Application of PCSK9 inhibitor in preparation of anti-aging and tissue repair products

Abstract

The invention belongs to the technical field of medicines and skin care products, and particularly relates to application of a proprotein convertase subtilisin / kexin9 type (PCSK9) inhibitor in anti-aging and tissue repair products. The PCSK9 gene is characterized in that the PCSK9 belongs to a proprotein converting enzyme family. The PCSK9 inhibitor is a PCSK9 small molecule compound inhibitor, a PCSK9 monoclonal antibody, a PCSK9 small interfering RNA (Ribonucleic Acid), a PCSK9 mimic peptide inhibitor, a PCSK9 mimic antibody protein, a PCSK9 antisense oligonucleotide or a PCSK9 vaccine, and is applied to preparation of anti-aging and tissue repair products. A systematic or external PCSK9 inhibitor product can be further developed and is used for resisting aging and repairing tissues.

Description

Technical Field

[0002] The present invention belongs to the technical fields of medicine and skin care products, and particularly relates to the application of PCSK9 inhibitors in anti-aging and tissue repair products. Background Art

[0003] The pathogenesis of aging is complex. Aging is an all-round, multi-factor, systematic and irreversible process under the combined influence of genetics and environment, often accompanied by the decline of the body's stress ability and structural degeneration. The skin is the outermost organ of the human body and can reflect the health and aging status of the body. The occurrence of skin aging is considered to be the result of the combined action of internal and external factors. Skin aging often manifests as thinning of the epidermis, shrinkage of the dermis, reduction in thickness, thinning of collagen fibers and even disordered arrangement and breakage. During the aging process, aging cells often show a trend of decreasing proliferation ability due to the decline in protein synthesis ability, dysregulation of immune system-related gene expression, etc. The decrease in cell proliferation ability is an important feature of aging.

[0004] Autophagy is a "self-phagocytosis" phenomenon of cells, mainly referring to the process in which cells use lysosomes to degrade their own damaged organelles and macromolecular substances under the regulation of autophagy-related genes, in order to achieve the management and quality control of intracellular proteins and organelles. The accumulation of intracellular damaged substances is a common feature of all aging cells and can lead to a decrease in the survival ability of the living organism. Autophagy can degrade damaged or aging organelles, proteins and other structures in cells, and is the main catabolic pathway in cells, and can participate in aging and various physiological and pathological processes related to aging. Research has confirmed that autophagy is a key link among various factors regulating aging and aging-related diseases. In aging brain cells, the expression of autophagy-related proteins decreases, and the decline in autophagy activity can cause the enrichment of aging-related modified proteins and damaged organelles in cells, inducing aging.

[0005] Telomeres are special heterochromatin nucleoprotein complexes at the ends of eukaryotic cell chromosomes. They can shorten with cell division and are crucial for maintaining chromosome integrity and cell stability. Their shortening can lead to cell aging. The anti-aging effect is related to promoting telomere stability, and the main mechanisms include antioxidant, activating telomerase activity in the biological characteristics of organism aging.

[0006] The sebum film is a transparent film covering the skin surface, mainly formed by the emulsification of sebum secreted by sebaceous glands, lipids produced by the disintegration of stratum corneum cells, and sweat secreted by sweat glands. It is weakly acidic, and its main components include squalene, linoleic acid, linolenic acid, and lipid components, which have the functions of locking in moisture and certain anti-inflammatory effects. The skin barrier is composed of the sebum film, keratin in the stratum corneum, lipids, "sandwich" structure, "brick wall" structure, dermal mucopolysaccharides, and mucopolysaccharides, etc., which can resist the entry of harmful substances, irritants, and sunlight from the outside world, and at the same time have the functions of moisturizing and regulating anti-inflammation. Damage to the skin barrier will cause skin dryness, skin aging, pigmentation, dermatitis, etc. Tissue repair includes wound healing, repair of skin barrier function, etc. Skin tissue repair is also beneficial to preventing local bacterial infections of the skin, assisting in the treatment of acute and chronic skin inflammations, and preventing the recurrence of skin inflammations, including seborrheic dermatitis, glucocorticoid-dependent dermatitis, eczema, atopic dermatitis, and psoriasis, etc.

[0007] Maintaining skin health, beauty, and delaying aging have always been the ideals of mankind, and more safe and effective products need to be found to meet the huge demand.

[0008] Proprotein convertase subtilisin / kexin type 9 (PCSK9) is a member of the proprotein convertase family. Proprotein convertases are secreted as inactive zymogens in the liver. The cDNA of the PCSK9 gene is 3617 bp in size and encodes a PCSK9 protein consisting of 692 amino acids. The PCSK9 precursor undergoes intramolecular autocatalysis in the endoplasmic reticulum to cleave its N-terminal propeptide. The separated N-terminal proregion is connected to the catalytic region, allowing the mature PCSK9 protein to leave the endoplasmic reticulum and enter the secretory pathway. After PCSK9 is secreted extracellularly, it binds to the low-density lipoprotein (LDL) receptor in the first epidermal growth factor-like region on the cell surface. The PCSK9-LDL receptor complex can enter the lysosome for degradation, resulting in a decrease in the LDL receptor on the cell surface, that is, the PCSK9 level is negatively correlated with the LDL receptor. And a number of studies have shown that the loss-of-function mutations of the PCSK9 gene can significantly reduce the LDL-C level and the incidence of coronary heart disease in different ethnic groups. In view of the significant effect of inhibiting PCSK9 on reducing the LDL-C level and the incidence of coronary heart disease, a number of treatment regimens are currently being developed to block PCSK9 drugs for reducing the LDL-C level and the incidence of coronary heart disease.

