Propionibacterium acnes and use of metabolites thereof for regulating skin barrier

Abstract

The invention provides propionibacterium acnes and application of metabolites of the propionibacterium acnes to regulation of skin barriers. Specifically, the invention provides application of propionibacterium acnes and / or propionibacterium acnes metabolites in preparation of drugs, and the drugs are used for at least one of the following: activating an AhR signal channel; the expression of the ALDH1A3 is promoted or the activity of the ALDH1A3 is improved; the synthesis of retinoic acid is promoted; the expression of skin barrier functional genes is promoted; the epidermal barrier integrity is promoted. The discovery of the relationship between the propionibacterium acnes and the AhR provides a new mechanism for the subsequent research on the promotion of the completeness of the host skin barrier function by the skin symbiotic bacteria.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to the use of Propionibacterium acnes and its metabolites in regulating the skin barrier. Background Art

[0002] Aryl Hydrocarbon Receptor (AHR or AhR) is a transcriptional regulatory factor that can bind ligands within cells. Under normal circumstances, AhR exists in the cytoplasm and forms a complex with multiple chaperone proteins. However, after binding to a ligand, AhR enters the nucleus through the nuclear pore and forms a dimer with Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT). The formed dimer can bind to the Xenobiotic Response Element (XRE) in DNA, promoting the expression of downstream genes. The AhR signaling pathway is of great significance for clearing exogenous harmful substances and maintaining the body's homeostasis.

[0003] The Human Microbiome Project is another major scientific project after the Human Genome Project and is of great significance for interpreting the relationship between human health and diseases. In recent years, the research on the human microbiome, including the skin microbiome, has been a hot topic at the forefront of world science and technology. Using skin symbiotic microorganisms to treat skin diseases is a brand-new treatment idea different from the conventional one and is considered to have great potential for the development of clinical drugs and personal skin care products. Top scholars in this field from many countries around the world have invested in clinical intervention research, including live bacterial preparations, skin microbiota transplantation, phage therapy, etc. However, at present, there are unclear effective strains, unclear mechanisms and targets in this field, resulting in a serious lag in the development and implementation of intervention strategies and products. Retinoic acid is an important bioactive molecule in the body. Retinoid drugs are widely used for the intervention of various skin conditions, including:

[0004] (1) Anti-aging: Retinoic acid can effectively inhibit the expression of matrix metalloproteinases induced by light radiation, thus improving light radiation-induced skin aging; (2) Treating a series of skin diseases such as psoriasis, acne, rosacea, etc.; in psoriasis, abnormal hyperplasia of keratinocytes can be seen, and topical application of retinoic acid can accelerate the terminal differentiation and exfoliation of keratinocytes, promoting normal keratinization of the skin; on the contrary, the lack of retinoic acid will lead to abnormal epidermal keratinization; (3) Anti-tumor: Retinoic acid interferes with tumorigenesis by anti-proliferation and promoting cell differentiation; (4) Anti-infection: The metabolism of retinoic acid is very important for resisting skin infections, and people with insufficient retinoic acid tend to have more skin infections.

[0005] Therefore, the present application discloses a new mechanism for commensal bacteria to maintain host skin homeostasis by promoting the generation of AhR, providing a new therapeutic target for refractory skin diseases. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0007] Previous research reports have shown that skin commensal bacteria can promote host skin homeostasis. However, to date, the target bacterial species that play this function have not been clearly identified, and the key regulatory mechanisms mediating the interaction between the target bacterial species and the host remain unclear. The inventors first discovered in previous research that after AhR is activated by environmental stimulants (benzo[a]pyrene BaP, and carbazole FICZ), it can target the transcription of ALDH1A3, thereby regulating the synthesis and metabolism of RA. Then, the inventors studied the relationship between skin commensal bacteria and AhR and found that Propionibacterium acnes can activate AhR in keratinocytes by secreting active substance A, target and up-regulate the expression of ALDH1A3, and thus promote the synthesis and metabolism of retinoic acid. It is known that retinoic acid can promote the transcription of skin barrier function genes and promote wound healing, thereby promoting the integrity of the skin barrier function.

