Application of troxerutin in preparation of medicine for preventing and / or treating skin keratinocyte aging and skin tissue aging

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of troxerutin in preparation of medicines for preventing and / or treating skin keratinocyte aging and skin tissue aging. The medicine can delay skin keratinocyte senescence by protecting skin keratinocytes from intracellular active oxygen accumulation, mitochondrial dysfunction and DNA damage caused by ionizing radiation, and the troxerutin can delay skin tissue senescence by protecting skin tissues from skin tissue thinning caused by ionizing radiation. The invention provides a new medicine source for preventing, protecting, delaying and researching the aging of skin keratinocytes and skin tissues. The medicine for controlling the aging of cells and organisms, provided by the invention, is safe, relatively strong in pharmacological action, small in side effect and definite in curative effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of troxerutin in the preparation of drugs for the prevention and / or treatment of aging of skin keratinocytes and skin tissue. Background Technology

[0002] The skin is the body's first line of defense against the external environment and is the most complex and multifunctional self-regulating organ. With economic development and changing awareness, skin health and youthfulness are increasingly valued. Like the aging of other organs, skin aging is characterized by the gradual loss of its protective and regenerative functions. This is a multifactorial process that affects almost every aspect of its biology. With age, the skin is constantly subjected to external and internal stimuli and influences, gradually exhibiting changes such as wrinkles, dryness, abnormal barrier function, thinning, and abnormal skin integrity. At the cellular level, these changes manifest as permanent cell cycle arrest and loss of proliferative capacity. Cellular senescence is mainly characterized by cell cycle inhibition and morphological changes such as flattening, enlargement, nuclear condensation, and increased senescence-associated β-galactosidase activity (SA-β-Gal), as well as the secretion of senescence-associated phenotypes (SASPs). If senescent cells are not promptly eliminated by the immune system, they can maintain metabolic activity and survive for several years. The accumulation of senescent cells and the secretion of SASPs can induce senescence in nearby cells, leading to tissue aging.

[0003] With the rapid development of the economy and technology, nuclear technology has been widely applied in many industries, from small-scale applications like electronic products and medical radiation to large-scale radiation exposure caused by nuclear accidents. The probability of people being exposed to ionizing radiation (IR) is constantly increasing. This threatens human health and affects social stability. Ionizing radiation can damage cellular DNA through both direct and indirect means. Firstly, it can directly act on macromolecules within cells, causing structural and functional abnormalities. Indirect damage is often caused by reactive oxygen species (ROS). Radiation can ionize water molecules within cells, causing them to produce large amounts of ROS within seconds. Large amounts of ROS can cause persistent oxidative stress, damaging the nucleic acid bases, lipids, and proteins of epicellular cells, leading to cellular senescence and even apoptosis, affecting cell division and proliferation. Intracellular ROS can also enter surrounding mitochondria, damaging the macromolecular structure within mitochondria and reducing mitochondrial membrane potential (MMP), thereby affecting mitochondrial function. When the body is exposed to ionizing radiation, the skin is the first barrier. Exposed skin can exhibit changes such as dryness, erythema, disordered skin texture, skin atrophy and thinning, and decreased elasticity. In severe cases, ulcers and erosions may occur, seriously affecting people's quality of life.

[0004] Troxerutin (Trx) is a semi-synthetic flavonoid compound, also known as vitamin P4, derived from rutin through hydroxyethylation. Originally extracted from the Japanese pagoda tree, it is also a component of tea, coffee, grains, and various fruits and vegetables. Its chemical formula is C33H4O19, and it is a water-soluble yellow or yellowish-green powder. It inhibits platelet aggregation and prevents thrombosis. It also counteracts vascular damage caused by serotonin and bradykinin, increases capillary resistance, and reduces capillary permeability, thus preventing edema caused by increased vascular permeability. However, there are currently no research reports on the effects of troxerutin on ionizing radiation-induced skin damage and aging. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides the application of troxerutin in the preparation of drugs for the prevention and / or treatment of skin keratinocyte aging and skin tissue aging. It can improve the safety of drugs for skin keratinocyte aging and skin tissue aging induced by ionizing radiation, has strong pharmacological effects, clear efficacy, and few side effects.

