Application of oleic acid in medicine for treating atopic dermatitis

Abstract

The invention provides application of oleic acid in a medicine for treating atopic dermatitis and relates to the technical field of biomedicine, oleic acid is common monounsaturated fatty acid, it is found for the first time through a non-targeted metabonomics technology that after peripheral sensory neurons are stimulated by an itching and inflammatory factor IL-31, the level of oleic acid (OA) in cells is remarkably increased (the VIP score gt; according to the present invention, the specific verification test results show that the oleic acid can significantly improve the symptoms of the pruritus model animal and regulate the related immune response under the specific concentration, and the new low-cost candidate scheme is provided for the treatment of the specific dermatitis;

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the application of oleic acid in drugs for the treatment of atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a common chronic inflammatory skin disease characterized by dry skin, itching, erythema, and exudation, severely impacting patients' quality of life. Its pathogenesis is closely related to immune dysregulation, impaired skin barrier function, and abnormal secretion of inflammatory factors. Current clinical treatments mainly include corticosteroids, immunosuppressants, and antihistamines, but these have significant side effects, decreased efficacy with long-term use, and a high relapse rate. For example, long-term use of corticosteroids may lead to adverse reactions such as skin atrophy and pigmentation, while immunosuppressants may affect the body's normal immune function.

[0003] Therefore, there is an important clinical need to develop new therapeutic drugs and targets that are safe, effective, and have few side effects. Summary of the Invention

[0004] The purpose of this application is to address the technical problems of existing drugs for atopic dermatitis, such as significant side effects and decreased efficacy with long-term use.

[0005] The use of oleic acid in the preparation of products for the treatment and / or prevention of itchy and inflammatory skin diseases.

[0006] Preferably, the product is any one of a pharmaceutical composition, a health product, or a functional food.

[0007] Preferably, the pruritus and inflammatory skin conditions are associated with activation of the IL-31 signaling pathway.

[0008] Preferably, the product is administered orally or topically.

[0009] Preferably, the product is administered orally via drinking water.

[0010] Preferably, the drug also includes other medically acceptable adjuvants.

[0011] This application also provides a product for treating or preventing pruritus and inflammatory skin diseases, the product comprising oleic acid.

[0012] This application also provides a method for verifying the use of oleic acid in the treatment or prevention of pruritus and inflammatory skin diseases, including in vivo efficacy verification and in vitro mechanism verification.

[0013] Preferably, the in vivo efficacy verification is performed by adding a certain concentration of oleic acid to the drinking water in a mouse atopic dermatitis pruritus model induced by MC903, and then evaluating the indicators of the mice.

[0014] Preferably, the in vitro mechanism verification is performed by treating macrophage cell line (RAW264.7) with oleic acid at a certain concentration range, and detecting LPS-induced inflammatory response without affecting cell viability.

[0015] Compared with the prior art, this application has at least the following beneficial effects:

[0016] 1. This application demonstrates through verification experiments that oral administration of low-dose oleic acid can significantly improve the appearance of skin inflammation and the core symptom of itching (scratching behavior) in an animal model of atopic dermatitis, with remarkable effects.

[0017] 2. This application is the first to link changes in oleic acid levels with the signaling of the pruritus-inducing and inflammatory factor IL-31, and confirms the anti-inflammatory and antipruritic effects of oleic acid, providing direct evidence for targeted fatty acid metabolism therapy for pruritus.

[0018] 3. Since oleic acid is a naturally occurring fatty acid, it is well tolerated by the human body, making it highly safe as a pharmaceutical or health supplement ingredient. Furthermore, the raw materials are readily available and the production cost is low. Attached Figure Description

[0019] Figure 1 This is the molecular structure of oleic acid.

[0020] Figure 2 The flowchart for non-targeted metabolomics analysis and the significant increase in oleic acid (OA) and palmitic acid (PA) induced by IL-31 stimulation of sensory neurons are shown. (A) Experimental flowchart: Cell processing → Metabolite extraction → LC-MS analysis → Data processing. (B) Principal component analysis (PCA) plot, showing good intra-group reproducibility. (C) VIP score table of differentially expressed metabolites, highlighting OA and PA.

[0021] Figure 3 To improve the skin inflammation phenotype in MC903 model mice with oral oleic acid (OA). Representative facial skin photographs of MC903 model mice in the Vehicle and OA groups on days 4, 6, and 8 were compared, showing that the skin inflammation was reduced in the OA group.

[0022] Figure 4 Oral administration of oleic acid (OA) can alleviate pruritus behavior in MC903 model mice. (A) A quantitative statistical graph of spontaneous scratching behavior in MC903 mice during the period when OA was added to their drinking water, showing a reduction in scratching frequency in the OA group. (B) A curve showing the change in mouse body weight during the experiment.