[0008] Therefore, in the first aspect of the present invention, the present invention proposes the use of Propionibacterium acnes and / or Propionibacterium acnes metabolites in the preparation of a product for at least one of the following: activating the AhR signaling pathway; promoting the expression of ALDH1A3 or increasing the activity of ALDH1A3; promoting retinoic acid synthesis; regulating the transcription of skin barrier function genes; promoting the integrity of the skin barrier function; promoting wound healing; treating or preventing pathogenic bacterial infections or skin infections.

[0009] Based on the discovered relationship between Propionibacterium acnes and / or Propionibacterium acnes metabolites and AhR, the relationship between AhR and ALDH1A3, the relationship between ALDH1A3 and retinoic acid, and the relationship between retinoic acid and skin barrier function, the inventors proposed the above-mentioned use. When using Propionibacterium acnes and / or Propionibacterium acnes metabolites, it can simultaneously promote the activation of the AhR signaling pathway, the synthesis of retinoic acid, the regulation of the transcription of skin barrier function genes, and the promotion of wound healing, thereby maintaining the integrity of the skin barrier.

[0010] According to an embodiment of the present invention, the use of Propionibacterium acnes and / or Propionibacterium acnes metabolites in the preparation of a product may further include at least one of the following additional technical features:

[0011] According to an embodiment of the present invention, the metabolites of Propionibacterium acnes are selected from at least one of 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid or 3-indoleethanol. The inventors found through experiments that 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid or 3-indoleethanol can regulate the AhR signaling pathway, the expression or activity of ALDH1A3, the synthesis of retinoic acid, and the transcription of skin barrier function genes.

[0012] According to an embodiment of the present invention, the metabolite of Propionibacterium acnes is selected from 3-indoleethanol.

[0013] According to an embodiment of the present invention, the product is selected from drugs, skin care products or health products.

[0014] According to an embodiment of the present invention, the skin barrier function genes are selected from at least one of CLDN1 (Claudin 1, tight junction protein 1), KRT16 (Keratin16, keratin 16), KRT4 (Keratin4, keratin 4), KRT75 (Keratin75, keratin 75), GRHL3 (Grainyhead Like Transcription Factor 3, granular transcription factor 3), KLK6 (Kallikrein Related Peptidase 6, kallikrein-related peptidase 6), DSC1 (Desmocollin 1, desmoglein 1), TGM1 (Transglutaminase 1, transglutaminase 1), OVOL1 (Ovo LikeTranscriptional Repressor 1, oocyte-like transcriptional repressor 1) and COL6A3 (Collagen Type VIAlpha 3Chain, type VI collagen alpha 3 chain).

[0015] It should be noted that the product can up-regulate CLDN1, KRT16, KRT75, GRHL3, DSC1, TGM1, OVOL1 and COL6A3 in the skin barrier function genes; the product can down-regulate KRT4 and KLK6.

[0016] In a second aspect of the present invention, the present invention provides the use of a reagent in the preparation of a product, the reagent being used to regulate the production of metabolites of Propionibacterium acnes, and the product being used for at least one of the following: regulating the AhR signaling pathway; regulating the expression or activity of ALDH1A3; regulating retinoic acid synthesis; regulating the transcription of skin barrier function genes; promoting wound healing. Based on the discovered relationships between Propionibacterium acnes and / or its metabolites and AhR, between AhR and ALDH1A3, between ALDH1A3 and retinoic acid, and between retinoic acid and skin barrier function genes, the inventors proposed the above-mentioned use. When using Propionibacterium acnes and / or its metabolites, it can simultaneously promote the activation of the AhR signaling pathway, the synthesis of retinoic acid, the transcription of skin barrier function genes, and promote wound healing, thereby maintaining the integrity of the skin barrier.

