Application and method of lauryl sodium sulfate in aspect of inducing skin keratinocyte senescence

Abstract

The invention relates to the technical field of cell research, in particular to application of lauryl sodium sulfate in the aspect of skin keratinocyte senescence induction and a method. The invention proves that SDS as a novel chemical inducer can effectively induce skin keratinocyte aging in vitro for the first time. By adjusting the concentration of SDS, obvious senescence phenotypes, such as cell morphological change, expression increase of senescence-related secretion phenotypes (SASP) and the like, can be induced on the premise of not influencing cell survival. Besides, in SDS-induced senescence cells, the expression levels of key senescence markers such as p53, p21 and p16 are remarkably increased, and the expression level of Lamin B1 is remarkably decreased, which indicates that SDS can activate senescence signal pathways in the cells. Compared with the most common light aging model, the SDS can generate an approximate aging induction effect, and a new experimental tool is provided for researching a skin aging mechanism.

Description

Technical Field

[0001] The present invention relates to the technical field of cell research, and specifically to an application and method of sodium dodecyl sulfate in inducing senescence of skin keratinocytes. Background Art

[0002] As the largest organ in the human body, the skin is not only the first line of defense against external microorganisms, ultraviolet rays and mechanical damage, but also participates in a variety of physiological functions such as temperature regulation and sensory transmission. Skin aging is mainly divided into natural aging (endogenous aging) and photoaging (exogenous aging). Keratinocytes are located in the outermost layer of the skin and are the main component of the skin barrier. Their health status is directly related to the overall function of the skin. Therefore, in-depth research on the aging mechanism of keratinocytes will not only help reveal the deep causes of skin aging, but also provide an important theoretical basis and technical support for the development of methods to delay skin aging, prevent and treat related skin diseases.

[0003] Studies have shown that in addition to the irreversible factor of aging, a variety of internal and external pressures, such as environmental pollution and bad living habits, can also accelerate the aging process of the skin. At the keratinocyte level, these pressures can lead to slower renewal, decreased cell function, increased oxidative stress, and accumulated DNA damage, which in turn weaken the skin's barrier function, reduce water retention, and trigger a series of physiological changes. More importantly, senescent cells accumulate and secrete the senescence-associated secretory phenotype (SASP), which has a negative impact on the surrounding healthy cells, forming a vicious circle and further aggravating the degree of skin aging. Although research in the field of skin aging has made significant progress in recent years, the specific mechanism of cell aging and its connection with diseases still need to be further explored.

[0004] In order to simulate the complex aging process in vivo, researchers have developed a variety of in vitro techniques to induce keratinocyte aging, mainly including the following:

[0005] 1. Oxidative stress

[0006] Creating an oxidative environment by adding hydrogen peroxide (H2O2), exposing to ultraviolet (UV) radiation, or using free radical generators can lead to intracellular DNA damage, protein and lipid oxidation, and ultimately induce cell senescence;

[0007] 2. Replicative senescence For primary keratinocytes, it can be achieved by repeated subculturing until they reach the Hayflick limit, i.e. the cells can no longer divide and enter an irreversible state of growth arrest;

[0008] 3. DNA damage

[0009] Using chemical substances such as bleomycin, or physical means such as γ-ray irradiation, to cause DNA double-strand breaks, trigger the DNA damage response mechanism in cells, and then induce aging;

[0010] 4. Telomere Shortening

[0011] Each time a cell divides, the telomeres at the ends of the chromosomes gradually become shorter. When the telomere length falls below a certain threshold, the cell stops dividing and enters a state of senescence. This process can be accelerated by inhibiting telomerase activity or directly manipulating telomere length;

[0012] 5. Inflammatory factors

[0013] Chronic low-grade inflammation is a characteristic of cell aging. Proinflammatory cytokines (such as TNF-α, IL-1β, IL-6, etc.) can be used to treat cells to simulate the long-term inflammatory environment in the body and promote cell aging.

[0014] 6. Metabolic stress

[0015] Cell culture conditions can be altered, such as by reducing glucose concentration, adding fatty acids, changing pH or oxygen levels (e.g., hyperoxia or hypoxia), to create metabolic stress, which can also induce cellular senescence.

[0016] 7. Drug-induced

[0017] Certain drugs or compounds can directly or indirectly affect cell cycle regulation, signal transduction pathways, or other biological processes associated with aging, such as targeting the mTOR pathway with rapamycin;

[0018] 8. Gene Editing

[0019] Use gene editing technologies such as CRISPR / Cas9 to knock out or overexpress specific genes, such as p53, p21, p16, etc.