[0023] Figure 5Oral administration of oleic acid (OA) can regulate the expression of skin inflammatory factors in MC903 model mice. A bar chart showing the relative mRNA expression levels of IL-6, TNF-α, and CD206 in skin tissue of MC903 mice after 10 days of drinking water supplemented with OA (50 μM) (compared to the Vehicle group, *P < 0.05, **P < 0.01).

[0024] Figure 6 The effects of oleic acid (OA) on macrophage viability and concentration selection are shown in Figure (A). (A) Cell viability assays of RAW264.7 cells treated with different concentrations of OA for different time periods. (B) Results of the effects of OA (20 μM) and LPS (200 ng / ml) on the inflammatory response of RAW264.7 cells. Detailed Implementation

[0025] This application discloses the use of oleic acid (OA) in the preparation of products for the treatment and / or prevention of pruritus and inflammatory skin diseases, wherein the chemical structural formula of the intracellular oleic acid is as follows: Figure 1 As shown. The product is any one of a pharmaceutical composition, health supplement, or functional food. The itching and inflammatory skin conditions are associated with activation of the IL-31 signaling pathway.

[0026] In one embodiment, the product is administered orally or topically. Preferably, the product is administered orally via drinking water.

[0027] In one embodiment, the drug also includes other medically acceptable adjuvants.

[0028] In addition, this application also provides a product for treating or preventing pruritus and inflammatory skin diseases, the product comprising oleic acid.

[0029] The above content will be elaborated below with specific verification experiments:

[0030] I. Experimental Materials and Sources

[0031] Serial Number Experimental materials source 1 C57 mice Nantong University Animal Center 2 Calcipotriol (MC903) Sigma 3 Oleic acid Sigma 4 Von Frey Filament Stoeling 5 physiological saline Azure Sky 6 Trizol White Shark 7 Isopropanol Xilong Science 8 Anhydrous ethanol Xilong Science 9 Evo M-MLV Reverse Transcription Mixture Kit Aikerui Biotechnology 10 SYBR Green Premix Pro Taq HS Reagent Kit Aikerui Biotechnology 11 RNAase-Free EP Tubes Corning, AxygenR 12 IL-6 primers Sangon Biotech (Shanghai) Co., Ltd. 13 TNF-α primers Sangon Biotech (Shanghai) Co., Ltd. 14 CD206 primers Sangon Biotech (Shanghai) Co., Ltd. 15 PBS buffer Xavier Bio 16 CCK-8 reagent kit Azure Sky

[0032] II. Implementation Examples

[0033] Example 1: Non-targeted metabolomics reveals that IL-31 causes elevated oleic acid levels.

[0034] Please see Figure 2To investigate the effects of pruritogenic and inflammatory factors on the metabolism of peripheral sensory neurons, the ND7-23 sensory neuron cell line was used and treated with vehicle and IL-31, respectively. Cell lysates were analyzed by LC-MS non-targeted metabolomics. Principal component analysis (PCA) showed good clustering and high reproducibility of the quality control samples. Differentially metabolized substances were screened using variable importance projection (VIP) analysis. Results are as follows: Figure 2 As shown, compared with the Vehicle group, the level of oleic acid (VIP=28.55) was increased in the IL-31 treatment group, suggesting that free fatty acid metabolic reprogramming is an important downstream event of IL-31 signaling.

[0035] Example 2: Therapeutic effect of oral oleic acid on MC903-induced atopic dermatitis model mice

[0036] A chronic pruritus / atopic dermatitis model was established in mice by repeated application of MC903 (calcipotriol) to the skin of the ears or face and neck. Mice were randomly divided into a vehicle group and a treatment group (OA). From the start of the modeling process, mice in the treatment group received continuous supplementation of 50 μM oleic acid in their drinking water. Observations and records were recorded.

[0037] Skin lesions ( Figure 3 Compared with the Vehicle group, the OA group mice showed significantly reduced inflammatory symptoms such as redness and thickening of the facial skin on days 4, 6, and 8 after modeling.

[0038] Scratching behavior ( Figure 4 A): The number of spontaneous scratching incidents in mice was analyzed through video recordings. The results showed that the number of scratching incidents in the OA group was significantly lower than that in the Vehicle group, indicating that the itching was relieved.

[0039] Weight monitoring ( Figure 4 B): There was no significant difference in body weight between the OA group and the Vehicle group, indicating that the oral administration of this dose was safe.

