Skin injury repair composition containing sphingosine as active ingredient and application thereof

Abstract

The invention discloses a skin injury repairing composition containing sphingosine as an active ingredient and application of the skin injury repairing composition. Specifically, the invention provides a composition for promoting skin injury repair, and the composition contains a metabolite of prevotella Copri as an active ingredient. The invention further provides application of the composition for repairing the skin injury to preparation of a product for repairing the skin injury. The skin injury repairing composition containing sphingosine has the effect of remarkably promoting regeneration of injured skin and can be well used in the skin repairing process.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to a skin damage repair composition containing sphingosine as an active ingredient and its application. Background Art

[0002] The skin, as the largest organ of the human body, is an important barrier against the invasion of foreign pathogens. A large number of microorganisms such as bacteria, fungi, and viruses existing in the environment will attach to the skin and form a precise and complex skin microbial community. This microbial community interacts with the host to form a dynamic balance relationship. In the case of normal and undamaged skin, microorganisms interact with each other, occupy ecological niches by competing for nutrients, and at the same time produce antimicrobial peptides to resist foreign pathogens, maintaining the homeostasis of the skin microbiota.

[0003] However, when the skin is damaged, the skin microbial homeostasis is disrupted, and the skin will undergo a series of highly complex and overlapping stages of hemostasis, inflammation, re-epithelialization, and remodeling at the moment of injury. This complex, dynamic, and continuous process depends on the interaction and synergy among cell components, growth factors, and cytokines. Skin microorganisms play an important role in the wound healing process, and their role far exceeds the traditional understanding of infection sources.

[0004] On the one hand, skin resident bacteria can secrete bioactive substances such as antimicrobial peptides and cytokines to regulate the wound immune inflammatory response, promote the infiltration and clearance of inflammatory cells, and at the same time stimulate the proliferation and migration of fibroblasts and endothelial cells, accelerating wound closure and the formation of new tissue. On the other hand, the dysregulation of the microbial community leads to the overgrowth and infection of pathogenic bacteria, triggers chronic inflammation, inhibits the wound healing process, and causes wound deterioration and complications.

[0005] In recent years, with the development of metagenomics technology, the research on the wound microbiota has gradually deepened, providing a new perspective for understanding the relationship between microorganisms and wound healing.

[0006] Treat wounds by using microorganisms beneficial to wound healing. For example, Lactobacillus can prevent and promote the wound healing of burns and is a potential probiotic for anti-burn wound infection. The aspergillus produced by Alcaligenes faecalis can promote the re-epithelialization of wound keratinocytes, promote the regeneration period of wound healing, and accelerate the wound healing process. Phage therapy can effectively reduce the infections of Staphylococcus aureus and Pseudomonas aeruginosa, improve the content of wound microorganisms, reduce the impact of pathogenic microorganisms on the wound during wound healing, and promote skin damage repair. Based on this, people can use the interaction between microorganisms-microorganisms and the promoting effect of microorganisms on wound healing as a new target for this treatment strategy to avoid the occurrence of antibiotic resistance.

[0007] Currently, existing probiotics (such as Lactobacillus) mainly inhibit pathogenic bacteria by secreting organic acids or antimicrobial peptides, but lack direct regulation of key signal pathways for wound repair (such as the sphingolipid metabolism pathway). The metabolites are single and cannot meet multiple requirements such as anti-inflammation, promoting keratinocyte migration, and angiogenesis at the same time. Moreover, the wound environment (such as high oxidative stress and low pH) may inhibit the survival of exogenous microorganisms. Existing strains lack adaptive modification (such as oxygen tolerance or biofilm formation ability), and the microbial colonization efficiency is low.

[0008] Therefore, there is an urgent need in the art to develop a composition or its application that can effectively promote wound healing and / or skin repair. Summary of the Invention

[0009] In view of the above problems, the object of the present invention is to provide a composition for repairing skin damage containing sphingosine as an active ingredient and its application, wherein sphingosine that can achieve excellent damage repair effect is used as the active ingredient.

