CN110464714B - Application of undecylenic aldehyde in relieving oxidative stress skin injury - Google Patents

Application of undecylenic aldehyde in relieving oxidative stress skin injury Download PDF

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CN110464714B
CN110464714B CN201910783502.9A CN201910783502A CN110464714B CN 110464714 B CN110464714 B CN 110464714B CN 201910783502 A CN201910783502 A CN 201910783502A CN 110464714 B CN110464714 B CN 110464714B
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undecanal
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王华威
郑希
黄泽彬
刘俊磊
张蓝月
张焜
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Guangdong University of Technology
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
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    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of undecanal in preparation of a product for relieving oxidative stress skin injury. The research of the invention finds that the undecanal can improve H2O2Induced decreased HaCaT cell activity, decreased H-supply2O2Induced apoptosis of cells, on H2O2The induced oxidative stress injury of the HaCaT cells has good protection effect, and in addition, the activity of ARE can be enhanced, the expression level of HO-1 and Nrf 2 proteins can be promoted, and a new way is provided for researching and developing new anti-oxidative stress skin injury medicines.

Description

Application of undecylenic aldehyde in relieving oxidative stress skin injury
Technical Field
The invention belongs to the technical field of medicines. More particularly, it relates to the use of undecanal for the relief of oxidative stress skin lesions.
Background
Oxidative stress refers to a series of stress reactions caused by imbalance of oxidative and antioxidant homeostasis of the body under the stimulation of internal and external factors and excessive accumulation of Reactive Oxygen Species (ROS), and the generation of excessive ROS can cause cell damage and induce various diseases. The skin covers the surface of the body, is the first important barrier for protecting the body from external damage, and is more easily affected by internal and external source stimulation compared with other organs, so that oxidative stress is formed. The generation of excessive ROS can oxidatively decompose proteins, activate matrix metalloproteinases, and enzymatically hydrolyze elastin and collagen of the skin, resulting in dry, loose, dark, aged skin and the occurrence of various skin diseases, such as inflammatory skin diseases, epidermal tumors, etc.
At present, the protection of the oxidative stress injury of the skin is insufficient, and a plurality of problems exist in related treatment methods, including large dosage, large toxic and side effect and high treatment price. Although many studies indicate that natural essential oils have a certain relieving effect, the lack of research on specific active ingredients thereof makes it urgent to find a protective agent that can effectively relieve skin damage caused by oxidative stress, has small toxic and side effects, is low in administration dose, and is relatively inexpensive.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the existing technology for protecting skin from oxidative stress injury, provides application of undecanal in preparation of products for relieving oxidative stress skin injury, and provides a protective agent with small toxic and side effects, low administration dosage and relatively low price for effectively relieving skin injury caused by oxidative stress.
It is another object of the present invention to provide a process for the preparation of undecalaldehyde with reduced H2O2Use in a product for inducing apoptosis.
It is another object of the present invention to provide the use of undecanal in the preparation of a product that enhances the activity of ARE.
Another object of the present invention is to provide the use of undecanal for the preparation of a product promoting the expression level of HO-1 protein.
Another object of the invention is to provide the use of undecanal in the preparation of a product that promotes the expression level of Nrf 2 protein.
It is another object of the present invention to provide a product for alleviating oxidative stress skin damage.
The above purpose of the invention is realized by the following technical scheme:
according to the invention, through researching the influence of the undecanal on the activity of the HaCaT cells, the undecanal with different concentrations is used for treating the cells, so that the activity of the HaCaT cells is hardly influenced, and the undecanal has no obvious toxicity to the HaCaT cells; the invention further researches and discovers that the undecanal can improve H2O2Induced decrease in HaCaT cell Activity on H2O2Induction ofThe HaCaT cells have good protection effect on oxidative stress injury; further study of undecalaldehyde on H2O2Effect of induced HaCaT apoptosis, the results show that pretreatment of cells with undecanal strongly reduced the level of apoptosis caused by H2O2Induced apoptosis. Namely, the experimental result shows that the undecanal has a better protective effect on oxidative stress skin injury.
The invention therefore claims the use of undecanal for the preparation of a product for relieving oxidative stress skin damage.
The invention also claims the reduction of H in the preparation of undecanal2O2Use in a product for inducing apoptosis.
The invention also claims a product for relieving oxidative stress skin injury.
Preferably, the undecalaldehyde is administered at a concentration of 80-120 μ M.