[0040] Skin cytokine detection ( Figure 5 On day 10 of modeling, diseased skin tissue was collected and detected by qPCR or ELISA. The results showed that the mRNA or protein levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the skin of the OA group were significantly lower than those in the Vehicle group, while the expression of CD206, a marker of M2 macrophages (anti-inflammatory), showed regulatory changes.

[0041] Example 3: Effects of oleic acid on macrophages. Primary macrophages were extracted from the peritoneal cavity of mice or cultured using the RAW264.7 macrophage cell line.

[0042] RAW264.7 cells were incubated with different concentrations (0, 20, 100 μM) of OA for 6 h, 12 h, and 24 h; cell viability was detected by CCK-8 assay.

[0043] The results are as follows Figure 6 As shown in Figure A, 20 μM OA had no significant toxic effect on cell viability after 24 h of treatment, so this concentration was chosen for subsequent experiments.

[0044] Please see Figure 6 B. RAW264.7 macrophages were stimulated with 20 μM OA and / or 200 ng / ml LPS for 24 h. Cell supernatant and lysate were collected, and the expression of relevant inflammatory factors was detected.

[0045] The results showed that OA pretreatment or co-treatment could alleviate LPS-induced macrophage inflammatory response. After OA treatment, the skin condition, scratching behavior, and inflammatory factor levels of atopic dermatitis were significantly improved compared to the model group (Vehicle), demonstrating the therapeutic effect of OA.

[0046] III. Experimental Procedure:

[0047] (a) Changes in metabolite expression after IL-31 stimulation of peripheral sensory neurons using non-targeted metabolomics analysis

[0048] Untargeted metabolomics using LC-MS was employed to detect metabolites in the IL-31-stimulated peripheral neuronal cell line ND7-23. Differential metabolites were screened using variable importance for the projection (VIP) values ​​obtained from a partial least squares discriminant analysis (PLS-DA) model. Higher VIP values ​​indicated greater importance, with oleic acid and palmitic acid ranking among the top three.

[0049] (II) Validation of the MC903-induced atopic dermatitis model

[0050] 2.1 Preparation of Atopic Dermatitis Model Mice

[0051] Calcipotriol (MC903) is a synthetic vitamin D3 analog with high affinity for vitamin D receptors. In this application, MC903 was used to induce a mouse model of facial atopic dermatitis.

[0052] C57 mice (female) were purchased from the Experimental Animal Center of Nantong University. They were 6-8 weeks old and housed in an SPF environment with a 12-hour light / dark cycle, maintained at a temperature of 22 ± 2℃, and had free access to food and water. All experimental procedures were approved by the Animal Ethics Committee of Nantong University (IACUC20211215-1001) and strictly followed the "Regulations on the Management of Laboratory Animals" and the 3R principle of laboratory animals.

[0053] The facial model was established by removing hair from the left side of the mouse face (1 cm × 1 cm), followed by application of 200 μM MC903 after acclimatization. Atopic dermatitis was successfully induced by repeated application of MC903 for 10 consecutive days.

[0054] 2.2 Behavioral testing of mice

[0055] Three days prior to the behavioral test, mice were allowed to acclimatize, with the same experimental personnel and environment maintained, and a double-blind method was used for the test.

[0056] For itch behavior testing, mice were placed in a transparent nine-square grid on a metal frame and allowed to acclimatize for 30 minutes, followed by 1 hour of video recording. The number of scratches was counted as one scratch: the mouse scratched its face with its hind paws, then licked, bit its paws, or placed back on the base; this cycle was counted as one scratch. If the mouse rubbed its face with its front paws, this was not counted as itch behavior. Behavioral test results are as follows: Figure 4 It can be clearly seen that compared with the OA groups using different doses, the OA group had a shorter scratching time and improved body weight.

[0057] (III) Detection of skin inflammatory factor expression

[0058] 3.1 RNA extraction, reverse transcription, and Real-time PCR process from animal tissue samples:

[0059] 3.1.1 RNA Extraction

[0060] (1) The DRG obtained by perfusion with physiological saline was placed into a 1.5 mL RNAase FreeEP tube containing 100 μL Trizol, placed on ice, and homogenized once for 60 seconds using a micro homogenizer for a total of 6 times. After homogenization, 900 μL Trizol was added and the tube was placed on ice for 5 min.

[0061] (2) Add 200 μL of chloroform to each tube, shake vigorously for 15 s, and let stand for 2 min;

[0062] (3) Centrifuge at 4℃ and 12000 rpm for 15 min;

[0063] (4) Aspirate the supernatant into a 1.5 mL RNAase-Free EP tube, add an equal volume of isopropanol, mix gently until no visible filaments remain, and let stand for 10 min.