[0010] In the first aspect of the present invention, a composition for promoting skin damage repair is provided, which contains the metabolite of Prevotella_Copri as an active ingredient.

[0011] In another preferred embodiment, the metabolite of Prevotella_Copri includes: Niflumic Acid, Sphingosine, Cordycepin, or Cer(d18:1 / 16:0).

[0012] In another preferred embodiment, the metabolite of Prevotella_Copri is Sphingosine.

[0013] In another preferred embodiment, the Prevotella_Copri is selected from Prevotella_Copri with the catalog number BNCC361375 from BeiNa Biological.

[0014] In another preferred embodiment, the composition further comprises one or a combination of non-inactivated Prevotella_Copri, inactivated Prevotella_Copri, and freeze-dried Prevotella_Copri strains.

[0015] In another preferred embodiment, the composition further comprises other drugs for promoting skin damage repair.

[0016] In another preferred embodiment, the other drugs for promoting skin injury repair include an antibiotic mixture.

[0017] In another preferred embodiment, the antibiotic mixture includes: 50 - 200 mg / kg ampicillin, 10 - 100 mg / kg vancomycin, 50 - 200 mg / kg metronidazole, and 50 - 200 mg / kg neomycin; preferably 100 mg / kg ampicillin, 50 mg / kg vancomycin, 100 mg / kg metronidazole, and 100 mg / kg neomycin.

[0018] In another preferred embodiment, the composition contains sphingosine at a concentration of 0.125 - 16 μmol / L.

[0019] In another preferred embodiment, the composition further includes a solvent, and the solvent is selected from one or more of PBS, DMSO, sterilized water, physiological saline, ethanol, propylene glycol, polyethylene glycol, glycerol, or vegetable oil.

[0020] In another preferred embodiment, the composition promotes skin repair and / or wound healing.

[0021] In another preferred embodiment, the composition has a significant effect of promoting the regeneration of damaged skin.

[0022] In another preferred embodiment, the composition is a cosmetic composition or a pharmaceutical composition.

[0023] In another preferred embodiment, the dosage form of the cosmetic composition is selected from at least one of the following groups: milk, skin softener, toner, astringent, nutrient cream, eye cream, liquid foundation, powder, essence, oil, gel, lotion, beauty liquid, mask, mask essence, soap, cleansing cream, facial cleansing foam, and body cleanser.

[0024] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from at least one of the following groups: granule, powder, syrup, elixir, emulsion, suspension, tablet, capsule, tincture, pill, transdermal absorbent, extract, aerosol, ointment, paste preparation, freeze-dried preparation, suppository, and injection.

[0025] In another preferred embodiment, the composition is an external preparation including sphingosine, and the external preparation is selected from any one of tincture, cream, paste, ointment, cream, emulsion, oil, mask, gel, smear, aerosol, or solution.

[0026] In the second aspect of the present invention, a microbial inoculum is provided. The microbial inoculum is the supernatant of a bacterial liquid prepared by activating strains of Prevotella_Copri, transferring the strains, and culturing the strains in liquid. The supernatant of the bacterial liquid is the metabolite of Prevotella_Copri.

[0027] In another preferred embodiment, the metabolites of Prevotella_Copri include: Niflumic Acid, Sphingosine, Cordycepin, or Cer(d18:1 / 16:0).

[0028] In another preferred embodiment, the metabolite of Prevotella_Copri is Sphingosine.

[0029] In another preferred embodiment, the Prevotella_Copri is selected from Prevotella with the catalog number BNCC361375 from Beina Biology.

[0030] In the third aspect of the present invention, a method for preparing the microbial inoculum as described in the second aspect of the present invention is provided, including the steps:

[0031] (1) Activating the strain: Smear Prevotella_Copri on a Columbia blood agar plate, place it in an anaerobic gas-generating bag for anaerobic culture, and incubate at a constant temperature of 35-38°C for 5-7 days;

[0032] (2) Transferring the strain: Pick a single colony and inoculate it onto a Columbia blood agar plate, and subculture it 2-5 times;

[0033] (3) Preparing the supernatant of the bacterial liquid: Pick the colony after subculture in step (2), inoculate it into a liquid medium containing Brain Heart Infusion Broth (BHI), place it in an anaerobic gas-generating bag for anaerobic culture, and incubate at a constant temperature of 35-38°C for 5-7 days; obtain the supernatant of the bacterial liquid, thereby obtaining the microbial inoculum.

