A low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 and its applications

Abstract

The present invention provides a low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 and its applications. According to the general cell autophagy regulation mechanism and its theory, a small-molecule synthetic peptide is newly designed and synthesized, which can regulate the autophagy activity and effect improvement of human epidermal cells. The low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 of the present invention adopts a peptide sequence composed of 9 amino acids of five types of amino acids (threonine, proline, tyrosine, asparagine, and arginine), and its amino acid composition and sequence are SEQ ID No.1: TPYNRRRRY-NH2. After applying it to human immortalized epidermal cells (HaCat), it is confirmed that it can up-regulate the expression levels of positive autophagy-related proteins Beclin-1, LC3-II, LAMP1, and TFEB, and can down-regulate the expression level of the negative autophagy-related protein p62. The autophagy regulatory peptide DKX-9 of the present invention has positive significance and medical value for future intervention in skin aging and prevention and treatment of certain skin diseases, and plays an important role in promoting the research and application of synthetic peptides in related disciplines such as cell autophagy in the future.

Description

Technical Field

[0001] The present invention belongs to the interdisciplinary field of bioengineering technology, fine chemicals and skin anti-aging cosmetics, and specifically relates to the category of cell autophagy regulating peptides, in particular to a low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 and its application. Background Art

[0002] The epidermal tissues and cells of the human skin not only protect the body from external aggressors through the epidermal barrier, but also delay the aging of skin tissue by preventing the evaporation of water from the skin tissue through water retention or moisturizing mechanisms. The barrier construction of the skin epidermal tissue and the moisturizing and anti-aging effects of the skin epidermal tissue are both crucial and indispensable for the survival of the body and tissue protection. Like other cell types in the human body, skin tissue cells will also age and decline in structure, composition, and function over time and with the aging of the body. Many metabolic wastes and inactivated, aging cells and subcellular structures cannot be effectively removed. The physiological cell normality and homeostasis are constantly out of balance, accelerating skin aging and skin diseases.

[0003] Autophagy can effectively degrade and remove aged, degenerated, or mutated proteins, synthetically incorrect and mutated nucleic acids, as well as damaged and useless cellular substructures and organelles within skin tissue and its cells, and recycle the degraded proteins, nucleic acids, and organelles for cell survival, growth, proliferation, differentiation, regeneration, and repair. Therefore, maintaining normal autophagy is related to prolonging the lifespan of the body. Recently, a correlation between autophagy and longevity was discovered in nematodes, which provides corresponding genetic evidence for the relationship between autophagy and aging. The autophagy effect of epidermal cells plays an important role in skin physiology, and plays an important role in maintaining the normal structural state (normal state), cell stability (homeostasis), and metabolic balance (equilibrium) of skin epidermal tissue cells. A crucial part of these effects is achieved through the autophagic effect of human epidermal cells, which actively digests and degrades denatured or mutated metabolic proteins and their aggregates, as well as damaged or aged organelles (such as mitochondria, Golgi apparatus, and endoplasmic reticulum), genetically mutated nuclei, and their DNA. This is particularly crucial for the proper terminal differentiation of human epidermal keratinocytes. Furthermore, modern cytological research has confirmed that epidermal autophagy plays a crucial role in the terminal differentiation of the epidermal granular layer. Furthermore, current research also suggests that nuclear retention in epidermal cells is closely related to impaired autophagic function, contributing to the characteristic changes seen in skin aging and certain skin diseases.

[0004] Autophagy is crucial for maintaining normal metabolic balance in skin tissue. Severe disruption of autophagy can lead to decreased epidermal cell proliferation, one of the most important hallmark functions of epithelial stem cells. However, autophagic activity generally declines with aging. This gradual decline in autophagy with aging has significant implications for the structure and function of epidermal cells. In particular, the widespread and often misused use of certain chemical corrosives or exfoliants (such as hydroxy acids, fruit acids, retinoic acid, and trichloroacetic acid), physical abrasion, laser treatments, electrocautery, and cryotherapy in cosmetic medical procedures for "skin exfoliation," "chemical peels," or "whitening and freckle removal" has resulted in physical and chemical damage to the human epidermis, disruption of the normal skin barrier structure and function, thinning of the epidermis, decreased stratum corneum water content, and severe skin aging.

[0005] HaCaT cells are immortalized human epidermal cells, a non-tumor-derived immortalized keratinocyte cell line derived from normal human skin. They have similar differentiation characteristics to normal human keratinocytes and possess strong proliferation, growth, and differentiation capabilities. Therefore, in order to prevent human skin damage, disruption of normal skin barrier structure and function, thinning of epidermal tissue and reduction of stratum corneum water content, and severe skin aging, the development of more efficient and specific human epidermal cell autophagy regulatory peptides will undoubtedly have positive significance and medical value in promoting or promoting skin anti-aging, intervening in, and preventing certain skin diseases. Furthermore, it will play an important role in promoting or facilitating the development and application of synthetic peptides in the field of cellular autophagy and related disciplines. Summary of the Invention

[0006] To address the above-mentioned issues, the present invention provides a low-molecular-weight human epidermal cell autophagy-regulating peptide, DKX-9. Based on universal autophagy regulation mechanisms and theories, this newly designed and synthesized small-molecule synthetic peptide can modulate the autophagy activity and efficacy of human epidermal cells (emphasizing HaCat cells, a well-recognized human immortalized epidermal keratinocyte cell line, for validation). The present autophagy-regulating peptide, DKX-9, is synthesized artificially, featuring mature technology, convenient sourcing, batch uniformity, stable performance, balanced effects, and easily controlled quality and quantity. Furthermore, its low molecular weight, good water solubility, and safety are beneficial for its use without side effects. In the future, it could be used as an effective ingredient for epidermal cell autophagy regulation in topical preparations and cosmetics for human skin. This offers advantages and features over currently available autophagy regulators, such as those that rely on rare and expensive miRNAs from the transcriptome or genome, monomeric or complex components that are tediously extracted and difficult to purify from natural plants or Chinese herbal medicines, or the cytotoxic rapamycin.

[0007] Another purpose of the present invention is to lay the theoretical and material foundation for the future batch artificial synthesis of this low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9, and to use it as an epidermal cell autophagy regulator in the early intervention or clinical prevention and treatment of certain skin diseases, as well as to provide a new functional ingredient in the application research of skin anti-aging preparations and beauty cosmetics.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9, the amino acid sequence of the autophagy regulating peptide DKX-9 is: threonine (Thr)-proline (Pro)-tyrosine (Tyr)-asparagine (Asn)-arginine (Arg)-arginine (Arg)-arginine (Arg)-arginine (Arg)-tyrosine (Tyr)-NH2, and the general abbreviation sequence is: TPYNRRRRY-NH2 (as shown in SEQ ID No. 1).

