CN115844871A - Application of hispidulin in preparation of targeted Th17 cell medicine - Google Patents
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- CN115844871A CN115844871A CN202211243737.7A CN202211243737A CN115844871A CN 115844871 A CN115844871 A CN 115844871A CN 202211243737 A CN202211243737 A CN 202211243737A CN 115844871 A CN115844871 A CN 115844871A
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Abstract
The invention relates to a natural compound Hispidulin (Hispidulin), the molecular weight of which is 300.263g/mol, and the invention discloses application of the Hispidulin in preparation of a targeted Th17 cell medicament. The invention discloses for the first time that the hispidulin has the function of obviously inhibiting the differentiation of Th17 cells in vitro and has the application prospect of preparing targeted medicaments in diseases with main function of the Th17 cells.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to application of hispidulin in preparation of a targeted Th17 cell medicine.
Background
Th17 cell is a subtype of CD4+ T lymphocyte, has important relation with autoimmune inflammatory diseases and clearance of extracellular infection, mainly releases cytokines such as IL-17 and the like to play a role, and drives diseases such as multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease and the like.
Activation of Th17 cells is characterized by expression of the Retinoic Acid Receptor (RAR) associated orphan receptor (ROR) gamma t transcription factor, which is encoded by the gene Rorc. STAT3 is an activator of ROR γ t, which is regulated by interaction with the first intron of Rorc, the Il17A promoter, and the intergenic region of the Il17A site, and IL-17A. Th17 transcriptional modulators have been the focus of targeted therapy for Th17 cell-related diseases, especially the ROR γ t-IL-17 axis. ROR gamma t is used as a new target, so far, no target medicine widely applicable to Th17 cell related diseases exists, AUR-101 is the first ROR gamma t inverse agonist for clinical evaluation, but has important curative effect only in psoriasis. Therefore, there is an urgent need to find new and broadly beneficial Th17 cell targeting agents.
Hispidulin (HPDL), a natural flavone with a wide range of biological activities, has been widely noticed for its anticancer activity and can reduce the resistance of cancer cells when used in combination with anticancer drugs. Most importantly, in colorectal cancer, hispidulin inhibits cancer cells via JAK2/STAT3 signaling. In addition, in BV2 microglia stimulated by lipopolysaccharide, the hispidulin can reduce the phosphorylation of IL-6 and STAT3 protein expressions, so the hispidulin is expected to be a lead compound for regulating and controlling Th17 cells and Th17 related diseases and preparing a medicament for targeting the Th17 cells.
Disclosure of Invention
The invention aims to provide application of hispidulin in preparation of a medicament for targeting Th17 cells. Experiments show that: the compound has the function of obviously inhibiting Th17 cell differentiation in vitro and has good research and application prospects.
The molecular formula of the hispidulin is C 16 H 12 O 6 Molecular weight 300.263, structural formula:
experiments prove that the hispidulin has the function of inhibiting Th17 cell differentiation in vitro and can be used for preparing a medicament for targeting the Th17 cell.
The invention also provides a Th17 cell-targeted pharmaceutical composition which takes the hispidulin as an active ingredient and further comprises pharmaceutically acceptable pharmaceutic adjuvant.
Optionally, the pharmaceutically acceptable pharmaceutical excipients include excipients and additives selected from solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickening agents, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, and release retardants.
Optionally, the pharmaceutical composition is in the form of an external preparation or an internal preparation.
Optionally, the pharmaceutical composition is in the form of oral liquid, mixture, tablet, tincture, powder, capsule, pill, powder, paper, injection solvent, oil, suspension, crystal, ointment, tincture, liniment, lotion, drop, suppository, plastics, patch, or slow-dissolving tablet.
The invention also provides a Th17 cell targeted pharmaceutical composition which contains a plant extract containing the hispidulin and pharmaceutically acceptable pharmaceutic adjuvant.
The invention has the advantage of providing a novel inhibiting compound for the differentiation of Th17 cells.
Drawings
The attached figure is a diagram of the Th17 cell flow ratio results of WT mice, in which DMSO is the control group and hispidulin is the experimental group.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments, in which, unless otherwise specified, the experimental procedures used are conventional and materials, reagents and the like used are commercially available from biological or chemical companies.
Materials and instruments described in the following examples:
1. the compound hispidulin (MedChemexpress, USA) is a natural flavone with wide biological activity and the purity is 99.75%.
WT mice were C57/B6J (Viton, beijing).
DMSO (solarbio, china), flow through antibodies (CD 4-FITC, IL-17A-PE), mouse anti-CD 3, mouse anti-CD 28, anti-IFN-. Gamma.anti-IL-4 (Biolegend, USA), IL-6, TGF-. Beta.PeproTech, USA, phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A (Sigma, germany).
MidiMACS sorter (Miltenyi, germany), flow cytometer (Beckman, USA).
Example 1 in vitro inhibition of Primary CD4+ T cell differentiation to Th17 cells in WT mice by hispidulin
1. The experimental method comprises the following steps:
1) anti-CD 3 and anti-CD 28 were coated in 24-well plates in advance overnight.
2) After the WT mice were sacrificed, the mice were wrapped with alcohol-sprayed paper, and the spleen and lymph nodes (thigh, arm, armpit, neck) were dissected and separated in a super clean bench to a filter screen.
3) After grinding with a 5mL syringe rubber head, washing and filtering with MASC buffer solution, and collecting the filtrate to a 15mL centrifuge tube.
4) Centrifuge at 600 × g for 4 min at 4 ℃, discard the supernatant, resuspend the cells in 500uL of MASC buffer, mix well.