[0009] PCSK9 inhibitors include two major categories: 1. Prevent the binding of PCSK9 to LDL-R, such as monoclonal antibodies, mimetic peptides (polypeptide inhibitors), and antibody-mimetic protein drugs; 2. Inhibit the expression of PCSK9 molecules or interfere with the secretion of PCSK9, such as small interfering RNA, antisense oligonucleotides, small molecule compound inhibitors, etc. Currently, all the PCSK9-targeted monoclonal antibody drugs on the global market are drugs that prevent the binding of PCSK9 to LDL-R. These include evolocumab jointly developed by Amgen and Astellas, with the trade name Repatha; alirocumab jointly developed by Sanofi and Regeneron, with the trade name Praluent, as well as LY3015014 from Eli Lilly, the recombinant humanized anti-PCSK9 monoclonal antibody (JS002) from Junshi Biosciences, and the recombinant fully human anti-PCSK9 monoclonal antibody injection from Sunway Biopharma Co., Ltd. Clinical studies have found that the above drugs have good tolerance in the treatment of hypercholesterolemia, and there is no significant difference in the incidence of adverse reactions between the placebo group and the active treatment group. In addition, Inclisiran is an siRNA (small interfering RNA) drug. Different from monoclonal antibodies, it can inhibit the expression of the PCSK9 gene, preventing the LDL receptor from being degraded by PCSK9, thereby improving the uptake of LDL particles by hepatocytes and reducing the LDL level in the blood. Inclisiran from Novartis and Alnylam is composed of a lipid nanoparticle conjugated with GalNAc (N-acetylgalactosamine) using proprietary technology, and GalNAc can bind to the asialoglycoprotein receptors ASGR1 and ASGR2 expressed on the surface of hepatocytes. This technology allows subcutaneous administration and targeting of the liver. ALN-PCS and ALN-PCSsc from Affiris also belong to siRNA (small interfering RNA) drugs. The PCSK9 vaccine drugs designed by Pfizer, as well as the AT04A and AT06A vaccines from Affiris, enable patients to achieve long-term LDL reduction with just one vaccination per year, reducing the frequency of use. The research and development of PCSK9 mimetic peptides and PCSK9 antibody-mimetic protein drugs include MK-0616 from Merck Sharp & Dohme Corp., DS9001 from Pieris, and 1G08 from Merck & Co., Inc. The PCSK9 antisense oligonucleotide drugs include SPC5001 from Santaris Pharma A / S. Summary of the Invention

[0010] The problems to be solved by the present invention are as follows: to clarify the role of the PCSK9 gene in the anti-aging and tissue repair mechanisms through animal and cell models, and the application of PCSK9 inhibitors in the preparation of products for anti-aging and tissue repair. By establishing aging animal and cell models, the present invention discovers that the PCSK9 gene plays a key role in the pathogenesis of aging, as well as the application value of PCSK9 inhibitors in the preparation of anti-aging products. The present invention also discovers through cell scratch repair experiments that PCSK9 inhibitors have tissue repair functions.

[0011] The technical solution of the present invention: Among the preparations that can significantly block PCSK9, the present invention respectively selects representative PCSK9 monoclonal antibodies, PCSK9 polypeptide inhibitors, PCSK9 small molecule compound inhibitors, and PCSK9 small interfering RNAs. Experiments confirm that inhibiting PCSK9 has an obvious delaying effect on mouse aging. Based on the above research, the present invention relates to the application of PCSK9 inhibitors (blockers) in anti-aging products, wherein the PCSK9 belongs to the proprotein convertase family (genebank serial number: 255738); the causes of aging and tissue damage include endogenous and exogenous factors, etc.

[0012] The PCSK9 inhibitor described in the present invention can be any product or method that can inhibit the expression or secretion of the PCSK9 gene by conventional molecular biology or medicinal chemistry means, for example, but not limited to knocking out or silencing the PCSK9 gene through existing molecular biology techniques; in some embodiments, it can also be or adopt a PCSK9 inhibitor. Preferably, the above PCSK9 inhibitor (blocker) is a PCSK9 small molecule compound inhibitor or PCSK9 interfering RNA or PCSK9 monoclonal antibody or PCSK9 mimetic peptide inhibitor or PCSK9 mimetic antibody protein or PCSK9 antisense oligonucleotide or PCSK9 vaccine.

[0013] In some examples, the PCSK9 small molecule inhibitor described in the present invention includes but is not limited to the product R-IMPP of Selleck, chemical formula: C24H27N3O2, molecular weight: 389.49, structural formula: or the product PF-06446846 of Selleck, chemical formula: C22H21N7O, molecular weight: 434.5, structural formula: or the product SBC-115076 of Selleck, chemical formula: C31H33N3O5, molecular weight: 527.61, structural formula: or the product SBC-110736 of Selleck, chemical formula: C26H27N3O2, molecular weight: 413.51, structural formula: .

