Use of 12-ketolithocholic acid for the preparation of a medicament for the prevention or treatment of atopic dermatitis

Abstract

The application discloses application of 12-ketolithocholic acid in preparation of a medicine for preventing or treating atopic dermatitis, and the 12-ketolithocholic acid can inhibit NUAK2 kinase activity expression and activity, and then negatively regulates a downstream TNF inflammatory signal path, significantly down-regulates IL-1beta, IL-8 and various proinflammatory factor secretion, and inhibits keratinocyte excessive inflammatory activation. The 12-ketolithocholic acid and ginsenoside F2 are compounded and used in combination to produce synergistic effect, and the anti-inflammatory effect is further enhanced; the active substance has low cytotoxicity and good safety, can be prepared into an external ointment or an oral preparation, and has a wide application prospect in the field of atopic dermatitis prevention and treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of 12-ketolithocholic acid in the preparation of drugs for the prevention or treatment of atopic dermatitis. Background Technology

[0002] Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease characterized by dry skin, intense itching, and eczematous rashes, often accompanied by immune dysregulation and impaired skin barrier function. Current routine clinical treatment primarily involves corticosteroids, antihistamines, and immunosuppressants. However, long-term use of these drugs can easily lead to side effects such as skin atrophy, drug resistance, and systemic immunosuppression, making it difficult to meet the needs for long-term safe medication use.

[0003] Existing research confirms that ginsenoside F2 can alleviate atopic dermatitis lesions by inhibiting the NF-κB pathway, downregulating Th2-type inflammatory factors, and regulating gut microbiota. However, when ginsenoside F2 is used alone, it suffers from insufficient transdermal penetration and slow onset of action. Furthermore, its activity as a single ingredient is relatively weak, and its preparation cost is high, making it difficult to simultaneously meet the needs of rapid symptom control in the acute phase of atopic dermatitis and long-term safe medication.

[0004] Ketolithocholic acid (12-KLCA) is a secondary bile acid derivative produced by gut microbiota metabolism. It exhibits excellent lipid solubility and strong transdermal penetration. Existing research indicates that 12-KLCA can slow the development of colitis in mice by inhibiting the secretion of the pro-inflammatory cytokine IL-17A by type 3 innate lymphocytes; and that 12-KLCA can significantly counteract CCl4-induced liver damage by inhibiting IL-17 and TNF-mediated inflammatory pathways. However, current technologies do not disclose the regulatory effects of 12-KLCA on inflammatory factors and pathways in skin tissue, nor do they reveal its medicinal value in the prevention and treatment of atopic dermatitis. Summary of the Invention

[0005] The present invention is based on the discovery that 12-ketolithocholic acid has the activity of preventing or treating atopic dermatitis, and thus the invention relates to the application of 12-ketolithocholic acid in the preparation of drugs for the prevention or treatment of atopic dermatitis.

[0006] The technical solution of the present invention is the application of 12-ketolithocholic acid in the preparation of drugs for the prevention or treatment of atopic dermatitis.

[0007] The preferred application of the 12-ketolithocholic acid is in the preparation of drugs that inhibit the activity of NUAK2 kinase in skin tissue.

[0008] The preferred application of the 12-ketolithocholic acid is in the preparation of drugs that inhibit the TNF inflammatory signaling pathway.

[0009] The preferred application of the 12-ketolithocholic acid is in the preparation of drugs that inhibit the secretion of pro-inflammatory factors IL-1β and / or IL-8 by keratinocytes in the skin.

[0010] The preferred application is a combination of the 12-ketolithocholic acid and ginsenoside F2.

[0011] The preferred mass ratio of 12-ketolithocholic acid to ginsenoside F2 is 1:0.1~10.

[0012] The preferred molar ratio of 12-ketolithocholic acid to ginsenoside F2 is 3:10.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1.12-KLCA can specifically inhibit the abnormal activation of NUAK2 kinase in skin tissue, thereby inhibiting the TNF-mediated inflammatory signaling pathway. It has strong targeting and lower off-target side effects.