[0006] The technical solution provided by this invention is as follows: This invention provides the use of troxerutin in the preparation of medicaments for the prevention and / or treatment of aging of skin keratinocytes and skin tissue.

[0007] Furthermore, the aging of skin keratinocytes and skin tissue is induced by ionizing radiation.

[0008] Furthermore, troxerutin delays skin keratinocyte aging by protecting skin keratinocytes from intracellular reactive oxygen species accumulation, mitochondrial dysfunction, and DNA damage caused by ionizing radiation, and delays skin tissue aging by protecting skin tissue from skin tissue thinning caused by ionizing radiation.

[0009] Furthermore, the method of delaying the aging of skin keratinocytes involves reducing the proportion of β-galactosidase-positive cells in skin keratinocytes upregulated by ionizing radiation, as well as the expression levels of aging markers CDKN1A and CDKN2A.

[0010] Furthermore, the method of delaying the aging of skin keratinocytes is to increase the expression level of Ki67, a proliferation marker downregulated by ionizing radiation.

[0011] Furthermore, the method of delaying the aging of skin keratinocytes involves reducing the expression levels of aging-related secretory phenotypic factors in skin keratinocytes that are upregulated by ionizing radiation.

[0012] Furthermore, the method of delaying the aging of skin keratinocytes is to reduce the ROS level in skin keratinocytes that are upregulated by ionizing radiation.

[0013] Furthermore, the aforementioned delay in the aging of skin keratinocytes is achieved by reducing the damage to skin keratinocyte DNA upregulated by ionizing radiation.

[0014] Furthermore, the drug comprises an effective dose of troxerutin, as well as pharmaceutically acceptable excipients.

[0015] Furthermore, the drug is introduced into the body tissues via intraperitoneal injection.

[0016] Furthermore, the drug is an injectable preparation made by dissolving troxerutin in PBS buffer.

[0017] Furthermore, the concentration of troxerutin is 1 mg / kg.

[0018] Furthermore, the intraperitoneal injection is performed more than twice, and the injection begins before the ionizing radiation.

[0019] Furthermore, the dosage of troxerutin for each injection is 20 mg / kg.

[0020] The term "effective dose" refers to the amount of a compound that is sufficient to treat a disease when administered to a subject. The effective dose can vary depending on the severity of the disease and the physical condition, age, weight, and sex of the subject to be treated.

[0021] The term "pharmaceuticalally acceptable excipient" refers to any formulation or carrier medium capable of delivering an effective dose of the active substance of the present invention without interfering with the biological activity of the active substance and without toxic side effects on the host or subject.

[0022] The term “prevention” refers to the reduction of the risk of acquiring a disease or disorder (i.e., stopping the development of at least one clinical symptom of the disease in a subject who may be facing or predisposed to facing the disease, but has not yet experienced or exhibited symptoms of the disease).

[0023] The term "treatment" in some embodiments refers to improving a disease or condition (i.e., slowing, stopping, or alleviating the development of a disease or at least one of its clinical symptoms). In other embodiments, it refers to alleviating or improving at least one bodily parameter, including bodily parameters that may not be perceptible to the subject. In still other embodiments, it refers to regulating a disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing bodily parameters) or both. In still other embodiments, it refers to preventing or delaying the onset, flare-up, or worsening of a disease or condition. Beneficial effects

[0024] This invention demonstrates, through simulations and clinical models of ionizing radiation-induced skin keratinocyte senescence and skin aging, that intraperitoneal injection of troxerutin into mice can significantly improve the occurrence and progression of ionizing radiation-induced skin aging. Simultaneously, this invention utilizes an in vitro cell senescence model: human skin keratinocytes (HaCaT). A skin cell senescence model was constructed using ionizing radiation (6 Gy), and troxerutin treatment of the cells alleviated the accumulation of intracellular reactive oxygen species, mitochondrial dysfunction, and DNA damage caused by ionizing radiation, ultimately delaying the senescence of skin keratinocytes. Therefore, this invention is the first to propose the application of troxerutin in the development and preparation of drugs for the prevention of clinically common ionizing radiation-induced skin aging.