[0014] In some examples, the PCSK9 monoclonal antibody inhibitors of the present invention include, but are not limited to, evolocumab jointly developed by Amgen and Astellas, or alirocumab jointly developed by Sanofi and Regeneron, or the recombinant humanized anti-PCSK9 monoclonal antibody (JS002) of Junshi Biosciences, or the recombinant fully human anti-PCSK9 monoclonal antibody injection of Sunway Biopharma Co., Ltd., or LY3015014 of Eli Lilly and Company, etc.

[0015] In some examples, the PCSK9 siRNA (small interfering RNA inhibitor) of the present invention includes, but is not limited to, Inclisiran of Novartis and Alnylam, ALN-PCS and ALN-PCSsc of Affiris, etc.

[0016] In some examples, the PCSK9 polypeptide inhibitors and protein inhibitors of the present invention include, but are not limited to, MK-0616 of Merck Sharp & Dohme Corp., DS9001 of Pieris, and 1G08 of Merck. The amino acid sequences of the PCSK9 polypeptide and protein inhibitors include, but are not limited to, the following amino acid sequences: glutamic acid-tyrosine-proline-tyrosine-lysine-histidine-serine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EYPYKHSGYYHRP; or glutamic acid-tyrosine-threonine-phenylalanine-aspartic acid-tyrosine-serine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EYPYDYSGYYHRP; or glutamic acid-tyrosine-threonine-phenylalanine-lysine-histidine-serine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EYTFKHSGYYHRP; or glutamic acid-tyrosine-proline-tryptophan-lysine-glycine-serine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EYPWKGSGYYHRP; or glutamic acid-tyrosine-threonine-tyrosine-aspartic acid-glycine-alanine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EYT YNGAGYYHRP; or glutamic acid-phenylalanine-proline-phenylalanine-lysine-tryptophan-serine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EFPFKWSGYYHRP; or glutamic acid-phenylalanine-aspartic acid-phenylalanine-valine-glycine-alanine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EFDFVGAGYYHRP; or glutamic acid-tyrosine-threonine-tyrosine-lysine-glycine-serine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EYT YKGSGYYHRP; or glutamic acid-phenylalanine-proline-phenylalanine-lysine-tryptophan-serine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EFPFKWSGYYHRP; or glutamic acid-phenylalanine-proline-tryptophan-proline-histidine-alanine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EFPWPHAGYYHRP; or glutamic acid-tyrosine-alanine-phenylalanine-glutamic acid-glycine-alanine-glycine-tyrosine-tyrosine-histidine-arginine-proline, abbreviated as: EYAFEGAGYYHRP. Chinese Name English Name Three-Letter Abbreviation One-Letter Symbol Glycine Glycine Gly G Alanine Alanine Ala A Valine Valine Val V Leucine Leucine Leu L Isoleucine Isoleucine Ile I Proline Proline Pro P Phenylalanine Phenylalanine Phe F Tyrosine Tyrosine Tyr Y Tryptophan Tryptophan Trp W Serine Serine Ser S Threonine Threonine Thr T Cysteine Cystine Cys C Methionine Methionine Met M Asparagine Asparagine Asn N Glutamine Glutarnine Gln Q Aspartic Acid Asparticacid Asp D Glutamic Acid Glutamicacid Glu E Lysine Lysine Lys K Arginine Arginine Arg R Histidine Histidine His H

[0017] In some examples, the PCSK9 antisense oligonucleotide inhibitor described in the present invention includes, but is not limited to, SPC5001 of Santaris Pharma.

[0018] In some examples, the PCSK9 vaccine inhibitors described in the present invention include, but are not limited to, AT04A and AT06A of Affiris, and the PCSK9 vaccine drugs designed by Pfizer.

[0019] Ki67 is an important indicator of cell proliferation ability. The present invention constructs a natural aging model of mice. The research shows that the percentage of Ki67-positive cells in the old group is lower than that in the young group. After inhibiting PCSK9, the expression of Ki67 in mouse tissues increases, suggesting that inhibiting PCSK9 can promote cell proliferation by upregulating the expression of intracellular Ki67, and thus play an anti-aging role. By detecting the morphological changes of the skin through HE and Masson staining, it is found that the old group is significantly lower than the young group, the dermis thickness becomes thinner, and the positive percentage of total collagen decreases; compared with the old group, the epidermal thickness of the skin of mice in the PCSK9 inhibition group increases, the dermis thickens, and the positive percentage of total skin collagen increases. It shows that inhibiting PCSK9 can delay the natural aging of mice.

[0020] Cellular senescence is an irreversible state of arrested proliferation. Senescent cells show characteristic phenotypes and molecular characteristics both in vitro and in vivo. Senescence-specific SA-β-Gal and oxidative stress biochemical indicators are currently commonly used for detecting senescence characteristics. In this study, a method of inducing senescence of human skin fibroblasts with D-gal was used to construct a senescent cell model. In this study, representative different types of PCSK9 inhibitors were used on D-gal-induced senescent fibroblasts, and it was found that PCSK9 inhibitors could significantly increase the survival rate of senescent cells.

[0021] A large amount of reactive oxygen species (ROS) can be produced in senescent cells, leading to oxidative stress and causing cell damage. MDA, SOD, and GSH-Px are commonly used oxidative stress biochemical indicators. MDA is the product of lipid peroxidation in the body and can reflect the degree of cell damage caused by excessive ROS; SOD can inhibit the production of MDA and free radical metabolites, scavenge superoxide anions in cells, and slow down the aging of the body; GSH-Px is an important antioxidant and free radical scavenger in the body. In this study, PCSK9 inhibitors can restore the activities of SOD and GSH-Px in cells of the senescent model group and significantly reduce the content of MDA.

