Method for relieving injury induced by high-dose uvb through low-dose caffeine
By co-treating low-dose caffeine with UVB irradiation, the problem of difficulty in alleviating UVB radiation damage in the prior art has been solved, which significantly improves cell viability and autophagy response, improves skin cells damage, and achieves effective protection of UVB radiation .
Patent Information
- Application Number
- CN202411114062.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-14
- Publication Date
- 2024-12-17
AI Technical Summary
The prior art is difficult to effectively alleviate the damage to skin cells by high-dose UVB radiation, and there is a lack of effective intervention measures.
Co-treatment of low-dose caffeine (1000ng/ml) and UVB irradiation was performed to significantly improve cell viability, reduce oxidative stress damage, regulate metabolic response, maintain mitochondrial membrane potential, increase the expression of autophagy-related proteins, and promote cell autophagy.
By improving cell viability, reducing oxidative stress damage, maintaining the stability of mitochondrial membrane potential, increasing autophagy response, significantly improving skin cell damage caused by UVB radiation, and promoting cell autophagy to play a protective role.
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Figure CN119139316A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for alleviating high-dose UVB-induced damage by using low-dose caffeine. Background Art
[0002] 91% of sunlight on the earth's surface is visible light with a wavelength of 400 to 800 nanometers; 8.7% is ultraviolet A, 320 to 400 nanometers; and 0.3% is ultraviolet B, too short at 280 to 320 nanometers, UVC, all of which are absorbed by the ozone layer. The penetration of UVA and UVB into the skin at different depths will have different effects, inducing different overlapping biological responses in the epidermis and dermis. Ultraviolet B (UVB) is one of the exogenous aging factors, which causes skin aging through the accumulation of external stimuli. Several studies have reported that UVB irradiation increases intracellular reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals, and hydrogen peroxide. ROS stimulate various signaling pathways and initiate biological processes, including cell death, cell senescence, and inflammation. UVB radiation causes the production of reactive oxygen species (ROS) in the skin, which is considered to be the initial inducing factor of irradiation-induced tissue damage. UV radiation has been shown to trigger changes in mitochondrial morphology and function. There have been a lot of studies but few satisfactory interventions, so it is necessary to develop effective mitigation measures and apply them in clinical practice.
[0003] Caffeine is a methylxanthine (1,3,7-trimethylxanthine) that can be synthesized and added to foods and beverages, including soft drinks and energy drinks, as well as pills on the market for reducing fatigue. Caffeine has a variety of biological activities, including harmful and beneficial effects. Caffeine has effects on various tissues, organs and systems in the human body, can improve metabolism and reduce the risk of chronic diseases. Moderate intake of caffeine (3-6mg / kg) has an energizing effect, but very high blood concentrations of 80μg / ml (412μM) or higher can cause adverse effects. Although it is difficult to determine the exact physiological concentration of caffeine, studies have found that caffeine from 0.1mM (19.4μg / ml) to 5mM (970.95μg / ml) inhibits cell proliferation and migration in a dose-dependent manner, leading to delayed epithelialization and wound closure. Summary of the invention
[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for alleviating high-dose UVB-induced damage by using low-dose caffeine.
[0005] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is:
[0006] A method for alleviating high-dose UVB-induced damage with low-dose caffeine, with a caffeine concentration of 1000 ng / ml affecting 50 mJ / cm2 UVB irradiation induces photochemical protection in human epidermal keratinocytes (HaCaT).
[0007] In the present invention, caffeine improves cell viability and alleviates oxidative stress damage induced by UVB irradiation.
[0008] In the present invention, UVB irradiation and caffeine incubation are used together to enhance the metabolic activity of hacat.
[0009] In the present invention, UVB irradiation and caffeine incubation are used together to cause changes in the mitochondrial membrane potential of hacat, and caffeine incubation can reduce the mitochondrial depolarization reaction.
[0010] In the present invention, UVB irradiation and caffeine incubation are used together to increase the autophagic flux in hacat.