[0025] The drug provided by this invention for delaying the aging of skin keratinocytes and skin tissue is safe, has strong pharmacological effects, clear efficacy, and few side effects. This invention provides a new drug source for the prevention, protection, treatment, and research of skin keratinocyte and skin tissue aging, and is easily applicable to clinical practice, with the potential to generate significant clinical application prospects and social benefits in a short period. Attached Figure Description

[0026] Figure 1 af is a schematic diagram of the results of RT-qPCR detection of mRNA levels of aging-related genes in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention (mean±SD, (a)n=6, (b)n=4, (c,e,f)n=5, (d)n=7).

[0027] Figure 1 g is a schematic diagram of the results (mean±SD, n=5) of the detection of p21 and GAPDH protein levels by Western blot in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention.

[0028] Figure 1 h is a schematic diagram of the results of Ki67 detection by immunofluorescence staining (IF) in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention (the left image is a representative image, and the right image is the percentage of Ki67 positive cells, mean±SD, n=5).

[0029] Figure 1 i is a schematic diagram of the results of aging-related β-galactosidase staining in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention (the left image is a representative photo, and the right image is the percentage of SA-β-Gal positive cells, mean±SD, n=4).

[0030] Figure 2 a is a schematic diagram of the results of mitochondrial membrane potential detection by flow cytometry in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention, using the mitochondrial membrane potential decrease (MMP) indicator Rhodamine 123. (mean±SD, n=5)

[0031] Figure 2 b is a schematic diagram of the results of mitochondrial membrane potential detection by JC-1 probe in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention (mean±SD, n=5).

[0032] Figure 2 c is a schematic diagram showing the results of ROS level detection (mean±SD, n=5) in Example 1 of this invention, using mitoSOX RED mitochondrial (mitoROS) indicator combined with flow cytometry in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group.

[0033] Figure 2 d is a schematic diagram of the results of ROS level detection by dihydroethidium (ROS) indicator combined with flow cytometry in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention (mean±SD, n=5).

[0034] Figure 3 a is a schematic diagram showing the results of mitochondrial quality detection (mean±SD, n=5) in Example 1 of this invention, using mitotracker deep red FM indicator combined with flow cytometry, for the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group.

[0035] Figure 3 b is a schematic diagram of the PCR array analysis results of mitochondrial energy metabolism in cells in Example 1 of the present invention.

[0036] Figure 3 c represents the first two genes upregulated and downregulated in the PCR array in Example 1 of this invention.

[0037] Figure 3 dg represents the mRNA levels of the first two upregulated and downregulated genes in the PCR array detected by RT-qPCR in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group of Example 1 of this invention (mean±SD, (d,e) n=5, (f,g) n=4).

[0038] Figure 4 a shows the results of immunofluorescence staining (IF) detection of 53BP1 in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of the present invention (the left image is a representative image, and the right image is the percentage of ≥5 cell nucleus damage points, mean±SD, n=5).

[0039] Figure 4 b shows the results of Western blot detection of pH2AX and GAPDH protein levels in the control group, troxerutin treatment group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 1 of this invention (mean±SD, n=4).

[0040] Figure 5 a is a schematic diagram of the skin phenotype results of the control group, the ionizing radiation treatment group, and the ionizing radiation + troxerutin treatment group in Example 2 of the present invention.

