Application of autoinducible factor 2 in preparation of acne drugs

Abstract

The invention provides application of an autoinducible factor 2 in preparation of a product for preventing or treating acne. Experiments show that the expression quantity of the autoinducible factor 2 in diseased skin tissues and sebum secretions of acne patients is obviously higher than that of healthy people; in an acne cell model, the autoinducible factor 2 can significantly up-regulate expression of inflammatory factors such as IL-1, IL-6, TNF-alpha and the like by activating an NF-kappa B pathway, and meanwhile, the secretion amount of sebum in sebaceous gland cells is promoted. Therefore, the autoinducible factor 2 serves as a new target for acne treatment, related inhibition products of the autoinducible factor 2 have definite prevention and treatment effects on acne, the autoinducible factor 2 has huge potential of being developed into corresponding drugs, medical instruments or skin care products, and meanwhile a new technical direction is provided for solving the problems of drug resistance, side effects and the like of existing acne treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of self-inducing factor 2 in the preparation of acne drugs. Background Technology

[0002] Acne is a common chronic inflammatory disease of the pilosebaceous unit, mainly manifested as comedones, papules, pustules, cysts, nodules, and scars on the face, chest, and back. It is often accompanied by seborrhea and is prevalent in adolescents, affecting their appearance and mental health. Its incidence rate is extremely high among teenagers, approximately 85%. Its core pathogenesis involves four key aspects: abnormal follicular keratinization, excessive sebum secretion, proliferation of Propionibacterium acnes, and the resulting inflammatory response. The colonization of Propionibacterium acnes plays a crucial role; it breaks down triglycerides in sebum, producing free fatty acids that stimulate the hair follicles and surrounding tissues, triggering an inflammatory response. Furthermore, it activates the body's immune response, further aggravating redness, swelling, pustules, and other inflammatory manifestations. This is a key step in the formation of inflammatory acne lesions.

[0003] Acne presents with diverse clinical manifestations. Early stages often involve whiteheads and blackheads, which can develop into inflammatory papules, manifesting as red papules and pustules. In more severe cases, nodules may appear, which are hard in texture and accompanied by pain. Cysts containing pus and blood may also form, easily leaving scars after rupture, including atrophic and hypertrophic scars. It commonly occurs on the face, chest, back, and other areas with high sebum secretion, often symmetrically distributed. Recurrent flare-ups can significantly impact the quality of life for some patients.

[0004] Currently, acne treatment follows the principles of individualization and stratification, mainly including drug therapy, physical therapy, and photodynamic therapy. Drug therapy includes topical and oral medications. Topical retinoids are commonly used to improve follicular keratosis and reduce comedones; benzoyl peroxide can kill Propionibacterium acnes and reduce inflammation; antibiotic ointments can control local inflammation. Oral medications include isotretinoin, which reduces sebum secretion and improves keratinization; and oral antibiotics, which inhibit Propionibacterium acnes and inflammation. In addition, physical therapy can be combined, such as phototherapy and chemical peels. Photodynamic therapy uses a combination of photosensitizers and specific light to destroy Propionibacterium acnes and reduce sebum secretion.

[0005] Antibiotic resistance in acne is currently a challenge, with major issues including antibiotic resistance, side effects, and limited treatment efficacy. In particular, it is not effective in regulating both inflammation and sebum secretion, necessitating the development of new therapeutic targets and strategies. Summary of the Invention

[0006] To address the problems of insufficient inflammation regulation, limited sebum secretion inhibition, and easy development of drug resistance in existing acne treatments, this invention provides the application of self-inducing factor 2 in the preparation of acne products.

[0007] The present invention adopts the following technical solution: Application of self-inducing factor 2 in the preparation of products for the prevention or treatment of acne.

[0008] The Autoinducer-2 (AI-2) described in this invention is a bacterial quorum sensing signaling molecule that participates in the activation of inflammatory responses and the regulation of sebum secretion during the pathogenesis of acne.

[0009] According to one embodiment of the invention, the application is to administer an effective amount of a competitive inhibitor of autoinducible factor 2 to a patient requiring treatment for acne. Preferably, the competitive inhibitor of autoinducible factor 2 is D-ribose.

[0010] According to one embodiment of the present invention, the application is to reduce the inflammatory response of acne.

[0011] According to one embodiment of the present invention, the application is to reduce the amount of sebum secreted by sebaceous gland cells.

[0012] According to one embodiment of the present invention, the application is the activation of inflammatory pathways in acne sebaceous gland cells.

[0013] According to one embodiment of the present invention, the product is a pharmaceutical product or a skin care product.

