CN111617074B - Application of artemisinin or derivatives artesunate and dihydroartemisinin - Google Patents
Application of artemisinin or derivatives artesunate and dihydroartemisinin Download PDFInfo
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- CN111617074B CN111617074B CN202010631485.XA CN202010631485A CN111617074B CN 111617074 B CN111617074 B CN 111617074B CN 202010631485 A CN202010631485 A CN 202010631485A CN 111617074 B CN111617074 B CN 111617074B
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
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Abstract
The invention belongs to the technical field of biomedicine, and relates to new application of artemisinin or derivatives artesunate, dihydroartemisinin chloroquine or derivatives artesunate and dihydroartemisinin drugs, in particular to application of artemisinin or derivatives artesunate, dihydroartemisinin chloroquine or derivatives artesunate and dihydroartemisinin in drugs for treating Graves eye diseases. The invention proves that the artemisinin or the derivatives artesunate and dihydroartemisinin have obvious inhibition effects on excessive proliferation, adipogenic differentiation, hyaluronic acid secretion and fibrosis of orbital fibroblasts, the superposition of the effects shows that the artemisinin chloroquine or the derivatives artesunate and the dihydroartemisinin effectively regulate and control the pathophysiology of the orbital fibroblasts which are the pathological leading factors of the Graves eye diseases, and the pathological physiology shows that the artemisinin chloroquine or the derivatives artesunate and the dihydroartemisinin have potential treatment values on the Graves eye diseases, so that the invention is a new application of the drug, and can save social and scientific research resources.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and relates to new application of artemisinin or derivatives artesunate and dihydroartemisinin, in particular to application of artemisinin or derivatives artesunate and dihydroartemisinin in a medicine for treating Graves eye diseases.
Background
Graves Ophthalmopathy (GO) is the most common extrathyroid manifestation of Graves disease, accounting for about 50%, and is manifested by eyelid contracture, exophthalmos, pain, redness and swelling, even resulting in optic nerve compression, causing blindness, and seriously affecting the health and life quality of patients. Currently, the effective rate of clinically applied glucocorticoids is 63% (oral administration) to 88% (venous shock), but the glucocorticoid is usually only applied to acute or moderate patients, and cannot reverse the invasive exophthalmos of GO, the application is limited by side effects such as edema, obesity, osteoporosis and the like, and surgery and radiotherapy are only used as complementary treatment, so a new treatment strategy aiming at the etiology needs to be explored urgently. The excessive proliferation, lipogenic differentiation and secretion of hydrophilic mucopolysaccharide of Orbital Fibroblasts (OFs) increase the Orbital content, increase the intra-Orbital pressure and simultaneously secrete inflammatory factors to recruit inflammatory cells, which are the core factors of the exophthalmos and inflammatory infiltration, but the current medicines for relieving the exophthalmos aiming at the Orbital fibroblasts are limited.
Research finds that autophagy plays a crucial role in early proliferation and differentiation of orbital fibroblasts, so that inhibition of autophagy is a new strategy for treating inactive-stage exophthalmos of Graves eye diseases by aiming at the orbital fibroblasts. Goodall et al screened Artemisinin (ARS) and other antimalarial drugs with a tumor cell line expressing fluorescent LC3 to find that the antimalarial drugs all have inhibition effects on autophagy to different degrees, which indicates that inhibition of autophagy may be one of common mechanisms of the antimalarial drugs. The artemisinin and the derivatives thereof are old drugs widely applied to clinic and have good safety and tolerance. The method takes the periorbital fibroblasts separated and extracted from periorbital adipose tissues excised by a Graves eye disease patient as a research object, and proves that Artemisinin (ARS) and derivatives thereof, namely Artesunate (ART) and Dihydroartemisinin (DHA), can obviously inhibit various pathological mechanisms of the periorbital fibroblasts, including proliferation, differentiation, hyaluronic acid secretion and fibrosis, is expected to become a new therapy of the Graves eye disease, and has important scientific and social meanings.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects and technical shortcomings in the prior art and providing a new application of artemisinin or derivatives of artesunate and dihydroartemisinin in a medicine for treating Graves eye diseases.