[0017] According to an embodiment of the present invention, the use of the reagent in the preparation of a product may further include at least one of the following additional technical features:

[0018] According to an embodiment of the present invention, the reagent is used to promote the production of metabolites of Propionibacterium acnes, and the product is used for at least one of the following: activating the AhR signaling pathway; promoting the expression of ALDH1A3 or increasing the activity of ALDH1A3; promoting retinoic acid synthesis; promoting the transcription of skin barrier function genes; promoting wound healing. The inventors found that the ability of Propionibacterium acnes to activate the AhR signaling pathway can depend on its metabolites. Therefore, the above-mentioned use was proposed, by regulating the production of metabolites of Propionibacterium acnes, and further regulating the AhR signaling pathway, the expression or activity of ALDH1A3, the synthesis of retinoic acid, and the transcription of skin barrier function genes, to promote wound healing.

[0019] According to an embodiment of the present invention, the metabolites of Propionibacterium acnes are selected from at least one of 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid, or 3-indoleethanol. The inventors found through experiments that 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid, or 3-indoleethanol can regulate the AhR signaling pathway, the expression or activity of ALDH1A3, the synthesis of retinoic acid, and the transcription of skin barrier function genes.

[0020] According to an embodiment of the present invention, the metabolite of Propionibacterium acnes is selected from 3-indoleethanol.

[0021] It should be noted that the "Propionibacterium acnes metabolites: 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid or 3-indole ethanol" described in the present invention can be isolated from the metabolites of Propionibacterium acnes or obtained by purchase. Whether obtained by isolation or purchase, 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid or 3-indole ethanol can activate the AhR signaling pathway; promote the expression of ALDH1A3 or increase the activity of ALDH1A3; promote retinoic acid synthesis; promote the transcription of skin barrier function genes; and promote wound healing.

[0022] According to an embodiment of the present invention, the product is selected from drugs, skin care products or health care products.

[0023] According to an embodiment of the present invention, the skin barrier function genes are selected from at least one of CLDN1, KRT16, KRT4, KRT75, GRHL3, KLK6, DSC1, TGM1, OVOL1 and COL6A3.

[0024] It should be noted that the product can up-regulate CLDN1, KRT16, KRT75, GRHL3, DSC1, TGM1, OVOL1 and COL6A3 in the skin barrier function genes; the product can down-regulate KRT4 and KLK6.

[0025] In the third aspect of the present invention, the present invention provides a method for activating the AhR signaling pathway in cells. According to an embodiment of the present invention, the method includes co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells. Based on the relationship between the above-mentioned Propionibacterium acnes and / or Propionibacterium acnes metabolites and AhR, by using the method of the present invention, the generation of AhR in cells can be promoted, and then the expression of ALDH1A3 protein in cells can be up-regulated, thereby promoting the synthesis of retinoic acid in cells. Therefore, it lays a foundation for maintaining the integrity of the skin barrier function subsequently.

[0026] In the fourth aspect of the present invention, the present invention provides a method for up-regulating the expression of ALDH1A3 protein in cells. According to an embodiment of the present invention, it includes co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells. Based on the relationship between the above-mentioned Propionibacterium acnes and / or Propionibacterium acnes metabolites and AhR, and the relationship between AhR and ALDH1A3, by using the method of the present invention, the expression of ALDH1A3 protein in cells can be up-regulated, thereby promoting the synthesis of retinoic acid in cells. Therefore, it lays a foundation for maintaining the integrity of the skin barrier function subsequently.

[0027] In the fifth aspect of the present invention, the present invention provides a method for promoting the production of retinoic acid in cells. According to an embodiment of the present invention, it includes co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells. Based on the relationships among Propionibacterium acnes and / or Propionibacterium acnes metabolites, AhR, AhR and ALDH1A3, and ALDH1A3 and retinoic acid, the method of the present invention can promote the synthesis of retinoic acid in cells. Therefore, it lays a foundation for subsequently maintaining the integrity of the skin barrier function.

