CN116445306A - Schizosaccharomyces pombe and its application in improving skin condition - Google Patents
Schizosaccharomyces pombe and its application in improving skin condition Download PDFInfo
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- CN116445306A CN116445306A CN202310706432.3A CN202310706432A CN116445306A CN 116445306 A CN116445306 A CN 116445306A CN 202310706432 A CN202310706432 A CN 202310706432A CN 116445306 A CN116445306 A CN 116445306A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a schizosaccharomyces pombe and application thereof in improving skin conditions, wherein the strain is schizosaccharomyces pombe @ the whole yeastSchizosaccharomyces pombe) SEUNEU-120 has been deposited in China center for type culture Collection with a deposit number of CCTCC NO: M20221432. The schizosaccharomyces pombe provided by the invention has the functions of promoting cell proliferation, repairing skin barrier, moisturizing and resisting aging, and can be used for preparing foods, medicines, cosmetics and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to schizosaccharomyces pombe and application thereof in improving skin conditions.
Background
The stratum corneum formed by the basal layer keratinocytes of the skin after undergoing directional differentiation can prevent various physical and chemical damages. The skin barrier function maintains normal operation of the skin physiological function by preventing loss of moisture, nutrients and the like, and simultaneously ensures that the organ tissues in the body are prevented from being affected by external harmful substances, thereby playing an important role in maintaining the homeostasis of the internal environment of the body.
Skin aging is a part of the aging process of the body and is the most intuitive organ for the aging process. The current research on skin aging is mainly on the aging of skin fibroblasts and keratinocytes. The histological changes of the aged dermis of the skin are mainly changes in the matrix composition of the skin, i.e. reduced collagen composition and abnormal elastic fiber deposition. The molecular change is mainly the abnormal expression of the epidermal hyperplasia and differentiation markers, and the expression of Matrix Metalloproteinase (MMP) is increased, for example, MMP1 is collagen degrading enzyme, is also a main factor for decomposing skin collagen fibers, can cause the aging of skin dermis, and is one of the indexes of skin aging. In the research of aging mechanism, it has been found that reducing apoptosis, reducing cell inflammation, increasing synthesis of extracellular matrix, reducing degradation thereof, improving antioxidant capacity of cells, etc. have all been demonstrated to slow aging of skin cells.
Skin is a complex and dynamic ecological system populated with bacteria, archaea, fungi and viruses. These microorganisms are collectively referred to as the skin microbiota and are the basis for skin physiology and immunity. Most microorganisms living on the skin appear to be symbiotic or reciprocal symbiotic under steady state conditions. Bacteria inhabit all layers of the skin, from the outer stratum corneum to the inner dermis. Microorganisms maintain skin barrier integrity critical to homeostasis and disease prevention. Therefore, the skin microecological related product developed by utilizing the microbial technology has wide application value in improving the skin state.
Disclosure of Invention
In view of this, the present invention provides a merozoite strain of Schizosaccharomyces pombe and its use for improving skin conditions.
The invention discloses a millet wineSchizosaccharomyces cerevisiaeSchizosaccharomyces pombe) In particular to schizosaccharomyces pombeSchizosaccharomyces pombe) SEUNEU-120 has been deposited in China center for type culture Collection with a deposit number of CCTCC NO: M20221432.
The invention also discloses application of the schizosaccharomyces pombe in preparing products for improving skin conditions.
Further, the above application, wherein the improving the skin condition comprises at least one of promoting cell proliferation, repairing skin barrier, moisturizing, and anti-aging.
In the present invention, repairing the skin barrier includes repairing SDS-induced cell damage and/or up-regulating expression of a barrier repair-related gene; the barrier repair related gene comprisesFLG、IVL、OVOL1AndOCLNat least one of them.
Furthermore, the invention takes HaCaT keratinocytes as a test object, and researches on the SDS damage repair capability of the schizosaccharomyces pombe prove that the schizosaccharomyces pombe can repair cell damage caused by SDS, improve the cell proliferation rate and up-regulate the expression of barrier repair related genes of non-damaged cells.
In the present invention, the moisturizing includes, but is not limited to, up-regulating expression of a moisturizing-related gene, which isGBA. The invention takes HaCaT keratinocyte as a test object, researches the moisturizing effect of the schizosaccharomyces pombe on the skin, and results show that the schizosaccharomyces pombe can up regulate the moisturizing related genesGBAIs effective in promoting skin cell moisture retention.