[0041] Figure 5 bc is a schematic diagram showing the AST(b) and ALT(c) levels in the control group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 2 of this embodiment.

[0042] Figure 5d shows the results of hematoxylin-eosin staining (HE staining) of the back skin and the thickness of the control group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 2 of the present invention (mean±SD, n=3).

[0043] Figure 5 The image ek shows the results of RT-qPCR detection of mRNA levels of aging-related genes in the control group, ionizing radiation treatment group, and ionizing radiation + troxerutin treatment group in Example 2 of this invention (mean±SD, (e,gj) n=5, (f,k) n=4). Detailed Implementation

[0044] The present invention will now be described in further detail with reference to the accompanying drawings: Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0045] All reagents and materials used in this example can be purchased routinely. The quantitative experiments involved in the examples were all repeated at least three times, and the results were averaged.

[0046] The C57BL / 6 mice used were purchased from the Comparative Medicine Center of Yangzhou University.

[0047] The troxerutin used was purchased from MCE Company, product number HY-N139.

[0048] The sequences of human primers used are shown in Table 1, the sequences of mouse primers are shown in Table 3, and the information on antibodies and probes used is shown in Table 2.

[0049] Ionizing radiation modeling was performed using a medical electron linear accelerator (CLINAC 23EX) at appropriate doses.

[0050] Example 1 In vitro experiment

[0051] 1. Experimental materials and grouping

[0052] 1.1 Culture of HaCaT cells

[0053] The human skin keratinocyte cell line HaCaT (provided by the Stem Cell Bank of the Chinese Academy of Sciences) was cultured in RPMI 1640 basal medium supplemented with glutamine, 10% fetal bovine serum (FBS, Procell), and 1% penicillin / streptomycin (Solarbio). Cell culture was conducted at 37°C and 5% CO2.

[0054] 1.2 Experimental Grouping

[0055] Control group (Ctrl): The control group consisted of normal HaCaT cells that had not been exposed to ionizing radiation and had not been given troxerutin.

[0056] Ionizing radiation group (IR): Normal HaCaT cells were exposed to ionizing radiation of 6 Gy for approximately 10 minutes, and the culture medium was changed immediately after irradiation. Cells were collected 2 days after irradiation for RT-qPCR analysis, and other analyses were performed 3 days after irradiation.

[0057] Troxerutin group (Trx): The troxerutin group consisted of normal HaCaT cells that had not undergone ionizing radiation, treated with 5 μM troxerutin for approximately 10 minutes. Cells were collected 2 days after treatment for RT-qPCR analysis, and other analyses were performed 3 days after treatment.

[0058] Ionizing radiation + troxerutin group (IR+Trx): After pretreatment with 5 μM troxerutin for 24 h, cells were subjected to 6 Gy of ionizing radiation for about 10 minutes. Immediately after irradiation, the culture medium was replaced and 5 μM troxerutin was added again for about 10 minutes. Cells were collected 2 days after irradiation for RT-qPCR analysis, and other analyses were performed 3 days after irradiation.

[0059] 2. RNA extraction and real-time quantitative PCR (RT-qPCR)

[0060] RNA extraction was performed using Tiangen reagent (catalog number: DP424); RNA was reverse transcribed into cDNA using TAKARA reverse transcription kit (catalog number: RR036A); RT-qPCR experiments were performed using YEASEN kit (catalog number: 11201ES); PCR array analysis was provided by Woji Gene (catalog number: wc-mRNA0277-H).

[0061] The primer sequences for RT-qPCR are shown in Table 1. The expression level of the target mRNA was normalized using the co-expression levels of PGK1 and HPRT1.

