A tgf-beta modulating peptide and compositions, articles of manufacture and uses thereof

Abstract

The present application relates to the technical field of TGF-beta regulating peptide, and particularly relates to a TGF-beta regulating peptide, a composition, a product and an application thereof.The amino acid sequence of the TGF-beta regulating peptide comprises: (a) any one of the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 20; (b) an amino acid sequence obtained by modifying the N terminal and / or C terminal of the TGF-beta regulating peptide defined in (a) and having the same or similar function.The TGF-beta regulating peptide provided by the present application can effectively activate the TGF-beta / Smad signal pathway, promote the synthesis of collagen and inhibit the generation of sebum, play the roles of resisting skin aging and regulating oil secretion, and has a wide application prospect in the fields of medicine, food and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of TGF-β regulatory peptide technology, and more particularly to a TGF-β regulatory peptide, its composition, products and applications. Background Technology

[0002] The transforming growth factor-β (TGF-β) signaling pathway is a highly conserved regulatory network that is widely involved in cell proliferation, differentiation, ECM (extracellular matrix) remodeling, immune homeostasis, and tissue repair.

[0003] In skin anti-aging, TGF-β-Smad2 / 3 is the central axis for maintaining fibroblast ECM homeostasis. Photoaging and deterioration of the mechanical microenvironment weaken the signaling efficiency of this axis, leading to ECM breakage and repair imbalance, and amplification of aging phenotypes. With age and the accumulation of photoaging, TGF-β receptor expression, Smad transcriptional coupling and mechanical feedback are remodeled, and the collagen regeneration capacity required for anti-aging decreases.

[0004] In sebaceous gland cells, TGF-β1 downregulates key genes for lipid production / differentiation such as FADS2 and PPARγ through the TβRII-Smad2 pathway, maintaining sebaceous cells in a state of "low differentiation and low lipid synthesis," thereby reducing sebum synthesis and secretion. However, with age or inflammatory stimulation, TGF-β signaling activity decreases, leading to increased sebum secretion, manifested as excessive oil and skin problems such as acne.

[0005] Therefore, how to effectively regulate the TGF-β signaling pathway and improve TGF-β signaling activity has become a technical problem that urgently needs to be solved in the industry. Summary of the Invention

[0006] The purpose of this invention is to solve the problem of insufficient or blocked activation of the TGF-β signaling pathway. A TGF-β regulatory peptide is proposed, which can effectively activate the TGF-β / Smad signaling pathway, promote collagen synthesis and inhibit sebum production, thereby playing a role in anti-skin aging and regulating sebum secretion.

[0007] According to a first aspect of this application, a TGF-β regulatory peptide is provided, wherein the amino acid sequence of the TGF-β regulatory peptide is: (a) any one of the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 20; or (b) any one of the amino acid sequences that have the same or similar function obtained by modifying the N-terminus and / or C-terminus of the TGF-β regulatory peptide defined in (a).

[0008] Optionally, the amino acid sequence of the TGF-β regulatory peptide described in (a) is any one of the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.20.

[0009] Optionally, the amino acid sequence of the TGF-β regulatory peptide described in (a) is any one of the amino acid sequences shown in SEQ ID NO.17 and SEQ ID NO.20.

[0010] According to a second aspect of this application, a composition comprising the above-described TGF-β regulatory peptide is provided.

[0011] According to a third aspect of this application, an article comprising the above-described TGF-β regulatory peptide or the above-described composition is provided.

[0012] Optionally, the article may also include acceptable excipients.

[0013] Specifically, the excipients are other basic components that do not affect the structure or properties of the TGF-β regulatory peptide.

[0014] Optionally, the product may be a pharmaceutical, daily chemical product, health product, or scientific research reagent.