The invention further determines the influence of the undecalaldehyde on the activity of an anti-oxidation reaction element (ARE) through a luciferase reporter molecule, and the research finds that the undecalaldehyde can enhance the activity of the ARE; in addition, the effect of undecanal on the expression levels of HO-1 and Nrf 2 proteins was determined by Western blot analysis, and it was found that treatment of HaCaT cells with undecanal significantly increased their levels of HO-1 and Nrf 2 protein expression.
The invention therefore also claims the use of undecanal for the preparation of a product that enhances the activity of ARE.
The invention also claims the application of undecanal in the preparation of products promoting the expression level of HO-1 protein.
The invention also claims the application of the undecanal in the preparation of products for promoting the expression level of the Nrf 2 protein.
The invention has the following beneficial effects:
1. the invention provides a new application of undecanal in skin oxidative stress injury protection, wherein the undecanal can improve H2O2The induced HaCaT cell activity is reduced, and H can be greatly inhibited2O2Induced HaCaT apoptosis on H2O2The induced oxidative stress injury of HaCaT cells hasGood protection effect, and can also enhance the activity of ARE and promote the expression level of HO-1 and Nrf 2 proteins. And the undecanal is a natural plant component, is colorless to yellowish oily liquid, is naturally and slightly existed in essential oil such as lemon oil, sweet orange oil, etc., has strong fresh lipid wax smell and rose fragrance, has almost no influence on cell activity as skin oxidative stress injury protective agent, and has no toxicity.
2. The protective agent provided by the invention has an obvious protective effect on oxidative stress skin injury, and a good effect can be obtained by adding a small amount of undecanal, so that the toxic and side effects of the medicine are reduced. In addition, the price of the protective agent provided by the invention is relatively low, and the economic burden of a patient is effectively reduced.
3. The protective agent provided by the invention lays a theoretical foundation for researching the action mechanism of the natural plant antioxidant for protecting oxidative stress skin injury.
Drawings
FIG. 1 is a graph of the effect of different concentrations of undecanal treatment on HaCaT cell activity in example 1;
FIG. 2 is a graph of different concentrations of undecanal treatment on H in example 22O2The effect of treating cell activity;
FIG. 3 is a graph of different concentrations of undecanal treatment vs. H in example 32O2To address the effects of apoptosis;
FIG. 4 is the effect of different concentrations of undecanal on the activity of the Antioxidant Response Element (ARE) in example 4;
FIG. 5 is a graph of the effect of different concentrations of undecanal on HO-1 and Nrf 2 protein expression levels in example 5.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The undecanal is colorless to yellowish oily liquid, is naturally and slightly existed in essential oil such as lemon oil, sweet orange oil, and orange oil, has strong fresh greasy wax gas and rose fragrance, and has the following structural formula:
Figure BDA0002177286310000031
HaCaT cells are biologically similar to the epithelial keratinocytes of the body, and have the characteristics of simple source, easy culture, good stability and strong proliferation capacity, and thus are used as human epidermal cells. The invention selects HaCaT cells cultured in vitro as a representative, uses undecanal as a pre-protective agent, and then uses H2O2Induce oxidative damage thereof by evaluating H of undecalaldehyde on HaCaT cells2O2The effect of oxidative damage and the effect of the expression level of the related proteins to investigate the protective effect of undecanal on oxidative stress skin damage.
Example 1 determination of the Effect of different concentrations of undecanal on HaCaT cell Activity by the MTT method
1. Experimental methods
HaCaT cells were digested from the flask with trypsin, centrifuged for 5min, and added to DMEM medium containing 10% FBS and antibiotics (100 units/mL penicillin, 100. mu.g/mL streptomycin) to prepare a cell suspension to inoculate 1X 104Each/mL of the cells was inoculated into a 96-well plate (100. mu.L/well), and incubated at 37 ℃ for 24 hours in 5% CO 2. Concentrations of undecalaldehyde were set at 160. mu.M, 120. mu.M, 80. mu.M, 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M, with 6 duplicate wells per concentration. 5% CO2After 24 hours of incubation at 37 ℃, the medium was carefully aspirated, and 150. mu.L of MTT solution (0.5mg/mL) prepared in serum-free medium was added to each well; after further incubation for 4h, the culture medium was carefully aspirated from the wells. Adding 150 mu L DMSO into each hole, and standing in an incubator for ten minutes; the absorbance of each well at 570nm was measured using a microplate reader.
2. Results of the experiment
As shown in FIG. 1, treatment of cells with different concentrations of undecanal had little effect on reducing cell viability. At the highest concentration (160. mu.M), undecanal reduced cell activity by only 14%. Therefore, the experimental result shows that the undecanal has no obvious toxicity to HaCaT cells.
Example 2 undecanal on H2O2Pre-protection experiment for induced oxidative stress injury of HaCaT cells
1. Experimental methods
