SOD (superoxide dismutase) for controlling abnormal skin grease secretion and application of superoxide dismutase

Abstract

The invention discloses superoxide dismutase for controlling skin grease secretion abnormity and application thereof, belongs to the field of biological medicines, and relates to application of superoxide dismutase in medicines or medical instruments for treating diseases such as seborrheic dermatitis, seborrheic alopecia and the like by controlling skin grease secretion. The invention also discloses application of the composition in cosmetics with an oil control effect. At present, most medicines for treating abnormal skin grease secretion are human hormone disruptors, are poor in safety and strong in side effect and cannot be used for a long time; oil control components in cosmetics are generally multi-component mixtures, and the safety and the batch stability of the oil control components need to be optimized. The invention provides a biomedical material which is high in safety, good in stability and single in composition, and can be used for developing medicines, medical instruments or cosmetics for controlling abnormal skin grease secretion. The invention provides therapeutic activity of superoxide dismutase in skin oil control and application of the superoxide dismutase.

Description

Technical Field

[0001] Superoxide dismutase for controlling abnormal skin sebum secretion and its application belong to the field of biomedicine. Background Art

[0002] Excessive sebum secretion by the skin is a typical feature of oily skin. Oily skin is usually accompanied by problems such as shiny skin, enlarged pores, acne, seborrheic dermatitis, and seborrheic alopecia.

[0003] Sebum secreted by the skin is roughly composed of two parts. One is secreted by sebaceous glands, and the other is produced by the rupture of stratum corneum cells (. Skin Pharmacology & Applied Skin Physiology, 2000, 13(6): 372 - 389.). They have different sources, compositions, and influencing factors. Sebum secreted by sebaceous glands is mainly affected by body fat percentage and androgen levels, while the sebum produced by the rupture of stratum corneum cells is usually related to factors such as ultraviolet radiation, bacterial infection, and damaged skin barrier.

[0004] However, current treatment methods for excessive skin sebum secretion are mostly botulinum toxin injection, photodynamic therapy, laser therapy, etc. These treatment methods require professional doctors and equipment, are not only expensive but often require multiple treatments. Spironolactone is a first-line drug for clinically treating excessive skin sebum secretion. It inhibits skin sebum secretion by interfering with the androgen signal in the body and has strong side effects, so it cannot be used as a long-term solution for ordinary oily skin conditioning.

[0005] Reported raw materials for cosmetics with sebum control effects, such as extracts of Oroxylum indicum (CN119454539A), Dipsacus asperoides extracts (CN115006302A), etc., are mostly mixed components extracted from plants. Their action mechanisms are unclear, their safety lacks verification, and it is difficult to ensure the consistency and stability between extraction batches, which greatly limits their application in the treatment of diseases such as acne, seborrheic dermatitis, and seborrheic alopecia. Summary of the Invention

[0006] The technical problem to be solved by the present invention is: to overcome the deficiencies of the prior art and provide a superoxide dismutase for controlling abnormal skin sebum secretion and its application to solve the problems in the above background art.

[0007] The technical solution adopted by the present invention to solve its technical problems is as follows: The superoxide dismutase for controlling abnormal skin sebum secretion is a protein that converts superoxide anions into hydrogen peroxide and oxygen molecules; or it includes one of copper-zinc superoxide dismutase, manganese superoxide dismutase, and iron superoxide dismutase, or an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids, and having at least 80% homology with its natural amino acid sequence and having the same or similar function.

[0008] Further, the superoxide dismutase is copper-zinc superoxide dismutase, and its amino acid sequence includes its natural sequence or an amino acid sequence having at least 90% sequence homology with its natural sequence and having the function of superoxide dismutase.

[0009] Further, the protein is a protein having the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues.

[0010] Further, a protein having the same function is obtained by tandem expression or chemical coupling of a short peptide or a protein domain at any one or both ends of the protein sequence of the protein.

[0011] The application of the above-mentioned superoxide dismutase for controlling abnormal skin sebum secretion in skin oil-control products realizes the inhibition of sebum secretion in the skin epidermis by diluting or compounding with superoxide dismutase as a raw material and then applying it to the skin or introducing it through microneedles.

[0012] The application of the above-mentioned superoxide dismutase for controlling abnormal skin sebum secretion in cosmetics with oil-control efficacy.

[0013] Further, the activity range of superoxide dismutase in products with oil-control efficacy is 5 - 5000 U / mL.