[0034] In the fourth aspect of the present invention, there is provided the use of the composition as described in the first aspect of the present invention or the microbial inoculum as described in the second aspect of the present invention in the preparation of a product for promoting wound healing and / or promoting skin injury repair.

[0035] In another preferred embodiment, the product includes cosmetics, food, or medicine.

[0036] In another preferred embodiment, the dosage form of the medicine is an external dosage form including Sphingosine.

[0037] In another preferred embodiment, the external preparation is selected from any one of tinctures, creams, pastes, ointments, emulsions, lotions, oils, facial masks, gels, paints, aerosols or solutions.

[0038] In another preferred embodiment, the external preparation is used at least twice a day.

[0039] Compared with the prior art, the present invention has the following technical effects:

[0040] (1) The composition for repairing skin damage containing sphingosine of the present invention has the ability to significantly promote the repair of damaged skin.

[0041] (2) The composition for repairing skin damage containing sphingosine of the present invention provides a new raw material option for skin damage repair products.

[0042] (3) The present invention provides a dynamic repair scheme that is more in line with the wound microecology through the bifunctional synergy of "microorganism - sphingosine". Description of the Drawings

[0043] Figure 1 The figure shows the results of ABX promoting the healing of mouse skin wounds in the examples of the present invention; among them, Figure A shows a schematic diagram of the mouse skin injury model; Figure B shows the healing situation of the wounds of mice treated with ABX, and WD0 - 13 respectively represent the 0th day to the 13th day of wound healing; Figure C shows a line statistical chart of the wound healing rate of mice treated with ABX; among them, n = 4, *P < 0.05, ***P < 0.001.

[0044] Figure 2 The figure shows the mRNA expression levels of related inflammatory factors (TNF-α, IL-6) and growth factors (IGF-1, FGF-7) in mouse skin wounds after ABX treatment in the examples of the present invention; among them, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0045] Figure 3 The figure shows a statistical chart of the contents of related growth factors (EGF, KGF, VEGF) in mouse skin wounds after ABX treatment in the examples of the present invention; among them, n = 4, **P < 0.01, ****P < 0.0001.

[0046] Figure 4 The figure shows an analysis diagram of the skin microbial species composition of mice after ABX treatment of wounds in the examples of the present invention; among them, Figure A shows a comparative analysis diagram of key species differences; Figure B shows a bar chart of species composition.

[0047] Figure 5 Shown is the skin wound healing diagram of a mouse wound treated with the supernatant of Prevotella copri bacterial solution in an embodiment of the present invention; among them, Figure A shows the healing condition diagram of a mouse wound treated with the supernatant of Prevotella copri bacterial solution, BHI represents Brain Heart Infusion Broth, a commonly used nutrient medium; P.copri represents Prevotella Copri; Figure B shows the line statistical chart of the wound healing rate of a mouse wound treated with the supernatant of Prevotella copri bacterial solution; among them, n = 4, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0048] Figure 6 Shown is the metabolite analysis diagram of the supernatant of Prevotella copri bacterial solution in an embodiment of the present invention; among them, Figure A shows the principal component analysis diagram, among which, the red frame includes 3 principal components of the control group BHI, and the green frame includes 3 principal components of the metabolites of the supernatant of Prevotella copri bacterial solution; Figure B shows the heat map of metabolome differential analysis.

[0049] Figure 7 Shown is the differential metabolite analysis diagram of the supernatant of Prevotella copri bacterial solution in an embodiment of the present invention; among them, Figure A shows the bar chart of metabolite content; Figure B shows the differential analysis diagram of metabolites.