[0022] p53 and p16 are the most commonly used senescence markers at present. p53 induces the expression of the cell cycle inhibitor p21, resulting in cell growth arrest. The expression of p16 increases in senescent cells. In this study, under the induction of D-gal, the expressions of p53 and p16 proteins were significantly up-regulated compared with the control group. After adding the PCSK9 inhibitor, the protein expressions of p53 and p16 were significantly down-regulated compared with the senescence model group. The PCSK9 inhibitor can achieve the purpose of delaying senescence by reducing the activity of SA-β-Gal and weakening cellular oxidative stress.

[0023] In in vitro efficacy models, cell migration or translocation experiments are relatively mature and effective in vitro evaluation methods for tissue repair and reconstruction functions. Fibronectin has a strong effect on promoting cell migration, so it was used as a positive control in this experiment. Based on the in vitro Balb / c 3T3 cell scratch repair experimental model, it was detected whether inhibiting the PCSK9 gene is beneficial to cell recognition and translocation, so as to achieve the effect of tissue repair and reconstruction. When the cells grew to a confluent monolayer state, an artificial blank area was created on the confluent monolayer cells, which was called a "scratch". The cells at the scratch edge would gradually enter the blank area to heal the "scratch". Images were captured at the beginning and regularly during cell migration, and the cell migration rate was determined by comparing the images. The experimental results showed that inhibiting the PCSK9 gene has the ability to promote cell migration, indicating that inhibiting the PCSK9 gene has a tissue repair effect.

[0024] It is well known to those skilled in the art that based on the above action mechanism of PCSK9, PCSK9 inhibitors have a repair effect on senescence and tissue damage caused by various reasons. PCSK9 inhibitors can be used alone or in combination with other drugs or methods, including drugs and skin care products.

[0025] The present invention also provides a composition, which is composed of the compound or its pharmaceutically acceptable salt described in the present invention as the active ingredient or the main active ingredient, supplemented with an acceptable carrier.

[0026] The compound or composition described in the present invention can be prepared into any pharmaceutically acceptable dosage form, for example, a preparation suitable for any administration method such as topical skin application, oral administration, parenteral administration, intraperitoneal administration, intravenous administration, intraarterial administration, transdermal administration, sublingual administration, intramuscular administration, rectal administration, buccal administration, intranasal administration, inhalation administration, vaginal administration, intraocular administration, topical administration, subcutaneous administration, intra-adipose administration, intra-articular administration, intraperitoneal administration or intrathecal administration.

[0027] In a preferred embodiment, the dosage form described in the present invention is a solution, tincture, spirit, liniment, spray, emulsion, ointment, gel, patch, aqueous solution, tablet, infusion, oral liquid, capsule, dripping pill, enema, film or injection.

[0028] The "product" described in the present invention can be any preparation suitable for administration to a patient according to the methods commonly used in the pharmaceutical field, including pharmaceuticals, skin care products, health care products, etc.

[0029] The product described in the present invention can be administered to mammals, such as rats, mice, livestock, humans, etc.; in some specific examples, it is humans.

[0030] The beneficial effects of the present invention compared with the prior art are as follows: The present invention provides a new and better method for inhibiting aging and tissue repair. Through the disclosure of the present invention, PCSK9 inhibitor (blocker) products can be further prepared, and then monomer or compound products containing various PCSK9 inhibitors can be developed for improving aging and tissue damage caused by various reasons. A series of new products with good effects and safety can be provided for the market. Description of the Drawings Figure 1 Cell states of the blank control group (NC), fibronectin group (FN), and si-PCSK9 group at 0 h and 24 h (40×): It can be seen that almost no cell migration was observed in the blank control group (NC), and the cell migration rates of the fibronectin group (FN) and the si-PCSK9 group were 100% and 72.26% respectively. Detailed Embodiments The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0033] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific embodiments, rather than for limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly specified in the text, the singular forms "a", "an", and "the" include the plural forms.

[0034] When embodiments give numerical ranges, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field of the present invention. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the technical field of the present invention and the description of the present invention, any methods, devices, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention, and all belong to the protection scope of the present invention.

[0035] Example 1 Effect of PCSK9 gene knockout on the senile mouse model 1. Experimental method 1.1 Animal grouping and model establishment Experimental animals: SPF-grade Kunming mice, PCSK9- / - Kunming mice (using CRISPR gene editing technology to knockout the Pcsk9 gene exon 2-3 to establish a Pcsk9 gene knockout mouse model).

[0036] The mice of different genotypes were divided into three groups: young mouse group, old mouse group, and old PCSK9- / - mouse group, with 10 mice in each group, 5 males and 5 females. The young group was fed with a normal diet for 8 months, and the two old groups were fed with a normal diet for 20 months to construct a natural aging model of mice.

[0037] 1.2 Observation indexes and test methods After the feeding was completed, the back skin tissues of the mice in each group were taken for HE staining to detect the thickness of the skin epidermis and dermis; Masson staining to detect the change of total collagen in the skin; immunohistochemistry to detect the change of the expression of the proliferation protein Ki67; Western blot to detect the change of the expression of autophagy-related proteins Beclin1, LC3Ⅱ / LC3Ⅰ, and p62. One-way analysis of variance was used for comparison between groups, and LSD-t test was used for pairwise comparison between groups.