[0020] Therefore, establishing a new and effective in vitro senescence induction model is crucial to explore the nature of cellular senescence. Summary of the invention

[0021] In order to solve the above technical problems existing in the prior art, the present invention provides an application and method of sodium dodecyl sulfate in inducing senescence of skin keratinocytes.

[0022] Application of sodium dodecyl sulfate in inducing senescence of skin keratinocytes, wherein the molecular formula of the sodium dodecyl sulfate is C 12 H 25 SO4Na, molecular weight is 288.38 g / mol.

[0023] Furthermore, the sodium dodecyl sulfate is composed of a hydrophilic sulfate group and a hydrophobic dodecyl group. This amphiphilic structure gives SDS good surface activity and emulsification ability. SDS is inexpensive, easy to obtain, and has the following characteristics:

[0024] 1. Chemical properties: The molecular formula is C12H25SO4Na, and the molecular weight is 288.38g / mol;

[0025] 2. Physical properties: usually white to slightly yellow crystalline powder, slightly toxic, slightly soluble in alcohol, insoluble in chloroform and ether, and easily soluble in water;

[0026] 3. Toxicological properties:

[0027] 1) Acute toxicity: oral LD50 (median lethal dose) for rats is 1288 mg / kg; intraperitoneal LD50 for rats is 210 mg / kg; intravenous LD50 for rats is 118 mg / kg; intraperitoneal LC50 for mice is 250 mg / kg; percutaneous LD50 for rabbits is 10 mg / kg; intravenous LC50 for mice is 118 mg / kg;

[0028] 2) Inhalation toxicity: Rat LD50>3900mg / m3 / 1H.

[0029] Furthermore, the induction of skin keratinocyte senescence is to induce an obvious senescence phenotype without affecting cell survival.

[0030] Furthermore, the inducing senescence of skin keratinocytes is to activate the senescence signaling pathway within the cells.

[0031] Furthermore, the induction of skin keratinocyte senescence significantly increases the mRNA levels of p53, p21, and p16, significantly decreases the mRNA level of Laminb-1, and significantly increases the protein levels of p53, p21, and p16.

[0032] A method for inducing senescence of skin keratinocytes, using sodium lauryl sulfate to stimulate the skin keratinocytes.

[0033] Furthermore, the stimulation with sodium dodecyl sulfate is such that the concentration of sodium dodecyl sulfate in the environment of skin keratinocytes is 0.006% to 0.008%, and the stimulation time is 12-36 hours.

[0034] Furthermore, the method comprises the following steps:

[0035] ① Add sodium dodecyl sulfate to HaCaT culture medium to obtain SDS-HaCaT culture medium;

[0036] ②According to 2×10 4~2×10 6 cells / mL for plating;

[0037] ③Discard the original culture medium, rinse with PBS, and finally aspirate the PBS;

[0038] ③Add the prepared SDS-HaCaT medium and stimulate for 12-36 hours.

[0039] ④After stimulation, discard the SDS-containing culture medium, rinse twice with ordinary HaCaT culture medium, and finally add fresh culture medium for culture.

[0040] SDS is also widely used in biological experiments, for example,

[0041] 1. Protein denaturation: SDS can destroy the secondary and tertiary structures of proteins, making them completely deformed; in SDS-PAGE (polyacrylamide gel electrophoresis), SDS can bind to proteins, making them negatively charged, and thus separate them according to their molecular weight in an electric field;

[0042] 2. Membrane protein extraction: SDS can dissolve cell membranes and help extract and purify membrane proteins;

[0043] 3. Protein extraction: SDS can destroy cell membranes and organelle membranes, helping to extract intracellular proteins. For example, the commonly used protein lysis RIPA contains 0.1% SDS;

[0044] 4. Buffer: SDS can be used to prepare various buffers to help maintain a stable pH value of the buffer.

[0045] 5. Detergent: SDS is a highly effective detergent, widely used in cleaning products;

[0046] During the experiment, we accidentally discovered that SDS may have the property of inducing senescence of skin epidermal cells, and designed the following experiment to verify it. We selected the immortalized keratinocyte cell line (HaCaT) as the experimental subject. HaCaT cells can simulate the differentiation process of keratinocytes, including the ability to produce keratin and other epidermal barrier-related proteins, and are often used to study skin differentiation, barrier function, and molecular mechanisms related to skin diseases.