[0050] Figure 8 Shown is the analysis diagram of sphingosine and ceramide inhibitor on mouse skin wound in an embodiment of the present invention; among them, Figure A shows the healing condition diagram of a mouse wound treated with sphingosine and ceramide inhibitor; Figure B shows the line statistical chart of the wound healing rate of a mouse wound treated with sphingosine and ceramide inhibitor; among them, Sph represents Sphingosine, P053 represents an effective, non - competitive and selective ceramide synthase 1 (CerS1) inhibitor (MCE, catalog number: HY - 126015); n = 4, *P < 0.05, ***P < 0.001, ****P < 0.0001. Detailed implementation manners

[0051] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. All kinds of commonly used reagents used in the embodiments are commercially available products.

[0052] Experimental methods

[0053] 1 Establishment of a mouse skin injury model:

[0054] The specific steps are as follows:

[0055] (1) Intraperitoneally inject 400 μL of anesthetic avertin into male C57BL / 6N mice at 8 weeks old.

[0056] (2) Use an electric hair clipper to shave the hair on the back of the mice, and wipe off the remaining hair with an alcohol cotton ball.

[0057] (3) Use a 6 mm skin biopsy punch to create a wound contour on the back of the mice, mark the wound contour with a marker pen, and use ophthalmic surgical scissors to cut off the back skin of the mice according to the marked contour. ( Figure 1 A)

[0058] (4) After the mice recover from anesthesia, house each mouse individually in a single cage. Fix the camera with a triangular bracket at a height of 10 cm, take pictures of the mice's wounds every other day, use ImageJ to calculate the wound healing area, and calculate the wound healing rate.

[0059] Wound healing rate = [Initial injury area - Unrepaired area at corresponding time point] / Initial injury area × 100%

[0060] (5) Decapitate the mice, cut out a skin tissue sample of the wound with a size of 1.5 cm × 1.5 cm × 0.2 cm, and store it at -80 °C.

[0061] 2 Medication application and experimental grouping for mouse wounds:

[0062] The specific steps are as follows:

[0063] Experimental grouping for detecting wound healing rate: The experiment is divided into 2 groups, namely the experimental group (treated with antibiotic ABX) and the control group (treated with PBS), with 4 replicates in each group. Among them, ABX refers to the Antibiotics antibiotic mixture, including: 100 mg / kg ampicillin, 50 mg / kg vancomycin, 100 mg / kg metronidazole, and 100 mg / kg neomycin. Use a 1 mL medical syringe to aspirate 50 μL of the reagent and drop it onto the wound of each mouse. Treat the mouse wounds with the drug every day, take pictures and record the wound area every other day, and calculate the wound healing rate.

[0064] Experimental grouping for qPCR and ELISA: The experiment is divided into 2 groups, namely the experimental group ABX and the control group PBS, with 4 replicates in each group. Use a 1 mL medical syringe to aspirate 50 μL of the reagent and drop it onto the wound of each mouse. Treat the mouse wounds with the drug every day.

[0065] 3 RNA extraction from mouse skin wound tissues:

[0066] The specific steps are as follows:

[0067] (1) Take the wound skin tissue of the mouse and place it in a 1.5 mL centrifuge tube. Add 1000 μL of Trizol and 2 steel beads, and grind the sample with a grinder at 65HZ for 60 S.

[0068] (2) Add 200 μL of chloroform. Mix well by inverting the tube up and down and let it stand at room temperature for 10 min.

[0069] (3) Centrifuge at 4 °C, 12000 rpm for 15 min, and transfer the supernatant to a new EP tube.

[0070] (4) According to the ratio of the upper layer liquid: isopropanol = 1:1, add the same volume of isopropanol. Mix well by inverting the tube up and down, let it stand at -20 °C for 30 min, and then centrifuge at 4 °C, 12000 rpm for 30 min.

[0071] (5) Add 1000 μL of 75% ethanol, and gently blow the precipitate with a pipette tip without dispersing the precipitate. Then centrifuge at 4 °C, 12000 rpm for 1 min.

[0072] (6) Repeat step (5).

[0073] (7) Air-dry at room temperature for 10 min, perform the operation on ice, and resuspend the precipitate with 30 μL of RNA-free water.

[0074] (8) Measure the RNA concentration of the sample.