Further, the above application, wherein the anti-aging comprises at least one of the following:
up-regulating expression of genes involved in extracellular matrix synthesis, includingCOL1A1、COL13A1、SPTSSA、SMAD3AndMKXat least one of (a) and (b);
down-regulating expression of genes associated with degradation of extracellular matrix; the genes related to the degradation of the extracellular matrix compriseMMPAt least one of the family genes；
Upregulating expression of a cellular antioxidant-associated gene comprisingNRF2、PTENAndSIRT-1at least one of (a) and (b);
up-regulating cell immune regulator related geneMORIs expressed by (a);
down-regulating apoptosis-related genesBAXAnd/orCaspase-9Is expressed by (a);
upregulation of cell autophagy-related genesLC3BIs expressed by (a);
up-regulating apoptosis-inhibiting related genesBCL-2Is expressed by (a);
up-regulating expression of a cell growth factor related gene comprisingFGF1、FGF2、FGF7、FGF21AndHGFat least one of (a) and (b);
scavenging DPPH free radical;
oxidation resistance.
In order to explore the anti-aging effect of schizosaccharomyces pombe, the invention detects the indexes related to cell aging, wherein the indexes comprise at least one of extracellular matrix synthesis, extracellular matrix degradation, cell oxidation resistance, cell immunity regulating factors, apoptosis, autophagy, apoptosis inhibition, cell growth factors, DPPH free radical removal and oxidation resistance.
In the present invention, the cell proliferation promoting means promotes proliferation of skin keratinocytes. The invention takes HaCaT keratinocytes as a test object to study the effect of promoting cell proliferation of the schizosaccharomyces pombe. The result shows that the schizosaccharomyces pombe can promote repair of HaCaT keratinocyte injury caused by SDS, improve the cell proliferation rate, and promote the proliferation rate to be 10.16-13.45%.
The invention takes HaCaT keratinocytes as a test object, and researches the influence of the schizosaccharomyces pombe on genes related to the synthesis of HaCaT extracellular matrix and genes related to degradation. The results show that the schizosaccharomyces pombe can up-regulate the genes related to the synthesis of extracellular matrixCOL1A1Promote synthesis of extracellular matrix, down-regulate degradation of extracellular matrix-related genesMMP1Is inhibited by expression of (a)Degradation of the extracellular matrix.
The invention takes HaCaT keratinocytes as a test object, and researches the influence of the schizosaccharomyces pombe on the autophagy of the HaCaT keratinocytes. The results show that the schizosaccharomyces pombe can up-regulate the cell autophagy related genesLC3BPromotes autophagy to clear aged cells.
The invention takes HaCaT keratinocytes as a test object, and researches the influence of the schizosaccharomyces pombe on the expression of the gene related to the oxidation resistance of the HaCaT keratinocytes. The results show that the schizosaccharomyces pombe can up-regulate the antioxidant related genesNRF2Enhancing the antioxidant capacity of the cells.
The invention takes HFF fibroblasts as a test object to study the influence of the schizosaccharomyces pombe on genes related to HFF extracellular matrix synthesis and genes related to degradation. The results show that the schizosaccharomyces pombe can up-regulate the genes related to the synthesis of extracellular matrixCOL13A1、SPTSSA、SMAD3AndMKXpromote synthesis of extracellular matrix, down-regulate degradation of extracellular matrix-related genesMMP3Inhibit degradation of extracellular matrix.
The invention takes HFF fibroblasts as a test object to study the influence of the schizosaccharomyces pombe on HFF apoptosis. The results show that the schizosaccharomyces pombe can down regulate the apoptosis related genesBAXAndCaspase-9is effective in inhibiting apoptosis.
The invention takes HFF fibroblasts as a test object, and researches the influence of the schizosaccharomyces pombe on the HFF cells to inhibit apoptosis. The results show that the schizosaccharomyces pombe can up-regulate and inhibit the apoptosis related genesBCL-2Is effective in inhibiting apoptosis.
The invention takes HFF fibroblasts as a test object to study the influence of the schizosaccharomyces pombe on the expression of the gene related to the HFF cell antioxidation. The results show that the schizosaccharomyces pombe can up-regulate the antioxidant related genesPTENAndSIRT-1enhancing the antioxidant capacity of the cells.