[0064] (5) Centrifuge at 4℃ and 12000 rpm for 15 min;

[0065] (6) Discard the supernatant, add 1 mL of anhydrous ethanol and mix lightly, then centrifuge at 4°C and 12000 rpm for 15 min.

[0066] (7) Discard the supernatant, invert it onto filter paper and air dry at room temperature until the precipitate is translucent;

[0067] (8) Add 10 μL of RNase-free H2O to each tube, incubate in a water bath at 60℃ for 10 min to promote dissolution, and place on ice after dissolution;

[0068] (9) Detect RNA concentration using an OD instrument.

[0069] 3.1.2 Total RNA was reverse transcribed into cDNA

[0070] (1) Removal of genomic DNA (10 μl)

[0071]

[0072] (2) RNA was reverse transcribed into cDNA (20 μL)

[0073]

[0074] After reverse transcription to cDNA, dilute 8-fold with ddH2O and store at -20℃ for later use.

[0075] (3) Primer sequence

[0076] Primer design: The mRNA sequences of mouse IL-6, TNF-α, CD206, and Gapdh were selected from the NCBI database. Primers were designed on the NCBI website and their specificity was verified by BLAST. The size of the amplification product was predicted. The specificity of the primers was further verified by melting curves and agarose gel running results. The primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0077] The primer sequences required for the experiment are shown in the table below.

[0078]

[0079] 3.3.3 Real-time PCR experiment

[0080] (1) Prepare the Real-time PCR reaction system (10 μl) according to the following table:

[0081]

[0082] (2) The reaction was performed using a PCR instrument (Life Technology) under the following conditions:

[0083]

[0084] (iv) Macrophage viability detection

[0085] This application uses the CCK8 reagent to detect macrophage viability. The specific detection steps are as follows:

[0086] (1) Macrophages extracted from the peritoneal cavity were seeded into 96-well plates and cultured in a CO2 incubator at 37 ℃ for 24 h;

[0087] (2) Wash three times with PBS, add OA at concentrations of 50 μM, 100 μM, 200 μM, and 500 μM, and continue culturing for 6 h;

[0088] (3) Add CCK8 reagent and incubate for 30 min;

[0089] (4) Then turn on the preheated microplate reader and read the OD value at 450 nm.

[0090] Based on the above verification experiments, this application has for the first time discovered through non-targeted metabolomics technology that after peripheral sensory neurons are stimulated by the pruritogenic and inflammatory factor IL-31, the intracellular oleic acid (OA) level is significantly increased (VIP score > 25), suggesting that free fatty acid metabolism is involved in the regulation of pruritus and inflammatory signals.

Claims

1. The use of oleic acid in the preparation of products for the treatment and / or prevention of pruritus and inflammatory skin diseases.

2. The use of oleic acid according to claim 1 in the preparation of products for treating and / or preventing pruritus and inflammatory skin diseases, characterized in that: The product is any one of a pharmaceutical composition, health product, or functional food.

3. The use of oleic acid according to claim 1 in the preparation of products for treating and / or preventing pruritus and inflammatory skin diseases, characterized in that: The pruritus and inflammatory skin conditions mentioned above are associated with activation of the IL-31 signaling pathway.

4. The use of oleic acid according to claim 1 in the preparation of products for treating and / or preventing pruritus and inflammatory skin diseases, characterized in that: The product can be administered orally or topically.

5. The use of oleic acid according to claim 4 in the preparation of products for treating and / or preventing pruritus and inflammatory skin diseases, characterized in that: The product is administered orally via drinking water.

6. The use of oleic acid according to claim 1 in the preparation of products for treating and / or preventing pruritus and inflammatory skin diseases, characterized in that: The drug also includes other medically acceptable adjuvants.

7. A product for treating or preventing pruritus and inflammatory skin diseases, characterized in that: The product includes oleic acid.

8. A method for verifying the application of oleic acid in the treatment or prevention of pruritus and inflammatory skin diseases, characterized in that: It includes in vivo efficacy verification and in vitro mechanism verification.

9. A method for verifying the application of oleic acid in the treatment or prevention of pruritus and inflammatory skin diseases according to claim 8, characterized in that: The in vivo efficacy verification was conducted by adding a certain concentration of oleic acid to the drinking water of a mouse atopic dermatitis pruritus model induced by MC903, and then evaluating the indicators of the mice.

10. A method for verifying the application of oleic acid in the treatment or prevention of pruritus and inflammatory skin diseases according to claim 8, characterized in that: The in vitro mechanism was verified by treating macrophages (RAW264.7) with oleic acid at a certain concentration range, and detecting LPS-induced inflammatory responses without affecting cell viability.

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