CN112618548A - Application of SRT1720 in preparation of medicines for treating Graves' disease hyperthyroidism inflammation - Google Patents

Abstract

The invention relates to application of SRT1720 in preparation of drugs for treating Graves' hyperthyroidism.

Description

Application of SRT1720 in preparation of medicines for treating Graves' disease hyperthyroidism inflammation

Technical Field

The invention belongs to the field of application of SRT1720, and particularly relates to application of SRT1720 in preparation of drugs for treating Graves' disease hyperthyroidism inflammation.

Background

Graves' disease (GD) is the most common cause of hyperthyroidism in non-iodine deficient areas, and this patient develops an autoimmune response against the thyroid gland, including lymphocyte infiltration and the production of autoantibodies specific for thyroglobulin, thyroid peroxidase and Thyroid Stimulating Hormone Receptor (TSHR). In addition to being influenced by genetic and environmental factors, immune disorders are also involved in the development of the Graves' disease hyperthyroidism. It has been suggested that antigen-presenting cells, the interaction between autoreactive T cells and thyroid follicular cells, elicit an immune response against thyroid autoantigens. Although the development of antibodies against autoantigens has been revealed both to break immune tolerance and to disrupt the adaptive immune system of the body, the underlying mechanism by which the Graves' disease is hyperthyroidically developed is still unclear.

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of SRT1720 in preparation of medicines for treating inflammation of Graves' disease hyperthyroidism patients.

The chemical structural formula of the SRT1720 is

The SRT1720 is prepared into a preparation by using pharmaceutically acceptable auxiliary materials or auxiliary components.

The composition is selected from one of tablets, powder, granules, capsules, oral liquid and sustained release agents.

Advantageous effects

The invention explores new medical application of SRT 1720;

the SRT1720 of the invention can treat inflammation in peripheral blood mononuclear cells of Graves' hyperthyroidism patients, and has a remarkable effect.

Drawings

FIG. 1 is a graph of SRT1720 reducing the inflammatory response in peripheral blood mononuclear cells from patients with Graves' disease hyperthyroidism; wherein (A) luciferase reporter gene detects NF-kB promoter activity, (B-K) peripheral blood mononuclear cells are added with SRT1720(2 mu M) -SIRT1 agonist for 24 hours, (B) Western Blotting detects P65 acetylation level, (C) peripheral blood mononuclear cells are added with lipopolysaccharide (50ng/mL) for 5 hours of final incubation, and fluorescent quantitative PCR detects TNF-alpha (D), IL-6(E), IL-8(F) and MCP1(G) mRNA expression level, and (H-K) ELISA method is used for quantifying TNF-alpha (H), IL-6 (I), IL-8(J) and MCP1(K) protein level in cell supernatant.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

Peripheral blood mononuclear cells were treated with SRT1720 and then tested:

1. SRT1720 was dissolved using DMSO, mixed using a pipette, gently mixed, and then DMSO dissolved SRT1720 and DMSO used for control were filtered using Millipore disposable needle filters 0.22 μm.

2. Peripheral Blood Mononuclear Cells (PBMC) according to 1 x 10⁶The cells were seeded in a 12-well plate at a density of one ml, peripheral blood mononuclear cells were cultured in a serum-free medium for 12 hours, and the prepared SRT1720 was added to adjust the final concentration of the SRT1720 to 2. mu.M.

Luciferase enzyme activity detection:

1. cells were seeded one day in advance at a density of about 50% in 24-well plates, 400ul of antibiotic-free medium containing 10% FBS per well;

2. diluting plasmid NF-. kappa.BETA (0.4 ug/well) and plasmid Sv40 (0.01 ug/well) with 50ul of Opti-MEM, taking care that the mixing is gentle, preparing another EP tube, mixing the liquid in an amount of 50. mu.l of Opti-MEM and 1ul of Lipofectamine 2000 per well, and incubating in a super clean bench for 5 min;

3. uniformly mixing 2 culture solutions in the step 2 by using a liquid transfer device, paying attention to gentle force, incubating for 20min in an ultra-clean bench, adding the culture solutions into a 24-pore plate according to the amount of 400ul of each pore during incubation, adding 100ul of mixed solution into each pore after 20min, simultaneously using the SRT1720 dissolved by DMSO and the control DMSO, wherein the final concentration of the SRT1720 is 2 mu M, adding the cell culture plate which is gently shaken before and after the cell culture plate is added, and putting the cell culture plate into a 37 ℃ cell culture box; the plasmid-transfected cells were harvested after 24h for luciferase activity assays, according to the instructions of the Promega dual luciferase protocol.