[0075] 4 Detection of the mRNA expression level of healing-related factors by fluorescence quantitative PCR:

[0076] The specific steps are as follows:

[0077] 1) Reverse transcription to obtain cDNA.

[0078] The reverse transcription system is shown in Table 1 below:

[0079] Table 1

[0080]

[0081] 2) Fluorescence quantitative PCR:

[0082] A. The fluorescence quantitative PCR reaction system is prepared as shown in Table 2 below:

[0083] Table 2

[0084]

[0085] Among them, the primers for each healing-related factor are shown in Table 3 below:

[0086] Table 3

[0087]

[0088] B. Place the sample in a qPCR instrument and perform the reaction according to the procedure shown in Table 4 below:

[0089] Table 4

[0090]

[0091] 3) Collect experimental data and analyze the data according to the 2-△△CT method.

[0092] 5 ELISA testing of skin tissue

[0093] (1) Cut the collected skin tissue into even pieces, add an appropriate amount of physiological saline and grind at 4°C, 3000 rpm, for 10 min. Collect the supernatant in a new EP tube.

[0094] (2) Add 50 μL of standards of different concentrations to the standard wells; add 10 μL of the sample to be tested and then add 40 μL of sample diluent to the sample wells, but do not add anything to the blank wells.

[0095] (3) Add 100 μL of HRP-labeled detection antibody to the standard wells and sample wells, seal the wells with a sealing film, and incubate at 37 °C for 60 min.

[0096] (4) Shake off the liquid in each well, fill each well with washing solution, let it stand for 1 min, shake off the liquid, and repeat 5 times.

[0097] (5) Add 50 μL of substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0098] (6) Add 50 μL of stop solution to each well and measure the OD value at a wavelength of 450 nm.

[0099] 6. Collection of mouse skin wound microorganisms

[0100] The present invention focuses on the effect of microorganisms on wound healing during the inflammatory period, which is also a critical period for microorganisms to participate in wound healing. Therefore, on the first day of wound healing (WD1, the first day after modeling) and the third day of wound healing (WD3), a sterile cotton swab was soaked with PBS, swabbed back and forth on the wound 50 times, and the cotton swab was placed in a sterile 1.5 mL centrifuge tube, the cotton swab stick was broken, the centrifuge tube was closed, the shaker was fully shaken, and the tube was frozen at -80 °C.

[0101] 7 16S rRNA sequencing

[0102] The samples were submitted to Shenzhen BGI Co., Ltd. for sequencing analysis.

[0103] The specific steps are as follows:

[0104] (1) Extract the genomic DNA of the sample. Use the MagPure Stool DNA KF kit B, Qubit Fluorometer, and Quit dsdna br assay kit to extract and quantify the genomic DNA of the sample, respectively.

[0105] (2) Construct the library. Use the universal primers 341-F and 806-R to perform PCR amplification on the V3-V4 region of the bacterial 16S rRNA gene. Purify the PCR products using Agencourt AMPure XP beads. Detect the library fragments using an Agilent 2100 Bioanalyzer. Follow the standard Illumina protocol to sequence the verified library on the Illumina MiSeq platform (BGI).

[0106] (3) Data processing. First, use Readfq v8 to remove low-quality data. Delete adapter contamination and reads containing N. Then use the FLASH software to assemble the paired sequences of the paired-end sequencing reads into high-region tagged sequences using the overlapping relationship. Finally, use the USEARCH software to cluster the valid tags generated by the UCHIME algorithm into operational taxonomic units (OTUs).

[0107] (4) Data visualization. First, annotate the classification information using the representative sequences of the OTUs according to the Greengenes database and the mothur method. Second, perform the principal component analysis of the OUT using the ade4 software package in R. Then use the RDP classifier Bayesian algorithm to classify the representative sequences of the OUT. Calculate the community composition of each sample at the species level, use the ggplot2 software package in R to make a species richness histogram, and use R to make a species composition heatmap. Finally, use the motor software to analyze the Alpha diversity and use the ggplot2 software package in R to make an Alpha diversity box plot.

