Application of diosmetin and method for establishing animal model of diosmetin

Abstract

The invention discloses application of diosmetin and a method for establishing an animal model of the diosmetin, and relates to new application of the diosmetin, in particular to application of the diosmetin in treating systemic lupus erythematosus. Diosmetin is a natural flavonoid compound, due to the natural physicochemical property of diosmetin, compared with a biological agent, the diosmetin is higher in stability, lower in potential side effect, large in safe concentration range and obvious in effect on interferon-induced genes, in peripheral blood of lupus patients, the integral of interferon of the patients is remarkably reduced due to addition of diosmetin, and the effect of the diosmetin in the peripheral blood of the lupus patients is remarkably improved. The compound is a small-molecule inhibitor with good patent medicine potential, and is hopeful to relieve lupus patients of which the clinical'IFN signal 'is obviously increased.

Description

technical field

[0001] The invention relates to a new application of diosmin, in particular to the application of diosin for treating systemic lupus erythematosus. Background technique

[0002] Systemic Lupus Erythematosus (SLE) is a disease caused by autoimmune regulation disorder, which often accumulates in various tissues and organs of the body. The overactivation of the type I IFN signature (I IFN) pathway is an important pathogenesis in the pathogenesis of lupus. High levels of interferon (Interferon, IFN) in lupus patients are associated with nephritis, mucocutaneous manifestations, and autoantibody levels relevant. At present, the clinical treatment of systemic lupus erythematosus is mainly hormones and immunosuppressants. In recent years, some biological agents have begun to emerge. Compared with hormones and immunosuppressants, biological agents can specifically target the pathways of disease occurrence, and side effects Compared with traditional drugs, it is smal...

Examples

Embodiment 1

[0044] Embodiment 1, cell culture

[0045] THP1, raji, and jurkat cells were obtained from the American Type Culture Collection (ATCC). When recovering, take out the frozen cells from the liquid nitrogen tank, quickly place them in a 37°C water bath, and shake slightly to speed up the dissolution; add the frozen liquid to 9ml of preheated cell culture medium, mix them upside down; centrifuge at 20°C and 90G for 5 Minutes; remove the supernatant, add 1ml of cell culture medium to resuspend the cells, transfer the cells to a cell culture dish or flask, and indicate the cell type, generation, date and name. Culture in a 25cm cell culture flask containing 1640 (plus 10% calf serum) medium, and transfer to a 75cm cell culture flask when the number of cells increases enough. All cells were cultured in a 37°C incubator containing 5% CO2, subcultured and replaced every 3 days, and the number of cell generations used in the experiment was maintained within 20 passages.

Embodiment 2

[0046] Embodiment 2, real-time fluorescent quantitative PCR (Real-time PCR)

[0047] PBMC from healthy people were collected, and CD3+T cells, CD19+B cells, and CD14+monocytes were obtained by magnetic bead sorting. The experimental group was diosmin, and the control group was DMSO. Cells were cultured in 24 wells, and diosin and DMSO were added. The control was incubated for 30min, and then Type I IFN (1000u / ml, PBL company) was added to stimulate. After stimulation for 0, 2h, 4h and 6h, the supernatant was removed and extracted using phenol-chloroform method. Cells in each well were 0.5mL Trizol (Invitrogen company ) to collect RNA, add 100uL chloroform and mix well, let stand for 3 minutes, centrifuge at 12000g 4°C for 15 minutes, absorb the supernatant and add 300uL isopropanol, mix well, let stand for 10 minutes, centrifuge at 12000g 4°C for 10 minutes, remove the supernatant, Wash twice with 500uL of 75% ethanol, and centrifuge at 4°C for 5 minutes at 7500g each time. I...