The technical scheme of the invention is as follows:
the application of the artemisinin or the derivatives artesunate and dihydroartemisinin in the preparation of the drugs for treating and/or relieving Graves eye diseases is described.
The application of artemisinin or derivatives artesunate and dihydroartemisinin is characterized in that: the artemisinin or the derivatives artesunate and dihydroartemisinin are applied to the preparation of the medicine for inhibiting the proliferation of orbital fibroblasts.
The application of artemisinin or derivatives artesunate and dihydroartemisinin is characterized in that: the artemisinin or the derivatives artesunate and dihydroartemisinin are applied to the preparation of the medicine for inhibiting the adipogenic differentiation of the orbital fibroblasts.
The application of artemisinin or derivatives artesunate and dihydroartemisinin is characterized in that: the artemisinin or the derivatives artesunate and dihydroartemisinin are applied to the preparation of the medicine for inhibiting the synthesis and secretion of the hyaluronic acid of the orbital fibroblast.
The application of artemisinin or derivatives artesunate and dihydroartemisinin is characterized in that: the artemisinin or the derivatives artesunate and dihydroartemisinin are applied to the preparation of the medicine for inhibiting the fibrosis of the orbital fibroblasts.
In addition, in the scheme, the artemisinin or the derivatives artesunate and dihydroartemisinin are prepared into a pharmaceutical preparation, and the preparation form is tablets or injection.
In the medicinal preparation, the content of the artemisinin in the preparation is 62.5 mg/tablet, the content of the dihydroartemisinin in the preparation is 20 mg/tablet, the content of the artesunate in the preparation is 50 mg/tablet, and the injection is 60 mg/injection.
The invention takes fibroblasts separated and extracted from periorbital adipose tissues excised by a Graves eye disease patient in an operation as a research object, under the action of artemisinin or derivatives thereof, namely artesunate and dihydroartemisinin, the excessive proliferation, adipogenic differentiation, hyaluronic acid secretion and fibrosis of the orbital fibroblasts are found to be obviously inhibited, and the effects are superposed to show that the artemisinin or the derivatives thereof, namely the artesunate and the dihydroartemisinin, effectively regulate and control the pathophysiology of the fibroblast which is a pathological leading factor of the Graves eye disease.
The invention has the following beneficial effects:
different from the current hyperthyroidism exophthalmos therapy, the artemisinin or the artesunate derivatives and the dihydroartemisinin exert the functions aiming at the pathophysiological mechanism of the orbital fibroblasts, have a certain immunoregulation function but not an immunosuppression function, are clinically applied for many years, and have high safety and low price.
The invention regulates and controls all pathogenesis of Graves ophthalmopathy exophthalmos, has easily obtained raw materials, has wide application in other diseases clinically, and has great significance for saving social resources and the like when being used as an old medicine for new use.
Drawings
FIG. 1 shows that artemisinin and its derivatives inhibit the proliferation of orbital fibroblasts in patients with Graves' eye disease.
FIGS. 2A-2B show that artemisinin and its derivatives inhibit adipogenic differentiation of orbital fibroblasts in patients with Graves' eye disease.
FIGS. 3A-3C show that artemisinin and its derivatives inhibit hyaluronic acid synthesis and secretion from orbital fibroblasts of Graves eye disease patients.
FIGS. 4A-4B are graphs showing that artemisinin and its derivatives inhibit the fibrosis of orbital fibroblasts in patients with Graves' eye disease.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the present invention are commercially available.
Materials and kits used:
1) proliferation culture medium: DMEM-F12(Gibco Laboratories, USA) containing 10% (vol/vol) fetal bovine serum.
2) Differentiation medium and oil red O staining solution: commercial induced adipogenic medium (selela, guangzhou, china).
3) Proliferation-related assay kit: cell cycle detection kit (Kaiky, Guangzhou, China).
4) RNA reverse transcription kit (Promega, usa);
5) hyaluronic acid ELISA kits (Echelon Bioscience inc., usa).
6) Artemisinin raw material (sigma, usa): in a sterile environment, 56.5mg of the powder was taken and 1.0mL of dimethyl sulfoxide (DMSO) was added to prepare a 200.0mM stock solution (1000X), which was diluted to 50.0mM stock solution (1000X) at a ratio of 1:4, and the latter was diluted to 10.0mM stock solution (1000X) at a ratio of 1:5, and the solutions were separately stored at-20 ℃ and diluted to 200.0. mu.M, 50.0. mu.M and 10.0. mu.M in the corresponding media at a ratio of 1:1000 to treat the cells.