[0062]

[0063] 3. Western Blot Experiment

[0064] HaCaT cells were lysed in RIPA buffer, and protein concentration was determined using a BCA kit (Thermo Fisher Scientific). Equal amounts of protein were separated by SDS-PAGE electrophoresis after boiling each sample. The samples were then transferred to a PVDF membrane (Bio-Rad). Incubation was performed with TBS-tween buffer (5% milk powder and 0.05% Tween-20), followed by detection with anti-GAPDH antibody, anti-p21 antibody, and anti-pH2AX antibody (as shown in Table 3), incubating overnight at 4°C. The next day, after rinsing, the membrane was incubated for 1 hour at room temperature with a peroxidase-bound secondary antibody. Protein blots on the membrane were then detected using the ECL Plus chemiluminescence system (Bio-Rad) according to the manufacturer's instructions.

[0065] 4. Immunofluorescence staining (IF)

[0066] HaCaT cells were seeded in 12-well plates (Solarbio) with cell spreaders. After treatment, cells on the spreaders were washed with 1xPBS and fixed with pre-chilled methanol at -20°C for 10 min. Cells were then blocked with PBS-Tween containing 20% ​​fetal bovine serum for 30 min and incubated overnight at 4°C with anti-Ki67 antibody and anti-53BP1 antibody (as shown in Table 3). The next day, cells were washed and incubated with secondary antibody at room temperature for 1 h. Cell nuclei were stained with DAPI Fluoromount G (SouthernBiotech, USA). Images were taken using a Nikon fluorescence microscope and NIS software (Nikon, Japan), and fluorescence intensity was analyzed using ImageJ software.

[0067] 5. Staining of aging-related β-galactosidase (SA-β-gal)

[0068] SA-β-Gal staining reference (Dimri et al, 2005). HaCaT cells in 12-well plates were washed once with 1xPBS and then fixed for 5 minutes with a fixative containing 0.2% glutaraldehyde and 2% formaldehyde. The cells were then incubated overnight at 37°C in CO2-free conditions using SA-β-Gal staining solution. The next day, the SA-β-Gal staining solution was discarded, and the cells were rinsed with 1xPBS and photographed under a microscope.

[0069] 6. Mitochondrial-related tests

[0070] 6.1 Mitochondrial membrane potential

[0071] 6.1.1 Detection of mitochondrial membrane potential using Rhodamine 123, an indicator of decreased mitochondrial membrane potential (MMP), in conjunction with flow cytometry.

[0072] HaCaT cells, after appropriate treatment, were incubated at 37°C for half an hour with the mitochondrial membrane potential decrease (MMP) indicator Rhodamine 123 (as shown in Table 3). Subsequently, the cells were washed and stored in PBS for flow cytometry analysis. Data were obtained using CytExpert for DxFLEX (BECKMAN COULTER, USA) software and analyzed using FlowJo v10 software.

[0073] 6.1.2 JC-1 detection of mitochondrial membrane potential

[0074] HaCaT cells were seeded in 12-well plates (Solarbio) with cell spreaders. After treatment, cells on the spreaders were washed with 1xPBS and fixed with pre-chilled methanol at -20°C for 10 min. They were then incubated with JC-1 dye (as shown in Table 3) at room temperature for 1 h. After washing, cells were photographed using a Nikon fluorescence microscope and NIS software (Nikon, Japan), and fluorescence intensity was analyzed using ImageJ software.

[0075] 6.2 Cell ROS Detection

[0076] 6.2.1 Detection of ROS using mitoSOX RED mitochondrial (mitoROS) indicator combined with flow cytometry

[0077] HaCaT cells, after appropriate treatment, were incubated at 37°C for half an hour with 1 mitoSOX RED mitochondrial (mitoROS) indicator (as shown in Table 3). Subsequently, the cells were washed and stored in PBS for flow cytometry analysis. Data were obtained using CytExpert for DxFLEX (BECKMAN COULTER, USA) software and analyzed using FlowJo v10 software.