[0038] 2. Experimental results 2.1 PCSK9 gene knockout can delay skin aging in mice HE staining showed that the epidermal thickness of the old group was lower than that of the young group (P < 0.05), while the epidermal thickness of the PCSK9 gene knockout group was higher than that of the old group (P < 0.05). The dermal thickness of the old group was thinner than that of the young group (P < 0.05), while the dermal thickness of the PCSK9 gene knockout group was higher than that of the old group (P < 0.05). The results of Masson staining showed that the positive percentage of total collagen in the skin of the old group was significantly lower than that of the young group (P < 0.01), while that of the PCSK9 gene knockout group was higher than that of the old group (P < 0.01). The results are shown in Table 1.

[0039] 2.2 PCSK9 gene knockout increases the proliferation ability of skin cells The immunohistochemical results of skin cells showed that the percentage of Ki67-positive cells in the old group was lower than that in the young group (P < 0.01), while that in the PCSK9 gene knockout group was higher than that in the old group (P < 0.01), and there was no significant difference between the PCSK9 gene knockout group and the young group (P > 0.05) (Table 1).

[0040] Table 1 Comparison of epidermal and dermal thickness, positive percentage of total skin collagen, and percentage of Ki67-positive cells in each group of mice (x ± s) Group Epidermal Thickness (um) Dermal Thickness (um) Total Skin Collagen Positive Percentage (%) Ki67 Positive Cell Percentage (%) Young Group 29.56±0.27* 271.41±9.63* 46.27±1.85* 30.15±3.23* Old Group 15.62±0.33 85.15±10.75 23.29±1.73 15.27±2.86 Old PCSK9 - / - Group 26.61±0.31* 206.26±8.49* 38.42±1.65* 26.63±2.46* * Indicates significant difference compared with the old group (P<0.01) 2.3 Effect of PCSK9 gene knockout on the expression of skin autophagy-related proteins Compared with the young group, the expression of Beclin-1 in the skin of the old group decreased (P < 0.05), the ratio of LC3Ⅱ / LC3Ⅰ decreased (P < 0.01), while the expression of p62 increased (P < 0.01); compared with the old group, the expression of Beclin-1 in the PCSK9 gene knockout group increased (P < 0.01), the ratio of LC3Ⅱ / LC3Ⅰ in the skin of mice increased (P < 0.01), while the expression of p62 decreased (P < 0.01). See Table 2.

[0041] Table 2 Expression of skin autophagy-related proteins in each group of mice (x ± s) Group Beclin1 LC3Ⅱ / LC3Ⅰ P62 Young Group 1.69±0.18* 3.6±0.37* 1.65±0.12* Old Group 0.36±0.12 1.83±0.06 2.46±0.32 Old PCSK9 - / - Group 1.68±0.19* 2.92±0.29* 1.67±0.23* * Indicates P<0.01 compared with the old group 3. Experimental conclusions PCSK9 gene knockout can delay skin aging in mice, and its mechanism may be related to increasing the proliferation ability of mouse skin cells and promoting the autophagy level of skin cells.

[0042] Example 2 Effect of PCSK9 inhibitor on senescent cells 1. Experimental methods 1.1 Materials (1)PCSK9 small interfering RNA-1 sequence and modification Gene 5'-3' Sense 5'-3' Antisense siPCSK9 - 1 GccuGGAGuuuAuucGGAAdT*dT UUCCgAAuAAACUCcAGGCdT*dT siPCSK9 - 2 AGGuGuAucuccuAGAcAcdT*dT GUGUCuAGGAGAuAcPCSK9UdT*dT After mixing siPcsk9-1 and 2 in equal amounts and diluting them to 20 µM with normal saline, the diluted siRNA was evenly mixed with the emollient lotion.

[0043] PCSK9 small interfering RNA-2: The RNA sequence is the same as Inclisran of Alnylam; PCSK9 small interfering RNA-3: The RNA sequence is the same as ALN-PCS of Affiris.

[0044] Compound inhibitor 1: R-IMPP from Selleck, chemical formula: C24H27N3O2, molecular weight: 389.49, structural formula: PCSK9 small molecule inhibitor 2: PF-06446846 from Selleck, chemical formula: C22H21N7O, molecular weight: 434.5, structural formula: PCSK9 small molecule inhibitor 3: SBC-115076 from Selleck, chemical formula: C31H33N3O5, molecular weight: 527.61, structural formula: PCSK9 small molecule inhibitor 4: SBC-110736 from Selleck, chemical formula: C26H27N3O2, molecular weight: 413.51, structural formula: PCSK9 small molecule inhibitor 5: CVI-LM001 from Civi Pharmaceutical; PCSK9 small molecule inhibitor 6: DC371739 from Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0045] (3)PCSK9 monoclonal antibody 1: evolocumab co-developed by Amgen and Astellas, trade name Repatha; PCSK9 monoclonal antibody 2: alirocumab co-developed by Sanofi and Regeneron, trade name Praluent.