[0015] Specifically, when the product is a tablet, excipients may include fillers such as starch or other pharmaceutically acceptable excipients; when the product is an injectable drug or other liquid product, excipients may include solvents, buffers, solubilizers, chelating agents, etc., which do not affect the structure or properties of the TGF-β regulatory peptide; excipients in cosmetics may include emollients, emulsifiers, thickeners, humectants, pH adjusters, skin protectants, preservatives, etc.; of course, excipients may also be the basic components in other dosage forms that do not affect the structure or properties of the TGF-β regulatory peptide.

[0016] Specifically, the medication is in the form of tablets, capsules, gels, granules, powders, injections, films, sprays, creams, or topical patches.

[0017] Optionally, the daily chemical products are cosmetics, shampoos, oral hygiene products, or bath products.

[0018] Specifically, cosmetics are available in the form of creams, lotions, emulsions, serums, or patches; health supplements are available in the form of oral liquids or capsules.

[0019] According to a fourth aspect of this application, an application is provided for the above-mentioned TGF-β regulatory peptide, composition or product in the prevention, improvement or treatment of diseases related to skin aging, abnormal sebum secretion, and extracellular matrix metabolic imbalance.

[0020] Skin aging-related problems include: photoaging, wrinkles, skin laxity, and skin atrophy; abnormal sebum secretion-related problems include: acne, seborrhea, poor wound healing, and chronic ulcers (such as diabetic foot ulcers, venous ulcers, and pressure injuries); extracellular matrix metabolic imbalance-related problems include: bone and joint diseases related to extracellular matrix metabolic imbalance.

[0021] The beneficial effects of using the embodiments of the present invention are as follows: The TGF-β regulatory peptides and their compositions and products provided in this application offer a variety of TGF-β regulatory peptides and their amino acid sequences, which can effectively activate the TGF-β / Smad signaling pathway, promote collagen synthesis and inhibit sebum production, thereby playing an anti-skin aging and sebum secretion regulating role. They have good safety and can not only be used to develop and treat bone and joint diseases related to skin aging, abnormal sebum secretion and extracellular matrix metabolic imbalance, but also can be widely used in functional cosmetics and health foods to meet the diverse needs of different groups for skin-related health. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] in: Figure 1 A schematic diagram illustrating the effects of different TGF-β regulatory peptides on the survival rate of human skin fibroblasts (BJ); Figure 2 A schematic diagram of ELISA detection showing the effects of different TGF-β regulatory peptides on type III collagen secretion in human skin fibroblasts (BJ); Figure 3 A schematic diagram of Western spectroscopy showing the effects of different concentrations of TG17 and TG20 on the expression of type I collagen in human skin fibroblasts (BJ). Figure 4 A schematic diagram of Western spectral analysis of the level of type III collagen expression in human skin fibroblasts (BJ) promoted by TG17 and TG20 at 40 µg / mL. Figure 5A schematic diagram of RT-qPCR detection of the mRNA expression levels of type I and type III collagen and fibronectin in human skin fibroblasts (BJ) promoted by TG17 and TG20 at 40 µg / mL. Figure 6 Schematic diagram of Western spectral analysis of the activity of TG17 and TG20 in activating the TGF-β signaling pathway in human skin fibroblasts at a concentration of 40 µg / mL. Figure 7 This is a schematic diagram illustrating the detection of the inhibition of adipogenesis in human sebaceous gland cells by 50 µg / mL TGB, TG16, TG17, and TG20. Detailed Implementation

[0024] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0025] This application provides a TGF-β regulatory peptide with the following amino acid sequence: (a) any one of the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 20; or (b) any one of the amino acid sequences obtained by modifying the N-terminus and / or C-terminus of the TGF-β regulatory peptide defined in (a).

[0026] Specifically, the amino acid sequences of SEQ ID NO. 1-SEQ ID NO. 20 are as follows: SEQ ID NO.1: SPLKRK.

[0027] SEQ ID NO.2: KPLKRQ.

[0028] SEQ ID NO.3: RSPLKR.

[0029] SEQ ID NO.4: SRLKRQ.

[0030] SEQ ID NO.5: SPRKRQ.

[0031] SEQ ID NO.6: KSPLKR.