[0011] In the present invention, UVB irradiation and caffeine incubation are used together to upregulate the expression of the autophagy-related protein band in hacat.
[0012] In the present invention, caffeine significantly improves the damage of HACAT cells irradiated by UVB, improves the damage of HACAT cells by reducing oxidative stress damage, regulating metabolic reactions, maintaining mitochondrial membrane potential and increasing the expression of autophagy-related proteins, improves the vitality of HACAT cells, improves mitochondrial aerobic respiration, maintains the stability of mitochondrial membrane potential and increases autophagy reaction, and caffeine reduces the oxidative stress damage of HACAT cells and accelerates mitochondrial aerobic respiration by improving the vitality of HACAT cells, thereby promoting cell autophagy and playing a protective role.
[0013] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.
[0014] The caffeine of the invention plays a protective role by increasing the activity of hacat cells, reducing oxidative stress damage of hacat cells and accelerating mitochondrial aerobic respiration, thereby promoting cell autophagy.
[0015] The specific implementation modes of the present invention are further described in detail below in conjunction with specific examples. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings are part of this application and are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:
[0017] Figure 1 :A.cck8 activity detection;B.ROS immunofluorescence analysis;C.ROS quantification;
[0018] Figure 2 :A. Time-OCR line graph of mitochondrial stress detection without UVB irradiation; B. Basal respiration OCR value without UVB irradiation; C. Maximum respiration OCR value without UVB irradiation; D. Time-OCR line graph of mitochondrial stress detection with co-treatment of UVB irradiation and caffeine incubation; E. Basal respiration OCR value with co-treatment of UVB irradiation and caffeine incubation; F. Maximum respiration OCR value with co-treatment of UVB irradiation and caffeine incubation;
[0019] Figure 3 :A. JC-1 immunofluorescence analysis; B. JC-1 quantification;
[0020] Figure 4 : Immunofluorescence analysis of autophagy reaction;
[0021] Figure 5 :A. Immunoblot analysis; B. LC3B quantification; C. ATG5 quantification; D. BECLIN1 quantification; E. UVRAG quantification.
[0022] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION
[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.
[0024] Example 1
[0025] Purpose
[0026] Numerous studies have shown that oxidative stress can lead to organelle defects, affect cellular metabolic activity and functional homeostasis, thereby activating autophagy and affecting cell survival or death. Mitophagy occurs when autophagy promotes the remodeling of damaged mitochondria. If the damage is below the lethal threshold, defective mitochondria are sequestered by autophagosomes. This process can support cell growth and proliferation by reducing ROS generation and damaged mitochondria.
[0027] In this study, we investigated the effects of caffeine (CAF) on UVB (50 mJ / cm 2) irradiation-induced photochemical protection of human epidermal keratinocytes (HaCaT). We chose a low dose of caffeine at a concentration of 1000ng / ml, and the results showed that caffeine significantly increased cell viability, reduced ROS generation and depolarization of mitochondrial membrane potential, and changes in metabolic activity in UVB-induced HaCaT cells. Here, we demonstrated that exposure to UVB radiation led to changes in oxygen consumption rate (OCR), which means that mitochondrial function and corresponding metabolic activities were altered. The results showed that caffeine significantly increased the basal respiratory OCR value and increased the maximum respiratory OCR value. In addition, increased autophagy and upregulation of autophagy-related protein expression were observed in UVB-irradiated caffeine-incubated HaCaT.
[0028] Experimental Materials and Methods
[0029] Antibodies and Reagents: The primary antibodies for Western Blotting were anti-gapdh (MAB374; Millipore), LC3B (ab14734; Abcam), ATG5, BECLIN-1 (ab57602;), UVRAG (ab56889; Abcam). The following secondary antibodies were used: rabbit anti-mouse (P0161; DAKO) and goat anti-rabbit (PO448; DAKO).