[0046] (4)PCSK9 series of polypeptide inhibitors were synthesized by Sangon Biotech. The amino acid sequence of PCSK9 polypeptide-1 is: EYPYKHSGYYHRP; the amino acid sequence of PCSK9 polypeptide-2 is: EYTFKHSGYYHRP; the amino acid sequence of PCSK9 polypeptide-3 is: EYPWKGSGYYHRP; the amino acid sequence of PCSK9 polypeptide-4 is: EYTNGAGYYHRP; the amino acid sequence of PCSK9 polypeptide-5 is: EFPFKWSGYYHRP; the amino acid sequence of PCSK9 polypeptide-6 is: EYTYKGSGYYHRP; the amino acid sequence of PCSK9 polypeptide-7 is EFPFKWSGYYHRP; the amino acid sequence of PCSK9 polypeptide-8 is: EFPWPHAGYYHRP; the amino acid sequence of PCSK9 polypeptide-9 is: EYAFEGAGYYHRP; the amino acid sequence of PCSK9 polypeptide-10 is: EYPYDYSGYYHRP.

[0047] 1.2 Cell culture and establishment of senescent cell model Human skin fibroblasts were cultured in high-glucose DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO2, and the medium was changed once every 2 - 3 days. Passage was carried out when the cell confluence reached 70% - 80%. When the confluence of the passage cells reached 50%, 10 g / L D-gal was used to induce for 72 h to prepare an aging cell model.

[0048] 1.3 Detection of cell viability by CCK-8 method Determine the optimal concentration and optimal action time of PCSK9 inhibitors in each group.

[0049] 1.4 Cell grouping and senescence-specific SA-β-Gal staining 1.4.1 Cell grouping The cells were divided into a control group, an aging model group, and an aging model + PCSK9 inhibitor group. The PCSK9 inhibitor group included compound group 1, compound group 2, compound group 3, compound group 4, monoclonal antibody 1 group, monoclonal antibody 2 group, PCSK9 small interfering RNA group - 1, PCSK9 small interfering RNA group - 2, PCSK9 small interfering RNA group - 3, and polypeptide group 1, polypeptide group 2, polypeptide group 3, polypeptide group 4, polypeptide group 5, polypeptide group 6, polypeptide group 7, polypeptide group 8, polypeptide group 9, polypeptide group 10, combined group 1 (compound group 1 + small interfering RNA inhibitor group - 1), and combined group 2 (compound group 2 + polypeptide group 1). When the cell confluence in the aging model group reached 50%, 10 g / L D-gal was added for induction for 72 h; when the cell confluence in the aging model + PCSK9 inhibitor group reached 50%, 10 g / L D-gal was used for induction for 72 h, and then the corresponding PCSK9 inhibitors were added for treatment. The control group cells were not induced by D-gal and not treated with PCSK9 inhibitors. The PCSK9 inhibitors in each group were treated with the optimal concentration and optimal action time.

[0050] 1.4.2 SA-β-Gal staining Take cells with a confluence of 80%, inoculate them into 6-well plates at a density of 2×104 cells / well, with 1 well for each group, and treat them according to the above cell grouping method. Perform SA-β-Gal staining. The specific operation refers to the instruction manual of the SA-β-Gal cytochemical staining kit. Incubate overnight in an incubator at 37 °C without CO2. Observe the cell staining situation under an ordinary optical microscope. Randomly select 5 fields of view for each well, count the blue-stained cells, calculate the proportion of positive cells, and take the average value.

[0051] 1.5 Determination of SOD, GSH-Px activities and MDA content Human skin fibroblasts were cultured in cell culture flasks. The grouping and treatment methods were as described above. After trypsin digestion, the cells were centrifuged at 1000×g for 5 min. The cells in each group were collected and ultrasonically disrupted on ice bath to obtain cell lysates. According to the steps of the kit instruction manual, the OD values of each group at 550 nm, 340 nm, and 532 nm were measured using an enzyme-labeling instrument, and the activities of SOD and GSHPx and the content of MDA were calculated.

[0052] 1.6 Detection of the expression of hTERT, p53, and p16 proteins by Western blotting Total proteins of the cells in each group were extracted using RIPA lysis buffer. The protein concentration of the lysis supernatant was measured by the BCA method, and then electrophoresis, membrane transfer, and blocking were performed overnight on a shaker at 4 °C. Primary antibodies β-actin (1:3000), p53 (1:1000), p16 (1:500), and TERT (1:500) were added. The membranes were washed with TBST, secondary antibody (1:8000) was added, and the incubation was carried out at room temperature for 1 h. The membranes were washed with TBST, developed, and the gray values of the bands were analyzed using ImageJ software.

[0053] 1.7 Statistical analysis SPSS 22.0 software was used for statistical analysis, and GraphPad 7.0 software was used for statistical graphing. Each experiment was repeated 3 times. Measurement data were expressed as x±s. One-way ANOVA was used for comparison among multiple groups, and the LSD method was used for further pairwise comparison. P<0.05 was considered statistically significant.

[0054] 2. Experimental results 2.1 Effects of the PCSK9 inhibitor group on the activity of senescence-specific SA-β-Gal in each group of cells The results of SA-β-Gal staining showed that compared with the control group, the proportion of SA-β-Gal positive cells in the senescence model group was significantly increased (P<0.001); compared with the senescence model group, the proportion of SA-β-Gal positive cells in the senescence model + PCSK9 inhibitor group was significantly decreased (both P<0.001). See Table 3.