[0028] In the sixth aspect of the present invention, the present invention provides a method for promoting gene expression in cells, and the gene is related to skin wound healing. According to an embodiment of the present invention, it includes co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells, and the gene is SERPINB2. Based on the relationships among Propionibacterium acnes and / or Propionibacterium acnes metabolites, AhR, and AhR and Serpinb2, the method of the present invention can up-regulate the expression of the Serpinb2 gene in cells, thereby promoting skin wound healing. Therefore, it lays a foundation for subsequently maintaining the integrity of the skin barrier function.

[0029] According to an embodiment of the present invention, the method for activating the AhR signaling pathway in cells, the method for up-regulating the expression of ALDH1A3 protein in cells, the method for promoting the production of retinoic acid in cells, and the method for promoting the expression of genes related to skin wound healing in cells further include at least one of the following additional technical features:

[0030] According to an embodiment of the present invention, the Propionibacterium acnes metabolites are selected from at least one of 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid, or 3-indoleethanol.

[0031] According to an embodiment of the present invention, the co-culturing time is 20 to 26 hours.

[0032] In the seventh aspect of the present invention, the present invention provides a Propionibacterium acnes which was deposited with the Guangdong Provincial Microbial Culture Collection Center on November 24, 2023, with the deposit number GDMCC No: 64063, and the deposit address: Guangdong, China. The Propionibacterium acnes of the present invention can simultaneously promote the activation of the AhR signaling pathway, the synthesis of retinoic acid, regulate the transcription of skin barrier function genes, and promote wound healing, thereby maintaining the integrity of the skin barrier.

[0033] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the following description of embodiments in conjunction with the accompanying drawings, wherein:

[0035] Figure 1 is the gel electrophoresis pattern of BaP activating AhR to up-regulate the expression of ALDH1A3 according to an embodiment of the present invention;

[0036] Figure 2 is the result graph showing the dose-effect dependence of BaP regulating ALDH1A3 transcription through AhR according to an embodiment of the present invention (WT: wild cell line; KO: AhR knockout cell line. **: P < 0.01; ***: P < 0.001);

[0037] Figure 3 is the result graph showing the dose-effect dependence of FICZ regulating ALDH1A3 transcription through AhR according to an embodiment of the present invention (WT: wild cell line; KO: AhR knockout cell line. **: P < 0.01; ***: P < 0.001);

[0038] Figure 4 is the result graph of BaP and FICZ regulating the intracellular concentration of all-trans retinoic acid (atRA) through AhR according to an embodiment of the present invention, wherein

[0039] Retinal was added in A;

[0040] Retinol was added in B;

[0041] Figure 5 is the result graph of the BaP-AhR-atRA pathway causing epidermal thickening in mouse keratinocytes according to an embodiment of the present invention, wherein

[0042] The mouse in A is AhR + / + mouse and AhR - / - mouse;

[0043] The mouse in B is AhR + / + K14-cre mouse and AhR - / - K14-cre mouse;

[0044] Figure 6 is the expression of ALDH1A3 after stimulation of three commensal bacteria filtrates of Staphylococcus epidermidis, Propionibacterium acnes and Moraxella osloensis on HaCaT (A) and NHEK (B) according to an embodiment of the present invention (RNA-seq);

[0045] Figure 7It shows the expression levels of CYP1A1 and ALDH1A3 after treating different cells with filtrates of three symbiotic bacteria, namely Staphylococcus epidermidis (abbreviated as SE in the figure), Propionibacterium acnes (abbreviated as C in the figure), and Moraxella osloensis (abbreviated as M in the figure), according to an embodiment of the present invention. Among them, WT: wild cell line, KO: AhR knockout cell line;

[0046] Figure 8 It is a LC-MS / MS detection result graph of differential metabolites of Propionibacterium acnes according to an embodiment of the present invention;

[0047] Figure 9 It shows the expression levels of CYP1A1 and ALDH1A3 after IEt stimulates HaCaT according to an embodiment of the present invention;

[0048] Figure 10 It shows the expression levels of CYP1A1 and ALDH1A3 after ILA stimulates HaCaT according to an embodiment of the present invention;