[0030] Cell culture and treatments: Immortalized human keratinocytes HaCaT cells were maintained in DMEM containing 10% fetal bovine serum and 5% antibiotics at 37°C, 95% humidity and 5% CO2. Subcultures were performed before the cells reached confluence by adding trypsin and EDTA. During the experiments, cells were incubated with different treatments of caffeine and UVB irradiation.
[0031] Human immortalized keratinocyte-derived (HaCaT) cell line was cultured in modified DMEM medium containing caffeine (1000 ng / ml), 10% fetal bovine serum (FBS) and 0.5% antibiotic solution. Cells were maintained in a humidified incubator at 37°C, 5% CO2. Cells were seeded to approximately 80% confluency 36 h before incubation in caffeinated medium for the indicated time or UVB irradiation for the indicated dose and time at the time of treatment or irradiation. UVB irradiation was performed using a UVB irradiation chamber and UV-mat dosimeter
[0032] Cell viability assay: Cell Counting Kit-8 (CCK-8, DojindoLaboratories, Japan) was used to detect cell viability according to the instructions. HaCaT cells were seeded in 96-well plates at a density of 5 × 10 3cells / well, 100 μL cell culture medium, 5% CO2, 37°C incubation. At the designated time points, 10 μL CCK-8 was added to each well. After 2 h of incubation, the absorbance at 450 nm was measured using a microplate reader (Molecular Devices Inc., San Jose, CA, USA).
[0033] Mitochondrial stress assay: The Agilent Seahorse cell mitochondrial stress test measures key parameters of mitochondrial function by directly measuring the OCR of the cells. One hour before the experiment, the culture medium was replaced with unbuffered DMEM (Seahorse Biosciences) supplemented with 10 mM glucose, and the cells were then equilibrated in a CO2-free incubator for 1 hour. Substrates (oligomycin, FCCP, and a mixture of Rote / AA) were added to the cells sequentially to measure ATP production, maximal respiration, and non-mitochondrial respiration, respectively. These parameters and basal respiration were then used to calculate proton leak and spare respiratory capacity. After analysis, the cells in each well were counted and the results were normalized to values per 5000 cells.
[0034] Mitochondrial membrane potential detection: Mitochondrial membrane potential was detected by laser confocal microscopy using JC-1 fluorescent dye. The maximum excitation wavelength of Jc-1 is 549 nm and the maximum emission wavelength is 574 nm. It appears red or green under a fluorescent microscope. Cells were collected and detected by laser confocal microscopy (Leica, STELLARIS 8, Weizler, Germany). Fluorescence intensity was measured by ImageJ software. Fluorescence intensity can reflect mitochondrial membrane potential. When the fluorescence intensity decreases, the membrane potential also decreases.
[0035] Oxidative stress detection: HaCaT cells were seeded in confocal culture dishes and treated with UVB the next day. Six hours after UVB, 3,30-dihexyloxacbocyanine iodioc6 (3)) (Invitrogen) was added to the culture medium at a final concentration of 40 nM, and the cells were further incubated at 37°C for 30 min. After incubation, the cells were washed with PBS and analyzed by immunofluorescence staining. Before imaging, the cells were stained with DAPI mounting medium.
[0036] Mitophagy detection: Confocal microscopy was used to evaluate mitochondrial morphology and autophagy. HaCaT cells were seeded in confocal-specific culture dishes. Six hours after UVB, cells were stained with 100 nM Mitotracker Red CMXRos (Thermo Fisher Scientific) at 37°C for 30 min. Cells were washed with PBS, fixed with 3.7% paraformaldehyde solution for 10 min at room temperature, and permeabilized with 0.2% Triton X-100 for 10 min. Blocking was performed with 1% BSA in PBS at 37°C for 1 h. Cells were incubated with Alexa Fluor 488-conjugated TOM20 antibody diluted with LAMP1 antibody in 1% BSA (1:50) at 4°C in a humid chamber overnight. The next day, cells were washed three times with PBS. Before imaging, cells were stained with DAPI mounting medium and analyzed by confocal microscopy with a 100x oil immersion objective.