[0014] According to one embodiment of the present invention, the effective dose of the self-inducing factor 2 inhibition-related reagent is a dose that can reduce the activity of self-inducing factor 2 at the lesion site by more than 30% or downregulate its expression by more than 25%.

[0015] In this invention, the reagent used to suppress self-inducing factor 2 has a purity of ≥95.0% by weight.

[0016] The term "treatment" as used in this article includes slowing down, stopping or reversing the progression of symptoms such as inflammatory papules, pustules, and nodules in acne patients, reducing excessive sebum secretion, and improving abnormal skin keratinization.

[0017] The term "patient" used in this article refers to a person.

[0018] The term "effective dose" as used in this article refers to the quantity or dosage of an agent that inhibits autoinducible factor 2, which, when administered to a patient in single or multiple doses, provides the expected anti-inflammatory and oil-controlling effects without producing significant toxic side effects. Beneficial effects

[0019] This invention provides the application of autoinducible factor 2 (AGF2) in the preparation of products for the prevention or treatment of acne. Experiments showed that the expression level of AGF2 in the lesioned skin tissue and sebum secretions of acne patients was significantly higher than that in healthy individuals. In an acne cell model, AGF2 significantly upregulated the expression of inflammatory factors such as IL-1, IL-6, and TNF-α by activating the NF-κB pathway, while simultaneously promoting sebum secretion in sebaceous gland cells. Therefore, AGF2, as a novel target for acne treatment, has clear preventive and therapeutic effects on acne, and its related inhibitory products have great potential for development into corresponding drugs, medical devices, or skincare products. It also provides a new technological direction for solving the problems of drug resistance and side effects in existing acne treatments. Attached Figure Description

[0020] Figure 1 This is a comparison chart of the AI-2 content in the sebum secretions of acne patients' skin lesions and the contents of hair follicles in normal individuals.

[0021] Figure 2 This is a graph showing the changes in cell activity after AI-2 treatment of sebaceous gland cells.

[0022] Figure 3 This is a graph showing the changes in sebum secretion after AI-2 treatment of sebaceous gland cells.

[0023] Figure 4 This is a graph showing the changes in the expression of inflammatory factors IL-1, IL-6, and TNF-α after AI-2 treatment of sebaceous gland cells.

[0024] Figure 5 This is a graph showing the changes in p-p65 and p65 expression levels after AI-2 treatment of sebaceous gland cells. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Unless otherwise specified, any range described in the present invention includes the endpoint value, any value between the endpoint values, and any sub-range formed by the endpoint value or any value between the endpoint values. Unless otherwise specified, all percentages mentioned in the present invention are mass percentages. Example 1

[0026] Acne patients were selected as the study subjects. All study subjects were outpatients at the Third Affiliated Hospital of Chongqing Medical University. According to statistical calculation requirements, at least 40 patients clinically diagnosed with grade III-IV acne vulgaris and healthy individuals with no prior history of acne, no family history of acne, and who passed a recent physical examination were included. All participants signed informed consent forms.

[0027] GCF Sample Collection: Five ruptured pustules were selected from each patient (for healthy individuals, comedones or blackheads were selected). The sampler, wearing sterile gloves, used an iodine swab from a sterile sterilization kit to disinfect the skin in a spiral motion from the inside out, centered on the marked lesion, covering an area approximately 2-3 cm in diameter. After waiting 30 seconds, the iodine was removed using a 75% alcohol swab in the same manner. A sterile comedone needle was used to apply pressure to the skin surrounding the lesion along the hair follicle direction, causing the keratin plug and secretions within the affected hair follicle to drain from the follicle opening. The secretions were collected using a disposable sterile sampling swab and stored in an EP tube.

[0028] Place the swab tip downwards into the corresponding sterile centrifuge tube, cut the swab rod at the tube opening, and immediately tighten the tube cap. Weigh the swab in an EP tube, converting the mass of the hair follicle contents to volume at a rate of 1 g / ml, and record the value. Elute the sample with AB medium, vortex for 15 min, and then centrifuge at 4°C and 13000 rpm for 10 min. Filter the collected supernatant through a 0.22 μm sterile filter and transfer it to a new EP tube, storing it at -80°C until use.

[0029] The expression level of AI-2 in the samples was detected by the V. harvestyi BB170 bioluminescence assay.