[0032] SEQ ID NO.7: RPLKRQ.

[0033] SEQ ID NO.8: SPLKRR.

[0034] SEQ ID NO.9: SKLKRQ.

[0035] SEQ ID NO.10: SPLKRM.

[0036] SEQ ID NO.11: SPKKRQ.

[0037] SEQ ID NO.12: HPLKRQ.

[0038] SEQ ID NO.13: QPLKRQ.

[0039] SEQ ID NO.14: ERPLKR.

[0040] SEQ ID NO.15: APLKRQ.

[0041] SEQ ID NO.16: HSPLKR.

[0042] SEQ ID NO.17: NSPLRKR.

[0043] SEQ ID NO.18: YPLKRQ.

[0044] SEQ ID NO.19: EKPLKR.

[0045] SEQ ID NO.20: LPLKRQ.

[0046] The polypeptides corresponding to the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 20 are named TG1-TG20 respectively.

[0047] It should be noted that the mechanism by which TGF-β affects related physiological phenomena in this protocol is that it can restore ECM synthesis and inhibit MMP expression by improving pathway efficiency (such as receptor presentation and nuclear translocation). Further human and cellular evidence shows that activating upstream TGF-β signaling in an "early, short-term, low-dose" manner can initiate Smad signaling and improve photoaging phenotypes.

[0048] Activated TGF-β signaling further phosphorylates Smads proteins, which then enter the nucleus to regulate the transcription of downstream genes, including collagen, matrix metalloproteinases (MMPs), cell cycle factors, and various cytokines. This classical Smad pathway can also extend its signaling pathway through non-classical branches, such as the MAPK signaling pathway (including extracellular regulated protein kinase ERK, stress-activated protein kinase JNK, and p38MAPK), the PI3K-AKT-mTOR signaling pathway, and the Rho GTPases signaling pathway, thereby conferring a wider range of biological effects on TGF-β, including cell proliferation, migration, cell motility, adhesion, inflammatory responses, cell survival, anti-apoptosis, ECM remodeling, and metabolic regulation.

[0049] In skin anti-aging, after TGF-β ligand binds to TβRII / ALK5 (TβRI), the Smad2 / 3-Smad4 complex enters the nucleus, upregulates extracellular matrix proteins COL1A1 / COL3A1 and FN1, and inhibits MMP-1 / 3, thereby enhancing the integrity of the collagen and elastin network. In sebaceous gland cells, TGF-β1 downregulates key genes for lipid production / differentiation such as FADS2 and PPARγ through the TβRII-Smad2 pathway, maintaining sebaceous cells in a state of "low differentiation and low lipid synthesis", thereby reducing sebum synthesis and secretion.

[0050] Specifically, the amino acid sequence of the TGF-β regulatory peptide may include any one of the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 20, or may be based on any one of the amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 20, with addition, substitution, insertion, deletion or modification operations on its sequence, and the number of amino acid residues changed is one or more, preferably two, three or four.

[0051] The above-mentioned addition refers to the amino acid addition treatment, which means adding an amino acid to the C-terminus or N-terminus of any one of the above-mentioned amino acid sequences, as long as the TGF-β regulatory peptide has the same function.

[0052] The term "substitution" refers to amino acid substitution treatment, which means that an amino acid residue at any position in the above amino acid sequence is replaced by another amino acid residue, as long as the TGF-β regulatory peptide has the same function.

[0053] The above-mentioned insertion, namely amino acid insertion treatment, refers to the insertion of an amino acid residue at an appropriate position in any of the above-mentioned amino acid sequences. The inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other, as long as the TGF-β regulatory peptide has the same function.

[0054] The above-mentioned deletion, i.e., amino acid deletion treatment, refers to the deletion of one, two or three or more amino acids in any of the amino acid sequences, as long as the TGF-β regulatory peptide has the same function.

[0055] The above-mentioned modification, namely amino acid modification, refers to any modification of any one or more amino acids, as long as the TGF-β regulatory peptide has the same function.