The invention uses HFF fibroblast as test pairThe effect of the schizosaccharomyces pombe on the expression of genes related to HFF cell immunomodulators was examined, for example. The results show that the schizosaccharomyces pombe can up-regulate the genes related to the immune regulatorMOREnhancing the immunomodulatory ability of the cell.
The invention takes HFF fibroblast as a test object, and researches the influence of the schizosaccharomyces pombe on the expression of HFF cell growth factor related genes. The results show that the schizosaccharomyces pombe can up-regulate the cell growth factor related genesFGF1、FGF2、FGF7、FGF21AndHGFenhancing the growth capacity of the cells.
The invention takes schizosaccharomyces pombe as a test object, and researches the detection of the schizosaccharomyces pombe on DPPH free radical removal and total antioxidant capacity. The results show that the schizosaccharomyces pombe can remove DPPH free radicals and has antioxidant capacity, so that oxidative damage of cells is reduced.
Further, the above application, wherein the product is a food, a pharmaceutical or a cosmetic.
The invention also discloses a product for improving skin condition, which is prepared from the raw materials including the schizosaccharomyces pombe.
Further, the above products include supernatant of schizosaccharomyces pombe and thallus lysate.
Further, the dosage forms of the product of the present invention include, but are not limited to, at least one of creams, emulsions, oils, aqueous solutions, gels, powders, and freeze-drying.
The invention also provides a method of improving skin condition comprising using the slave product of the invention. The application method comprises smearing, external application, fumigation or injection, and the invention is not limited to the method.
The invention provides schizosaccharomyces pombeSchizosaccharomyces pombe) The culture medium is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M20221432. Experiments show that the schizosaccharomyces pombe provided by the invention has the effects of promoting cell proliferation, repairing skin barrier and preserving moistureHas antiaging effect, and can be used for preparing foods, medicines, cosmetics, etc.
Description of biological preservation
Schizosaccharomyces pombeSchizosaccharomyces pombe) SEUNEU-120 was deposited at China center for type culture Collection (China, university of Wuhan, wuhan) with a deposit number of CCTCC NO: M20221432 at 9 and 14 of 2022.
Detailed Description
The invention provides schizosaccharomyces pombe and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The schizosaccharomyces pombe strain SEUNEU-120 of the invention is derived from sugar cane molasses of guests of Guangxi Zhuang nationality, and is identified as schizosaccharomyces pombe through 26S rDNA identificationSchizosaccharomyces pombe). The strain SEUNEU-120 is round or oval under a microscope; growing on a wort solid flat plate to form milky white, matt colonies and tidy edges; the strain grows in a malt juice liquid culture medium in a uniform turbidity mode, the strain is white in precipitation after long-term placement, and the optimal growth temperature is 28 ℃.
Schizosaccharomyces pombeSchizosaccharomyces pombe) SEUNEU-120, accession number: china center for type culture Collection, address: in the Jiuqiu No. 299 university of Wuhan, hubei province, the date of preservation: and 2022, 9 and 14 days, wherein the preservation number is CCTCC NO: M20221432.
Furthermore, the present invention provides the use or product of the schizosaccharomyces pombe SEUNEU-120 in the form of a live or dead or sterilized intermittently, or in the form of a lysate and/or extract, or in the form of a yeast product, or in the form of a supernatant or derivative. Preferably, schizosaccharomyces pombe SEUNEU-120 is present in a form selected from the group consisting of supernatant or bacterial lysate. The derivative form is preferably selected from: metabolites, metabolic biological products, exosomes, probiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components.
The test materials used in the present invention are all commercially available products, and the present invention is further described below with reference to examples.
EXAMPLE 1 SEUNEU-120 isolation
Sampling in cane molasses. After the sample is properly treated, shaking and uniformly mixing the sample in normal saline, streaking the supernatant on a wort solid plate, culturing the wort solid plate at the constant temperature of 28 ℃ for 48 hours, and then picking white colonies, repeatedly inoculating and screening until uniform single colonies are obtained, and the colony is named as SEUNEU-120.
And (5) microscopic examination: the strain SEUNEU-120 is round or oval under a microscope; growing on a wort solid flat plate to form milky white, matt colonies and tidy edges; the strain grows in a malt juice liquid culture medium in a uniform turbidity manner, and the strain is white in precipitation after long-term placement.