[0037] Immunoblot analysis: Cells were washed twice with PBS at 4°C, added with RIPA buffer supplemented with protease inhibitors at a ratio of 1:1000, and lysed on ice. After incubation on ice for 5 min, the supernatant was obtained by centrifugation at 15,000 rpm for 10 min at 4°C. Protein concentration was determined using the Pierce BCA assay kit (Thermo Fisher Scientific). Lysates were mixed with 5X loading buffer (bromophenol blue (0.25%), β-mercaptoethanol (5%), glycerol (50%), SDS (sodium dodecyl sulfate; 10%), Tris-HCl (0.25 M, pH 6.8)), boiled at 100°C for 10 min, and subjected to 7.5%, 10%, or 12.5% SDS polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% bovine serum albumin (BSA) in TBST (0.05% Tween 20 in TBS buffer). The membrane was then incubated with the following primary antibodies: anti LC3 (1:1000, Abcam, USA), antiATG5 (1:1000, Abcam), antiBECLIN1 (1:1000, Abcam), antiUVRAG (1:1000, Abcam), and anti-GAPDH (1:50, Santa Cruz.) at 4°C overnight. After washing with TBST, horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit IgG (Bio-Rad) was used as a secondary antibody for 1 h at room temperature. Proteins were visualized by ECL Prime Western blotting detection reagent (ThermoFisher Scientific). Bands were quantified using ImageJ open source software
[123] (version 1.51k, National Institutes of Health, Bethesda, MD).
[0038] Statistical analysis: GraphPad Prism 6.0 statistical software (GraphPad software, CA, USA) was used for statistical evaluation using Student's t-test or one-way or two-way analysis of variance. P < 0.05 was considered statistically significant. All error bars represent mean ± SEM. The results are representative of at least three independent experiments.
[0039] Experimental Results
[0040] Caffeine improves cell viability and alleviates UVB-induced oxidative stress damage, such as Figure 1As shown in A, UVB irradiation reduced HaCaT cell viability in a dose-dependent manner. Importantly, caffeine treatment significantly increased UVB-stimulated HaCaT cell viability ( Figure 1 A), however, when UVB reached 100 mj / cm2, the effect of coffee on cell viability was not statistically significant ( Figure 1 A), so in the following experiments our UVB irradiation dose was 50 mj / cm2. ROS is a hallmark of skin damage caused by UVB irradiation
[10] . We found that UVB-induced ROS increased in HACAT cells, while incubation with caffeine significantly reduced oxidative stress damage in HACAT cells ( Figure 1 B and C).
[0041] Co-treatment of UVB irradiation and caffeine incubation alters hacat metabolic activity: To determine how cells metabolically regulate UVB treatment, we provided evidence that co-treatment of UVB irradiation and caffeine resulted in changes in oxygen consumption rate (OCR). We measured OCR values for basal and maximal respiration of hacat cells using an Agilent Seahorse FX analyzer. OCR data showed that caffeine increased both basal and maximal respiration (Figure 2A-C). Caffeine significantly increased basal respiration and increased maximal respiration after UVB treatment and caffeine incubation (Figure 2A-C). Figure 2 DF). These results suggest that co-treatment with UVB irradiation and caffeine incubation alters hacat metabolic activity.
[0042] Co-treatment with UVB irradiation and caffeine incubation caused changes in mitochondrial membrane potential in hacat: At the molecular level, mitochondria convert mitochondrial membrane potential into ATP via mitochondrial respiration. As assessed by JC-1 staining, mitochondrial membrane potential rapidly dissipated under UVB stimulation ( Figure 3 A and B). Interestingly, caffeine incubation maintained mitochondrial membrane potential ( Figure 3 A and B). UVB irradiation induced changes in mitochondrial membrane potential of hacat, and incubation with caffeine reduced the mitochondrial depolarization response.