[0030] Fresh BB170 culture cultured overnight (OD600nm = 0.7-1.2) was diluted 1:5000. 180 μL of the diluted BB170 culture was then mixed with 20 μL of the treated supernatant and incubated at 30°C and 120 rpm. Chemiluminescence intensity was measured every half hour using a multi-mode microplate reader for 5 hours. AB medium served as a negative control; the lowest value measured in the negative control was taken as the experimental result. Each sample was tested in triplicate. OD values ​​in the microplates were measured using a multi-mode microplate reader. Statistical analysis revealed a significant difference in AI-2 levels between the contents of acne lesions and the contents of hair follicles from healthy individuals.

[0031] Sebaceous gland cell culture: Primary human sebaceous gland cells frozen in liquid nitrogen were rapidly thawed in a 37°C water bath. The thawed cell suspension was transferred to a 10 mL centrifuge tube containing 5 mL of DMEM complete medium (containing 1% penicillin and 10% fetal bovine serum), gently pipetted several times, and centrifuged at 1200 rpm for 5 min. The centrifuge tube was gently removed, the supernatant was discarded, 4 mL of DMEM complete medium was added, and the cells were gently pipetted several times. The cell suspension was then transferred along the wall of the tube to a culture flask containing an appropriate amount of medium. The cells were evenly distributed at the bottom of the flask using a figure-eight motion. Cell growth was observed, and the medium was changed every 2 days. Once the cell density reached a certain level, the cells were passaged. One flask of well-grown human sebaceous gland cells was selected, the medium containing cell debris was discarded, and the cells were washed twice with PBS to remove residual serum. Add 1 mL of 0.25% trypsin solution to completely submerge the cells. Incubate at 37°C for 1-2 minutes, or until the cells become rounded as observed under a microscope. Stop digestion. Add an appropriate amount of DMEM complete medium along the cell surface, gently tap the culture flask a few times, and then pipette to disperse the cells. Centrifuge to remove trypsin, passage at a 1:3 ratio, and incubate at 37°C with 5% CO2. Replace the medium after a monolayer has formed for further experiments.

[0032] CCK8: The safe concentration range of AI-2 was determined using the CCK8 method. Healthy human sebaceous gland cells were collected, treated with trypsin, centrifuged, and the cell suspension concentration was adjusted to 6000-8000 cells / mL with complete culture medium. 200 µL was added to each well of a 96-well plate. After cell attachment, the cells were washed twice with PBS. Control groups (basal DMEM medium + human sebaceous gland cells) and drug groups (basal DMEM medium containing different concentrations of AI-2 + human sebaceous gland cells) were set up, with three replicates for each group. After drug administration, the cells were incubated at 37°C for 12 h. Subsequently, 20 µL of MTT solution (5 mg / mL, i.e., 0.5% MTT) was added to each well under light-protected conditions, and the cells were cultured for another 4 h. Then, the culture medium was removed, and 150 µL of dimethyl sulfoxide was added to each well. The plates were shaken slowly for 10 min to fully dissolve the formazan crystals within the cells. The absorbance of each well was measured at 490 nm using a microplate reader to calculate cell viability. Compared with other groups, treatment with 50 μmol / L AI-2 significantly reduced the survival rate of human sebaceous gland cells. Treatment with 2.5, 5, and 10 μmol / L AI-2 showed no statistically significant difference in the survival rate of human sebaceous gland cells. Based on these results, the optimal low, medium, and high concentrations of AI-2 for subsequent treatments were determined to be 2.5, 5, and 10 μmol / L.

[0033] Cell viability = (OD value of drug group - OD value of blank group) / (OD value of model group - OD value of blank group) × 100%.

[0034] Oil Red O staining: Human sebaceous gland cells were washed with phosphate-buffered saline (PBS) and fixed in 10% formaldehyde for 10 min. The fixed cells were then stained with filtered 0.7% Oil Red O solution (Sigma, St. Louis, MO, USA) in propylene glycol for 30 min. The stained cells were washed with distilled water, reverse-stained with hematoxylin, and observed under a microscope. As the AI-2 concentration increased, the sebum secretion from sebaceous gland cells gradually increased.

[0035] ELISA: Select healthy human sebaceous gland cells, wash with PBS, digest with trypsin, and adjust the cell concentration to 6000-8000 cells / mL. Spread the cells evenly in 96-well plates, adding 200 µL of cell suspension to each well, and incubate at 37°C. After cell attachment, treat human sebaceous gland cells with different concentrations of AI-2 and continue incubation at 37°C for 24 h. After incubation, collect the supernatant and use an enzyme-linked immunosorbent assay (ELISA) kit to detect the levels of IL-1, IL-6, and TNF-α in the supernatant.

[0036] The results showed that as the concentration of AI-2 increased, the levels of IL-1, IL-6, and TNF-α gradually increased.