[0010] In the present invention, the present invention newly designed and synthesized this low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 (hereinafter referred to as DKX-9) for human epidermal cells and applied it to a human epidermal cell experimental model. That is, when HaCat cells reached 80% confluence in vitro, the culture medium was replaced, and DKX-9 was added to these cell culture dishes at final concentrations of 25 μmol / L, 50 μmol / L, and 100 μmol / L, respectively. After co-culture for 24 hours, the morphology was observed using an inverted microscope, and the expression levels of several important autophagy marker molecules closely related to cell autophagy, autophagy-related factors, and lysosome-related proteins were detected by immunoglobulin blotting (Western blotting) and confocal immunofluorescence microscopy to observe its actual effect and effect on autophagy regulation when applied to human epidermal keratinocytes. In this way, the optimal action time and effective dose of this low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 on the autophagy level of human epidermal keratinocytes (HaCat cells) were explored and found.

[0011] The low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 of the present invention adopts a peptide sequence composed of five types of amino acids (threonine, proline, tyrosine, asparagine and arginine) and a total of 9 amino acids. Its amino acid composition and sequence are SEQ ID No. 1: TPYNRRRRY-NH2, with a purity of 97.54% and a measured molecular weight of 1280.97 Daltons, which is within a range of only 0.1% of the theoretical molecular weight.

[0012] As a preferred embodiment of the present invention, the autophagy regulating peptide DKX-9 is synthesized by an artificial synthesis method.

[0013] The above-mentioned autophagy regulating peptide DKX-9 is used to regulate autophagy in human epidermal keratinocytes.

[0014] As a preferred embodiment of the present invention, the autophagy regulating peptide DKX-9 is used in a skin external dosage form or a skin anti-aging preparation.

[0015] As a preferred embodiment of the present invention, the autophagy-regulating peptide DKX-9 can be used in facial cosmetics. The amino acids used in this invention are all essential amino acids abundant in skin tissue. Theoretically, they are non-toxic or non-toxic to skin tissue and its cells, and are non-irritating or non-allergenic. Furthermore, they possess mild physical and chemical properties, stable synthesis quality, low molecular weight, good water solubility, and are easily compatible with other natural medicines and their raw materials. They also exhibit good compatibility and compatibility, as well as a mild reaction, a pronounced onset of action, and a long-lasting effect. Furthermore, they offer the potential for covalently linking peptides or bioactive ingredients with low transdermal permeability or absorption to penetrate the skin and exert their effects. This provides a novel method and favorable conditions for their future use as a bioactive ingredient, or in combination with certain synergistic and synergistic natural medicines, ingredients, and active substances in topical skin preparations (including medical cosmetics and anti-aging skin preparations). The autophagy-regulating peptide DKX-9 has demonstrated significant efficacy in regulating autophagy in human epidermal keratinocytes.

[0016] As a preferred embodiment of the present invention, the autophagy regulating peptide DKX-9 with the optimal dosage and action time was detected, analyzed and identified by Western blotting, and it was found that it could significantly upregulate the expression levels of the positively regulating autophagy-related proteins Beclin-1 and LC3-II in human epidermal keratinocytes, while downregulating the expression of the negatively regulating autophagy protein p62, thereby achieving autophagy regulation in human epidermal keratinocytes.

[0017] As a preferred embodiment of the present invention, the autophagy regulating peptide DKX-9 with the optimal dosage and action time can achieve autophagy regulation in human epidermal keratinocytes by increasing the expression level of the autophagy lysosomal membrane protein LAMP1.

[0018] As a preferred embodiment of the present invention, the autophagy regulating peptide DKX-9 with the optimal dosage and action time achieves autophagy regulation in human epidermal keratinocytes by increasing the expression level of the key autophagy regulating transcription factor TFEB.

[0019] As a preferred embodiment of the present invention, the autophagy regulating peptide DKX-9 may be used in combination with drugs for delaying skin aging, preventing or treating skin diseases in the future.

[0020] As a preferred embodiment of the present invention, the effective concentration range of the autophagy regulating peptide DKX-9 is 25-100 μmol / L, and the optimal effective concentration range is 50 μmol / L.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1) The low-molecular-weight human epidermal cell autophagy-regulating peptide DKX-9 of the present invention was co-cultured with human epidermal keratinocytes (HaCat cells), a classical human epidermal cell experimental model recognized internationally in the same field, for 24 hours in vitro. Western blotting (WB) and confocal immunofluorescence microscopy were then used to detect the expression levels of several important autophagy marker molecules, autophagy-related factors, and lysosome-related proteins for efficacy testing and evaluation.

[0023] 2) The present invention not only designs, synthesizes, and detects and analyzes the sequence, amino acid composition, purity, and actual molecular weight of the low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9, but also experimentally studies and functionally identifies its actual role in regulating autophagy in human epidermal keratinocytes and its effects. Furthermore, through experimental research, the present invention explores and identifies the optimal duration and effective dose of this low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 for enhancing the autophagy level in human epidermal keratinocytes (HaCat cells).

[0024] 3) The present invention uses the low molecular weight human epidermal cell autophagy regulatory peptide DKX-9 to regulate the autophagy of human epidermal keratinocytes and observes its effect. At the same time, Beclin1, LC3, P62, lysosomal membrane protein LAMP1, TFEB and other indicators are selected to observe and evaluate its effect on the improvement of the autophagy level of human epidermal keratinocytes (HaCat cells). The above indicators are used to illustrate and explain that the low molecular weight human epidermal cell autophagy regulatory peptide DKX-9 of the present invention has a positive significance in regulating the autophagy mechanism of epidermal cells;

[0025] 4) The low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 of the present invention has a low molecular weight (approximately 1280 Daltons) and is rich in positively charged arginine, making it easy to bind to and interact with skin epidermal cells, especially keratinocytes in the epidermal cells. In addition, its low molecular weight facilitates skin penetration or absorption, while the positively charged arginine facilitates charge attraction between its arginine side groups and the negatively charged components on the epidermal cell membrane. Finally, through the assistance of epidermal cell membrane depressions, membrane receptors, and clathrin, it is more conducive to its non-covalent binding to the epidermal cell membrane and entering the cytoplasm of the epidermal cell to play its biological role in participating in and mediating epidermal cell autophagy regulation.