EXAMPLE 2 nucleic acid identification of SEUNEU-120
1. 26S rDNA gene sequence analysis:
single colony is selected and placed in malt juice culture medium, after the culture is carried out at 28 ℃ overnight, the culture medium is centrifuged for 2min at 12000 revolutions to collect thalli, and the operation is carried out according to the step of extracting the yeast genome DNA kit. The primers adopt yeast universal primers NL1 and NL4, the PCR amplification system is a 50 mu L system, and the primers are pre-denatured for 5min at 95 ℃;94℃1min,52℃1min,72℃90s,36 cycles; extending at 72℃for 10min.
2. Results
The PCR product sequencing result is compared with the published standard sequence in GenBank (BLASTN) to obtain the SEUNEU-120 strain which is schizosaccharomyces pombeSchizosaccharomyces pombe）。
Example 3 SEUNEU-120 promoting SDS-induced repair of HaCaT injury in human immortalized keratinocytes
1. Preparation of SEUNEU-120 supernatant and thallus lysate:
selecting single colony of schizosaccharomyces pombe SEUNEU-120 in malt wort liquid medium, shake culturing at 28 ℃ for 16-18 h, detecting by using a microplate reader, and diluting with PBS to adjust OD 600 The cells were separated and precipitated by centrifugation at 12000 rpm for 2min, the supernatant was removed, inactivated at 121 ℃ for 30min under high pressure, and filtered through a 0.22 μm filter membrane to obtain an inactivated supernatant. The method comprises the steps of washing the centrifugally precipitated thalli twice by PBS, adding liquid nitrogen, grinding and crushing, collecting crushed bacterial mud, re-suspending the crushed bacterial mud to the volume of the centrifugally precipitated thalli by PBS, crushing the crushed thalli by ultrasonic waves for 20 minutes, and inactivating the crushed thalli at 121 ℃ for 30 minutes under high pressure to obtain a thalli lysate.
2. Promoting HaCaT cell repair experiments
Inoculation of HaCaT cells (5×10) 4 cells/well) to 96-well plates, cultured overnight to cell attachment. 50. Mu.g/ml SDS was prepared, 100. Mu.l of each well was added thereto, and the mixture was incubated in a 5% carbon dioxide incubator at 37℃for 8 hours. 5% (V/V) supernatant and 10% (V/V) cell lysate (control group replaced supernatant or cell lysate with equal volume of PBS) were added to each well, respectively, and cultured for 24h. Mu.l of CCK-8 solution was added to each well, and incubated for 4 hours, and the absorbance at 450nm was measured for brightness A.
The cell proliferation rate was calculated as: cell proliferation rate= (experimental group a-control group a)/control group a x 100%, and the calculation results are shown in table 1.
TABLE 1 SEUNEU-120 promoting HaCaT cell repair
As shown in Table 1, both the SEUNEU-120 supernatant and the cell lysate promote repair of SDS-induced HaCaT keratinocyte injury, increase cell proliferation rate, and promote proliferation rate by 10.16-13.45%.
Example 4 SEUNEU-120 promotion of HaCaT Barrier maintenance-related Gene expression experiments
1. Preparation of SEUNEU-120 supernatant and thallus lysate:
the preparation is described in example 3.
2. Promote HaCaT barrier repair related gene expression experiment
Inoculated human immortalized keratinocyte HaCaT (2 ml/well, contained5×10 5 Cells) to 6-well plates, 5% carbon dioxide incubator at 37 ℃ overnight until cells adhere. Respectively adding 5% (V/V) supernatant and 10% (V/V) thallus lysate (the control group respectively replaces supernatant or thallus lysate with equal volume PBS), culturing for 24 hr, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNA to obtain final productGAPDHAs reference gene, real-time qPCR detection was usedFLG、IVL、OVOL1AndOCLNexpression of the genes. The control group (relative gene expression fold f=1) was treated with PBS added in equal volume, using 2 -ΔΔCT The F value of each sample was calculated by the method. The results of the expression calculation of the SEUNEU-120 supernatant and the thallus lysate up-regulation repair gene are shown in Table 2 and Table 3 respectively.