The immunofluorescence staining specifically comprises the following steps:

1. and taking out the unused glass slide, and placing the slide throwing device in a position to be placed in the cell slide throwing instrument according to the instruction.

2. SRT1720 treated peripheral blood mononuclear cells and DMSO treated peripheral blood mononuclear cells were subjected to cell number adjustment to 10⁶After being mixed uniformly, 100ul of the mixture is sucked by a liquid transfer device and added to a flail apparatus;

3. setting the speed of the centrifuge to 800rpm at 25 ℃, centrifuging for 5min at the speed, and adopting a low-speed mode to participate in acceleration and deceleration processes in the centrifuging process;

4. after completion, the slides were air dried in a dry and ventilated place.

5. Putting 4% paraformaldehyde into a refrigerator at 4 ℃ in advance, and circling the glass slide dried in the step 4 by using a grouping pen

The cells were incubated for 15 min.

6. The slides were washed three times with PBS, 5min each time at room temperature and gently shaken on a shaker.

7. Immersed in 0.2% PBST for 10min and shaken gently in a shaker at room temperature.

8. PBS was washed once for 5min and shaken gently on a shaker at room temperature.

9. Sections were blocked with antibody dilutions for 1 hour.

10. Primary antibody (SIRT 11: 100) diluted with antibody diluent in corresponding proportion was incubated at 4 deg.C

Overnight.

11. Taking out at 4 deg.C, and rewarming at room temperature for 30 min.

12. PBS was washed three times for 10min each time, and shaken gently in a shaker at room temperature.

13. Donkey anti-rabbit fluorescent secondary antibody (1: 500 dilution in PBS) was added and incubated in the wet box for 1 hour at room temperature.

14. PBS was washed three times for 10 minutes each time with gentle shaking on a shaker at room temperature.

15. Add anti-quench mounting agent mounting containing DAPI.

16. The images were taken by confocal microscopy.

Fluorescent real-time quantitative PCR

Fluorescent real-time quantitative PCR reaction system:

volume of reagent (μ l) for reaction

SYBR Premix Ex TaqTM(2×)5

cDNA template 2

Upstream primer (10. mu.M) 0.5

RNase-free Water 2

Downstream primer (10. mu.M) 0.5

Conditions are as follows:

the first step is as follows: 95 ℃ for 10s

The second step is that: setting temperature at 95 deg.C for 5s and setting temperature at 60 deg.C for 31s, and repeating 40 cycles

And a last step: the dissociation curve was determined.

As a result:

luciferase reporter gene experiments demonstrated that SRT1720 treated HEK-293 cells have reduced NF- κ BETA transcriptional activity (FIG. 1A). Meanwhile, Western blot analysis proves that the level of acetylated p65 protein in peripheral blood mononuclear cells of patients with SRT 1720-treated Graves' disease hyperthyroidism is reduced (figure 1B), and immunofluorescence experiment results show that p65 enters cytoplasm from the nucleus after SRT1720 is added, which indicates that the activity of NF-kappa BETA pathway is reduced (figure 1C). Moreover, SRT1720 treatment resulted in NF- κ beta-targeted pro-inflammatory genes: the mRNA expression levels of TNF- α (FIG. 1D), IL-6 (FIG. 1E), IL-8 (FIG. 1F) and MCP1 (FIG. 1G) were decreased with or without lipopolysaccharide addition. In addition, TNF-. alpha. (FIG. 1H), IL-6 (FIG. 1I), IL-8 (FIG. 1J) and MCP1 (FIG. 1K) protein levels were significantly reduced in supernatants of monocytes cultured in vitro in patients with Graves' disease hyperthyroidism following SRT1720 treatment of peripheral blood mononuclear cells. In conclusion, the results of the invention show that SRT1720 activates SIRT1 activity, which is beneficial to relieving the inflammatory response of Graves' hyperthyroidism patients.

Claims (4)

1. An application of SRT1720 in preparing medicine for treating Graves' disease hyperthyroidism inflammation.

2. The use of claim 1, wherein the SRT1720 has the chemical formula

3. The use of claim 1, wherein the SRT1720 is formulated with a pharmaceutically acceptable excipient or adjuvant.

4. The use according to claim 3, wherein the medicament is one selected from the group consisting of tablets, powders, granules, capsules, oral liquids, and sustained release agents.

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