[0026] 5) The low-molecular-weight human epidermal cell autophagy-regulating peptide DKX-9 of the present invention does not damage or irritate skin tissue or its cells, nor does it have pharmacological irritation or biological allergenic properties. Furthermore, it has mild physical and chemical properties, stable synthesis quality, low molecular weight, good water solubility, and is easily compatible with other natural medicines and their raw materials. It also has good compatibility and compatibility, and exhibits a mild reaction, a pronounced onset of action, and a long-lasting effect. Furthermore, it has the potential to covalently link peptides or bioactive ingredients with low transdermal permeability or absorption rates to penetrate the skin and exert their effects. This provides a novel approach and favorable conditions, and lays the foundation and innovative possibilities for its future use as a bioactive ingredient, or in combination with certain synergistic and synergistic natural medicines, functional ingredients, and active substances, in topical skin preparations (including medical cosmetic and anti-aging preparations). BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of step 1) in Example 1.

[0028] Figure 2 It is a schematic diagram of step 2) to step 5) in Example 1.

[0029] Figure 3 It is a schematic diagram of step 6) to step 8) in Example 1.

[0030] Figure 4 It is a schematic diagram of step 9) to step 14) in Example 1.

[0031] Figure 5 It is a schematic diagram of step 15)-step 16) in Example 1.

[0032] Figure 6 The molecular weight of the autophagy regulating peptide DKX-9 of the present invention is identified by mass spectrometry analysis.

[0033] Figure 7 This is the HPLC analysis and identification of the actual purity of the autophagy regulating peptide DKX-9 of the present invention.

[0034] Figure 8 This is the morphological effect of the DKX-9 intervention of the present invention on human epidermal cells HaCat cells.

[0035] Figure 9 The expression levels of autophagy-related proteins Beclin 1, P62, LAMP1, LC3I, LC3II and internal reference GAPDH were changed.

[0036] Figure 10 It is the change in the expression levels of the autophagy regulatory factor TFEB and the internal reference GAPDH.

[0037] Figure 11 Confocal immunofluorescence technology was used to detect the effects of different concentrations of low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 on the expression levels of autophagy-related protein P62 and lysosome-related membrane protein LAMP1 after co-culture with HaCat for 24 hours.

[0038] Figure 12 Confocal immunofluorescence technology was used to detect the effects of different concentrations of low molecular weight human epidermal cell autophagy regulatory peptide DKX-9 on the expression of autophagy-related protein LC3-II and lysosome-related membrane protein LAMP1 after co-culture with HaCat for 24 hours.

[0039] Figure 13 Confocal immunofluorescence technology was used to detect the effects of different concentrations of low molecular weight human epidermal cell autophagy regulatory peptide DKX-9 on the expression level of the autophagy lysosome regulatory factor TFEB after co-culture with HaCat cells for 24 hours. DETAILED DESCRIPTION

[0040] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention, in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, and do not constitute a complete set of embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0041] The low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 of the present invention adopts a peptide sequence composed of five types of amino acids (threonine, proline, tyrosine, asparagine and arginine) and a total of 9 amino acids. Its amino acid composition and sequence are SEQ ID No. 1: TPYNRRRRY-NH2, with a purity of 97.54% and a measured molecular weight of 1280.97 Daltons, which is within a range of only 0.1% of the theoretical molecular weight.

[0042] The resin carrier used in this invention is AM resin, whose active sites are amino groups. Peptide solid-phase synthesis first requires swelling the resin, then reacting the C-terminal carboxyl group of the first amino acid with the active site amino group on the resin. After the first amino acid is attached to the resin, dehydration condensation is performed to attach the second amino acid. After condensation is complete, the Fmoc protection is removed. This process is repeated according to the designed amino acid sequence, and the remaining amino acids are attached in sequence. Finally, the peptide is cleaved from the resin using a cleavage reagent.

[0043] The main raw materials and reagents required for the synthesis of low molecular weight autophagy regulating peptide DKX-9:

[0044] Fmoc-L-Tyr(Tbu)-OH (tyrosine), Fmoc-L-Arg(Pbf)-OH (arginine), Fmoc-L-Asn(Trt)-OH (asparagine), Fmoc-L-Pro-OH (proline), and Fmoc-L-Thr(Tbu)-OH (threonine). The above protected amino acids were purchased from Chengdu Chengnuo New Technology Co., Ltd., Rink Amide-AM Resin (AM resin) was purchased from Zhejiang Puer Resin Co., Ltd., DMF (N,N-dimethylformamide) was purchased from Nanjing Runkai Chemical Glass Instrument Co., Ltd., DCM (dichloromethane) was purchased from Nanjing Runkai Chemical Glass Instrument Co., Ltd., acetonitrile was purchased from Nanjing Runkai Chemical Glass Instrument Co., Ltd., HBTU (benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate) was purchased from Suzhou Haofan Biological Co., Ltd., DIEA (N,N-diisopropylethylamine) Purchased from Suzhou Haofan Biotechnology Co., Ltd., TFA (trifluoroacetic acid) was purchased from Jinan Ruifu Chemical Co., Ltd., TIS (triisopropylsilane) was purchased from Shanghai Chuqing New Material Technology Co., Ltd., EDT (1,2-ethanedithiol) was purchased from Shanghai Yishi Chemical Co., Ltd., ether was purchased from Nanjing Runkai Chemical Glass Instrument Co., Ltd., piperidine was purchased from Nanjing Gutian Chemical Co., Ltd., ethanol was purchased from Nanjing Runkai Chemical Glass Instrument Co., Ltd., ninhydrin was purchased from Shanghai Qiangshun Chemical Reagent Co., Ltd., phenol was purchased from Jinan Huishi Chemical Co., Ltd., pyridine was purchased from Nanjing Runkai Chemical Glass Instrument Co., Ltd., and acetic anhydride was purchased from Nanjing Wanqing Chemical Glass Instrument Co., Ltd.

[0045] The main instruments and equipment required for the synthesis of low molecular weight autophagy regulating peptide DKX-9:

[0046] TDL-50 desktop low-speed large-capacity centrifuge (Changzhou Meixiang Instrument Co., Ltd.), HY-2 speed-adjustable multi-purpose oscillator (Changzhou Langyue Instrument Manufacturing Co., Ltd.), FD-1A-50 vacuum freeze dryer (Nanjing Pusen Instrument Equipment Co., Ltd.), SHZ-D(III) circulating water vacuum pump (Bangxi Instrument Technology Co., Ltd.), electronic balance (Changzhou Ruipin Precision Instrument Co., Ltd.), artificially designed peptide synthesizer, industrial nitrogen, LC3000 high-performance liquid chromatograph (Beijing Keruihai Scientific Instrument Co., Ltd.), Agilent 6120LC / MS (Agilent Technologies Co., Ltd.), etc.