[0014] Further, the enzyme activity range of the superoxide dismutase in cosmetics with oil-control efficacy is 50 - 500 U / mL.

[0015] Further, the enzyme activity range of the superoxide dismutase in drugs or medical devices with oil-control efficacy is 500 - 5000 U / mL.

[0016] In the present invention, using superoxide dismutase as the active ingredient for skin oil control can not only inhibit the accumulation of lipids in human sebaceous gland cells, reduce the sebum secretion of sebaceous gland cells, but also scavenge free radicals in human immortalized keratinocytes and inhibit the death of keratinocytes caused by external stimuli, thereby inhibiting the sebum release caused by keratinocyte lysis. Superoxide dismutase can simultaneously intervene in the sebum release from both sebaceous gland cells and keratinocytes, and can inhibit sebum secretion and release at an activity concentration of 10 - 100 U / mL.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) High safety. As a biogenic material, superoxide dismutase has been authorized by national authoritative institutions for use in many industries such as cosmetics, food, and medical devices, and its medicinal safety has been widely verified.

[0018] (2) Good oil control effect. Using superoxide dismutase as the active ingredient for skin oil control can not only inhibit the accumulation of oil in sebaceous gland cells but also inhibit the release of lipids in keratinocytes, and can simultaneously inhibit the lipid secretion from two sources in the epidermis, showing a good oil control effect.

[0019] (3) Single component; clear structure; good stability, suitable for product research and development in the medical field.

[0020] Currently, most of the oil control ingredients for cosmetics are mixed ingredients extracted from plants, making it difficult to ensure the consistency and stability between extraction batches. Moreover, medicinal oil control ingredients often have strong irritation and many side effects, and cannot be used as a long-term solution for ordinary oily skin conditioning. Superoxide dismutase already has relatively mature quality standards and risk prevention and control systems in different industries. Under the current quality standards and risk management systems, the developed products have high safety and low potential risks, and can be widely used in the treatment of diseases such as seborrheic alopecia and seborrheic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the staining result after protein denaturing electrophoresis of recombinant human superoxide dismutase used in the present invention; Figure 2 is the standard curve for detecting the protein concentration of recombinant human superoxide dismutase used in the present invention by the BCA method; Figure 3 is the standard curve for detecting the endotoxin content in the recombinant human superoxide dismutase used in the present invention; Figure 4 is the relative accumulation amount of intracellular lipids in SZ95 cells after adding a specified dose of SOD1, *** indicates p < 0.01; Figure 5 is the relative change in cell viability of SZ95 after treatment with a specified concentration of SOD1; Figure 6 is the change in relative lipid content per unit viability of SZ95 cells after treatment with SOD1, *** indicates p < 0.01; Figure 7 is the ROS staining result of HACAT cells after pretreatment with SOD1, scale bar 50 μm; Figure 8It shows the viability changes of HACAT cells after pretreatment with a specified dose of SOD1 followed by UVB irradiation. * indicates p < 0.05, *** indicates p < 0.01, and ns indicates no statistical difference. Detailed implementation manners

[0022] Figures 1 to 8 This is the best embodiment of the present invention. The following further explains the present invention in conjunction with the attached Figures 1 to 8 drawings.

[0023] The following further explains the present invention with specific embodiments. However, those skilled in the art should understand that the detailed description given here in conjunction with the drawings is for better explanation. The structure of the present invention necessarily goes beyond these limited embodiments. For some equivalent replacement schemes or common means, no detailed description is given herein, but they still fall within the protection scope of this application.

[0024] Example 1 This example mainly focuses on the quality detection of superoxide dismutase.

[0025] The recombinant human superoxide dismutase 1 (https: / / e.tb.cn / h.TueyXy6MN9JBPza?tk=48bCekZSyka) used in the present invention is optimized and produced by our company. Table 1 shows the quality standards for microbial content and heavy metal content in product production quality control. Figure 1 Figure 1 is the Coomassie brilliant blue staining diagram of recombinant superoxide dismutase 1 after protein electrophoresis. After software scanning and calculation, it can be known that the purity of superoxide dismutase 1 in the sample used is about 97%. After diluting the sample 20 times and detecting it by the BCA method (Biosharp; BL521A), it can be known that the protein concentration in the original sample is 5.2 mg / mL. The standard curve is as Figure 2 shown. The detected value of the diluted sample is 0.366, and the regression equation is y = 0.6477x + 0.023, and the regression coefficient R 2 = 0.995. After diluting the sample 20 times, the residual amount of endotoxin in the sample was calculated using an endotoxin detection kit (Genscript; L00350). It can be known that the endotoxin content in the original sample is less than 1 EU / ml. The standard curve is as Figure 3 shown. The detected value of the diluted sample is 0.138, and the regression equation is y = 3.7799x - 0.0203, and the regression coefficient R 2 = 0.094.