[0014] 2. By regulating the TNF signaling pathway, the overexpression of key pro-inflammatory factors such as IL-1β and IL-8 in the skin inflammation model can be significantly downregulated, thereby achieving targeted and precise inhibition of local skin inflammation.

[0015] 3. When 12-KLCA and ginsenoside F2 are combined, they exhibit synergistic inhibitory effects on NUAK2 kinase and key molecules in the TNF signaling pathway, with anti-inflammatory regulatory effects superior to the two active substances used alone. At the same dosage concentration, the toxicity of the combination to keratinocytes is significantly lower than that of 12-KLCA or ginsenoside F2 alone, with a wider safety window and less irritation, making it suitable for long-term topical intervention for atopic dermatitis.

[0016] 4. Both 12-KLCA alone and 12-KLCA combined with ginsenoside F2 can be processed into topical or oral preparations. Topical preparations rely on good lipid solubility to penetrate to the site of skin inflammation to take effect, while oral preparations can exert a regulatory effect through the body's pathways. The formulation is flexible and has broad prospects for industrial application. Attached Figure Description

[0017] Figure 1 This is a quantitative bar chart showing the effects of 12-ketolithocholic acid, ginsenoside F2, and their combination on the secretion of IL-1β and IL-8 by HaCaT cells.

[0018] Figure 2 Volcano diagrams showing differential gene expression in HaCaT cells after treatment with ginsenoside F2 and 12-ketolithocholic acid, respectively.

[0019] Figure 3 for Figure 2 The bubble chart shows the KEGG pathway enrichment analysis of differentially expressed genes.

[0020] Figure 4 A schematic diagram illustrating the inhibitory effects of 12-ketolithocholic acid, ginsenoside F2, and their combination on the NUAK2 and TRAF2 gene expression for qRT-PCR verification.

[0021] Figure 5 The toxicity test results of 12-ketolithocholic acid, ginsenoside F2, and the combination of the two in equal amounts are shown in the figure. Detailed Implementation

[0022] Unless otherwise specified, all reagents were commercially purchased (F2: SMB00685, Sigma-Aldrich; 12-KLCA: HY-113430, MedChemExpress).

[0023] Experiment Example 1: TNF-α-induced HaCaT keratinocyte inflammation model and monomeric / combination anti-inflammatory experiments Cell culture: After HaCaT cells were revived, they were cultured in DMEM complete medium (10% FBS + 1% antibiotics) at 37°C and 5% CO2. When the cells reached 80% confluence, they were passaged by trypsin digestion.

[0024] Preliminary experiment on safe concentration (CCK-8 method): HaCaT cells were used at a rate of 1×10 4 Cells were seeded per well in a 96-well plate and cultured for 24 h; or ginsenoside F2 at concentrations of 3, 10, 30, 100, 300, and 1000 μM were added, with 3 replicates per group, and a blank group (culture medium only) and a normal control group (cells + culture medium) were set up; after culturing for another 24 h, 10 μL of CCK-8 reagent was added to each well and incubated for 1 h, and the OD value was measured at 450 nm; cell viability was calculated as follows: cell viability (%) = (OD of drug group - OD of blank group) / (OD of control group - OD of blank group) × 100%; the experimental concentrations were determined based on the results: F2 30 μM, 12-KLCA 100 μM.

[0025] Cell grouping and modeling (6-well plate, for qPCR detection): Cells at 3.5 × 10 5 One sample per well was inoculated into a 6-well plate and cultured for 23 h. The plates were then divided into 6 groups, with 3 replicates per group. The specific groupings are as follows: Control group: cultured in standard culture medium; Model group: 50 ng / mL recombinant human TNF-α was added; F2 monotherapy group: pretreatment with 30 μM F2 for 1 h + 50 ng / mL TNF-α; 12-KLCA monotherapy group: pretreatment with 100 μM 12-KLCA for 1 h + 50 ng / mL TNF-α; F2+12-KLCA combination group: pretreated with 30 μM F2 + 100 μM 12-KLCA for 1 h + 50 ng / mL TNF-α; all groups were cultured for another 24 h before cell collection.