[0047] Compared with the prior art, the technical effects created by the present invention are embodied in:

[0048] The present invention confirms for the first time that SDS, as a new type of chemical inducer, can effectively induce senescence of skin keratinocytes in vitro. By adjusting the concentration of SDS, obvious senescence phenotypes such as changes in cell morphology and increased expression of the senescence-associated secretory phenotype (SASP) can be induced without affecting cell survival. In addition, in SDS-induced senescent cells, the expression levels of key senescence markers such as p53, p21, and p16 were significantly upregulated, and the expression level of Lamin B1 was significantly downregulated, indicating that SDS can activate the senescence signaling pathway in cells. Compared with the most commonly used photoaging model, SDS can produce similar senescence-inducing effects, providing a new experimental tool for studying the mechanism of skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a graph showing the results of screening the SDS concentration suitable for HaCaT cell culture.

[0050] Figure 2 This is a result diagram to verify the effect of SDS-induced senescence. The results show that SDS induction leads to an increase in the proportion of HaCaT senescent cells; A. SA-β-Gal staining detection: expression of β-galactosidase positive cells in HaCaT after treatment with SDS at different concentrations (0.006%, 0.007%, 0.008%) in the control group; B. Statistical diagram of the proportion of β-galactosidase positive cells (n=3, ****P<0.0001)

[0051] Figure 3 This is a comparison of the results of SDS induction and photoaging models. The results show that SDS induction can produce an aging effect similar to that of UVB irradiation; A. RT-qPCR detection of the relative expression of aging markers: the relative expression levels of aging markers (Laminb-1, p53, p21, p16) in HaCaT at 48 hours after 0.007% SDS stimulation and 3 days after UVB irradiation (n=3, **P<0.01, ***P<0.001, ****P<0.0001); B. Western Blotting detection of protein expression of aging markers: the expression of aging proteins (p53, p21, p16) in HaCaT at 0 hours, 24 hours, 48 ​​hours after 0.007% SDS stimulation and 3 days after UVB irradiation. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0053] The experimental methods in the following examples are all conventional methods unless otherwise specified; the biological and chemical reagents used are all conventional reagents in the art unless otherwise specified.

[0054] 1. Preliminary screening of SDS concentration

[0055] ① Weigh 0.010 g SDS powder, add 50 mL HaCaT culture medium to prepare 0.02% SDS-HaCaT culture medium, and dilute into a concentration gradient (1000 μL) according to the following table;

[0056] 0.000％ 0.001％ 0.002％ 0.003％ 0.004％ 0.005％ 0.006％ 0.007％ 0.008％ 0.009％ 0.010％ 0.011％ 0.02％ 0 50 100 150 200 250 300 350 400 450 500 550 0％ 1000 950 900 850 800 750 700 650 600 550 500 450

[0057] ② One day in advance according to 2×10 5 cells / mL, 300L / well, for plating in 48-well plates;

[0058] ③Discard the culture medium, rinse twice with PBS, and aspirate the PBS in the last rinse;

[0059] ④ Add the prepared SDS-HaCaT medium, 300L / well, and three replicate wells for each concentration;

[0060] Place in a 37°C, 5% CO2 incubator for 24 h;

[0061] ⑤After 24 hours, discard the SDS-containing medium, rinse twice with PBS, and finally add an appropriate amount of PBS and observe under a microscope;

[0062] The results showed that when the concentration reached 0.006%, the cells began to show obvious signs of aging, such as enlarged volume and irregular cell morphology; when the concentration reached 0.008%, obvious cell death occurred; and when the concentration reached 0.009%, the cells could not survive; 2. Verify the effect of SDS-induced aging

[0063] ① Select and use three concentrations of SDS-HaCaT culture medium: 0.006%, 0.007%, and 0.008%;

[0064] ② One day in advance according to 2×10 5 cells / mL, 1 mL / well, for plating in 12-well plates;

[0065] ③Discard the original culture medium, rinse twice with PBS, and aspirate the PBS in the last wash;

[0066] ③ Add the prepared SDS-HaCaT medium, 1 mL / well, three replicate wells for each concentration, and place in a 37°C, 5% CO2 incubator for stimulation for 24 hours;

[0067] ④After 24 hours, discard the medium containing SDS, rinse twice with ordinary HaCaT culture medium, and finally add 1 mL of fresh culture medium to each well for culture;

[0068] ⑤After 8 days, by detecting the proportion of beta-galactosidase-positive cells, it was found that with the increase of SDS concentration, the proportion of senescent cells also increased significantly, showing an obvious concentration gradient; 3. Comparison with the photoaging model

[0069] Ultraviolet light is the most commonly used method to induce keratinocyte senescence. We compared SDS-induced and UV-induced methods and found that both methods were effective.