[0026] The activity of superoxide dismutase in the sample was detected according to the method of a superoxide dismutase activity detection kit (Solarbio, BC0170). It can be calculated that the activity of SOD in the original sample is 15000 U / mg.

[0027] Table 1 Partial quality standards of the recombinant superoxide dismutase used in this patent

[0028] Example 2 Example 2 was used for superoxide dismutase treatment to inhibit lipid accumulation in human sebaceous gland cells.

[0029] After mixing DMEM and F12 medium at a ratio of 1:1, 10% fetal bovine serum, 1% mixed antibiotics, and 5 ng / mL human epidermal growth factor (MCE, HY-P7109G) were added, and human immortalized sebaceous gland cells SZ95 were cultured in a cell incubator at 37 °C and 5% carbon dioxide. The medium was changed every 2 - 3 days.

[0030] The SZ95 cells were respectively inoculated into 2 96-well plates (about 5*103 cells / well) and cultured overnight. The superoxide dismutase in Example 1 was filtered through a 0.22 mm sterile filter and medium was used to prepare media with SOD activities of 10 U / mL, 100 U / mL, 1000 U / mL, and 5000 U / mL. Using the medium as a control, the media containing different SOD enzyme activities were respectively added to the 2 96-well plates inoculated with cells, and 5 replicates were set for each concentration, and the cells were cultured for another 24 hours.

[0031] After the culture was completed, the medium in the first 96-well plate was removed, and the cells were washed 3 times with PBS solution, and then fixed with 4% paraformaldehyde solution at room temperature for 15 minutes. After fixation, the fixing solution was discarded, and the cells were washed 3 times with PBS solution, and then the prepared Oil Red O staining solution (Beyotime, C0157S) was added and stained at room temperature in the dark for 30 minutes. After staining, the staining solution was removed, and the cells were washed 3 times with PBS solution, 200 μL of isopropanol was added, and after shaking on a shaker for 10 minutes, the absorbance of the sample at 560 nm was measured with a multifunctional microplate reader (BioTek, Synergy HTX), and the data was statistically analyzed ( Figure 4 ) The MTT stock solution (Beyotime, C0009S) was diluted with medium to a final concentration of 0.5 mg / mL MTT working solution. The medium in the plate was discarded, 0.1 mL of MTT working solution was added to each well, and then the culture plate was put back into the incubator and cultured for 6 - 8 hours. After the culture was completed, the liquid in the wells was carefully discarded, 0.1 mL of DMSO was added to each well, and after shaking on a shaker for 15 minutes, the absorbance at 490 nm was detected with a multifunctional microplate reader (BioTek, SynergyHTX). The results are as Figure 5As shown, even at a dose as high as 5000 U / ml, SOD1 has no inhibitory effect on the cell viability of SZ95 cells, but rather has a certain promoting effect, indicating that SOD1 is a biomaterial with high biosafety. Under the same viability treatment, the absorbance value obtained by Oil Red O staining is divided by the absorbance value obtained by MTT treatment to obtain the lipid content in cells under the average cell viability. The mean value of the lipid content under the average cell viability of the control group is set to 1, and the relative changes in the lipid content of cells under different treatments can be obtained. The results are as Figure 6 shown. Adding 10 U / mL of superoxide dismutase 1 to the culture medium can inhibit lipid accumulation in SZ95 cells. As the dose increases, the inhibitory effect becomes more obvious.

[0032] Example 3 Example 3 is used for superoxide dismutase treatment to inhibit the accumulation of free radicals in human keratinocytes.

[0033] Add 10% fetal bovine serum and 1% mixed antibiotics to DMEM medium, and culture human immortalized keratinocytes HACAT in a cell culture incubator at 37 °C and 5% carbon dioxide. Replace the medium every 2 days.