[0037] Wb: Collect cultured human sebaceous gland cells from the culture dish, wash twice with PBS buffer to remove the culture medium, and add an appropriate amount of RIPA lysis buffer and protease inhibitor (PMSF) mixture at a ratio of 1000:1. Incubate on ice for 30 minutes, with intermittent shaking to mix. Homogenize the cell lysate to ensure complete cell lysis. Then centrifuge at 12,000 g for 15 minutes at 4°C and collect the supernatant. Next, heat at 95°C for 5–10 minutes to completely denature the proteins. The denatured proteins can be stored long-term at -80°C.

[0038] Prepare 10 ml of 10% gel using the Beyotime SDS-PAGE gel preparation kit. First, prepare the lower gel by adding 2.7 ml distilled water, 3.3 ml 30% Acr-Bis, 3.8 ml Tris, 0.1 ml 10% SDS, 0.1 ml 10% gel polymerase catalyst, and 0.004 ml TEMED to a centrifuge tube. After adding the lower gel to the electrophoresis apparatus, add distilled water to smooth the prepared gel. Let it stand for 20 minutes to solidify. Then, prepare the upper gel (2.7 ml distilled water + 0.67 ml 30% Acr-Bis + 0.5 ml Tris + 0.04 ml 10% SDS + 0.04 ml 10% gel polymerase catalyst + 0.004 ml TEMED). Add the upper gel to the electrophoresis plate and quickly insert the sample comb. Let it stand for 10 minutes to solidify.

[0039] Prepare 10X electrophoresis buffer (Tris Base 30.3 g + Glycine 144 g + SDS 10 g + 1 L distilled water). To use, add 9 times the amount of distilled water to prepare 1X electrophoresis buffer. Place the electrophoresis glass plate in the electrophoresis tank, add 10-20 µL of sample protein, set the voltage to 80 V, and allow the protein to slowly separate in the separating gel for 30 minutes. Then, set the voltage to 110 V to completely separate the target protein.

[0040] Prepare a 1X transfer buffer (Tris Base 3.03 g + Glycine 14.4 g + SDS 1 g + methanol 200 ml), and finally add distilled water to make the transfer buffer volume 1 L. Cut the PVDF membrane into gel-sized pieces and let it rest in methanol for 2 minutes. Use clamps to attach the PVDF membrane to the gel and place it in the transfer buffer for transfer. Set the voltage to 200 mA. After 2 hours of transfer, gently remove the PVDF membrane.

[0041] Prepare a 5% skim milk blocking buffer (5 g skim milk powder + 1X TBST). Immerse the PVDF membrane in the blocking buffer and block on a shaker at room temperature for 1 hour. Remove the blocked PVDF membrane, immerse it in 1X TBST, and gently wash off the blocking buffer three times on a shaker for 10 minutes each time. Prepare the primary antibody for the desired protein using TBST buffer and incubate overnight at 4°C. The next day, remove the PVDF membrane and wash it three times with TBST buffer for 10 minutes each time. Prepare the secondary antibody for the desired protein, incubate overnight at 4°C, remove the PVDF membrane, and wash it again three times with TBST buffer for 10 minutes each time. Visualize the protein bands using an Odyssey infrared imaging system and perform grayscale analysis of the bands using an ImageJ system.

[0042] The results showed that as the concentration of AI-2 increased, the expression level of p-p65 gradually increased, and this increase could be reversed by D-ribose, a competitive inhibitor of AI-2.

Claims

1. Application of self-inducing factor 2 in the preparation of products for the prevention or treatment of acne.

2. The application as described in claim 1, characterized in that, The application involves administering an effective amount of a competitive inhibitor of autoinducing factor 2 to patients who require acne treatment.

3. The application as described in claim 2, characterized in that, The competitive inhibitor of the self-inducing factor 2 is D-ribose.

4. The application as described in any one of claims 1-3, characterized in that, The application is to reduce the inflammatory response of acne.

5. The application as described in any one of claims 1-3, characterized in that, The application is to reduce the amount of sebum secreted by sebaceous gland cells.

6. The application as described in any one of claims 1-3, characterized in that, The application activates the inflammatory pathways of acne sebaceous gland cells.

7. The application as described in any one of claims 1-3, characterized in that, The product in question is a pharmaceutical or skincare product.

8. The application as described in claim 7, characterized in that, The effective dose of the self-inducing factor 2 inhibition reagent is a dose that can reduce the activity of self-inducing factor 2 at the lesion site by more than 30% or downregulate its expression by more than 25%.

9. The application as described in claim 8, characterized in that, The reagent used to inhibit self-inducing factor 2 has a purity of ≥95.0% by weight.

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