[0108] 8 Culture of Prevotella

[0109] 1) Strain activation

[0110] (1) Dissolve the freeze-dried tube containing the freeze-dried powder of the strain (Beijing Na Biological Technology Co., Ltd., product number: BNCC361375) in a 37 °C water bath and shake it quickly and evenly.

[0111] (2) Wipe the outer wall of the freeze-dried tube with an alcohol cotton ball and perform deoxygenation on a Columbia blood agar plate.

[0112] (3) Open the freeze-dried tube in a safety cabinet and add 500 μL of sterile water to dissolve and mix well.

[0113] (4) Aspirate all the liquid, and add 200 μL of the bacterial solution to each Columbia blood agar plate (Qingdao Haibo Biotechnology, product number: HBPM0153).

[0114] (5) Use a spreading rod to spread evenly, place it in an anaerobic gas-generating bag, and incubate at 37 °C anaerobically for 5 - 7 days.

[0115] 2) Strain subculture

[0116] Use a sterile pipette tip to pick up the milky white raised round colonies with neat edges in the safety cabinet, and transfer them by streaking onto a new Columbia blood agar plate. Repeat the operation for 3 passages.

[0117] 3) Liquid culture of the strain

[0118] For the plate that has been passaged 3 times, use a sterile pipette tip to pick up the milky white raised round colonies with neat edges in the safety cabinet and transfer them to a T25 culture flask containing liquid medium of brain heart infusion broth (BHI), and incubate at 37 °C anaerobically in an anaerobic gas-generating bag for 5 - 7 days. Collect the supernatant of the bacterial solution and store it at -80 °C.

[0119] Example 1

[0120] Through the above experimental method, this example detects the effect of skin microorganisms on wound healing. The results are as follows:

[0121] 1. ABX treatment promotes wound healing

[0122] Figure 1 The results shown indicate that in the mouse skin injury model prepared with a 6 mm skin biopsy punch, it is found that from the 1st day (WD1) to the 3rd day (WD3) of the wound is the wound inflammation period, from the 5th day (WD5) to the 9th day (WD9) of the wound is the wound regeneration period, and from the 11th day (WD11) to the 9th day (WD9) of the wound is the wound remodeling period ( Figure 1 B). In the PBS control group, the surface of the mouse skin wound showed thickening of the scab, the scab shedding speed was slow, and the wound healing rate was slow; while in the ABX treatment group, the scab shedding speed of the mouse skin wound was fast, the wound healing rate was fast, and at the 9th day, the scab in the ABX group had completely fallen off ( Figure 1 B). On the 3rd day, 5th day, 7th day, and 9th day of wound healing, the wound healing rate of the mice in the ABX treatment group was significantly higher than that of the PBS control group ( Figure 1 C). It can be thus explained that ABX treatment accelerates wound healing.

[0123] 2. ABX promotes the expression and increase in the content of healing-related factors in wound tissues

[0124] By detecting inflammatory factors during the wound inflammation period (day 3 after wounding) and growth factors during the wound regeneration period (day 7 after wounding) using qPCR, it was found that ABX significantly upregulated the mRNA expression levels of inflammatory factors (TNF-α, IL-6) during the inflammation period and growth factors (FGF-7, IGF-1) during the regeneration period ( Figure 2 ).

[0125] By detecting growth factors related to the regeneration period (KGF, EGF, VEGF) on day 7 after wounding using ELISA, it was found that after treating the wounds of mice with ABX, the content of growth factors in the wound skin tissue was significantly increased ( Figure 3 ).

[0126] 3. Prevotella will be enriched during ABX-promoted wound healing in mice

[0127] To further explore the effect of microorganisms on wound healing, the inventor found through species composition analysis that the species abundance of Prevotella Copri (abbreviated as P. copri) increased with the progress of wound healing during the control group treated with BHI ( Figure 4 A-4B); at the same time, during the process of ABX-promoted wound healing, the abundance of Prevotella also increased, and the increase in the abundance of Prevotella was greater than that of the control group (BHI) ( Figure 4 A-4B). This indicates that Prevotella is closely related to wound healing and plays a beneficial and key role in wound healing.