7) Artesunate feedstock (APExBIO, usa): 19.0mg of powder was taken in a sterile environment and 1.0mL of DMSO1 was added to prepare a stock solution with a concentration of 50.0mM, and the stock solutions were diluted with DMSO in a ratio of 1:5, 1:25, and 1:100, respectively, to obtain three stock solutions of 1000X: 10.0mM, 2.0mM and 0.5mM, separately dispensed, stored at-20 ℃, diluted to 10.0. mu.M, 2.0. mu.M and 0.5. mu.M in the respective media at a ratio of 1:1000, respectively.
8) Dihydroartemisinin starting material (APExBIO, usa): 12.4mg of the powder was added with DMSO1mL in a sterile environment to prepare a stock solution with a concentration of 50.0mM, and the stock solutions were diluted with DMSO in a ratio of 1:2.5, 1:12.5, and 1:50 to three stock solutions of 1000 Xconcentration: 20.0mM, 5.0mM and 1.0mM, dispensed, stored at-20 ℃, diluted to 20.0. mu.M, 5.0. mu.M and 1.0. mu.M respectively in the corresponding medium at a ratio of 1: 1000.
Example 1: the method for inhibiting the orbital fibroblast proliferation of Graves eye disease patients by artemisinin and derivatives comprises the following steps:
A) extracting orbital fibroblasts (n ═ 5) of Graves eye disease patients, and carrying out the experiment by passage to the third generation in proliferation culture medium;
B) after synchronous treatment with a basal medium, the cells of each group are digested for 20 hours and fixed overnight by 75 percent ethanol after being divided into a control group (DMSO), Artemisinin (ARS) (50.0 and 200.0 mu M), Artesunate (ART) (2.0 and 10.0 mu M) and Dihydroartemisinin (DHA) (5.0 and 20.0 mu M) treatment groups with different concentrations;
C) after centrifugation at 4 ℃ to remove supernatant, PBS was resuspended, stained with PI and the proportion of each cell cycle was determined by flow cytometry.
As a result: as shown in figure 1, after the orbital fibroblasts of patients with Graves eye disease are respectively treated with artemisinin and derivatives thereof with different concentrations in a proliferation medium, the cell cycle is detected by a flow cytometry technology, and the difference of the G0/1 phase cell ratio and the S phase cell ratio of the 200.0 mu M artemisinin, 10.0 mu M artesunate and 20.0 mu M dihydroartemisinin treatment group is statistically significant compared with the control group, which indicates that the artemisinin and the derivatives thereof inhibit the orbital fibroblast proliferation of the patients with Graves eye disease.
Example 2: artemisinin and its derivatives can inhibit lipidogenic differentiation of orbit fibroblast of Graves eye disease patient
The method comprises the following steps:
A) taking orbital fibroblasts (n ═ 5) of Graves eye disease patients, and carrying out passage in a proliferation medium to a third generation;
B) after the cell density reached 100% and the contact inhibition was performed for 48h, the induction medium was replaced, and the cells were divided into control group (DMSO), Artemisinin (ARS) (10.0, 50.0 and 200.0. mu.M), Artesunate (ART) (0.5, 2.0 and 10.0. mu.M) and Dihydroartemisinin (DHA) (1.0, 5.0 and 20.0. mu.M) treatment groups;
C) after induced differentiation, oil red O staining quantitative analysis, a real-time fluorescence quantitative PCR experiment and a protein immunoblotting experiment are respectively carried out to detect the influence of the medicament on the adipogenic differentiation of the orbital fibroblast.
As a result: as shown in FIGS. 2A-2B, induced differentiation culture was performed on orbital fibroblasts of Graves eye disease patients, and simultaneously, treatment with artemisinin and its derivatives at different concentrations, oil red O staining method, quantitative experiment, real-time fluorescence quantitative PCR experiment, and immunoblotting experiment all showed that artemisinin drugs inhibited the adipogenic differentiation ability of orbital fibroblasts.