[0056] In an optional embodiment, the amino acid sequence of the TGF-β regulatory peptide is any one of the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.20.

[0057] In an optional embodiment, the amino acid sequence of the TGF-β regulatory peptide is any one of the amino acid sequences shown in SEQ ID NO.17 and SEQ ID NO.20.

[0058] This application provides a composition comprising the above-mentioned TGF-β regulatory peptide.

[0059] This application provides an article comprising a TGF-β regulatory peptide or a composition thereof.

[0060] In an optional embodiment, the article of manufacture may also include acceptable excipients.

[0061] In optional embodiments, the product is a pharmaceutical, daily chemical product, health product, or scientific research reagent.

[0062] In an optional implementation, the daily chemical product is a cosmetic, shampoo, or bath product.

[0063] This application provides an example of the use of the above-mentioned TGF-β regulatory peptide, composition, or product in the prevention, improvement, or treatment of skin aging-related diseases.

[0064] In an optional embodiment, the above-mentioned TGF-β regulatory peptide, composition, or product is used in diseases related to abnormal sebum secretion.

[0065] In optional embodiments, the above-mentioned TGF-β regulatory peptide, composition, or product is used in the prevention, improvement, or treatment of diseases related to extracellular matrix metabolic imbalance.

[0066] In an alternative embodiment, the TGF-β regulatory peptide, composition, or article exerts its effect by modulating the activation of the TGF-β signaling pathway.

[0067] This application provides a method for preparing TGF-β regulatory peptide as described in the foregoing embodiments, the specific steps of which are as follows: (1) The synthesis sequence of TGF-β regulatory peptide is from C-terminus to N-terminus: 20 g of AM resin was placed in a reaction tube, 15 mL / g of DCM (dichloromethane) was added, and the mixture was shaken for 45 minutes. The DCM solvent was removed by vacuum filtration through a sintered glass core. 3 times the molar excess of Fmoc-Linker-OH (CAS: 145069-56-3) was added, followed by 6.5 times the molar excess of DIEA (N,N-diisopropylethylamine), and the mixture was shaken for 75 minutes. The mixture was blocked with methanol. DMF was removed by rotary evaporation, and 20 mL / g of 20% (V / V) piperidine-DMF solvent was added. After washing for 5 minutes, the solution was removed, and then 20 mL / g of 20% (V / V) piperidine-DMF solvent was added again. The mixture was washed for 20 minutes. The resin was rinsed twice with DMF (15 mL / g), DCM (15 mL / g), and DMF (15 mL / g) respectively, and the mixture was shaken for 40 minutes.

[0068] (2) Inoculation of the first amino acid: Remove the solvent DCM by vacuum filtration through a sand core filter, add 3.5 molar excess of Fmoc-Ala-OH, dissolve in DMF (dimethylformamide), then add 6.5 molar excess of DIEA (N,N-diisopropylethylamine), and shake for 70 minutes. Block with methanol.

[0069] (3) Deprotection: Evaporate DMF by rotary evaporation, add 20 mL / g 20% ​​(V / V) piperidine-DMF solvent, wash for 5 minutes, remove the DMF, add 15 mL / g 20% ​​(V / V) piperidine-DMF solvent, and wash for 20 minutes.

[0070] (4) Detection: Take 15 resin grains from the piperidine solution, wash them three times with ethanol, and add the test reagent. Heat at 105-110℃ for 8 minutes. A positive reaction is indicated when the color turns dark blue.

[0071] (5) Rinse the resin: Rinse the resin twice in sequence with DMF (15mL / g), DCM (15mL / g), and DMF (15mL / g).

[0072] (6) Condensation: Add three times the excess of DMF to dissolve the next amino acid derivative (from the C-terminus to the N-terminus), dissolve 3.5 times the excess of HBTU (benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) in DMF in the reaction tube, and then immediately add sixteen times the molar amount of DIEA and react for 40 minutes.