5) The corresponding CD4+ beads were added, mixed well and incubated for 20 minutes at 4 ℃.
6) After incubation, cold MACS buffer was added to each tube, mixed well and centrifuged at 600 × g for 4 min at 4 ℃.
7) After centrifugation, the supernatant was discarded, resuspended in 1mL of MACS buffer, and CD4+ T cells sorted according to the midi MACS sorter instructions.
8) The cell suspension was centrifuged at 600 × g for 4 min at 4 deg.C, the cell fluid containing IL-6 (10 ng/mL), TGF-. Beta.s (1 ng/mL), anti-IFN-. Gamma.s (10 μ g/mL), anti-IL-4 (10 μ g/mL) was added, seeded at 0.5 × 10^6 cells per well in 24-well plates coated with mouse anti-CD 3 and mouse anti-CD 28, and compound was added simultaneously to intervene.
9) After 3 days of culture, phorbol 12-myristate 13-acetate (PMA, 50 ng/mL), ionomycin (1. Mu.g/mL), brefeldin A (5. Mu.g/mL) were added to stimulate Th17 cells for 4 hours at 37 ℃.
10 Cells were collected and subjected to Th17 cell flow assay.
2. The experimental results are as follows: the hispidulin can stably inhibit the differentiation of initial CD4+ T cells to Th17 cells in vitro, and the inhibition rate reaches over 50 percent. The results are shown in the figure.
Claims (9)
1. Application of hispidulin in preparing medicines for targeting Th17 cells is provided.
2. Use according to claim 1, characterized by the regulatory effect of hispidulin on Th17 cell differentiation.
3. Use according to claim 1, characterized by the regulatory effect of hispidulin on Th17 cell differentiation in diseases in which Th17 cells are involved.
4. The method of claim 2, wherein the effect is achieved by including a gene, protein, other biomolecule or pathway associated with roryt or associated with Th17 cells.
5. The Th17 cell-targeting pharmaceutical composition is characterized by taking the hispidulin as an active ingredient and further comprising pharmaceutically acceptable pharmaceutic adjuvants.
6. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable pharmaceutical excipients comprise excipients and additives selected from solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants.
7. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is in the form of an external preparation or an internal preparation.
8. The pharmaceutical composition of claim 7, wherein the pharmaceutical composition is in the form of oral liquid, mixture, tablet, tincture, powder, capsule, pill, paper, injectable solution, oil, suspension, crystal, ointment, tincture, liniment, lotion, drop, suppository, film, patch, or slow-dissolving tablet.
9. A Th17 cell-targeting pharmaceutical composition comprising a plant extract comprising hispidulin and a pharmaceutically acceptable pharmaceutical excipient.
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| CN202211243737.7A CN115844871A (en) | 2022-10-12 | 2022-10-12 | Application of hispidulin in preparation of targeted Th17 cell medicine |
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| CN202211243737.7A CN115844871A (en) | 2022-10-12 | 2022-10-12 | Application of hispidulin in preparation of targeted Th17 cell medicine |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107019793A (en) * | 2017-02-27 | 2017-08-08 | 新乡医学院 | Applications of the MBL in prevention or treatment Th17 cell initiation disease medicaments is prepared |
| CN111733233A (en) * | 2020-05-09 | 2020-10-02 | 中国人民解放军陆军军医大学 | Application of RORCE2 gene as drug target in preparation of drugs for treating Th17 cell-related diseases |
| CN112156091A (en) * | 2020-09-30 | 2021-01-01 | 中山大学附属第一医院 | Application of hispidulin in preparation of medicine for treating and/or preventing cardiovascular diseases |
| CN113876763A (en) * | 2021-08-17 | 2022-01-04 | 上海中医药大学附属曙光医院 | Application of hispidulin in preparation of anti-bile duct cancer drugs |
| CN114452275A (en) * | 2022-02-25 | 2022-05-10 | 中国科学技术大学 | Application of vitamin B5 in preparation of medicine or preparation for preventing and/or treating Th17 cell related diseases |
-
2022
- 2022-10-12 CN CN202211243737.7A patent/CN115844871A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107019793A (en) * | 2017-02-27 | 2017-08-08 | 新乡医学院 | Applications of the MBL in prevention or treatment Th17 cell initiation disease medicaments is prepared |
| CN111733233A (en) * | 2020-05-09 | 2020-10-02 | 中国人民解放军陆军军医大学 | Application of RORCE2 gene as drug target in preparation of drugs for treating Th17 cell-related diseases |
| CN112156091A (en) * | 2020-09-30 | 2021-01-01 | 中山大学附属第一医院 | Application of hispidulin in preparation of medicine for treating and/or preventing cardiovascular diseases |
| CN113876763A (en) * | 2021-08-17 | 2022-01-04 | 上海中医药大学附属曙光医院 | Application of hispidulin in preparation of anti-bile duct cancer drugs |
| CN114452275A (en) * | 2022-02-25 | 2022-05-10 | 中国科学技术大学 | Application of vitamin B5 in preparation of medicine or preparation for preventing and/or treating Th17 cell related diseases |
Non-Patent Citations (1)
| Title |
|---|
| NAMKYUNG KIM ET AL.: "Hispidulin alleviates imiquimod-induced psoriasis-like skin inflammation by inhibiting splenic Th1/Th17 cell population and keratinocyte activation", 《INTERNATIONAL IMMUNOPHARMACOLOGY》, 31 December 2020 (2020-12-31), pages 1 * |
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