[0047] Example 1

[0048] Chemical solid phase synthesis method and operation steps of low molecular weight autophagy regulating peptide DKX-9:

[0049] See also Figures 1 to 5 The specific method of the crude synthesis technology of low molecular weight autophagy regulating peptide DKX-9 is as follows:

[0050] (1) Resin swelling: Weigh 0.61 g of AM resin (0.078 mmol of the target peptide to be synthesized), with a degree of substitution of 0.9 mmol / g of AM resin (in multiple excess), place it in a reaction column, then add 20 ml of DCM to the column, shake for 30 min, and activate for later use;

[0051] (2) Deprotection: Remove the DCM solvent by filtration through a sand core, add 20 ml of 20% piperidine / DMF solution, and remove it after 5 min. Then add 20 ml of 20% piperidine / DMF solution and shake for 15 min.

[0052] (3) Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat at 105-110°C for 5 min. If the color turns dark blue, it is a positive reaction and you can proceed to the next amino acid. If it does not change color, it is a negative reaction and needs to be deprotected again.

[0053] (4) First wash: Wash twice with 15 ml of DMF, 15 ml of methanol, and 15 ml of DMF respectively;

[0054] (5) Connect the first amino acid: add 3 times the molar amount of Fmoc-L-Tyr(Tbu)-OH and 3 times the molar amount of HBTU, dissolve in a small amount of DMF, and immediately add 10 times the molar amount of DIEA, and react for 30 min.

[0055] (6) Detection: Remove the solvent, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat at 105-110°C for 5 min. If the reaction is colorless, it is a positive reaction. If it is blue, it needs to be re-condensed.

[0056] (7) Deprotection: Add 20 ml of 20% piperidine / DMF solution and remove after 5 min. Then add 20 ml of 20% piperidine / DMF solution and shake for 15 min.

[0057] (8) Detection: Drain the piperidine solution, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat at 105-110°C for 5 min. If the color turns dark blue, it is a positive reaction and you can proceed to the next amino acid. If it does not change color, it is a negative reaction and needs to be deprotected again.

[0058] (9) Washing: Wash twice with 15 ml DMF, 15 ml methanol, and 15 ml DMF respectively;

[0059] (10) Condensation: Add 3 times the molar amount of Fmoc-L-Arg(Pbf)-OH and 3 times the molar amount of HBTU, dissolve in a small amount of DMF, and immediately add 10 times the molar amount of DIEA, and react for 30 min.

[0060] (11) Detection: Remove the solvent, take a dozen resin pellets, wash them three times with ethanol, add one drop each of ninhydrin, pyridine, and phenol, and heat at 105-110°C for 5 min. If the reaction is colorless, it is a positive reaction. If it is blue, it needs to be re-condensed.

[0061] (12) Washing: Wash twice with 15 ml DMF, 15 ml methanol, and 15 ml DMF respectively;

[0062] (13) Peptide chain extension: Repeat the above steps to connect the remaining amino acids in sequence;

[0063] (14) Peptide contraction: Once the last amino acid is connected, the synthesis of the entire peptide is completed;

[0064] Entering the final shrinkage stage, the reaction was washed with DMF 3 times, DCM 3 times, and methanol 3 times, and finally the peptide resin was drained;

[0065] (15) Deprotection of amino acid side chains and resin cleavage: The synthesized amino acid sequence is TPYNRRRRY-NH2, which contains side chain protecting groups such as Pbf, Trt, and Tbu. These protecting groups are unstable under acidic conditions, and TFA is used to cleave the resin, so deprotection and resin cleavage can be carried out simultaneously;

[0066] (16) Prepare 15 mL of cutting solution, containing the following components in the following volume ratios: TFA (94.5%), water (2%), EDT (2.5%), and TIS (1%). Place the resin in a flask and shake at a constant temperature (30°C) for 2 h. Blow the lysate dry with nitrogen as much as possible, then pour it into a centrifuge tube and slowly add ether. Seal the tube and centrifuge for 5 min. Discard the supernatant, leaving a white solid below. Wash with ether six times, then evaporate to dryness at room temperature to obtain the crude peptide.

[0067] Specific method of the synthesis technology route of the pure low molecular weight autophagy regulating peptide DKX-9:

[0068] (a) Dissolution: Place the crude peptide in a dish and completely dissolve it in 30-50 mL of 50% acetonitrile in water. Slightly sonicate for 2 minutes.

[0069] (b) Filtration: Filter the solution through a 0.45 μm filter membrane;

[0070] (c) Analysis: Analyze the crude product by analytical HPLC using 3 μL of the solution for subsequent preparation. The mobile phase is water and acetonitrile, and the elution time is 30 min. The HPLC is equilibrated for 5 min with a starting gradient of 95% water, 5% acetonitrile, and the final gradient is 5% water, 95% acetonitrile.

[0071] (d) Preparation: Prepare the dissolved sample for injection. Equilibrate the preparative HPLC for 10 min with a starting gradient of 95% water, 5% acetonitrile, and a final gradient of 25% water, 75% acetonitrile over a 40-min gradient. Collect the sample from the detector.

[0072] Example 2

[0073] The low molecular weight autophagy regulating peptide DKX-9 prepared in Example 1 was subjected to high performance liquid chromatography and mass spectrometry analysis:

[0074] Table 1 shows the basic property indicators of amino acids used in the synthesis of DKX-9 and their protective modifications.

[0075] Table 1. Basic property indexes of amino acids used in DKX-9 synthesis and their protective modifications

[0076]

[0077] As shown in Table 1, a total of nine amino acids from five different species were used in the synthetic synthesis of DKX-9. All amino acid raw materials were protected with corresponding protecting groups. The original main-chain amino groups were all protected with Fmoc, while some amino side chains containing reactive groups were also protected with various side-chain protecting groups, such as Pbf, Trt, and Tbu, to prevent side reactions and interference during target peptide synthesis. Therefore, the molecular weights of the amino acids after adding protecting groups, as shown in Table 1, were increased compared to the corresponding unmodified molecular weights. Therefore, the relative molecular weights of the amino acids with protecting groups were used as the basis for calculating the theoretical amount of amino acid raw materials required.