The formula: f=2 -ΔΔCT Wherein:
△CT experiment =CT Experiment -CT Internal reference (experiment);
△CT control =CT Control -CT Internal control (control);
△△CT=△CT experiment -△CT And (3) controlling.
TABLE 2 expression of SEUNEU-120 supernatant up-regulating repair genes
TABLE 3 SEUNEU-120 bacterial lysate up-regulates the expression of repair genes
In vitro cell experiments show that the supernatant and the thallus lysate of the schizosaccharomyces pombe SEUNEU-120 have the up-regulated skin barrier repair related factor silk polymerized protein genesFLGEndothelial protein geneIVLOVO-like transcription factor 1 geneOVOL1And a claudin geneOCLNThe expression effect is that the gene expression quantity is up-regulated by 1.11-2.81 times. SEUNEU-120 has been shown to have an effect of promoting skin barrier repair.
Example 5 SEUNEU-120 up-regulation of HaCaT moisture-retention-related Gene expression experiments
1. Preparation of SEUNEU-120 thallus lysate:
the preparation is described in example 3.
2. Up-regulating HaCaT moisture-preserving related gene expression experiment
Inoculated human immortalized keratinocyte HaCaT (2 ml/well, 5X 10 contained therein) 5 Cells) to 6-well plates, 5% carbon dioxide incubator at 37 ℃ overnight until cells adhere. Adding 10% (V/V) thallus lysate (the control group is replaced by equal volume PBS), culturing for 24 hr, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHAs reference gene, real-time qPCR detection was usedGBAExpression of the genes. The control (relative gene expression fold f=1) was equal volume PBS treated group, using 2 -ΔΔCT The F value of each sample was calculated by the method. The test results are shown in Table 4.
TABLE 4 SEUNEU-120 bacterial lysate up-regulates expression of moisturizing genes
In vitro cell experiments show that the schizosaccharomyces pombe SEUNEU-120 has glucose cerebrosidase gene related to up-regulation and moisture preservationGBAThe expression effect is that the gene expression quantity is up-regulated by 1.15-1.30 times. SEUNEU-120 has been shown to have skin moisturizing effects.
Example 6 SEUNEU-120 Regulation of light aging HaCaT extracellular matrix/antioxidant/autophagy/degradation of extracellular matrix-related Gene expression experiments
1. Preparation of SEUNEU-120 supernatant and thallus lysate:
the preparation is described in example 3.
2. HaCaT cell preparation and ultraviolet injury
HaCaT cells were digested and then subjected to a reaction at 0.5 ml/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. The total dose of cells in the well was 2J/cm 2 Is damaged by ultraviolet UVB irradiation.
3. SEUNEU-120 addition
5% (V/V) supernatant and 10% (V/V) cell lysate were added to each stimulated HaCaT cell (control group replaced supernatant/cell lysate with equal volume of PBS, respectively). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of extracellular matrix/antioxidant/autophagy/degradation extracellular matrix related genes
Removing culture medium from above cells, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHAs reference genes, real-time qPCR was used to detect extracellular matrix-related genesCOL1A1Antioxidant related genesNRF2Cell autophagy-related genesLC3BAnd degradation of extracellular matrix-related genesMMP1Is expressed by (a). Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
Supernatant up-regulating cell autophagy related genesLC3BThe method comprises the steps of carrying out a first treatment on the surface of the Down-regulating degradation of extracellular matrix related genesMMP1. The results are shown in Table 5.
TABLE 5 SEUNEU-120 supernatant regulates the expression of genes involved in autophagy/degradation of extracellular matrix
Up-regulating extracellular matrix related genes by thallus lysateCOL1A1Antioxidant related genesNRF2Autophagy geneLC3BThe method comprises the steps of carrying out a first treatment on the surface of the Down-regulating degradation of extracellular matrix related genesMMP1. The results are shown in Table 6.
TABLE 6 SEUNEU-120 bacterial lysate regulates the expression of extracellular matrix/antioxidant/autophagy/degradation of extracellular matrix-related genes
In vitro cell experiments show that the schizosaccharomyces pombe SEUNEU-120 has the I-type collagen alpha 1 chain gene related with up-regulation of HaCaT extracellular matrixCOL1A1Antioxidant, antioxidationRelated nuclear factor E2 related factor 2 geneNRF2Cell autophagy-related microtubule-associated protein 1 light chain 3 beta geneLC3BThe expression function, the relative expression multiple of genes is 1.19-1.93; matrix metalloproteinase family genes associated with downregulation degradation of extracellular matrixMMP1The expression function, the relative expression multiple of genes is 0.18-0.82.