[0078] 6.2.2 Detection of ROS using dihydroethidium (ROS) indicator combined with flow cytometry

[0079] HaCaT cells, after appropriate treatment, were incubated with 2 dihydroethidium (ROS) indicator (as shown in Table 3) at 37°C for half an hour. Subsequently, the cells were washed and stored in PBS for flow cytometry analysis. Data were obtained using CytExpert for DxFLEX (BECKMAN COULTER, USA) software and analyzed using FlowJo v10 software.

[0080] 6.3 Mitochondrial quality

[0081] HaCaT cells, after appropriate treatment, were incubated at 37°C for half an hour with mitotracker deep red FM indicator (as shown in Table 3). Subsequently, the cells were washed and stored in PBS for flow cytometry analysis. Data were obtained using CytExpert for DxFLEX (BECKMAN COULTER, USA) software and analyzed using FlowJo v10 software.

[0082]

[0083] 7. Statistical Analysis

[0084] Data are expressed as mean ± standard deviation (SD). For statistical analysis, ANOVA was used to determine the significance of differences between the two groups. GraphPad Prism v.9.0 software (California) was used for statistical analysis. In all statistical tests, significance was set as *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant (p>0.05).

[0085] 8. Experimental Results

[0086] 8.1 Troxerutin delays ionizing radiation-induced skin aging

[0087] like Figure 1 As shown, in this embodiment, a HaCaT cell senescence model was established using a 6 Gy dose of ionizing radiation. These cells were treated with troxerutin (5 μM) before ionizing radiation, followed by approximately 10 minutes of ionizing radiation to assess the effect of troxerutin on senescence.

[0088] The results showed that, compared with the control group Ctrl, the upregulation of irradiation-induced aging markers CDKN1A and CDKN2A, the downregulation of the proliferation marker Ki67, and the upregulation of SASP factors IL1A, IL6, and IL8 were all reversed by the addition of troxerutin. Figure 1 a-1f, 1h). Furthermore, compared to the control group Ctrl, the upregulation of CDKN2A-encoded p21 protein by irradiation was alleviated after troxerutin treatment. Figure 1 g). Compared with the control group Ctrl, SA-β-Gal staining showed that troxerutin was able to inhibit the increase in the percentage of senescent cells induced by ionizing radiation (g). Figure 1 i). In summary, troxerutin delays ionizing radiation-induced senescence of skin keratinocytes.

[0089] 8.2 Troxerutin restores ionizing radiation-induced decrease in mitochondrial membrane potential and ROS accumulation

[0090] like Figure 2As shown, in order to study the mechanism by which troxerutin inhibits ionizing radiation-induced senescence of skin keratinocytes, a literature review revealed that troxerutin is involved in the regulation of mitochondrial oxidative stress, so the mitochondrial membrane potential (MMP) was measured.

[0091] The results showed that, compared with the IR treatment group, ionizing radiation induced a decrease in MMPs in HaCaT cells, while treatment with troxerutin restored the decreased MMPs: Rhodamine123 ( Figure 2 a) and JC-1 probe ( Figure 2 b) proves this phenomenon.

[0092] Since a decrease in MMPs triggers an increase in mitochondrial ROS (mitoROS) levels, ROS (mitoROS) levels were measured.

[0093] The results showed that after troxerutin treatment, the ionizing radiation-induced mitoROS accumulation and ROS generation were reversed compared with the IR of the treatment group. Figure 2 c, 2d).

[0094] In summary, troxerutin can restore the decline in MMPs and inhibit ionizing radiation-induced intracellular ROS accumulation.

[0095] 8.3 Troxerutin alleviates ionizing radiation-induced mitochondrial dysfunction

[0096] Since excessive ROS production in cells can lead to mitochondrial dysfunction, mitochondrial quality was assessed using mitoTracker. Mitochondrial energy metabolism was detected by PCR array assay in HaCaT cells with and without ionizing radiation treatment. A total of 90 mitochondrial energy metabolism-related genes were tested, and most of these genes (64 out of 90) showed downregulation after ionizing radiation. Figure 3 b) Subsequently, the top two downregulated genes, namely cytochrome c oxidase subunit 6c (COX6C) and ubiquitin-cytochrome c reductase core protein 2 (UQCRC2), and the top two upregulated genes, namely mitochondrial inner membrane protein (OXA1L) and endothelin 1 (EDN1), were selected from the PCR array for further validation. Figure 3 c).