[0078] Figure 6 It is a low molecular weight human epidermal cell autophagy regulating peptide DKX-9 identified by mass spectrometry analysis. Figure 6It can be seen that the actual molecular weight of the low-molecular-weight human epidermal cell autophagy-regulating peptide DKX-9 was determined by mass spectrometry analysis to determine the actual molecular weight of the synthetic peptide. Two target peaks with different charges were obtained through mass spectrometry analysis: a [M+2H]2+ mass-to-charge ratio of 641.43, with a measured molecular weight of 641.43*2-2=1280.86 Daltons, and a [M+3H]3+ mass-to-charge ratio of 427.99, with a measured molecular weight of 427.99*3-3=1280.97 Daltons. Compared with the theoretical molecular weight of 1280.42 Daltons, the errors of the actual molecular weights measured by mass spectrometry were within the allowable error of 0.1%, confirming that the two were basically consistent.

[0079] HPLC analysis:

[0080] The actual purity of the low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 synthesized in Example 1 was measured using high-performance liquid chromatography (HPLC), that is, the purity of the low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 was analyzed and identified using HPLC technology to measure the actual purity of the synthetic peptide. The LC3000 high-performance liquid chromatograph used had the following chromatographic analysis conditions: C18, reverse phase, 4.6mm*150mm, gradient elution. The starting gradient was 5% A+95% B, the ending gradient was 30% A+70% B, the time was 30min, the flow rate was 1.0mL / min, the UV detection wavelength was 214nm, and the injection volume was 10μL. Mobile phase A was 0.1% trifluoroacetic acid and 100% acetonitrile, and mobile phase B was 0.1% trifluoroacetic acid and 100% water. See the results. Figure 7 With Table 2.

[0081] Table 2. Experimental record and calculation of the purity of the low molecular weight human epidermal cell autophagy regulating peptide DKX-9

[0082] Serial number Peak time / min purity / % Peak area / mV*min 1 6.102 1.262 30918 2 6.895 97.54 2391112 3 7.628 0.233 5711 4 8.913 0.965 23888

[0083] From Table 2 and Figure 7 The results show that the low molecular weight human epidermal cell autophagy regulating peptide DKX-9 pure product was analyzed by HPLC, and its highest peak at the peak time of 6.895 was determined to be the peak with the largest area, which is also the target peak. This is the purity that the product can achieve. The product peak in Figure 7 can be seen through integration that its purity can reach 97.54%.

[0084] The present invention designs and synthesizes a low-molecular-weight human epidermal cell autophagy regulator DKX-9 based on cellular autophagy-related signaling pathways, particularly molecular mechanisms that moderately regulate the autophagy activity and function of human epidermal cells and their signaling pathways. The amino acid composition and sequence of the regulator DKX-9 are TPYNRRRRY-NH2, the purity is 97.54%, and the measured molecular weight is 1280.97 Daltons, with an error of only 0.1% from the theoretical molecular weight. This confirms that the low-molecular-weight human epidermal cell autophagy regulator DKX-9 synthesized by chemical solid-phase synthesis is successful. This provides a scientific basis and material foundation for modifying the regulator DKX-9 with a transdermal delivery carrier in the future to prepare different topical skin formulations, apply the regulator DKX-9 to studies on human epidermal cell autophagy regulation and skin aging and their related molecular mechanisms, and use the regulator DKX-9 to identify new targets for intervention, treatment, and delaying skin aging.

[0085] Example 3

[0086] In this example, a low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 was applied to a human epidermal cell experimental model, i.e., it was co-cultured with human epidermal keratinocytes (HaCat cells). Then, immunoblotting (Western blotting, WB) was used to detect the expression levels of several important cell autophagy marker molecules, autophagy-related factors, and lysosome-related proteins to observe its actual effect and effect on the autophagy regulation of human epidermal keratinocytes, thereby exploring and finding the optimal action time and effective dose of this low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 on the autophagy level of human epidermal keratinocytes (HaCat cells).

[0087] The raw materials, reagents, main instruments and equipment, main utensils and consumables required for implementation are as follows:

[0088] Human immortalized epidermal cells (HaCat) (Cell Bank, Chinese Academy of Sciences), autophagy-regulating peptide DKX-9 (synthesized by Shanghai Taopu Biotechnology according to our design), high-glucose DMEM medium (Gibco), high-quality fetal bovine serum (Gibco), trypsin (Gibco), RIPA lysis buffer (Biyuntian), PMSF (Biyuntian), 100× phosphatase inhibitor cocktail (Beijing Pulilai Gene Technology Co., Ltd.), BCA protein kit (Thermo); prestained standard protein marker (BIO-RAD), 2× protein loading buffer (BIO-RAD), 30% Acr-Bis (29:1) (Biosharp), 10% ammonium persulfate (LEGGENE), 1.5 M Tris solution (BIO-RAD), 1 M Tris solution (BIO-RAD), 10% SDS (VETEC), paraformaldehyde (Shanghai Shenggong), skim milk powder (BIO-RAD), TEMED (MP-1000) Biomedicals products), β-mercaptoethanol (MACKLIN products); ECL luminescent solution (BIO-RAD), TBS-T rinsing buffer (Boster Biotechnology), PBS phosphate buffer (Maixin Biotechnology Development Co., Ltd.), BSA bovine serum albumin (BioFROXX); GAPDH antibody (Proteintech), LAMP1 antibody (Mybiosource), TFEB antibody (Affinity), p62 antibody (Cell Signaling), Beclin 1 antibody (Affinity), LC3B antibody (Abcam), DAPI staining solution (Solarbio), anti-mouse FITC (MilliporeSigma), anti-rabbit CY3 (MilliporeSigma), HRP-conjugated goat anti-rabbit IgG (H+L) (Biyuntian Company), HRP-conjugated goat anti-mouse IgG (H+L) (Biyuntian Company).

[0089] CO2 cell culture incubator (Thermo), ultra-clean workbench (Thermo), inverted microscope (Leica), inverted fluorescence microscope (Leica), 1 / 10,000 electronic balance (METTLER TOLEDO), low-temperature centrifuge (Eppendorf, 5804R), enzyme-labeled analyzer (BIO-RAD), ultra-low temperature refrigerator (Thermo, 907), electrophoresis apparatus (BIO-RAD), membrane transfer apparatus (BIO-RAD), sample gun (Eppendorf), etc.

[0090] Disposable 25cm 2Plastic culture flasks (corning), 2 mL plastic cryovials (corning), 0.22 μm microporous filters (corning), 15 mL centrifuge tubes (corning), 6-well culture plates (corning), 24-well culture plates (Corning), pathology-grade microscope slides (Site Corporation), microscope cover slips (Site Corporation), PVDF membranes (Merck Millpore), and medical X-ray films (Carestream).