[0073] (7) Detection: Take 15 resin grains, wash them three times with ethanol, add the test reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction is a negative reaction.

[0074] (8) Rinse the resin: Rinse the resin twice in sequence with DMF (10mL / g), DCM (10mL / g), and DMF (10mL / g).

[0075] (9) Repeat steps (3) to (8) to connect the amino acids shown in SEQ ID No.1 from right to left.

[0076] (10) Drain and wash the resin as follows: DMF (15 mL / g) twice, methanol (15 mL / g) twice, DMF (15 mL / g) twice, DCM (15 mL / g) twice, and drain for 10 minutes.

[0077] (11) Cutting peptides from resin: The cutting solution used at 25°C consisted of 95% (V / V) TFA (trifluoroacetic acid), 1% (V / V) water, 2% (V / V) EDT (mercaptoethanol), and 2% (V / V) TIS (triisopropylsilane); the cutting time was 120 minutes, and the resulting lysate was obtained.

[0078] (12) Drying and washing: Dry the lysate with nitrogen, wash it six times with ether, and then let it evaporate at room temperature.

[0079] (13) Analysis, purification and freeze drying: The crude peptide was purified by high performance liquid chromatography; the peptide solution was collected and concentrated in a freeze dryer and freeze-dried to produce a white powder, thus obtaining an antimicrobial peptide with the structure SPLKRK.

[0080] The detection reagent is ninhydrin.

[0081] In this embodiment, TGF-β regulatory peptide SEQ ID NO:1 20, all of which can be directly synthesized using the methods described above.

[0082] Unless otherwise specified, the raw materials and instruments used in the embodiments of this application are as follows: instrument: ELISA kit: Jianglai Biotechnology, JL14257-96T; Super FastPure Cell RNA Isolation Kit: Novizan, RC102-01.

[0083] Universal high-sensitivity dye-based quantitative PCR detection kit (ChamQ Universal SYBR qPCRMaster Mix): Novozymes, Q711-02; Rapid Reverse Transcription Kit (cDNA Synthesis Kit): Yisheng, 11150ES60kit; Laser confocal microscope: NIKONAX; Oil Red O staining kit: Beyotime, C0158S.

[0084] Material: Human skin fibroblasts (BJ): Cell Bank of Chinese Academy of Sciences, GNHu49; Human sebaceous gland cells: SZ95, BluefBio, BFN60807569; Collagen Type I antibody: 67288-1-Ig, Proteintech, Wuhan Sanying Biotechnology Co., Ltd.; Collagen Type III antibody: 68320-1-Ig, Proteintech, Wuhan Sanying Biotechnology Co., Ltd.; p-Smad3(Ser425): Mouse, Santa Cruz Biotechnology, sc-517575, 1D9.

[0085] Example 1 This embodiment relates to the cytotoxicity detection of TGF-β regulatory peptide (hereinafter referred to as TG).

[0086] 1. Cell viability assay using human skin fibroblasts (BJ). ① Cell seeding plate: Human skin fibroblasts were taken and the cell density was adjusted to 1×10⁻⁶. 5 100 μL / well was seeded into a 96-well plate and cultured overnight until 80% confluence was reached. Then, 50 µg / mL of peptide was added and incubated at 37°C in a 5% CO2 incubator for 48 hours. ② Group culture: Blank control group: 200 μL of complete culture medium; Negative control group: Cells were added to 200 μL of complete culture medium; Positive control group: Cells were supplemented with 20 ng / mL TGF-β1; Experimental group: Cells were added with 50 µg / mL solutions of different peptides diluted with complete culture medium; ③ Cell viability assay: After incubation at 37℃ and 5% CO2 for 48 hours, collect the supernatant. For the remaining cells, use CCK-8 assay to detect cell viability: Add 20 μL of CCK-8 reagent to each well under dark conditions, gently vortex to mix, and incubate at 37℃ for 4 hours in the dark. Measure the absorbance (OD value) of each well at 450 nm using a microplate reader. If air bubbles or precipitates are present in the wells, centrifuge at 1000 rpm for 5 minutes before reading to eliminate interference. Perform three replicates for each concentration.