[0070] ① Using HaCaT cells, 1×10 5 After the cells adhered to the wall, they were irradiated with UVB, with an energy of 60 mJ / cm 2 , irradiate continuously for 4 seconds every day for three consecutive days;

[0071] ② On the third day, collect RNA and protein samples of cells;

[0072] ③At the same time, the cells were stimulated with 0.007% SDS-HaCaT medium, and protein samples of SDS-HaCaT cells were collected at 0h, 24h, and 48h after washing the SDS-HaCaT medium; RNA samples of SDS-HaCaT cells were collected at 48h;

[0073] The results showed that the mRNA levels of p53, p21, and p16 were significantly increased, the Laminb-1 mRNA level was significantly decreased, and the protein levels of p53, p21, and p16 were also significantly upregulated. Compared with the photoaging model, SDS can also induce aging.

[0074] Conclusion: This study confirmed for the first time that SDS, as a new type of chemical inducer, can effectively induce senescence of skin keratinocytes in vitro. By adjusting the concentration of SDS, obvious senescence phenotypes such as cell morphological changes and increased expression of the senescence-associated secretory phenotype (SASP) can be induced without affecting cell survival. In addition, in SDS-induced senescent cells, the expression levels of key senescence markers such as p53, p21, and p16 were significantly upregulated, and the expression level of Lamin B1 was downregulated, indicating that SDS can activate the senescence signaling pathway in cells. Compared with the most commonly used photoaging model, SDS can produce similar senescence-inducing effects, providing a new experimental tool for studying the mechanism of skin aging. Future studies will further explore the specific molecular mechanisms of SDS-induced senescence and evaluate its potential application value in the development of anti-aging products.

[0075] Finally, it should be emphasized that the above implementation cases are only highly representative examples of the present invention. Undoubtedly, the technical concepts covered by the present invention are far more than these specific examples, and its boundaries are broad, which can accommodate many variations and innovations. Therefore, any various variations and improvements that can be directly derived or reasonably associated with by any person skilled in the art based on the information disclosed in the present invention should be regarded as falling within the protection scope of the present invention without exception.

Claims

1. The use of sodium lauryl sulfate in inducing senescence of skin keratinocytes, characterized in that: The molecular formula of sodium dodecyl sulfate is C 12 H 25 SO4Na, molecular weight is 288.38 g / mol.

2. The use according to claim 1, characterized in that: The sodium dodecyl sulfate is composed of a hydrophilic sulfate group and a hydrophobic dodecyl group.

3. The use according to claim 1, characterized in that: The inducing senescence of skin keratinocytes is to induce obvious senescence phenotype without affecting cell survival.

4. The use according to claim 1, characterized in that: The inducing senescence of skin keratinocytes is to activate the senescence signaling pathway within the cells.

5. The use according to claim 1, characterized in that: The induction of skin keratinocyte senescence is to significantly increase the mRNA levels of p53, p21, and p16 in the cells, significantly reduce the mRNA level of Laminb-1, and significantly increase the protein levels of p53, p21, and p16.

6. A method for inducing senescence of skin keratinocytes, characterized in that: Sodium lauryl sulfate is used to stimulate skin keratinocytes.

7. The method according to claim 6, characterized in that The stimulation with sodium dodecyl sulfate is to make the concentration of sodium dodecyl sulfate in the environment of skin keratinocytes to be 0.006% to 0.008%.

8. The method according to claim 6, characterized in that The stimulation is carried out using sodium dodecyl sulfate, and the stimulation time is 12-36 hours.

9. The method according to claim 6, characterized in that The steps include: ① Add sodium dodecyl sulfate to HaCaT culture medium to obtain SDS-HaCaT culture medium; ②According to 2×10 4 ~2×10 6 cells / mL for plating; ③Discard the original culture medium, rinse with PBS, and finally aspirate the PBS; ③Add the prepared SDS-HaCaT medium and stimulate for 12-36 hours.

10. The method according to claim 9, characterized in that After stimulation, the SDS-containing culture medium was discarded, the cells were rinsed twice with ordinary HaCaT culture medium, and finally fresh culture medium was added for culture.

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