[0091] First, the culture and passage of HaCat cells

[0092] DMEM high glucose medium containing 10% fetal bovine serum (FBS) was used at 37°C and 5% CO2. 2 Culture the cells in a culture flask. Change the medium or subculture the cells according to their growth rate and status. Subculture the cells once they reach approximately 80% to 90% confluency. For subculture, add 0.25% trypsin to digest for approximately 1 minute, then add 2 mL of DMEM high-glucose medium containing 10% FBS to terminate the reaction. Transfer the entire solution in the culture flask to a centrifuge tube and centrifuge at 1000 rpm / min for 5 minutes. Discard the supernatant, re-add DMEM high-glucose medium containing 10% FBS, pipette to mix thoroughly, and then culture the cells in separate flasks. Subculture is complete.

[0093] Intervention with DKX-9:

[0094] Concentration and preparation: The DKX-9 stock solution concentration is 5 mmol / L; the final working concentrations of DKX-9 are: 25 μmol / L, 50 μmol / L, and 100 μmol / L. Usage: When human epidermal Hacat cells reach 80% confluency, replace the culture medium and add the corresponding concentration of drug to each culture dish. Gently shake in a figure-8 motion to ensure complete coverage. Protein extraction is performed 24 hours later.

[0095] Protein extraction and Western blotting detection

[0096] Extraction of total cell protein

[0097] Add 2 mL of PBS to gently rinse the cells, then add 120 μL of Lysis Buffer (with protease inhibitors added) and place on ice for 15 minutes. Scrape the cells with a cell scraper and centrifuge at 10,000 × g at 4°C for 10 minutes. Aspirate the supernatant and transfer it to a new EP tube to obtain the total cell protein.

[0098] Determination of protein concentration

[0099] (1) Use a BCA protein quantification kit (Thermo Scientific) containing Solution A, Solution B, and a protein standard solution (2 mg / mL). Mix (A+B) working solution is prepared at a ratio of 50:1. Prepare a gradient protein standard and a standard curve according to Table 3.

[0100] Table 3. Gradient protein standards

[0101]

[0102] (2) Prepare a clean, sterilized white 96-well plate. Add 199 μL of Mix (A+B) to each well, followed by 1 μL of the diluted standard protein solution. Mix thoroughly and place in a 37°C water bath for 30 min. Measure the absorbance of the protein sample at 562 nm using a UV spectrophotometer and calculate the standard curve.

[0103] (3) Take a clean, disinfected white 96-well plate and add 199 μL of Mix (A+B) to each well. Add 1 μL of the protein solution to be tested. Mix thoroughly and place in a 37°C water bath for 30 min. Use a UV spectrophotometer to measure the absorbance of the protein sample at 562 nm and calculate the protein concentration using the standard curve formula.

[0104] Western blotting

[0105] Reagent configuration and storage are as follows:

[0106] 1) For making glue

[0107] (1) 10% SDS

[0108] Add distilled water to 100 mL of SDS 10.0 g. If dissolving is difficult, dissolve in a 50°C water bath and store at room temperature.

[0109] (2) 10% ammonium persulfate (AP)

[0110] Prepare 1.0 g of ammonium persulfate and 10 mL of distilled water, divide into smaller portions, and store at -20°C in the dark.

[0111] (3) 0.5 M Tris HCl (pH 6.8), 1.5 M Tris HCl (pH 8.8), 30% acrylamide and TEMED were all stored at 4°C.

[0112] 2) Electrophoresis and transfer buffer

[0113] (1) 5× electrophoresis buffer

[0114] Prepare 30.0 g of Tris (MW 121.14), 144.0 g of glycine (MW 75.07), and 10.0 g of SDS by adding distilled water to 2000 mL. Dissolve the solution and store at room temperature. Dilute the solution 5-fold when needed. Usually, 200 mL is enough to make up 1000 mL.

[0115] (2) 10× transfer buffer

[0116] Add 144.0g of glycine (MW75.07), 30.3g of Tris (MW121.14), and 1.5g of SDS to 1000mL in distilled water. Dissolve and store at room temperature. Dilute 10-fold before use and add methanol to 20%. Typically, take 200mL of the mother liquor, add 1400mL of distilled water, and finally add 400mL of methanol to make 2000mL. Note that adding methanol first can easily cause precipitation.

[0117] 3) Membrane washing solution

[0118] (1) 5×TBS buffer

[0119] 22.4 g of Tris (MW 121.14) and 292.0 g of NaCl were added to 2000 mL with distilled water, and the pH was adjusted to 7.6 with concentrated hydrochloric acid. The mixture was dissolved and stored at room temperature.

[0120] (2) 1×TBST buffer

[0121] Mix 200 mL of 5× TBS buffer, 800 mL of distilled water, and 0.75 mL of Tween-20. Store at room temperature. Because Tween-20 is quite viscous, pipette slowly and cut off a piece of the pipette tip to prevent bubbles.

[0122] 4) Blocking solution

[0123] (1) Blocking solution (5% skim milk):

[0124] Dissolve 50 mL of 1× TBST buffer and 2.5 g of skim milk powder in the solution. Store at 4°C and use within one week.

[0125] SDS-PAGE protein electrophoresis, membrane transfer, immunoreaction and chemiluminescence detection:

[0126] 1) Casting a protein polyacrylamide gel (SDS-PAGE): Prepare a 5% stacking gel and a 15% separating gel. Steps: First, cast the separating gel. Allow the gel to polymerize for 30 minutes at room temperature before casting the stacking gel. Insert the comb and wait approximately 30 minutes for the stacking gel to fully polymerize before starting electrophoresis.

[0127] 2) Mix the sample with 5× loading buffer at a ratio of 1:4, incubate in a 100°C water bath for 5 minutes, cool on ice for 5 minutes, and add the sample to each well.

[0128] 3) Use a Bio-Rad vertical electrophoresis apparatus with the following electrophoresis parameters: constant voltage 80 V for stacking gel and V for separation gel.

[0129] 4) Protein transfer: After electrophoresis, remove the gel, cut away the stacking gel, and cut a PVDF membrane according to the size of the gel. Soak the PVDF membrane in methanol for 2 minutes, then transfer it to transfer buffer and soak it with the sponge and filter paper for 10-15 minutes. Add transfer buffer to an enamel tray, unfold the gel holder, and stack the sponge, a piece of filter paper, gel, PVDF membrane, a piece of filter paper, and sponge in order from the cathode (black) plate to the anode plate. Use a clean glass rod to remove any bubbles in the gaps. Place the holder vertically into the electrophoresis tank, place in an ice box and pre-chilled transfer buffer, and transfer at a constant voltage of 100V for 120 minutes, with a starting current of approximately 150-180mA and a termination current of less than 350mA.