[0043] Co-treatment with UVB irradiation and caffeine incubation activates autophagic flux in hacat: Mitochondrial autophagy plays an important role in clearing damaged mitochondria, thereby alleviating cell stress and death. We used TOM20 to stain mitochondria to evaluate mitochondria in autophagosomes. To further verify mitophagy, we used LAMP1 to stain lysosomes and observed the co-localization of mitochondria and lysosomes by immunofluorescence. We studied the effects of co-treatment with UVB irradiation and caffeine incubation on mitophagy-related signaling events. The results showed that co-incubation with UVB irradiation and caffeine caused co-localization of hacat autophagy ( Figure 4).
[0044] Co-treatment with UVB irradiation and caffeine incubation regulates the expression of autophagy-related protein bands in hacat: Autophagy is key to clearing damaged proteins or organelles, and the level of LC3II is proportional to the number of autophagic vacuoles
[22] . Many proteins play a key role in the formation of autophagosomes and the final autophagy. To study mitophagy, we measured the expression levels of mitochondrial LC3B-II, ATG5, BECLIN-1, and UVRAG. Although caffeine incubation alone increased the level of autophagy, caffeine could further upregulate the level of LC3II in UVB-stimulated cells ( Figure 5 A and B). The autophagy promoter ATG5 was also significantly increased in UVB-treated cells ( Figure 5 (A and C). We further evaluated the levels of the key upstream autophagy regulator beclin 1, UVRAG. Beclin 1 is responsible for initiating the formation of autophagosomes (17,18). Consistent with the increased formation of LC3 II, co-treatment with UVB irradiation and caffeine significantly increased the levels of beclin 1 in cells ( Figure 5 A and D). The expression of beclin 1 was also significantly increased after co-treatment with UVB irradiation and caffeine incubation compared with the UVB alone irradiation group. UVRAG (ultraviolet radiation resistance associated) is an important regulator of autophagy by interacting with BECN1 (11). UVRAG plays an indispensable role in the repair of UV-induced DNA damage (12). Consistent with the expression of beclin 1, the expression of UVRAG was significantly increased after co-treatment with UVB irradiation and caffeine incubation compared with the UVB alone irradiation group ( Figure 5 A and D). Caffeine alone significantly increased the level of UVRAG in cells compared to the control group ( Figure 5 A and D).
[0045] Experimental Summary
[0046] Our results show that caffeine significantly improves the damage of HACAT cells irradiated by UVB, and improves HACAT cell damage by reducing oxidative stress damage, regulating metabolic responses, maintaining mitochondrial membrane potential, and increasing the expression of related proteins such as autophagy. We found that caffeine increased the viability of HACAT cells, increased mitochondrial aerobic respiration, maintained mitochondrial membrane potential stability, and increased autophagic response. Therefore, our study suggests that caffeine may play a protective role by increasing the viability of HACAT cells, reducing oxidative stress damage in HACAT cells, and accelerating mitochondrial aerobic respiration, thereby promoting cell autophagy.
[0047] Since the epidermis is located in the outermost layer of the human body, it is chronically exposed to various external stimuli, and eliminating these stimuli is important for maintaining the body's homeostasis. Among the external stimuli, ultraviolet B (UVB) radiation is well known to have harmful effects on the skin, and some studies have reported UVB-induced carcinogenesis of keratinocytes. Therefore, protecting keratinocytes from UV damage is essential for maintaining skin health. It has been well documented that caffeine plays a key role in the pathophysiology of various cells in response to oxidative stress and autophagy. However, little is known about the connection between UVB-induced oxidative modifications of caffeine and mitochondrial metabolism in skin damage, which play an important role in regulating autophagy and cell growth. Our data show that caffeine can enhance the activity of HACAT cells after UVB irradiation and reduce the generation of ROS (e.g. Figure 1 AC). Caffeine can also improve mitochondrial metabolism after UVB radiation, increase basal respiration of HACAT cells and improve maximal aerobic respiration (such as Figure 2 AF).