[0049] Figure 11 It shows the expression levels of CYP1A1 and ALDH1A3 after 5-Me-IAA (abbreviated as 5-MIAA) stimulates HaCaT according to an embodiment of the present invention;

[0050] Figure 12 It shows the expression levels of CYP1A1 and ALDH1A3 after IEt stimulates NHEK according to an embodiment of the present invention;

[0051] Figure 13 It is a gene expression graph related to skin barrier function after IEt treats NHEK cells according to an embodiment of the present invention;

[0052] Figure 14 It is a gene expression graph of wound healing gene SERPINB2 after IEt treats NHEK cells according to an embodiment of the present invention. Detailed implementation manners

[0053] The embodiments of the present invention are described in detail below. The described embodiments are exemplary and are only used to explain the present invention, and should not be construed as a limitation to the present invention. For those without specific technical or conditions noted in the embodiments, the techniques or conditions described in the literature in the field or according to the product specifications are followed. Those reagents or instruments without indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0054] Example 1: New target ALDH1A3 of AhR signaling pathway

[0055] Previously, the inventors successfully constructed an AhR-specific knockout human keratinocyte cell line (HaCaT) using the CRISPR / Cas9 technology, or the immortalized human keratinocyte cell line HaCaT was purchased from the China Center for Type Culture Collection (Wuhan, China). High-affinity ligands benzo[a]pyrene (BaP, 1 μM) or 6-formylindolo[3,2-b]carbazole (FICZ, 100 nM) were added to the wild-type cell line (WT cell line) and the AhR knockout cell line (KO cell line) for stimulation respectively. After 6 hours of cell stimulation, transcriptomic sequencing was performed. The RNA-seq results showed that the expression of ALDH1A3 was significantly downregulated in AhR-knockout cells. This result was verified by WB( Figure 1 ). The experimental results showed that after BaP treatment of the wild-type cell line (AhR + / + , WT cell line), the protein expression level of ALDH1A3 increased significantly; while the expression of ALDH1A3 in the AhR knockout cell line (AhR - / - , KO cell line) did not change significantly. Meanwhile, in the AhR reconstituted cell line (AhR - / - _pAhR), with the increase of AhR expression, the protein expression level of ALDH1A3 also recovered significantly; and after BaP stimulation, the expression of ALDH1A3 protein was significantly increased compared with the wild-type cell line. This indicated that BaP could activate AhR to induce the expression of ALDH1A3 protein.

[0056] After stimulating HaCat cells with BaP (A) and FICZ (B) respectively, the experimental results of the expression level of ALDH1A3 mRNA relative to GAPDH are as Figure 2 and Figure 3 shown: BaP and FICZ could activate AhR and induce the expression of ALDH1A3, and the expression level of ALDH1A3 showed obvious time- and dose-dependence with the increase of time and the doses of BaP and FICZ.

[0057] Example 2: Promote AhR-dependent retinoic acid synthesis through the new target ALDH1A3

[0058] High-affinity ligands benzo[a]pyrene (BaP, 1 μM) or 6-formylindolo[3,2-b]carbazole (FICZ, 100 nM) were added to the wild-type cell line (WT cell line) and the AhR knockout cell line (KO cell line) for stimulation respectively. After 24 hours of stimulation, the substrates of AHLD1A3, retinal (10 μM) or retinol (10 μM), were added and the cells were treated in the dark for 6 hours. The concentration of retinoic acid (RA) in the cells was detected by liquid chromatography-mass spectrometry (LC-MS). The results are as Figure 4As shown, after stimulating cells with BaP and FICZ, it can promote the synthesis of all-trans retinoic acid (atRA) dependent on AhR; the concentration of retinoic acid in the reconstituted cell line (KO-WT) increased significantly.