Embodiment 3

[0050] Embodiment 3, immunoblotting experiment (Western Blot)

[0051] THP1, raji, and jurkat cells were cultured in 6-well plates, diosmin and DMSO were added to incubate for 30min, and then Type I IFN (1000u / ml, PBL company) was added to stimulate for 0, 5min, 10min, 30min, 1h, and then removed The supernatant was extracted with the lysis method of RIPA Buffer (product of Thermo Company), and the obtained protein was diluted with 5×SDS-PAGE buffer solution (product of Beyotime Company), and denatured. The Bio-Rad vertical electrophoresis system was used for protein electrophoresis, and then the Bio-Rad wet transfer system and PVDF membrane were used for protein transfer, and then the PVDF membrane was blocked with BlockingBuffer (Thermo company product), the primary antibody was incubated overnight at 4°C, washed with PBST, and the secondary antibody Incubate at room temperature for 1 h, wash with PBST, and develop using Pierce ECL Western Blotting Substrate (product of Ther...

technical field

[0001] The invention relates to a new application of diosmin, in particular to the application of diosin for treating systemic lupus erythematosus. Background technique

[0002] Systemic Lupus Erythematosus (SLE) is a disease caused by autoimmune regulation disorder, which often accumulates in various tissues and organs of the body. The overactivation of the type I IFN signature (I IFN) pathway is an important pathogenesis in the pathogenesis of lupus. High levels of interferon (Interferon, IFN) in lupus patients are associated with nephritis, mucocutaneous manifestations, and autoantibody levels relevant. At present, the clinical treatment of systemic lupus erythematosus is mainly hormones and immunosuppressants. In recent years, some biological agents have begun to emerge. Compared with hormones and immunosuppressants, biological agents can specifically target the pathways of disease occurrence, and side effects Compared with traditional drugs, it is smal...

Examples

Embodiment 1

[0044] Embodiment 1, cell culture

[0045] THP1, raji, and jurkat cells were obtained from the American Type Culture Collection (ATCC). When recovering, take out the frozen cells from the liquid nitrogen tank, quickly place them in a 37°C water bath, and shake slightly to speed up the dissolution; add the frozen liquid to 9ml of preheated cell culture medium, mix them upside down; centrifuge at 20°C and 90G for 5 Minutes; remove the supernatant, add 1ml of cell culture medium to resuspend the cells, transfer the cells to a cell culture dish or flask, and indicate the cell type, generation, date and name. Culture in a 25cm cell culture flask containing 1640 (plus 10% calf serum) medium, and transfer to a 75cm cell culture flask when the number of cells increases enough. All cells were cultured in a 37°C incubator containing 5% CO2, subcultured and replaced every 3 days, and the number of cell generations used in the experiment was maintained within 20 passages.

Embodiment 2

[0046] Embodiment 2, real-time fluorescent quantitative PCR (Real-time PCR)

[0047] PBMC from healthy people were collected, and CD3+T cells, CD19+B cells, and CD14+monocytes were obtained by magnetic bead sorting. The experimental group was diosmin, and the control group was DMSO. Cells were cultured in 24 wells, and diosin and DMSO were added. The control was incubated for 30min, and then Type I IFN (1000u / ml, PBL company) was added to stimulate. After stimulation for 0, 2h, 4h and 6h, the supernatant was removed and extracted using phenol-chloroform method. Cells in each well were 0.5mL Trizol (Invitrogen company ) to collect RNA, add 100uL chloroform and mix well, let stand for 3 minutes, centrifuge at 12000g 4°C for 15 minutes, absorb the supernatant and add 300uL isopropanol, mix well, let stand for 10 minutes, centrifuge at 12000g 4°C for 10 minutes, remove the supernatant, Wash twice with 500uL of 75% ethanol, and centrifuge at 4°C for 5 minutes at 7500g each time. I...

Embodiment 3

[0050] Embodiment 3, immunoblotting experiment (Western Blot)

[0051] THP1, raji, and jurkat cells were cultured in 6-well plates, diosmin and DMSO were added to incubate for 30min, and then Type I IFN (1000u / ml, PBL company) was added to stimulate for 0, 5min, 10min, 30min, 1h, and then removed The supernatant was extracted with the lysis method of RIPA Buffer (product of Thermo Company), and the obtained protein was diluted with 5×SDS-PAGE buffer solution (product of Beyotime Company), and denatured. The Bio-Rad vertical electrophoresis system was used for protein electrophoresis, and then the Bio-Rad wet transfer system and PVDF membrane were used for protein transfer, and then the PVDF membrane was blocked with BlockingBuffer (Thermo company product), the primary antibody was incubated overnight at 4°C, washed with PBST, and the secondary antibody Incubate at room temperature for 1 h, wash with PBST, and develop using Pierce ECL Western Blotting Substrate (product of Ther...

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