[0048] Normally functioning mitochondria produce ATP and regulate cellular redox balance to ensure epidermal cell metabolism. Damaged mitochondria repair and clear damaged mitochondria through a variety of actions. For example, damaged mitochondria have low mitochondrial potential and cannot convert energy substrates into ATP. Our data show that UVB treatment alone reduces mitochondrial membrane potential, while co-treatment with UVB irradiation and caffeine incubation can increase mitochondrial membrane potential (e.g. Figure 3 A and B), indicating that the mitochondrial depolarization response is reduced, thereby maintaining mitochondrial function.
[0049] We found that mitochondria and lysosomes labeled with TOM20 and LAMP1, respectively, co-treated with UVB irradiation and caffeine incubation and observed autophagy co-localization in hacat cells using confocal microscopy (e.g. Figure 4 We also observed increased co-localization of lysosomes and mitochondria after caffeine treatment alone compared to untreated controls (e.g. Figure 4 These results provide evidence that UVB induces mitophagy in mitochondrial stress in HACAT cells co-treated with caffeine incubation.
[0050] Although the outcome of autophagosome formation and its effects on cell survival and death depend on the cell type and context (33,34), elevated expression of LC3B has been shown to trigger an autophagic response, and ATG5 is an initiator of autophagic responses, consistent with LC3B expression (e.g. Figure 5AC). Beclin 1 is associated with human diseases, including cancer, heart disease, pathogen infection, and neurodegeneration. It has been reported that UVRAG and Beclin 1 work together to induce autophagosome biogenesis. In this study, we found that caffeine significantly increased the expression of Beclin1 and UVRAG in HACAT cells. Therefore, these data suggest that caffeine improves UVB-induced damage to HACAT through autophagy. These results suggest that caffeine has a potential therapeutic effect on UV damage.
[0051] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.
Claims
1. A method for alleviating high-dose UVB-induced damage with low-dose caffeine, characterized in that: The effect of 1000 ng / ml caffeine concentration on 50 mJ / cm 2 UVB irradiation induces photochemical protection in human epidermal keratinocytes (HaCaT).
2. The method of claim 1 for alleviating high-dose UVB-induced damage with low-dose caffeine, characterized in that: Caffeine improves cell viability and alleviates UVB irradiation-induced oxidative stress damage.
3. The method of claim 1 for alleviating high-dose UVB-induced damage with low-dose caffeine, characterized in that: Co-treatment of UVB irradiation and caffeine incubation increased the metabolic activity of hacat.
4. The method of claim 1 for alleviating high-dose UVB-induced damage with low-dose caffeine, characterized in that: Co-treatment of UVB irradiation and caffeine incubation caused changes in mitochondrial membrane potential of hacat, and caffeine incubation could reduce the mitochondrial depolarization response.
5. The method of claim 1 for alleviating high-dose UVB-induced damage with low-dose caffeine, characterized in that: Co-treatment of UVB irradiation and caffeine incubation increased autophagic flux in Hacat.
6. The method of claim 1 for alleviating high-dose UVB-induced damage with low-dose caffeine, characterized in that: Co-treatment of UVB irradiation and caffeine incubation upregulated the expression of the autophagy-related egg band in Hacat.
7. The method of claim 1 for alleviating high-dose UVB-induced damage with low-dose caffeine, characterized in that: Caffeine significantly improves the damage of HACAT cells induced by UVB radiation by reducing oxidative stress damage, regulating metabolic responses, maintaining mitochondrial membrane potential and increasing the expression of autophagy-related proteins. Caffeine improves the vitality of HACAT cells, improves mitochondrial aerobic respiration, maintains the stability of mitochondrial membrane potential and increases autophagy response. Caffeine plays a protective role by improving the vitality of HACAT cells, reducing oxidative stress damage of HACAT cells and accelerating mitochondrial aerobic respiration, thereby promoting cell autophagy.