[0087] in: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of negative control group - OD value of blank group) × 100%.

[0088] Results Analysis: The test results are shown below. Figure 1 Different TG peptides had no significant effect on the survival rate of human skin fibroblasts, and the cell survival level was consistent with that of the negative control group. Therefore, the TG peptides at this concentration did not show obvious cytotoxicity and had good biosafety.

[0089] Example 2 This embodiment involves the detection of the regulatory effect of TGF-β regulatory peptide.

[0090] 1. Type III Collagen Secretion Test ① The cell seeding and grouping culture of human skin fibroblasts are the same as in Example 1; ②Detection of type III collagen content: After incubation at 37℃ and 5% CO2 for 48 hours, the supernatant was collected and the content of type III collagen was detected using an ELISA kit.

[0091] Results Analysis: The test results are shown below. Figure 2 All peptides can promote the secretion of type III collagen. Among them, TG17 and TG20 have better and more stable promoting effects. Therefore, in this scheme, TGF-β regulatory peptide can effectively promote the secretion of type III collagen, and TG17 and TG20 peptides will be further tested.

[0092] 2. Collagen expression detection ① Cell seeding: Human skin fibroblasts (BJ) were harvested and the cell density was adjusted to 1×10⁻⁶. 5 Inoculate 2 mL / well into a 6-well plate and incubate overnight until 50% confluence. ② Group culture: Blank control group: Cells were added to 1 mL of complete culture medium; Experimental group: Cells were added with 20, 40, and 80 µg / mL TG17 and TG20 peptide solutions diluted with complete culture medium; ③ Collagen content detection: After incubation at 37℃ and 5% CO2 for 48 hours, the supernatant was discarded, and the cells were washed twice with PBS. Cell lysis buffer was added, and the cells were lysed on ice to collect proteins. The content of type I and type III collagen was detected by Western blotting. Antibody information is as follows: Collagen Type I antibody (67288-1-Ig); Collagen Type III antibody (68320-1-Ig); β-tubulin. Using β-tubulin as a reference protein, the protein bands were semi-quantitatively analyzed using ImageJ software.

[0093] Results analysis: such as Figure 3 As shown, 20, 40, 80 µg / mL TG17 and TG20 peptide solutions can all promote the production of type I collagen, with 40 µg / mL TG17 and TG20 peptide solutions showing better promoting effects. like Figure 4 As shown, 40µg / mL TG17 and TG20 peptide solutions can promote the production of type III collagen.

[0094] 3. Detection of collagen and fibronectin mRNA expression ① Cell seeding: Human skin fibroblasts (BJ) were harvested and the cell density was adjusted to 1×10⁻⁶. 5 Inoculate 2 mL / well into a 6-well plate and incubate overnight until 50% confluence. ② Group culture: Blank control group: Cells were added to 1 mL of complete culture medium; Experimental group: Cells were added with 40 µg / mL TG17 and TG20 peptide solutions diluted with complete culture medium; ③ mRNA expression level detection: Incubate at 37℃ and 5% CO2 for 48 hours, discard the supernatant, wash twice with PBS, and extract RNA using an ultra-fast single-column animal cell total RNA extraction kit; reverse transcribe into cDNA using a rapid reverse transcription kit; use a universal high-sensitivity dye-based quantitative PCR detection kit to perform qPCR to detect the mRNA expression levels of COL1A1, COL3A1, and Fibronectin1 genes.

[0095] Results Analysis: The test results are shown below. Figure 5 40µg / mL TG17 and TG20 peptide solutions can increase the mRNA expression levels of type I and type III collagen and fibronectin.