[0059] Example 3: In Vivo Experiment on Mice

[0060] In the in vitro cell experiments of Example 2 and Example 3, it was found that after stimulating cells with BaP or FICZ, the expression of ALDH1A3 could be upregulated through AhR, and then the synthesis of intracellular atRA was increased. Next, the above signal pathway was further verified on a mouse model. Then, the mice were divided into 4 groups, including 2 groups of WT (AhR + / + ) mice and 2 groups of AhR - / - mice, with 5 - 6 mice in each group. BaP or FICZ was applied to the skin of the 4 groups of mice respectively (where BaP (33.3 mg / kg) was applied to the mouse skin for 7 days, and FICZ (0.01%) was applied to the mouse skin for 7 days) to detect the epidermal thickness of the mice. The experimental results are as Figure 5 shown in A below. BaP can increase the epidermal thickness of WT (AhR + / + ) mice through AhR, and promote the proliferation and differentiation of mouse skin.

[0061] Then, K14-cre conditional knockout mice were used for further verification. The K14-cre conditional knockout mice were divided into 4 groups, including 2 groups of AhR + / + K14-cre and 2 groups of AhR - / - K14-cre. The above operations were repeated to further confirm that BaP can increase the epidermal thickness of mice by activating AhR in keratinocytes. The experimental results are as Figure 5 shown in B below. BaP can increase the epidermal thickness of AhR + / + &K14-cre mice through AhR, and promote the proliferation and differentiation of mouse skin.

[0062] Example 4: Isolation and Collection of Skin Commensal Bacteria Species

[0063] The inventors sampled five healthy volunteers with no obvious acne on their faces and who had not used topical antibiotic drugs recently to isolate Staphylococcus epidermidis (S. epidermidis for short), Cutibacterium acnes (C. acnes for short), and Moraxella osloensis (M. osloensis for short). The volunteers' ages ranged from 23 to 56 years old, including 2 males and 3 females. Samples were collected from the forehead and cheeks using cotton swabs soaked in 0.15 M NaCl solution. Each sampling area was wiped 20 times. Facial swab specimens were sequentially inoculated onto R2A agar (Solarbio, catalog number: LA0700) containing 5% sheep blood (Solarbio, catalog number: TX0030) and tryptone soy agar (TSA) (Solarbio, catalog number: T8650), and placed under aerobic and anaerobic growth conditions. Colonies were selected based on phenotypic diversity and then preliminarily screened using specific primers (Sangon Biotech): M.osloensis-F (5'-GGTGAAAGGGGGCGCAAGC-3') and M.osloensis-R (5'-GTGCTTATTCTGCAGGTAACGTCTAATC-3'), C.acnes-F (5'-GGATAACTTCAGGAAACTGGGGCT-3') and C.acnes-R (5'-TACCCATTACCGTCACTCACGC-3'), S.epidermidis-F (5'-TTTCAAGAAGCTCACTCAGAG-3') and S.epidermidis-R (5'-ATTCCCACGCTAACGATTTACG-3'), and finally characterized by 16S rRNA gene sequencing. M. osloensis was streaked on R2A agar plates and incubated overnight at 30 °C. The next day, individual colonies of this strain were grown overnight in liquid R2A medium at 30 °C with shaking at 220 RPM. The culture from the activated strain was inoculated at 2% into fresh R2A medium (Solarbio, catalog number: LA0700). Further incubation was carried out for 24 hours (OD 600After reaching an OD of 0.8 - 1.0, spin the bacteria and filter the culture supernatant twice using a 0.22 μm Spin-X centrifuge tube filter (Corning). Store the supernatant at -80 °C for the following experiments. Collect the supernatant of Propionibacterium acnes (which has been deposited, deposit number: GDMCC No: 64063) (cultured using reinforced Clostridium medium (Solarbio, product number: LA4360), 37 °C, anaerobic) and Staphylococcus epidermidis in a similar manner. The difference is that their supernatants are collected 72 h and 12 h after inoculation respectively to reach the required OD 600 .