EXAMPLE 7 SEUNEU-120 experiments on the expression of genes involved in modulating oxidative damage of HFF extracellular matrix/antioxidant/immunomodulatory factors/inhibiting apoptosis/cell growth factors/degrading extracellular matrix/apoptosis
1. Preparation of SEUNEU-120 supernatant and thallus lysate:
the preparation is described in example 3.
2. HFF cell preparation and H 2 O 2 Induced oxidative damage
After digestion of HFF cells in DMEM culture, the cells were subjected to digestion at 0.5 ml/well (containing 2X 10 cells) 5 Cells) were inoculated into 24-well plates and incubated overnight at 37℃in a 5% carbon dioxide incubator. H was added to each well at a final concentration of 200. Mu.M 2 O 2 Stimulation was performed and the mixture was allowed to stand at 37℃for 1 hour.
3. SEUNEU-120 addition
5% (V/V) supernatant and 10% (V/V) cell lysate were added to stimulated HFF cells, respectively (control groups replaced supernatant/cell lysate with equal volumes of PBS, respectively). Each group was incubated overnight at 37℃in parallel with 3.
4. qPCR method for detecting relative expression fold of extracellular matrix/antioxidant/immunoregulatory factor/apoptosis inhibition/cell growth factor/degradation extracellular matrix/apoptosis-related gene
Removing culture medium from above cells, adding lysate, extracting total RNA of cells, detecting RNA concentration and purity, and reverse transcribing into cDNAGAPDHAs reference genes, real-time qPCR was used to detect extracellular matrix-related genesCOL13A1、SPTSSA、MKXAndSMAD3antioxidant related genesPTENAndSIRT-1immunomodulating factorsMORInhibiting apoptosis-related genesBCL-2Cell growth factor related genesFGF1、FGF2、FGF7、FGF21AndHGFdegradation of extracellular matrix-related genesMMP3And apoptosis-related genesBAXAndCaspase-9is expressed by (a). Relative expression fold f=1, using 2 for control group genes -ΔΔCT The F value of each sample was calculated by the method.
Supernatant up-regulates extracellular matrix-related genesSMAD3Antioxidant related genesSIRT-1Inhibiting apoptosis genesBCL-2The method comprises the steps of carrying out a first treatment on the surface of the Down-regulating degradation of extracellular matrix related genesMMP3Apoptosis-related genesBAXAndCaspase-9the results are shown in Table 7.
TABLE 7 SEUNEU-120 supernatant regulates extracellular matrix/antioxidant/apoptosis-inhibiting/degradation of extracellular matrix/apoptosis-related Gene expression
Up-regulating extracellular matrix related genes by thallus lysateCOL13A1、MKXAndSPTSSAimmunomodulatory factor-related genesMORAntioxidant related genesPTENAndSIRT-1inhibiting apoptosis-related genesBCL-2Cell growth-related genesFGF1、FGF2、FGF7、FGF21AndHGF. The results are shown in Table 8.
TABLE 8 SEUNEU-120 bacterial lysate up-regulates extracellular matrix/immunomodulator/antioxidant/inhibits apoptosis/cytostatic factor-related gene expression
In vitro cell experiments show that the schizosaccharomyces pombe SEUNEU-120 has the collagen-like membrane protein 13 alpha chain gene related with up-regulation of HFF extracellular matrixCOL13A1Serine palmitoyltransferase geneSPTSSAMo Huoke protein geneMKXAnd signal transduction protein geneSMAD3Immune modulator-related beta-endorphin receptor genesMORAntioxidant related No. 10 chromosome deleted phosphatase and tensin homolog genePTENAnd Sirtuins protein family baseBecause ofSIRT-1Inhibiting apoptosis-related B lymphocytoma-2 geneBCL-2And a fibroblast growth factor gene related to a cell growth factorFGF1、FGF2、FGF21Keratinocyte growth factor geneFGF7And hepatocyte growth factor geneHGFThe expression function, the relative expression times of genes are 1.06-2.81 times; matrix metalloproteinase family genes associated with downregulation degradation of extracellular matrixMMP3And apoptosis-related BCL2-Associated X protein genesBAXAnd cysteine protease family genesCaspase-9The relative expression multiple of genes is 0.43-0.80.