[0130] 5) After transfer, place the PVDF membrane in a 10-fold diluted Ponceau stock solution and stain for 5 minutes to observe whether the target protein band is transferred.

[0131] 6) Cut the Ponceau-stained PVDF membrane according to the required protein size, then wash it three times with distilled water and then three times with TTBS, each for 5 minutes.

[0132] 7) Block the membrane with 5% skim milk powder solution at room temperature for 1 hour, shaker speed 120 rpm, and then wash with TTBS three times, each time for 5 minutes.

[0133] 8) Add primary antibodies diluted in 5% BSA (rabbit LC3B antibody 1:3000, rabbit P62 antibody 1:3000, rabbit Beclin 1 antibody 1:2000, mouse GAPDH antibody 1:6000) and incubate at 4°C overnight (shaker speed 80 rpm, about 14-16 hours).

[0134] 9) Aspirate excess primary antibody and wash six times with TTBS on a shaker at 120 rpm for 5 minutes each time.

[0135] 10) Add secondary antibodies (goat anti-rabbit antibody 1:6000, goat anti-mouse antibody 1:6000), incubate with the membrane at room temperature for 2 hours, and wash with TTBS 6 times, 5 minutes each time.

[0136] 11) Prepare luminescent solution (1 mL of solution A: 1 mL of solution B), return to room temperature, and place the membrane in the luminescent solution in a dark room for reaction.

[0137] 12) Remove the membrane and wrap it in plastic wrap to prevent wrinkles. Use filter paper to absorb excess luminescent liquid. Place the membrane in a dark box and place the front of the membrane in contact with the film. Press firmly and expose the membrane. The exposure time depends on the brightness of the band.

[0138] 13) Develop and fix the film. After drying, scan the film into grayscale images and perform quantitative analysis using grayscale software (Image J, NIH). GAPDH was used as an internal reference to calculate the relative expression of total protein.

[0139] Immunofluorescence detection and analysis

[0140] Cells were fixed with 4% paraformaldehyde for 30 minutes and placed in a 24-well plate in a wash box. Approximately 150 μL of normal nonspecific goat serum blocking buffer was added to each well and incubated with shaking at room temperature for 1 hour. The primary antibody of interest (LAMP1, 1:100; beclin1, 1:100; P62, 1:100; LC3B, 1:100) was added at a concentration of approximately 200 μL per well. The plates were then incubated with shaking overnight at 4°C. The next day, the plates were removed and rinsed with PBS for 5 minutes x 6 at room temperature. Approximately 200 μL of the corresponding fluorescent secondary antibody (anti-mouse FITC and anti-rabbit CY3, 1:100) was added to each well. The plates were incubated with shaking at room temperature for 2 hours in the dark, followed by rinsing with PBS for 5 minutes x 3 times. Finally, the plates were mounted with aqueous mounting medium containing DAPI and observed and imaged under a fluorescence microscope.

[0141] Example 4

[0142] According to the detection method of Example 3, this example describes the effect of DKX-9 on the expression of autophagy-related proteins in HaCat cells by Western blotting. Figure 8 , Figure 9 and Figure 10 .

[0143] Figures 8 to 10 Figure 2 (A): Effects of DKX-9 on the morphology of human epidermal HaCat cells. Figure 2 (BF): Western blotting analysis of the expression levels of autophagy-related proteins Beclin 1, P62, LAMP1, LC3I, LC3II, and the internal reference protein GAPDH after 24 hours of coculture of DKX-9 and HaCat cells. Figure 2 (GH): Western blotting analysis of the expression levels of the autophagy regulatory factor TFEB and the internal reference protein GAPDH after 24 hours of coculture of DKX-9 and HaCat cells.

[0144] See also Figure 8 Different concentrations (final concentrations of 25 μmol / L, 50 μmol / L, and 100 μmol / L) of low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 were co-cultured with HaCat cells for 24 hours. Observation under an inverted microscope and film analysis showed no toxic reaction in the HaCat cells, and the cell morphology and structure were normal.

[0145] See also Figure 9 and Figure 10 After HaCat cells were treated with DKX-9 for 24 hours, DKX-9 at three concentrations, namely 25 μmol / L, 50 μmol / L, and 100 μmol / L, promoted the expression of positive autophagy-related protein beclin1 and autophagy regulatory factor TFEB protein; while DKX-9 at a concentration of 50 μmol / L could increase the level of lysosome-related protein LAMP1; in addition, DKX-9 at concentrations of 25 μmol / L and 100 μmol / L could downregulate the expression level of negative autophagy-related protein p62; at the same time, DKX-9 at concentrations of 50 μmol / L and 100 μmol / L could promote the expression level of positive autophagy-related protein LC3II.

[0146] Therefore, the low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 can regulate the expression levels of autophagy-related proteins in HaCat cells, promote the increase of autophagy activity in HaCat cells and enhance the autophagy effect.

[0147] Example 5

[0148] According to the detection method of Example 3, this example uses confocal immunofluorescence to detect the effect of DKX-9 on the expression of autophagy-related proteins in HaCat cells. Figure 11 , Figure 12 and Figure 13 .

[0149] Figure 11 Confocal immunofluorescence was used to examine the effects of different concentrations of the low-molecular-weight autophagy-regulating peptide DKX-9 on the expression of the autophagy-related protein P62 and the lysosomal-associated membrane protein LAMP1 in HaCat cells co-cultured for 24 hours. The figure shows the fluorescence intensity of LAMP1 and P62 in the control group (N), the DKX-9 25 μmol / L group, the DKX-9 50 μmol / L group, and the DKX-9 100 μmol / L group, respectively. Group N: control group; DKX-9: low-molecular-weight autophagy-regulating peptide; LMAP1: lysosomal-associated membrane protein 1; P62: Sequestosome-1, a selective autophagy substrate; DAPI: 4',6-diamidino-2-phenylindole.