[0055] 2.2 Effects of PCSK9 inhibitor on the activities of SOD, GSH-Px and the content of MDA in each group of cells The results of enzyme-labeling instrument detection showed that compared with the control group, the activities of SOD and GSH-Px in the senescence model group were significantly decreased (both P<0.001), and the content of MDA was significantly increased (P<0.001), indicating that the senescence model was successfully established by D-gal induction and the antioxidant defense ability of the cells was reduced. Compared with the senescence model group, the activities of SOD and GSH-Px in the senescence model + PCSK9 inhibitor group were significantly increased (both P<0.001), and the content of MDA was significantly decreased (both P<0.001). See Table 3.

[0056] Table 3 Proportion of SA-β-Gal positive cells in each group (%) and activities of SOD, GSH-Px, and content of MDA Group SA - β - Gal Positive Cells (%) SOD (U / ml) GSH - Px (U / mg) MDA (mmol / L) Negative Control Group 9.1 25.35 5.83 1.78 Aging Model Group 86.2 8.56 2.46 7.95 Compound Group 1 38.3* 12.25* 4.23* 4.82* Compound Group 2 26.6* 16.32* 4.85* 3.32* Compound Group 3 28.5* 14.12* 4.66* 4.16* Compound Group 4 35.3* 14.46* 4.35* 4.42* Monoclonal Antibody Group 1 23.6* 17.83* 5.23* 3.19* Monoclonal Antibody Group 2 25.1* 17.65* 5.28* 3.28* Interfering RNA Group 1 38.3* 11.21* 4.28* 4.22* Interfering RNA Group 2 22.2* 13.23* 5.26* 3.58* Interfering RNA Group 3 23.8* 12.52* 5.18* 3.83* Peptide Group 1 21.7* 15.63* 4.86* 3.67* Peptide Group 2 23.5* 13.56* 4.35* 3.95* Peptide Group 3 28.3* 11.26* 4.27* 4.26* Peptide Group 4 26.7* 15.62* 4.13* 4.15* Peptide Group 5 32.2* 12.45* 3.96* 4.28* Peptide Group 6 29.5* 13.58* 3.85* 3.76* Peptide Group 7 32.6* 11.42* 3.93* 3.58* Peptide Group 8 31.8* 10.36* 4.08* 4.08* Peptide Group 9 27.9* 10.59* 3.83* 4.13* Peptide Group 10 28.5* 10.76* 3.91* 4.26* Combined Group 1 19.6* 20.26* 5.35* 2.87* Combined Group 2 21.5* 21.85* 5.46* 2.65* Note: *P < 0.001 compared with the senescence model group.

[0057] 2.3 Effects of PCSK9 inhibitors on the protein expressions of hTERT, p53, and p16 in cells of each group The results of Western blotting showed that compared with the control group, the expression level of hTERT protein in the senescence model group was significantly decreased (P < 0.01), while the expression levels of p53 and p16 proteins were significantly increased (P < 0.01), indicating that the senescent cell model was successfully constructed; compared with the senescence model group, the expression level of hTERT protein in the senescence model + PCSK9 inhibitor group was significantly increased (P < 0.01 for all), and the expression levels of p53 and p16 proteins were significantly decreased (P < 0.01 for all). See Table 4.

[0058] Table 4 Relative expression levels of hTERT, p53, and p16 proteins in each group Group hTERT p53 p16 Negative Control Group 0.62* 0.56* 0.62* Aging Model Group 1.89 1.28 1.65 Compound Group 1 0.56* 0.82* 0.97* Compound Group 2 0.51* 0.65* 0.73* Compound Group 3 0.55* 0.71* 0.75* Compound Group 4 0.58* 0.73* 0.81* Monoclonal Antibody Group 1 0.49* 0.63* 0.71* Peptide Group 0.79* 0.89* 0.95* Interfering RNA Group 1 0.59* 0.71* 0.82* Interfering RNA Group 2 0.48* 0.78* 0.69* Interfering RNA Group 3 0.45* 0.73* 0.72* Peptide Group 1 0.65* 0.69* 0.69* Peptide Group 2 0.71* 0.85* 0.75* Peptide Group 3 0.58* 0.87* 0.78* Peptide Group 4 0.61* 0.69* 0.69* Peptide Group 5 0.65* 0.76* 0.71* Peptide Group 6 0.63* 0.78* 0.76* Peptide Group 7 0.59* 0.69* 0.81* Peptide Group 8 0.62* 0.68* 0.83* Peptide Group 9 0.76* 0.81* 0.91* Peptide Group 10 0.73* 0.78* 0.89* Combined Group 1 0.33* 0.59* 0.63* Combined Group 2 0.36* 0.56* 0.66* Note: *There were significant differences compared with the senescence model group (P < 0.01 for all) 3. Experimental conclusions All kinds of PCSK9 inhibitors can reduce the antioxidant defense ability of cells and inhibit cell senescence by increasing the activities of SOD and GSH-Px, increasing the expression of hTERT, decreasing the content of MDA, and downregulating the expressions of p53 and p16.

[0059] Example 3 Effect of siRNA-PCSK9 on scratch repair of fibroblasts in vitro 1. Experimental materials Positive control: Fibronectin (FN) siRNA-PCSK9: Small interfering RNA plasmid of PCSK9 Fibroblast cell line: Balb / c 3T3 cells Other reagents: Cell nutrient solution, serum-free medium, PBS, 0.25% trypsin Other items: 96-well cell culture plates, TIP heads, and micropipettes, which were sterilized or filtered and sterilized before the experiment.

