergic dermatitis, contact dermatitis, seborrheic dermatitis, neurodermatitis, drug eruption, acne, papules, redness of sensitive skin, and other skin inflammatory diseases. The function of inhibiting skin inflammation includes one or more functions selected from the group consisting of: (1) reducing the expression of inflammatory factors in epidermal cells; (2) reducing the infiltration of immune cells in the epidermis; (3) reducing epidermal hyperplasia and skin redness; and wherein (4) inhibiting the NF-κB and JAK-STAT signaling pathways mediating inflammatory responses in epidermal cells.

2. Use according to claim 1, characterized in that, The sponge-spicule complex contains miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof having a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function identical or substantially identical to that of miR-24 as an active ingredient; and a pharmaceutically acceptable carrier.

3. Use according to claim 1, characterized in that, The pharmaceutically acceptable carrier includes a buffer and sodium hyaluronate.

6. A method for preparing the sponge-spicule complex of claim 4, the method comprising the steps of: (S1) dissolving sponge spicules in a buffer to prepare a sponge spicule solution; (S2) adding an active ingredient selected from miR-24 or a modified miR-24 derivative, or a miRNA or a modified miRNA derivative thereof having a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function identical or substantially identical to that of miR-24 to the sponge spicule solution prepared in step (S1); (S3) adding sodium hyaluronate to the solution obtained in step (S2) to obtain the sponge-spicule complex.

4. A sponge-bone pin composite, characterized by, The pharmaceutical composition contains the sponge-spicule complex of claim 4, and a pharmaceutically acceptable carrier.

5. The sponge-bone needle composite of claim 4, wherein, ​ ​ ​ ​ ​ 7. A pharmaceutical composition, characterized by, ​ 8. A cosmetic composition characterized in that, The cosmetic composition contains miR-24 or a modified miR-24 derivative, or an miRNA or a modified miRNA derivative thereof having a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function identical or substantially identical to that of miR-24 as an active ingredient, or the sponge spine complex as claimed in claim 4; and a cosmetically acceptable carrier.

9. A kit characterized in that, The kit comprises: (Z1) a drug disposed in a container, the drug being selected from the group consisting of: The sponge spine complex as claimed in claim 4, or the pharmaceutical composition as claimed in claim 7; and (Z2) a surgical film of the adhesive type.

10. A microneedle array, characterized by, The microneedle contains an active ingredient selected from the group consisting of miR-24 or a modified miR-24 derivative, or an miRNA or a modified miRNA derivative thereof having a core sequence of 5'-GGCUCAG-3', a length of 16-28 nt, and a function identical or substantially identical to that of miR-24.

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