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Action mechanism of silybin for preventing liver apoptosis through FXR (Farnesoid X Receptor) path

A technology of silibinin and its mechanism of action, applied in the research progress field of silibinin exerting anti-hepatocyte apoptosis via FXR-dependent pathway

Inactive Publication Date: 2014-01-08
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of drugs with specific targets for the prevention and treatment of liver injury.

Method used

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  • Action mechanism of silybin for preventing liver apoptosis through FXR (Farnesoid X Receptor) path
  • Action mechanism of silybin for preventing liver apoptosis through FXR (Farnesoid X Receptor) path
  • Action mechanism of silybin for preventing liver apoptosis through FXR (Farnesoid X Receptor) path

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Embodiment 1.PCR experiment

[0012] Experimental Materials:

[0013] Human liver cancer cell line HepG2 cells were purchased from Shanghai Cell Bank and stored at 37°C in 5% CO 2 Under normal conditions, the culture medium was DMEM (Gibco) containing 10% calf serum; fetal bovine serum (Fetal bovine serum) was purchased from Hyclone (Logan, Utah, USA); trypsin (Trypsin) was purchased from Amersco (Solon, Ohio, USA); penicillin, streptomycin and dimethylthiodiphenyl tetrathiazole (MTT) were all purchased from Nanjing Shengxing Bioengineering Company (Jiangsu); FXR primers and internal reference GAPDH primers, Control SiRNA and FXR SiRNA, RNAiMAX transfection reagents were purchased from Invitrogen; Trizol Total RNA Extraction Reagent, RT-PCR Kit, Real-time PCR Master Mix (SYBR Green) were purchased from Takara (Dalian Bao Biology); FXR antibody was purchased from Santa Cruz Company, GAPDH antibody Purchased from Bioworld; RIPA cell lysate, Hoechst33342 staining solutio...

Embodiment 2

[0020] Embodiment 2.Western Blot experiment

[0021] Experimental material: with embodiment 1.

[0022] experimental method:

[0023] After HepG2 cells were seeded in six-well plates for 48 hours, they were stimulated with 0, 5, 10, 20, and 50 μM silibinin for 24 hours, respectively. In addition, HepG2 cells were stimulated with ActD (0.2 μM) and TNFα (20 ng / ml) 48 hours after seeding in six-well plates, and stimulated with different doses of silibinin (0, 10, 20 and 50 μM) for 12 hours. After washing the cells once with PBS, place them on an ice plate, scrape the cells with a cell scraper, and centrifuge at 8000rpm×1min to obtain the cell pellet. Add 100μL RIPA cell lysate to each 20μL cell volume, lyse on ice for 30min, and ultrasonicate Broken 5 times, 2s each time, centrifuged at 15000rpm×10min to obtain the supernatant which is the extracted total protein sample. Protein concentration was determined by BCA method. Protein samples were added to loading buffer at a volu...

Embodiment 3

[0024] Embodiment 3.MTT experiment

[0025] Experimental material: with embodiment 1.

[0026] experimental method:

[0027] 1.MTT experiment

[0028] HepG2 cells were seeded in 96-well plates, stimulated with ActD (0.2μM) and TNFα (20ng / ml) after 48h, and given different doses of silibinin (0, 10, 20 and 50μM). After 12 hours, add 20 μL (5 mg / mL) MTT to each well, incubate in a carbon dioxide incubator for 4 hours, remove the MTT solution, add DMSO (150 μL / well) to dissolve the crystals (37°C shaker, 50 r / min), and take it out after 30 minutes. The absorbance was measured in a microplate reader (measurement wavelength 490nm, reference wavelength 630nm). The cell survival rate of the administration group was calculated by the following formula:

[0029] Survival rate = absorbance of treatment group / absorbance of control group * 100%

[0030] 2. FXR gene silencing

[0031] According to the instruction manual of RNAiMAX transfection reagent (Invitrogen), add the reagent ...

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Abstract

The invention relates to the field of natural medicines, and in particular relates to an action mechanism of silybin for preventing liver apoptosis through an FXR (Farnesoid X Receptor) path. According to the invention, the silybin prevents apoptosis of HepG2 cells caused by ActD / TNF alpha through an FXR dependent path. Within human hepatoma cell line HepG2 cells, silybin can induce up-regulation of mRNA level and protein level of FXR in a dose-dependent manner, and can reverse down-regulation of FXR expression in a HepG2 cell apoptosis model caused by ActD / TNFalpha remarkably; the silybin has remarkable protective effect on the HepG2 cell apoptosis caused by the ActD / TNFalpha, but the anti-apoptosis function of the silybin is lost after the FXR SiRNA is used for silencing endogenous FXR genes.

Description

technical field [0001] The present invention relates to the field of natural medicines, in particular to the research progress of silibinin in anti-hepatocyte apoptosis via FXR-dependent pathway. Background technique [0002] Milk thistle (Silybin, SB) has been used to treat liver and gallbladder diseases for more than 2,000 years. Its main active ingredient silybin is flavonoid lignans, and its chemical structure is as follows: [0003] [0004] Silibinin can prevent liver damage caused by chemical toxins, food toxins and drugs. It enters the nucleus and acts on RNA polymerase, increases the formation of ribosomes, stimulates protein synthesis, and promotes the regeneration and repair of liver cells. It is known as "Natural liver protection drug"; it can maintain the fluidity of cell membrane, protect liver cell membrane, increase the level of GSH in the liver and other tissues; it can also act as an antioxidant to remove free radicals in the body and reduce the generati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/357A61P1/16
Inventor 王广基郝海平王洪颜婷婷赵敏
Owner CHINA PHARM UNIV
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