HaCaT cells were digested from the flask with trypsin, centrifuged for 5min, and added to DMEM medium containing 10% FBS and antibiotics (100 units/mL penicillin, 100. mu.g/mL streptomycin) to prepare a cell suspension to inoculate 1X 104Each/mL of the cells was inoculated into a 96-well plate (100. mu.L/well) and incubated at 37 ℃ in 5% CO 2. After 24H, medium, blank and H were discarded2O2The group was added with 100. mu.L of DMEM basal medium, and the experimental group was added with different concentrations of undecalaldehyde (160. mu.M, 120. mu.M, 80. mu.M, 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M), 3 multiple wells per concentration were set, and pre-treatment was performed. After 24H, 100. mu.L of H-containing solution was added to each well2O2(500. mu.M) DMEM basal medium, then placed at 37 ℃ with 5% CO2Culturing in an incubator. After 24h, the medium was carefully aspirated, and 150. mu.L of MTT solution (0.5mg/mL) in serum-free medium was added to each well; after further incubation for 4h, the culture medium was carefully aspirated from the wells. Adding 150 mu L DMSO into each hole, and standing in an incubator for ten minutes; the absorbance of each well at 570nm was measured using a microplate reader.
2. Results of the experiment
As shown in FIG. 2, using H2O2Treatment of the cells resulted in a reduction in cell viability of 53%. After the cells were pretreated with lower concentration of undecanal (5-40. mu.M), H was treated2O2The induced reduction of cell activity has little protective effect, and the concentration (80-160 mu M) of the undecanal is increased to enhance the effect of the undecanal on H2O2Protective effect inducing a decrease in cellular activity, wherein cellular activity is enhanced to 83% for H when the concentration of undecanal is 120. mu.M2O2The protective effect of the induced reduction of cell activity is strongest.
Example 3 undecanal to H2O2Pre-protection assay for induced HaCaT apoptosis
1. Experimental methods
HaCaT cells were digested from the flask with trypsin, centrifuged for 5min, and added to DMEM medium containing 10% FBS and antibiotics (100 units/mL penicillin, 100. mu.g/mL streptomycin) to prepare a cell suspension to inoculate 1X 104Each/mL of the cells was inoculated into a 96-well plate (100. mu.L/well) and incubated at 37 ℃ in 5% CO 2. After 24H, medium, blank and H were discarded2O2The group was added with 100. mu.L of DMEM basal medium, and the experimental group was added with different concentrations of undecalaldehyde (160. mu.M, 120. mu.M, 80. mu.M, 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M), 3 multiple wells per concentration were set, and pre-treatment was performed. After 24H, 100. mu.L of H-containing solution was added to each well2O2(500. mu.M) DMEM basal medium, then placed at 37 ℃ with 5% CO2Culturing in an incubator. After treatment, cytospins were prepared and cells were fixed with acetone/methanol (1:1) for 10 min at room temperature, then stained with propidium iodide (1. mu.g/mL in PBS) for 10 min and analyzed using a fluorescence microscope (Nikon Eclipse TE200, Japan). Apoptotic cells were identified by classical morphological features including nuclear condensation, cell shrinkage and the formation of apoptotic bodies.
2. Results of the experiment
As shown in FIG. 3, using H2O2(500. mu.M) treatment of the cells resulted in a strong increase in apoptosis. Pretreatment of cells with lower concentrations of undecalaldehyde (5-40 μ M) for H reduction2O2The induced apoptosis has a minor effect. The pretreatment of the cells with higher concentration of undecanal (80-160 μ M) can strongly reduce the free radical of H2O2Induced apoptosis of cells in which H was detected in the experimental groups at concentrations of 120. mu.M and 160. mu.M2O2The inhibition effect of inducing apoptosis is equivalent.
Example 4 Effect of undecanal on Activity of Oxidation reaction elements (AREs)
1. Experimental methods
The effect of undecanal on ARE activity was determined by luciferase reporter assay. In these experiments, HaCaT cells were transiently transfected with a firefly luciferase reporter plasmid containing the ARE-promoter plasmid p-ARE-Luc and the Renilla luciferase plasmid p-RL using Lipofectamine 2000 transfection reagent. 24h after transfection, cells were treated with different concentrations of undecanal for 24 h. After washing with PBS, cells were lysed and the supernatant was used for luciferase assay. Luciferase activity was measured by luminometer.
2. Results of the experiment
As shown in FIG. 4, the activity of ARE was enhanced by treating the cells with 5-20. mu.M of undecanal, which had little effect on the increase in ARE activity, and the increase in ARE activity was moderate by 40. mu.M of undesulfurization. Higher concentration of undecanal (80-160 μ M) has stronger effect on increasing the activity of ARE.
Example 5 Effect of undecanal on HO-1 and Nrf 2 protein expression levels
1. Experimental methods
The effect of undecanal on HO-1 and Nrf 2 levels was determined by western blot analysis. In these experiments, HaCaT cells were seeded in 100mm dishes and incubated for 24 h. The cells were then treated with different concentrations of undecanal for 24 h. At the end of the experiment, the cells were washed with PBS and lysed. Western blot analysis of cell lysates using anti-HO-1 and anti-Nrf 2 antibodies.
2. Results of the experiment
As shown in FIG. 5, treatment of cells with 80 μ M and 120 μ M undecanal increased the levels of HO-1 and Nrf 2. Undecanal at 120 μ M had a stronger effect than increasing the levels of HO-1 and Nrf 2 at 80 μ M.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (3)

1. Application of undecanal in preparing products for relieving oxidative stress skin injury is provided.
2. Use according to claim 1, characterized in thatThen, the oxidative stress skin injury is caused by H2O2Induced oxidative stress skin damage.
3. Reduction of H in the preparation of undecanal2O2Application in products of induced HaCaT cell apoptosis.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
US20120021932A1 (en) * 2008-07-31 2012-01-26 Andreas Mershin Multiplexed Olfactory Receptor-Based Microsurface Plasmon Polariton Detector

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120021932A1 (en) * 2008-07-31 2012-01-26 Andreas Mershin Multiplexed Olfactory Receptor-Based Microsurface Plasmon Polariton Detector

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