[0026] Total RNA extraction from cells and qPCR detection of inflammatory factors: RNA extraction: Add 1 mL of Trizol reagent to each well to fully lyse the cells. Extract the RNA using chloroform, precipitate with isopropanol, and wash with 75% ethanol according to standard methods to obtain total RNA. Detect RNA purity and concentration using Nanodrop.

[0027] cDNA synthesis: RNA was reverse transcribed into cDNA according to the cDNA synthesis kit instructions.

[0028] qPCR reaction: Real-time quantitative PCR was used to detect the expression levels of IL-1β and IL-8 mRNA, with GAPDH as the internal reference gene; The relative expression levels of genes were calculated, and the inhibition effects of each group were compared.

[0029] The results are as follows Figure 1 As shown, Figure A shows the inhibitory effect of 12-ketolithocholic acid, ginsenoside F2 and their combination on IL-1β secretion by HaCaT cells; Figure B shows the inhibitory effect of 12-ketolithocholic acid, ginsenoside F2 and their combination on IL-8 secretion by HaCaT cells.

[0030] The results showed that 12-ketolithocholic acid, ginsenoside F2 and the combination group all inhibited the secretion of IL-1β and IL-8 by HaCaT cells. The inhibitory effect of the combination group on IL-1β and IL-8 was significantly stronger than that of each single drug group (p<0.01).

[0031] Experiment Example 2: Molecular Mechanism Experiment of Single Drug Intervention in an Atopic Dermatitis Cell Model HaCaT cells treated with the Model group, F2 single-drug group, and 12-KLCA single-drug group in Experiment 1 were collected. Total RNA was extracted from each group of cells. After the RNA samples passed quality control, libraries were constructed using the NEBNext Ultra II RNA Library Kit and sequenced using the Illumina platform in PE150 mode. After data quality control, gene expression levels were statistically analyzed using HTSeq, and differentially expressed genes were screened using DESeq2 (|log2FoldChange|>1, P<0.05). The results are as follows: Figure 2The diagram shows the differential gene expression volcanoes of HaCaT cells after treatment with ginsenoside F2 and 12-ketolithocholic acid. Figure 2 Figures A and B in the middle are differential gene volcano plots of Model vs F2 and Model vs 12-KLCA, respectively.

[0032] The results showed that, compared with the Model group, both F2 monotherapy and 12-KLCA monotherapy resulted in highly enriched differentially expressed genes in cells on the NUAK2 kinase and TNF-mediated inflammatory signaling pathways. Both groups could specifically downregulate the expression of core pro-inflammatory factors in this pathway. Moreover, 12-KLCA showed a more significant inhibitory trend on skin-derived IL-1β and IL-8 pro-inflammatory factors, indicating that it has a stronger targeting ability than ginsenoside F2 in the context of skin inflammation.

[0033] based on Figure 2 The two sets of differentially expressed gene lists were subjected to KEGG pathway enrichment analysis. The enriched pathways were mapped into bubble charts based on rich factor, p-value, and number of enriched genes, as shown below. Figure 3 The bubble chart showing KEGG pathway enrichment analysis is shown below. Figure 3 Figures A and B in the middle are bubble charts of pathway enrichment analysis for Model vs F2 and Model vs 12-KLCA, respectively.

[0034] The results showed that the differentially expressed genes after single-drug intervention in both groups were highly enriched in the inflammatory signaling pathway, which is mainly mediated by TNF and secondarily by IL-17.