[0060] 2. Experimental steps (1) Divide into three groups: blank control group (NC), fibronectin group (FN), and siRNA-PCSK9 group.

[0061] (2)Culture Balb / c 3T3 cells and seed them in 6-well plates. When the cell density reaches 60 - 70%, perform siRNA-PCSK9 plasmid transfection.

[0062] (3)Use a marker pen to draw horizontal lines evenly on the back of the 6-well plate with a ruler aligned. Make a line approximately every 0.5 - 1 cm, crossing the wells horizontally. Add 2 ml of cell suspension at a concentration of 5×105 cells / ml to the wells of the 6-well plate.

[0063] (4)The next day, when it is observed that the cells in the 6-well plate have all grown into a monolayer, use a pipette tip against the ruler to make two scratches perpendicular to the horizontal lines on the back of each well. Wash the cells 3 times with PBS to wash away the detached floating cells.

[0064] (5)Add 1.8 ml of serum-free culture medium to each well, and then add 200 μl of the sample. Add an equal volume of PBS solution to the cell control well.

[0065] (6)Place it in a 37°C 5% CO2 incubator for culture. Take a photo at 0 h and record the photo-taking position in each well. Observe and take photos at the fixed position during subsequent observations.

[0066] 3. Experimental results The observation results of the blank control group (NC), fibronectin group (FN), and siRNA-PCSK9 group (si-PCSK9) at 0 h and 24 h are as Figure 1 shown.

[0067] Data processing: Area detection method (scratch distance measurement is an equivalent measurement) Average scratch width = scratch gap area / length Cell migration rate = (scratch width at 0 h - scratch width after culture) / scratch width at 0 h × 100% Calculate the in vitro cell migration promotion rate from this (see Table 5).

[0068] Table 5 Migration rates of Balb / c 3T3 cells in each group Sample Cell Migration Rate (%) Fibronectin (FN) 100 Blank Control Group (NC) 0 siRNA - PCSK9 Group 72.26 4. Experimental conclusions Inhibiting the PCSK9 gene can significantly improve the migration ability of cells and has a tissue repair function.

Claims

1. Use of a PCSK9 inhibitor in the preparation of a product for anti-aging and tissue repair, characterized in that The PCSK9 protein is proprotein convertase subtilisin / kexin type 9 and belongs to the proprotein convertase family.

2. The application according to claim 1, characterized in that, The PCSK9 inhibitors include, but are not limited to, small molecule compound inhibitors of PCSK9, PCSK9 monoclonal antibodies, PCSK9 small interfering RNA inhibitors, PCSK9 mimetic peptide inhibitors, PCSK9 mimetic antibody proteins, antisense oligonucleotides targeting PCSK9, or PCSK9 vaccines.

3. The application according to claim 1, wherein The anti-aging and tissue repair include the repair of aging or tissue damage caused by all reasons, including but not limited to aging and tissue damage caused by endogenous or exogenous factors.

4. The application according to claim 1, wherein The PCSK9 inhibitor is a small molecule compound inhibitor of PCSK9, or a PCSK9 mimetic peptide inhibitor, or a PCSK9 monoclonal antibody.

5. The application according to claims 1 and 2, characterized in that, The small molecule compound inhibitors of PCSK9 include, but are not limited to, compounds represented by the structural formulas such as formula (Ⅰ), formula (Ⅱ), formula (Ⅲ), or formula (Ⅳ); 6. Formula (Ⅰ) Formula (Ⅱ) Formula (Ⅲ) Formula (Ⅳ) The application according to claims 1 and 2, characterized in that, The PCSK9 monoclonal antibodies include, but are not limited to, evolocumab, alirocumab, recombinant humanized anti-PCSK9 monoclonal antibody JS002, the recombinant fully human anti-PCSK9 monoclonal antibody injection of Sino Biopharmaceutical Co., Ltd., or the PCSK9 inhibitor LY3015014.

7. The application according to claims 1 and 2, characterized in that, The mimetic peptide inhibitors and PCSK9 mimetic antibody protein inhibitors include, but are not limited to, the following amino acid sequences: EYPYKHSGYYHRP, or EYPYDYSGYYHRP;, or EYTFKHSGYYHRP, or EYPWKGSGYYHRP, or EYTNGAGYYHRP, or EFPFKWSGYYHRP, or EYTKGSGYYHRP, or EFDFVGAGYYHRP, or EFPWPHAGYYHRP, or EYAFEGAGYYHRP. The PCSK9 mimetic antibody proteins include, but are not limited to, the mimetic antibody protein drug DS-9001 or the human antibody-antigen binding fragment 1G08 acting on the carboxyl terminal domain of PCSK9.

8. The application according to claims 1 and 2, characterized in that, The PCSK9 small interfering RNA inhibitors include, but are not limited to, Inclisran injection, ALN-PCS injection, or ALN-PCSsc injection.

9. The application according to claims 1 and 2, characterized in that The PCSK9 inhibitor can be used alone or in combination with other treatment methods, drugs, or pharmaceutically acceptable carriers in the application of anti-aging and tissue repair.

10. The application according to claims 1 and 2, characterized in that, The PCSK9 inhibitor and the composition described in claim 9 can be prepared into any pharmaceutically acceptable dosage form or any administration method for use in anti-aging and tissue repair.

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