[0128] 4. Prevotella can promote wound healing

[0129] By culturing and collecting the metabolites of Prevotella Copri (P. copri), namely the supernatant of the bacterial solution, and treating the skin wounds of mice, it was found that the supernatant of the Prevotella bacterial solution could promote the healing of skin wounds in mice. Among them, in the BHI control group of mice, the surface of the skin wound showed thickening of the scab, the scab shedding speed was slow, and the wound healing rate was slow; while in the group of mice treated with P. copri, the scab shedding speed of the skin wound was fast, the wound healing rate was fast, and by day 9, the scab in the group of mice treated with P. copri had completely fallen off ( Figure 5 A). On days 3, 5, 7, and 9 of wound healing, the wound healing rate of the group of mice treated with P. copri was significantly higher than that of the control group BHI ( Figure 5 B). This indicates that the treatment with the supernatant of P. copri accelerated wound healing.

[0130] Example 2: Sphingosine specifically produced by Prevotella can promote wound healing in mice

[0131] In previous studies, Prevotella copri was found to be the dominant bacterium in wound healing; Prevotella copri can promote wound healing. This example examines how Prevotella copri distributed at the wound site promotes wound healing. Therefore, the mechanism of action of Prevotella copri on wound healing was explored next. The specific experimental procedure is as follows:

[0132] (1)Non-targeted metabolome detection of Prevotella copri

[0133] The bacterial liquid prepared by the above experimental method was centrifuged at 3000 rpm, and the supernatant of the bacterial liquid was collected and quickly transferred to a liquid nitrogen tank for rapid freezing. It was mailed to Shanghai Bioquark Biomedical Co., Ltd. for testing by dry ice.

[0134] (2)Grouping for non-targeted metabolome detection

[0135] There were 2 groups in the experiment, namely the BHI-Ctrl group and the P.copri group. The biological replicates corresponding to each group were 3 cases. Metabolomics detection and analysis based on LC-MS were performed, with a total of 6 samples.

[0136] (3)Metabolite extraction

[0137] (P.copri low-speed centrifugation) Pipette 100 μL of the sample into an EP tube, add 400 μL of the extraction solution (methanol:acetonitrile = 1:1 (V / V)), and the extraction solution contains isotope-labeled internal standard; vortex for 30 s and sonicate for 10 min (ice-water bath); let stand at -40 °C for 1 h; centrifuge the sample at 4 °C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; take the supernatant and transfer it to an injection vial for on-machine detection.

[0138] (4)On-machine detection

[0139] For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph to chromatographically separate the target compounds through a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) liquid chromatography column. The mobile phase A for liquid chromatography was an aqueous phase containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia water, and the mobile phase B was acetonitrile. The sample tray temperature was 4 °C, and the injection volume was 2 μL.

[0140] The Orbitrap Exploris 120 mass spectrometer can collect primary and secondary mass spectrometry data under the control of the control software (Xcalibur, version: 4.4, Thermo). The detailed parameters are as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320 °C, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, Spray Voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0141] (5)Data processing

[0142] After the raw data is converted into the mzXML format by the ProteoWizard software, a co-written R package is used for metabolite identification, and the database used is BiotreeDB (V3.0)7, and then a self-written R package is used for visual analysis.

[0143] Experimental results:

[0144] 1. Prevotella specifically produces sphingosine significantly

[0145] After confirming that the metabolites of Prevotella can promote wound healing, in order to verify which specific metabolites of Prevotella play a role, the inventor collected the supernatant of Prevotella culture broth for non-targeted metabolome sequencing analysis. Through principal component analysis, significant differences between groups and a heatmap of metabolite difference analysis were used to find that there were large differences between the BHI medium control group and the experimental group of the supernatant of P. Copri culture broth, and metabolites significantly enriched in the culture broth supernatant ( Figure 6 A-6B).

[0146] Through the analysis of the bar chart of metabolite species content, it was found that four compounds, niflumic acid, sphingosine (abbreviated as Sph), cordycepin, and ceramide Cer(d18:1 / 16:0), were specifically and significantly increased in the experimental group of the culture broth supernatant ( Figure 7 A).