[0035] Experiment Example 3: qPCR Validation of Core Gene Expression cDNA from each group of cells in Experiment 1 was used to perform qPCR detection for NUAK2 and TRAF2. The upstream and downstream primer sequences used for detecting NUAK2 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively, and the upstream and downstream primer sequences used for detecting TRAF2 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The relative expression level was calculated using the method, and the results are as follows: Figure 4 As shown. Figure 4 Figure A in the middle shows the effect of downregulating the NUAK2 gene in various cells; Figure 4 Figure B shows the effect of downregulating the TRAF2 gene in various cells. The results indicate that 12-ketolithocholic acid, ginsenoside F2, and the combined group can all inhibit NUAK2 kinase expression and TNF-mediated inflammatory signaling pathways, with the combined group showing a more significant inhibitory effect.

[0036] Experimental Example 4: Toxicity test of equal amounts of 12-ketolithocholic acid, ginsenoside F2, and combinations thereof HaCaT cells were used at a rate of 1×10 4 Cells were seeded per well in 96-well plates and cultured for 24 h. Then, 130 μM 12-KLCA, 130 μM ginsenoside F2, and 30 μM F2 + 100 μM 12-KLCA were added to each well, with three replicates per group. A blank control group (culture medium only) and a normal control group (cells + culture medium) were also included. After another 24 h of culture, 10 μL of CCK-8 reagent was added to each well, and the cells were incubated for 1 h. OD values ​​were measured at 450 nm. Cell viability was calculated as follows: Cell viability (%) = (OD of drug-treated group - OD of blank group) / (OD of control group - OD of blank group) × 100%. Results are as follows. Figure 5 As shown: The results showed that, at the same drug concentration, the toxicity of the two compound to keratinocytes was significantly lower than that of 12-KLCA alone or ginsenoside F2 alone.

[0037] All experimental data are expressed as mean ± standard deviation (x ± s) and analyzed using GraphPad Prism 10.0 software. The t-test was used for comparison between two groups, and one-way ANOVA was used for comparison of multiple groups. P < 0.05 was considered statistically significant, and P < 0.01 and P < 0.001 were considered highly significant.

[0038] The above experimental results indicate that 12-ketolithocholic acid, or the combination of 12-ketolithocholic acid and ginsenoside F2, can be used to prepare drugs for the prevention or treatment of atopic dermatitis. Specifically, it achieves its efficacy by inhibiting the activity of NUAK2 kinase in skin tissue, the TNF inflammatory signaling pathway, and the pro-inflammatory factors IL-1β and IL-8. When 12-ketolithocholic acid and ginsenoside F2 are used in combination, the mass ratio of 12-ketolithocholic acid to ginsenoside F2 is 1:0.1~10, preferably a molar ratio of 3:10. The drug can be an oral or topical preparation; preferred oral preparations are tablets, capsules, granules, or oral liquids; preferred topical preparations are creams, ointments, gels, or sprays.

Claims

1. Application of 1,2-ketolithocholic acid in the preparation of drugs for the prevention or treatment of atopic dermatitis.

2. The application according to claim 1, characterized in that: The 12-ketolithocholic acid is used in the preparation of drugs that inhibit the activity of NUAK2 kinase in skin tissue.

3. The application according to claim 2, characterized in that: The 12-ketolithocholic acid is used in the preparation of drugs that inhibit the TNF inflammatory signaling pathway.

4. The application according to claim 3, characterized in that: The 12-ketolithocholic acid is used in the preparation of drugs that inhibit the secretion of pro-inflammatory factors IL-1β and / or IL-8 by keratinocytes in the skin.

5. The application according to claim 1, 2, 3 or 4, characterized in that: The 12-ketolithocholic acid is used in combination with ginsenoside F2.

6. The application according to claim 5, characterized in that: The mass ratio of 12-ketolithocholic acid to ginsenoside F2 is 1:0.1~10.

7. The application according to claim 6, characterized in that: The molar ratio of 12-ketolithocholic acid to ginsenoside F2 is 3:10.

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