[0150] Figure 12Confocal immunofluorescence was used to examine the effects of different concentrations of the low-molecular-weight autophagy-regulating peptide DKX-9 on the expression of the autophagy-related protein LC3-II and the lysosomal-associated membrane protein LAMP1 in HaCat cells after 24 hours of coculture. The figure shows the fluorescence intensity of LAMP1 and LC3B in the control group (N), the DKX-9 25 μmol / L group, the DKX-9 50 μmol / L group, and the DKX-9 100 μmol / L group, respectively. Group N: control group; DKX-9: low-molecular-weight autophagy-regulating peptide; LMAP1: lysosomal-associated membrane protein 1; LC3B: autophagosome-associated protein; DAPI: 4',6-diamidino-2-phenylindole.

[0151] Figure 13 Confocal immunofluorescence was used to examine the effects of different concentrations of the low-molecular-weight human epidermal autophagy-regulating peptide DKX-9 on the expression of the autophagy-lysosomal regulatory factor TFEB in HaCat cells cocultured for 24 hours. The figure shows the intensity of TFEB fluorescence in the control group (N), the DKX-9 25 μmol / L group, the DKX-9 50 μmol / L group, and the DKX-9 100 μmol / L group. Group N: control group; DKX-9: low-molecular-weight autophagy-regulating peptide; TFEB: transcription factor EB; DAPI: 4',6-diamidino-2-phenylindole.

[0152] Figure 11 , Figure 12 and Figure 13 The results showed that confocal immunofluorescence detection revealed that after 24 hours of treatment with the low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9, the fluorescence intensity of TFEB, LAMP, and LC3B increased significantly in HaCat cells, and TFEB was transferred from the cytoplasm to the nucleus. At the same time, the results of fluorescent double-labeling detection showed that under the action of the low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9, the co-labeling of lysosome-associated membrane protein LMAP1 with autophagosome-associated proteins P62 and LC3B in HaCat cells increased, also suggesting that it promoted the increase in the level of autophagy in HaCat cells.

[0153] The experimental results of Western blotting detection showed that the low-molecular-weight cellular autophagy regulator DKX-9 at final concentrations of 25μmol / L, 50μmol / L, and 100μmol / L could upregulate the expression level of beclin-1 in HaCat cells; DKX-9 at a concentration of 50μmol / L could increase the expression level of lysosome-associated protein LAMP1; while DKX-9 at concentrations of 25μmol / L and 100μmol / L could reduce the level of p62 protein; at the same time, DKX-9 at concentrations of 50μmol / L and 100μmol / L could promote the increase of LC3II level, showing a trend of enhancing the regulatory effect on the autophagy effect of HaCat cells.

[0154] The low-molecular-weight autophagy regulator DKX-9 also upregulated TFEB total protein expression in HaCat cells at final concentrations of 25 μmol / L, 50 μmol / L, and 100 μmol / L. TFEB, through nuclear translocation, regulates the transcription of multiple key genes involved in autophagy and lysosome formation and maturation, thereby enhancing autophagic activity and function. This suggests that TFEB total protein expression may be one of the key factors contributing to DKX-9's enhanced autophagy effect in human epidermal keratinocytes.

[0155] Furthermore, confocal immunofluorescence experiments also demonstrated that 24 hours after DKX-9 treatment, the fluorescence intensities of TFEB, LAMP1, and LC3II increased, with TFEB gradually translocating from the cytoplasm to the nucleus. Furthermore, double-labeling revealed that co-labeling of LMAP1 with the autophagosome-associated proteins P62 and LC3II increased in HaCat cells following DKX-9 treatment, suggesting that DKX-9 treatment elevates autophagy in HaCat cells.

[0156] These results indicate that different doses of the low-molecular-weight human epidermal cell autophagy-regulating peptide DKX-9 can modulate the expression levels and fluorescence intensities of autophagy-related proteins in HaCat cells to varying degrees, effectively promoting increased autophagic activity and enhancing the autophagic effect in HaCat cells. The biological effects of DKX-9 in inducing enhanced autophagy in human epidermal keratinocytes are likely related to its regulatory mechanisms on the TFEB signaling pathway.

[0157] Within the acceptable range of existing preparations, the low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 of the present invention can be matched with other natural medicines and their raw materials, or it can be used alone or in combination with certain synergistic and synergistic natural medicines, functional ingredients and active substances in skin topical preparations (including medical beauty and skin anti-aging preparations, etc.). The effective concentration range of the low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 of the present invention is 25-100 μmol / L, and the optimal effective concentration range is 50 μmol / L.

[0158] As for how to prepare the low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 of the present invention, there are some conventional methods in the prior art, which will not be described in detail here.

[0159] The low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 of the present invention and its application undoubtedly have positive significance and medical value in promoting or promoting skin anti-aging and intervening in and preventing certain skin diseases, and also play an important role in promoting or facilitating the development and application of polypeptides in the field of cell autophagy and related disciplines.

[0160] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention. Sequence Listing <110> Guangzhou Haomei Bioengineering Technology Co., Ltd. <120> A low-molecular-weight human epidermal cell autophagy regulating peptide DKX-9 and its application <141> 2021-11-30 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 9 <212> PRT <213> Artificial sequence (DKX-9) <400> 1 Thr Pro Tyr Asn Arg Arg Arg Arg Tyr 1 5

Claims

1. A low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9, characterized in that, The amino acid sequence of the autophagy regulatory peptide DKX-9 is shown as SEQ ID No.

1.

2. The low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 according to claim 1, characterized in that, The autophagy regulatory peptide DKX-9 is synthesized by the artificial chemical solid-phase synthesis method.

3. Use of a low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 according to claim 1, characterized in that, The application of the low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 in the preparation of a facial beauty cosmetic composition for autophagy regulation of human epidermal keratinocytes, and the composition is a topical skin beauty cosmetic dosage form or a skin anti-aging beauty cosmetic preparation.

4. Use of a low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 according to claim 3, characterized in that, The autophagy regulatory peptide DKX-9 regulates autophagy of human epidermal keratinocytes by upregulating the expression levels of the positive autophagy-related proteins Beclin-1 and LC3-II and downregulating the expression level of the negative autophagy-related protein p62.

5. Use of a low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 according to claim 3, characterized in that, The autophagy regulatory peptide DKX-9 regulates autophagy of human epidermal keratinocytes by increasing the expression level of LAMP1.

6. Use of a low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 according to claim 3, characterized in that, The autophagy regulatory peptide DKX-9 regulates autophagy of human epidermal keratinocytes by increasing the expression level of TFEB.

7. Use of a low-molecular-weight human epidermal cell autophagy regulatory peptide DKX-9 according to claim 3, characterized in that, The effective concentration range of the autophagy regulatory peptide DKX-9 is 25 - 100 μmol / L.

Images