Example 3: artemisinin and its derivatives can inhibit hyaluronic acid synthesis and secretion of orbit fibroblast of Graves eye disease patient
The method comprises the following steps:
A) carrying out passage on orbital fibroblasts of patients with Graves eye disease in a proliferation culture medium to a third generation for experiment;
B) the treatment groups were divided into interleukin-1 β treatment group and non-treatment group, and further divided into control group (DMSO), Artemisinin (ARS) (10.0, 50.0 and 200.0. mu.M) treatment groups at different concentrations, Artesunate (ART) (0.5, 2.0 and 10.0. mu.M) and Dihydroartemisinin (DHA) (1.0, 5.0 and 20.0. mu.M);
C) after the cells are treated, the hyaluronic acid concentration and the expression of the hyaluronic acid synthase 2 and the hyaluronidase 3 are respectively detected by using an Enzyme-linked immunosorbent assay (ELISA) kit.
As a result: as shown in fig. 3A-3C, artemisinin and its derivatives significantly inhibited hyaluronic acid secretion in a concentration-dependent manner, and significantly inhibited the expression of hyaluronidase synthase 2, without affecting the expression of hyaluronidase 3.
Example 4: artemisinin and its derivatives inhibit fibrosis of orbital fibroblast of Graves eye disease patient
The method comprises the following steps:
A) carrying out passage on orbital fibroblasts of patients with Graves eye disease in a proliferation culture medium to a third generation for experiment;
B) under the transforming growth factor-beta 1 treatment, the samples were further divided into control group (DMSO), Artemisinin (ARS) (10.0, 50.0 and 200.0. mu.M), Artesunate (ART) (0.5, 2.0 and 10.0. mu.M) and Dihydroartemisinin (DHA) (1.0, 5.0 and 20.0. mu.M) treatment groups at different concentrations;
C) after the cells are treated, the expression of the fibrosis marker is detected through a real-time fluorescence quantitative PCR experiment and an immunoblotting experiment.
As a result: as shown in FIGS. 4A-4B, artemisinin and its derivatives showed concentration-dependent significant inhibition of the expression of the fibrosis marker.
The invention firstly discovers that the artemisinin and the derivatives thereof can simultaneously act on various pathological mechanisms of eye orbit fibroblasts of a hyperthyroidism eye disease patient, including proliferation, differentiation, hyaluronic acid formation and fibrosis, and determines the potential treatment effect of the artemisinin and the derivatives thereof on future clinical application.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
1. The application of artemisinin or artesunate and dihydroartemisinin is the application of artemisinin, artesunate or dihydroartemisinin in preparing a medicament for treating and/or relieving Graves eye diseases.
2. Use according to claim 1, characterized in that: the artemisinin, artesunate or dihydroartemisinin inhibits the proliferation of orbital fibroblasts.
3. Use according to claim 1, characterized in that: the artemisinin, artesunate or dihydroartemisinin inhibits the adipogenic differentiation of orbital fibroblasts.
4. Use according to claim 1, characterized in that: the artemisinin, the artesunate or the dihydroartemisinin can inhibit the synthesis and secretion of the hyaluronic acid of the orbital fibroblast.
5. Use according to claim 1, characterized in that: the artemisinin, artesunate or dihydroartemisinin inhibits orbital fibroblast fibrosis.
6. Use according to any one of claims 1 to 5, characterized in that: the artemisinin, the artesunate or the dihydroartemisinin is prepared into a pharmaceutical preparation, and the preparation form is tablets or injection.
7. The use of artemisinin or artesunate or dihydroartemisinin as claimed in claim 6, wherein the content of artemisinin in the pharmaceutical preparation is 62.5 mg/tablet of artemisinin.
8. The use of artemisinin or artesunate or dihydroartemisinin as claimed in claim 6, wherein the content of dihydroartemisinin in the pharmaceutical preparation is 20 mg/tablet.
9. The use of artemisinin or artesunate or dihydroartemisinin as claimed in claim 6, wherein the content of artesunate in the pharmaceutical preparation is 50 mg/tablet of artesunate and 60 mg/injection.
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| Title |
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| 青蒿素类化合物新用途相关专利现状分析;张雄辉等;《中国发明与专利》;20181031;第15卷(第10期);第6-10页 * |
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