[0147] It should be noted that the present invention discovers that sphingosine is specifically present in the supernatant of Prevotella, but not found in the culture medium. Other metabolites (such as niflumic acid, cordycepin, ceramide) are present in both the culture medium and the bacterial supernatant, but are present in greater amounts in the bacterial supernatant. To better illustrate that the substances derived from the bacteria promote wound healing, the present invention selects sphingosine for subsequent experiments.

[0148] Among them, niflumic acid and cordycepin mainly play an anti-inflammatory pain role, reducing the damage of oxidative stress to cells, regulating immunity to enhance the body's immunity, and inhibiting bacterial adenosine kinase to exert antibacterial and antiviral functions. Ceramide Cer (d18:1 / 16:0) is an endogenous bioactive ceramide ( Figure 7 B). Sphingosine produces ceramide by hydrolysis.

[0149] 2. Sphingosine specifically and significantly produced by Prevotella can promote wound healing

[0150] Mouse skin wounds were treated with sphingosine (1 μmol / L) and ceramide inhibitor (P053) (1 μmol / L) and verified in an in vitro mouse skin injury model. It was found that sphingosine could promote wound healing, while the ceramide inhibitor delayed wound healing ( Figure 8 A-8B). Thus, it can be shown that Prevotella copri can promote wound healing by exogenously providing sphingosine, and sphingosine produces ceramide by hydrolysis to regulate the biosynthesis of hyaluronic acid.

[0151] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but should not be construed as limiting the scope of the present invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. A composition for promoting skin damage repair, characterized in that: It contains metabolites of Prevotella Copri as active ingredients.

2. The composition according to claim 1, characterized in that The metabolites of Prevotella_Copri include: Niflumic Acid, Sphingosine, Cordycepin or Ceramide Cer (d18:1 / 16:0).

3. The composition according to claim 1, characterized in that The metabolite of Prevotella Copri is sphingosine.

4. The composition according to claim 1, characterized in that The composition further comprises a solvent, and the solvent is selected from one or more of PBS, DMSO, sterile water, physiological saline, ethanol, propylene glycol, polyethylene glycol, glycerol or vegetable oil.

5. The composition according to claim 1, characterized in that The composition is a cosmetic composition or a pharmaceutical composition.

6. The composition according to claim 1, characterized in that The dosage form of the pharmaceutical composition is selected from at least one of the following groups: granules, powders, syrups, elixirs, emulsions, suspensions, tablets, capsules, tinctures, pills, transdermal absorbents, lotions, aerosols, ointments, paste preparations, freeze-dried preparations, suppositories and injections.

7. The composition according to claim 1, characterized in that The composition is an external dosage form comprising sphingosine, and the external dosage form is selected from any one of tincture, cream, paste, ointment, cream, emulsion, oil, mask, gel, smear, aerosol or solution.

8. A microbial agent, characterized in that: The microbial agent is a bacterial liquid supernatant obtained by strain activation, strain transfer and strain liquid culture of Prevotella Copri, and the bacterial liquid supernatant is a metabolite of Prevotella Copri.

9. A method for preparing the microbial agent according to claim 8, characterized in that: Includes steps: (1) Activation of strains: Spread Prevotella Copri on Columbia blood agar plates, place in anaerobic gas-producing bags, and culture at 35-38°C for 5-7 days; (2) Strain transfer: pick a single colony and inoculate it into Columbia blood agar plate and subculture it 2 to 5 times; (3) Preparing bacterial supernatant: Pick the bacterial colonies after subculture in step (2), inoculate them into a liquid culture medium containing brain heart infusion broth (BHI), place them in an anaerobic gas production bag, and culture them at a constant temperature of 35-38° C. for 5-7 days; prepare the bacterial supernatant, thereby obtaining the microbial agent.

10. Use of the composition according to claim 1 or the microbial agent according to claim 8 in the preparation of a product for promoting wound healing and / or promoting skin damage repair.

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