[0097] turn out( Figure 3 Compared to the control group Ctrl, ionizing radiation promoted an increase in mitochondrial mass, while troxerutin reduced this increase. Figure 3 a). This indicates that ionizing radiation promotes changes in mitochondrial morphology; compared with the IR treatment group, troxerutin restored the expression of COX6C and UQCRC2, which were suppressed by ionizing radiation, as well as the expression of OXA1L and EDN1, which were induced by ionizing radiation. Figure 3 d-3g).

[0098] Therefore, troxerutin alleviates ionizing radiation-induced mitochondrial dysfunction during the aging process of skin keratinocytes.

[0099] 8.4 Troxerutin alleviates ionizing radiation-induced DNA damage

[0100] Mitochondrial dysfunction can promote DNA damage, so two DNA damage biomarkers, 53BP1 and p-H2AX, are used to detect DNA damage.

[0101] turn out( Figure 5 a and Figure 5 b) Compared with the IR treatment group, troxerutin was able to reduce DNA damage caused by ionizing radiation.

[0102] Example 2 In vivo experiment

[0103] 1. Experimental materials and grouping

[0104] 1.1 Constructing a model of ionizing radiation-induced aging of back skin

[0105] In in vivo experiments, starting four days before ionizing radiation exposure, mice were intraperitoneally injected with 20 mg / kg troxerutin (1 mg / ml) every two days. After hair removal from the backs of mice, the skin on their backs was subjected to two 5 Gy ionizing radiation doses (6-MV X photons, 2 Gy / min) on days one and four. Ten days later, the mice were sacrificed, and skin samples were collected from their backs for further experiments. The control group received the same treatment except for irradiation.

[0106] 1.2 Experimental Grouping

[0107] Control group (Ctrl): No further treatment was given after hair removal on the back;

[0108] Ionizing radiation group (IR): After hair removal on the back, the skin on the back was subjected to ionizing radiation of 5 Gy twice a day on the first and fourth days, each time for about 10 minutes;

[0109] Ionizing radiation + troxerutin group (IR+Trx): Four days before ionizing radiation, 20 mg / kg of troxerutin (1 mg / ml) was injected intraperitoneally every two days. After hair removal on the back, the skin on the back was given ionizing radiation twice a day, on the first and fourth days, each time for about 10 minutes.

[0110] 2. Detect skin phenotype

[0111] Observe the skin folds and elasticity on the back of the mice and take photos.

[0112] 3. Detect the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood.

[0113] Venous blood was collected from mice after anesthesia and centrifuged at 3000 rpm at room temperature for 15 min to separate the supernatant. Serum AST (catalog number: C010-2-1) and ALT (catalog number: C009-2-1) detection kits from Jiancheng Biotechnology Co., Ltd. were used to detect serum AST and ALT levels according to the manufacturer's instructions.

[0114] 4. Hematoxylin-eosin staining (HE staining)

[0115] After obtaining skin tissue from the dorsal side of mice, it was fixed in 4% paraformaldehyde and embedded in paraffin. Subsequently, the paraffin tissue was dewaxed and dehydrated, and stained with hematoxylin-eosin. Images were photographed and saved using a microscope.

[0116] 5. RNA extraction and real-time quantitative PCR (RT-qPCR)

[0117] RNA extraction was performed using Tiangen reagent (catalog number: DP424); RNA was reverse transcribed into cDNA using TAKARA reverse transcription kit (catalog number: RR036A); RT-qPCR experiments were performed using YEASEN kit (catalog number: 11201ES).