EXAMPLE 8 SEUNEU-120 DPPH free radical scavenging ability experiment
1. Preparation of SEUNEU-120 supernatant:
the supernatant preparation method is described in example 3.
2. DPPH free radical scavenging ability assay
The reagent preparation and detection method is carried out according to the instruction of a Soxhlet DPPH free radical scavenging capacity detection kit. The 515nm absorbance of each sample was measured, averaged and the radical scavenging rate of each sample was calculated. The calculation formula is as follows: d% = [ (a blank- (a assay-a control) ]/a blank×100% and the calculation results are shown in table 9.
TABLE 9 SEUNEU-120 DPPH radical clearance (%)
The results show that the schizosaccharomyces pombe SEUNEU-120 has DPPH free radical scavenging capacity, and the DPPH free radical scavenging rate is 84.26% -87.39%.
Example 9 SEUNEU-120 Total antioxidant Capacity assay
1. Preparation of SEUNEU-120 supernatant:
the supernatant preparation method is described in example 3.
2. Total antioxidant capacity assay
The reagent preparation and detection method is carried out according to the specification of a Soxhaust total antioxidant capacity (T-AOC) detection kit, wherein the total reaction volume is 0.204ml, and the sample volume in the reaction is 0.006ml. The 593nm absorbance A of each sample was determined according to the formula: a measurement-a blank= 6.1464X-0.0009 to obtain an X value, and further to obtain the total antioxidant capacity.
The total antioxidant capacity is calculated as follows: total antioxidant capacity (μmol/mL) =x×v inverse total/V sample, calculated results are shown in table 10.
TABLE 10 SEUNEU-120 supernatant Total antioxidant Capacity (μmol/mL)
The results show that the schizosaccharomyces pombe SEUNEU-120 has an antioxidant capacity, and the total antioxidant capacity is 5.610-5.814 mu mol/mL.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. Schizosaccharomyces pombeSchizosaccharomyces pombe) SEUNEU-120 has been deposited in China center for type culture Collection with a deposit number of CCTCC NO: M20221432.
2. Use of schizosaccharomyces pombe according to claim 1 for the preparation of products for improving skin conditions.
3. The use of claim 2, wherein the improving skin conditions comprises at least one of promoting cell proliferation, repairing skin barrier, moisturizing, and anti-aging.
4. The use according to claim 3, wherein said promotion of cell proliferation is promotion of skin keratinocyte proliferation.
5. The use according to claim 3, wherein the repair of the skin barrier comprises restoring cell viability and/or up-regulating expression of a barrier repair related gene comprisingFLG、IVL、OVOL1AndOCLNat least one of them.
6. The use according to claim 3, wherein the moisturizing is an up-regulating moisturizing related geneGBAIs expressed by (a).
7. The use according to claim 3, wherein said anti-aging comprises at least one of the following:
up-regulating expression of genes involved in extracellular matrix synthesis, includingCOL1A1、COL13A1、SPTSSA、SMAD3AndMKXat least one of (a) and (b);
down-regulating expression of genes associated with degradation of extracellular matrix; the genes related to the degradation of the extracellular matrix compriseMMPAt least one of the family genes;
upregulating expression of a cellular antioxidant-associated gene comprisingNRF2、PTENAndSIRT-1at least one of (a) and (b);
up-regulating cell immune regulator related geneMORIs expressed by (a);
down-regulating apoptosis-related genesBAXAnd/orCaspase-9Is expressed by (a);
upregulation of cell autophagy-related genesLC3BIs expressed by (a);
up-regulating apoptosis-inhibiting related genesBCL-2Is expressed by (a);
up-regulating expression of a cell growth factor related gene comprisingFGF1、FGF2、FGF7、FGF21AndHGFat least one of (a) and (b);
scavenging DPPH free radical;
oxidation resistance.
8. The use according to any one of claims 2 to 7, wherein the product is a food, a pharmaceutical or a cosmetic.
9. A product for improving skin conditions, characterized in that it is made from a raw material comprising schizosaccharomyces pombe according to claim 1.
10. The product according to claim 9, characterized by comprising the supernatant and the bacterial lysate of schizosaccharomyces pombe according to claim 1.
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