[0096] 4. Detection of TGF-β signaling pathway activity ① Cell seeding: Human skin fibroblasts (BJ) are harvested and the cell density is adjusted to 5~10×10⁻⁶.4 Inoculate 100 μL / well into a 12-well plate and incubate overnight until 50% confluence. ② Group culture: Blank control group: Cells were added to 1 mL of complete culture medium; Positive control group: Cells were supplemented with 20 ng / mL TGF-β1; Experimental group: Cells were added with 40 µg / mL TG17 and TG20 peptide solutions diluted with complete culture medium; ③ Immunofluorescence detection: Incubate at 37℃ and 5% CO2 for 15 min, discard the supernatant and wash twice with PBS, fix with 4% paraformaldehyde for 2 h, label p-Smad3 protein with immunofluorescence staining, observe and collect fluorescence distribution using laser confocal microscopy, antibody information is as follows: p-Smad3 (Ser425).

[0097] Results Analysis: The test results are shown below. Figure 6 Treatment with 40 µg / mL TG17 and TG20 peptide solutions increased p-Smad3 expression in BJ cells, indicating that 40 µg / mL TG17 and TG20 peptides can activate the TGF-β signaling pathway.

[0098] 5. Sebaceous gland fat production detection ① Cell seeding: Human sebaceous gland cells were evenly seeded into 96-well plates and cultured overnight until 80% confluence was achieved. Then, the culture medium was replaced with fractionated medium (DMEM + P / S + 100uM Linoleic Acid). ② Group culture: Set up a blank control group: Add 1 mL of LA differentiation medium to the cells; Positive control group: Cells were supplemented with 50 ng / mL TGF-β1; Experimental group: Cells were added with 50µg / mL TG8, TG16, TG17, and TG20 peptide solutions diluted with LA differentiation medium, respectively; ③ Lipid distribution detection: After incubation at 37℃ and 5% CO2 for 48 h, the supernatant was discarded, the mixture was washed twice with PBS, stained with Oil Red O staining kit, and observed and photographed using an electron microscope.

[0099] Results Analysis: The test results are shown below. Figure 7 LA can induce sebaceous gland cells to produce lipids. 50 µg / mL TG16, TG17, and TG20 peptides can significantly inhibit this effect, while 50 µg / mL TG8 has no significant inhibitory effect.

[0100] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A TGF-β regulatory peptide, characterized in that, The amino acid sequence of the TGF-β regulatory peptide contains a continuous PLKR tetrapeptide motif, and the amino acid sequence is any one of the following sequences: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 or SEQ ID NO.

20.

2. The TGF-β regulatory peptide according to claim 1, characterized in that, The amino acid sequence of the TGF-β regulatory peptide is any one of the amino acid sequences shown in SEQ ID NO.3, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.

20.

3. The TGF-β regulatory peptide according to claim 1, characterized in that, The amino acid sequence of the TGF-β regulatory peptide is any one of the amino acid sequences shown in SEQ ID NO.17 and SEQ ID NO.

20.

4. A composition, characterized in that, It contains the TGF-β regulatory peptide according to any one of claims 1-3.

5. An article, characterized in that, It contains the TGF-β regulatory peptide as described in any one of claims 1-3 or the composition as described in claim 4.

6. The article of claim 5, characterized in that, The product also includes acceptable excipients.

7. The article of claim 5, characterized in that, The product is a drug, a daily chemical product, or a scientific research reagent.

8. The article of claim 7, characterized in that, The daily chemical products mentioned are cosmetics, shampoos, or bath products.

9. The use of the TGF-β regulatory peptide as described in any one of claims 1-3, the composition as described in claim 4, or the product as described in claims 5-8 in the prevention, improvement, or treatment of skin aging-related diseases; And / or its use in the prevention, improvement or treatment of diseases related to abnormal sebum secretion; The skin aging-related diseases mentioned are photoaging, wrinkles, skin laxity, or skin atrophy; The diseases associated with abnormal sebum secretion are acne or seborrhea.

10. The application according to claim 9, characterized in that, The TGF-β regulatory peptide, the composition, or the article exerts its effect by regulating the activation of the TGF-β signaling pathway.

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