[0064] Example 5: Metabolites of skin commensal bacteria induce upregulation of ALDH1A3 expression in keratinocytes

[0065] Select one strain each of Staphylococcus epidermidis, Propionibacterium acnes, and Moraxella osloensis from Example 4, culture them until the stationary phase, filter out the microorganisms, and then dilute and add the bacterial culture medium to immortalized human keratinocytes (HaCaT, purchased from China Center for Type Culture Collection (Wuhan, China)) and normal primary epidermal keratinocytes (NHEK) (Lifeline Cell Technology, Cat: FC - 0007) cultured in vitro. After co-incubation for 24 hours, detect the transcriptome of the cells. The RNA-seq results are as Figure 6 shown. The results show that Propionibacterium acnes can stably and significantly upregulate the expression of ALDH1A3 in both types of cells, while the effects of the other two commensal bacteria are not significant.

[0066] Example 6: Supernatant of Propionibacterium acnes targets and upregulates the expression of ALDH1A3

[0067] Reagents: DMEM medium (Gibco, US), fetal bovine serum (FBS), antibiotic-antimycotic solution (100×), WT-HaCaT (wild-type HaCaT) cell line, AhR-KO-HaCaT (AhR-knockout HaCaT) cell line, RCM medium, LB medium, R2A medium.

[0068] Experimental method: Collect the supernatants of the three bacteria, filter them twice with a 0.22 μm filter head, and treat WT-HaCaT and AhR-KO-HaCaT cells with the supernatant of the bacteria at a concentration with a volume ratio of supernatant of bacteria to cell culture medium of 1:25. After 24 hours, collect the cells and perform QPCR to detect the expression of CYP1A1 (CYP1A1 is a target gene of AhR, and upregulation of CYP1A1 expression indicates activation of AhR) and ALDH1A3. All cells are cultured at 37 °C and 5% CO2.

[0069] The experimental results are as follows Figure 7 shown. The supernatant of C.acnes can specifically activate AhR in keratinocytes, targetedly upregulate the expression of ALDH1A3, that is, induce the expression of ALDH1A3 through AhR, while the supernatants of S.epidermidis and M.osloensis cannot induce the expression of ALDH1A3.

[0070] Example 7: Differential metabolites of Propionibacterium acnes

[0071] Experimental protocol: Collect the supernatant of Propionibacterium acnes, filter it twice with a 0.22 μm filter head, quickly freeze it in liquid nitrogen, store it at -80 °C, and detect the differential metabolites of Propionibacterium acnes by LC-MS / MS; quantify 31 tryptophan metabolites based on LC-MS / MS.

[0072] The experimental results are as follows Figure 8 shown. The differential metabolites of Propionibacterium acnes are: Indole-3-lactic acid (DL-indole-3-lactic acid, abbreviated as ILA), 5-Methoxyindoleacetic acid (5-methoxyindole-3-acetic acid, abbreviated as 5-Me-IAA or 5-MIAA), Tryptophol (3-indoleethanol, abbreviated as IEt).

[0073] Example 8: Targeted upregulation of the expression of ALDH1A3 by differential metabolites of Propionibacterium acnes

[0074] 1. Activate AhR in keratinocytes

[0075] Reagents: DMEM medium, Fetal Bovine Serum (FBS), antibiotic-antimycotic solution (100×), WT-HaCaT cell line, AhR-KO HaCaT cell line, DMSO, IEt / ILA / 5-Me-IAA.

[0076] Experimental method: Treat WT-HaCaT and AhR-KO HaCaT cells with different concentrations of IET / ILA / 5-Me-IAA, collect the cells after 24 hours, and detect the expression of CYP1A1 (CYP1A1 is a target gene of AhR, and the upregulation of CYP1A1 expression indicates that AhR is activated) and ALDH1A3 by QPCR. All cells are cultured at 37 °C and 5% CO2.

[0077] The experimental results are as follows Figure 9 、 Figure 10 or Figure 11Shown: IEt can activate AhR in keratinocytes, targetedly upregulate the expression of ALDH1A3, that is, induce the expression of ALDH1A3 through AhR. However, ILA / 5-Me-IAA cannot activate the expression of AhR and ALDH1A3.