[0118] The primer sequences for RT-qPCR are shown in Table 3. The expression level of the target mRNA was normalized using the expression level of GADPH.

[0119]

[0120] 6. Statistical Analysis

[0121] Data are expressed as mean ± standard deviation (SD). For statistical analysis, a one-way ANOVA test was used to determine the significance of differences between the two groups. GraphPad Prism software (v.9.0) was used for statistical analysis. In all statistical tests, significance was set as *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant (p>0.05).

[0122] 7. Experimental Results

[0123] Troxerutin alleviates ionizing radiation-induced skin aging

[0124] Based on Example 1, it was demonstrated that troxerutin can delay ionizing radiation-induced senescence of skin keratinocytes, and a model of ionizing radiation-induced senescence of back skin was constructed to study whether troxerutin plays a regulatory role in skin aging in vivo.

[0125] The results showed that, compared with the control group Ctrl, ionizing radiation caused wrinkles, dryness, and decreased elasticity in the skin when skin phenotypes were examined, while troxerutin inhibited these phenotypes. Figure 5 a) When measuring the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the blood, troxerutin inhibited the increase of AST and ALT induced by ionizing radiation compared with the treatment group IR, indicating that troxerutin may have a protective effect against ionizing radiation-induced liver damage. Figure 5 b, 5c); When measuring the thickness of the back skin after H&E staining, compared with the control group Ctrl, it was found that ionizing radiation could promote skin thinning, while troxerutin could reverse this phenomenon. Figure 5 d); RT-qPCR experiments showed that the upregulation of ionizing radiation-induced aging markers Cdkn1a and Cdkn2a, the downregulation of proliferation marker Ki67, and the upregulation of SASP factors Il1a, Il6, Nf-kb1, and Rela were partially restored after troxerutin treatment. Figure 5 e-5g).

[0126] In conclusion, troxerutin can delay ionizing radiation-induced skin aging phenotypes and SASP secretion in vivo. Troxerutin can improve skin aging phenotypes in ionizing radiation-induced skin aging models.

Claims

1. The use of troxerutin in the preparation of drugs for the prevention and / or treatment of aging of skin keratinocytes and skin tissue.

2. The application according to claim 1, characterized in that, The aging of skin keratinocytes and skin tissues is induced by ionizing radiation.

3. The application according to claim 2, characterized in that, The troxerutin mentioned above delays the aging of skin keratinocytes by protecting them from the accumulation of intracellular reactive oxygen species, mitochondrial dysfunction, and DNA damage caused by ionizing radiation. It also delays the aging of skin tissue by protecting it from the thinning of skin tissue caused by ionizing radiation.

4. The application according to claim 3, characterized in that, The method to delay the aging of skin keratinocytes involves reducing the proportion of β-galactosidase-positive cells in skin keratinocytes upregulated by ionizing radiation, as well as the expression levels of aging markers CDKN1A and CDKN2A.

5. The application according to claim 3, characterized in that, The method to delay the aging of skin keratinocytes is to increase the expression level of Ki67, a proliferation marker downregulated by ionizing radiation.

6. The application according to claim 3, characterized in that, The method to delay the aging of skin keratinocytes involves reducing the expression levels of aging-related secretory phenotypic factors in skin keratinocytes that are upregulated by ionizing radiation.

7. The application according to claim 3, characterized in that, The method to delay the aging of skin keratinocytes is to reduce the ROS level in skin keratinocytes that is upregulated by ionizing radiation.

8. The application according to claim 3, characterized in that, The method to delay the aging of skin keratinocytes is to reduce the damage to skin keratinocyte DNA caused by ionizing radiation.

9. The application according to claim 1, wherein the medicament comprises an effective dose of troxerutin and pharmaceutically acceptable excipients.

10. The application according to claim 9, characterized in that, The drug is introduced into the body tissues via intraperitoneal injection.

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