[0034] Inoculate HACAT cells into 24-well plates (about 1*105 cells / well) and culture overnight. Filter the superoxide dismutase in Example 1 with a 0.22 mm sterile filter and prepare media with SOD activities of 10 U / mL, 100 U / mL, and 1000 U / mL using the medium. Using the medium as a control, add the media containing different SOD enzyme activities to the 24-well plates inoculated with cells, 0.5 mL per well, and continue to culture for 8 hours. After the culture is completed, according to the method in the ROS kit (Beyotime, S0033S), first dilute the DCFH-DA probe 1000 times with the medium (final concentration 10 μM), remove the medium in the 24 wells, add the diluted DCFH-DA solution, and place it back in the 37 °C cell culture incubator for continued incubation for 20 minutes. After the incubation is completed, wash the cells 3 times with PBS, 2 minutes each time, to fully remove the residual DCFH-DA probe.

[0035] Then place the 24-well plate on the stage of an inverted fluorescence microscope (Nikon TI fluorescence microscopy), select the green fluorescence channel to observe and photograph the cells. The results are as shown in Figure 7. Adding 10 U / mL of superoxide dismutase 1 to the culture medium can inhibit the accumulation of free radicals in HUVEC cells. As the dose increases, the less ROS accumulates in the cells.

[0036] Example 4 Example 4 is used for superoxide dismutase treatment to inhibit the death of human keratinocytes induced by UVB.

[0037] Add 10% fetal bovine serum and 1% mixed antibiotics to phenol red-free DMEM medium, and culture human immortalized keratinocytes HACAT in a cell culture incubator at 37°C and 5% carbon dioxide. Replace the medium every 2 days. Inoculate HACAT cells into a 96-well plate (about 1×104 cells / well) and culture overnight. Filter the superoxide dismutase in Example 1 with a 0.22 mm sterile filter and prepare media with SOD activities of 0 U / mL, 10 U / mL, 100 U / mL, and 1000 U / mL using the medium. Additionally, set up a control group treated with only the medium. Add the media containing different SOD enzyme activities to the 96-well plate inoculated with cells, 0.1 mL per well, and continue to culture for 8 hours.

[0038] After the culture is completed, except for the control group, irradiate with a 310 nm ultraviolet light emitter at 400 mJ / cm2, and then place it back in the incubator to continue culturing for 24 hours. After the culture is completed, dilute the MTT stock solution with the medium to an MTT working solution with a final concentration of 0.5 mg / mL. Discard the medium in the plate, add 0.1 mL of the MTT working solution to each well, and then place the culture plate back in the incubator to culture for 6 - 8 hours. After the culture is completed, carefully discard the liquid in the wells, add 0.1 mL of DMSO to each well, shake on a shaker for 15 minutes, and then detect the absorbance at 490 nm using a multifunctional microplate reader (BioTek, SynergyHTX) and perform statistics. The results are as Figure 8 shown. In HACAT cells, UVB irradiation can significantly reduce cell viability. Exogenous addition of superoxide dismutase 1 treatment can significantly inhibit cell death caused by UVB irradiation at a dose of 10 U / mL, and can completely restore the decrease in cell viability caused by UVB irradiation when the dose reaches 1000 U / mL.

[0039] As described above, it is only a preferred embodiment of the present invention, and it is not a limitation of the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A superoxide dismutase for controlling abnormal skin oil secretion, characterized in that: A protein that converts superoxide anions into hydrogen peroxide and oxygen molecules; or includes one of copper-zinc superoxide dismutase, manganese superoxide dismutase, iron superoxide dismutase, or an amino acid sequence that is substituted, deleted or added with one or more amino acids, and the amino acids have at least 80% homology with their natural amino acid sequence and have the same or similar functions.

2. The superoxide dismutase for controlling abnormal skin oil secretion according to claim 1, characterized in that: The superoxide dismutase is copper-zinc superoxide dismutase, and its amino acid sequence includes its natural sequence or an amino acid sequence with at least 90% sequence homology to its natural sequence and having superoxide dismutase function.

3. The superoxide dismutase for controlling abnormal skin oil secretion according to claim 1, characterized in that: The protein is a protein with the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues.

4. The superoxide dismutase for controlling abnormal skin oil secretion according to claim 1 or 3, characterized in that: A protein with the same function is obtained by expressing in series or chemically coupling a short peptide or protein domain at any one or both ends of the protein sequence of the protein.

5. The use of superoxide dismutase for controlling abnormal skin oil secretion according to any one of claims 1 to 4 in skin oil control products, characterized in that: Superoxide dismutase is used as the raw material, which is diluted or compounded and then applied to the skin or introduced by microneedles to inhibit the secretion of skin epidermis oil.

6. Use of the superoxide dismutase for controlling abnormal skin oil secretion according to any one of claims 1 to 4 in cosmetics with oil-controlling efficacy.

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