[0078] 2. Activation of AhR in primary human keratinocytes

[0079] Reagents: DermaLife primary human keratinocyte medium (Lifeline Cell Technology, Cat: LL-0007), normal primary human keratinocytes (NHEK), DMSO, IEt.

[0080] Experimental method: Treat NHEK cells with different concentrations of IEt, harvest the cells after 24 hours, and detect the expression of CYP1A1 and ALDH1A3 by QPCR. All cells were cultured at 37°C and 5% CO2.

[0081] The experimental results are as Figure 12 Shown: IEt activates AhR in primary human keratinocytes, targetedly upregulates the expression of ALDH1A3, that is, induces the expression of AhR and ALDH1A3 in primary human keratinocytes.

[0082] Example 9: Regulation of the expression of skin barrier function genes by C. acnes metabolites

[0083] Treat NHEK cells with the metabolite IEt (500 μM) of C. acnes for 24 h. The RNAseq results are as Figure 13 Shown: Activation of AHR by IEt can regulate the expression of genes related to skin barrier function: among them, CLDN1, KRT16, KRT75, GRHL3, DSC1, TGM1, OVOL1, COL6A3 are upregulated, and KRT4 and KLK6 are downregulated.

[0084] Example 10: Promotion of the expression of the wound healing gene SERPINB2 by C. acnes metabolites

[0085] Treat NHEK cells with the metabolite IEt (500 μM) of C. acnes for 24 h. The RNAseq results are as Figure 14 Shown: Activation of AHR by IEt can increase the expression of the skin wound healing gene SERPINB2.

[0086] In the description of this specification, the descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0087] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of Propionibacterium acnes and / or Propionibacterium acnes metabolites in the preparation of a product for at least one of the following: Activating the AhR signaling pathway; Promoting the expression of ALDH1A3 or enhancing the activity of ALDH1A3; Promoting retinoic acid synthesis; Regulating the transcription of skin barrier function genes; Treating or preventing pathogenic or skin infections; Promoting wound healing.

2. Use of a reagent in the preparation of a product for regulating the production of Propionibacterium acnes metabolites, the product for at least one of the following: Regulating the AhR signaling pathway; Regulating the expression of ALDH1A3 or the activity of ALDH1A3; Regulating retinoic acid synthesis; Regulating the transcription of skin barrier function genes; Promoting wound healing.

3. The use according to claim 2, wherein the reagent is for promoting the production of Propionibacterium acnes metabolites, and the product is for at least one of the following: Activating the AhR signaling pathway; Promoting the expression of ALDH1A3 or enhancing the activity of ALDH1A3; Promoting retinoic acid synthesis; Promoting the transcription of skin barrier function genes; Promoting wound healing.

4. The use according to claim 1 or 2, characterized in that, The Propionibacterium acnes metabolites are selected from at least one of 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid or 3-indoleethanol; Optionally, the product is selected from drugs, skin care products or health products; Optionally, the skin barrier function genes are selected from at least one of CLDN1, KRT16, KRT4, KRT75, GRHL3, KLK6, DSC1, TGM1, OVOL1 and COL6A3.

5. A method for activating the AhR signaling pathway in cells, characterized in that, Comprising co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells.

6. A method for up-regulating the expression of ALDH1A3 protein in cells, characterized in that, Comprising co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells.

7. A method for promoting the production of retinoic acid in cells, characterized in that, Comprising co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells.

8. A method for promoting gene expression in cells, wherein the gene is related to skin wound healing, characterized in that, Comprising co-culturing Propionibacterium acnes and / or Propionibacterium acnes metabolites with the cells, and the gene is SERPINB2.

9. The method according to any one of claims 5 to 8, characterized in that The Propionibacterium acnes metabolites are selected from at least one of 5-methoxyindole-3-acetic acid, DL-indole-3-lactic acid or 3-indoleethanol; Optionally, the co-culture time is 20-26 h.

10. A Propionibacterium acnes, deposited with the Guangdong Provincial Microbial Culture Collection Center on November 24, 2023, with the deposit number GDMCC No: 64063.

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