Compound for preventing and treating metabolic diseases and application thereof
A technology for metabolic diseases, compounds, applied in the field of pharmacy
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[0232] 10) The preparation method of the CsCl gradient is as follows: add 2.0ml of CsCl solution with a density of 1.40g / ml (the same solvent as above), then add 3.0ml of a CsCl solution with a density of 1.30g / ml, and then add 5ml of virus suspension. Centrifuge at 20,000 rpm for 2 hours at room temperature.
[0233] 11) Collect the virus bands with a density between 1.30g / ml and 1.40g / ml into the dialysis bag (use 10mM EDTA-Na 2 boil for 10 minutes). In dialysis buffer (50g sucrose, 10ml 1M Tris-HCl, pH8.0, 2ml 1M MgCl 2 Dilute to 1 L), dialyze at 4°C for 24 hours, and change the dialysate once in the middle. The virus was collected, and the virus titer was determined (see the manual of the Stratagene Virus Titer Quantification Kit for the specific determination method).
[0234] Adenovirus-mediated SCAP gene silencing in mouse liver
[0235] The inventor adopts AdEasy TM The adenoviral expression vector (purchased from Stratagene) system mediated mouse liver SCAP gene ...
Embodiment 1
[0247] Example 1, Betulin is a specific inhibitor of the SREBP pathway
[0248] The active form of SERBP in the nucleus promotes the expression of downstream genes by combining with the sterol regulatory element (SRE) in the promoter region of the target gene, thereby promoting the biosynthesis of lipids (Wang, X. et al. (1993). Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization. J. Biol. Chem. 268, 14497-14504). The present inventors have constructed a luciferase reporter gene system, the promoter region of the luciferase contains a sterol regulatory element (SRE), and the luciferase is stably expressed in the human liver cancer cell line Huh-7 cells, and finally Huh -7 / SRE-Luc cells ( figure 1 A) Use as a screen for small molecule inhibitors of the SREBP pathway. After the cells are treated with different candidate compounds for a certain period of time, the cells are collected and the ch...
Embodiment 2
[0252] Example 2, Betulin down-regulates the expression of SERBP target genes and reduces cellular lipid synthesis
[0253] Since SREBP-2 is its own target gene (Sato, R. et al. (1996).Sterol-dependent transcriptional regulation of sterol regulatory element-binding protein-2.J.Biol.Chem.271,26461-26464), so SERBP -2 expression was downregulated by approximately 40% after betulin treatment. At the same time, the inventors detected the expression of other dozens of SREBP-2 target genes involved in the cholesterol synthesis pathway such as HMGCR, HMG-CoA synthetase (HMGCS), and Squalene Epoxidase (SE) after betulin treatment were significantly down-regulated ( Figure 4 A). At the same time, the expression levels of genes and enzymes involved in the cellular fatty acid synthesis pathway, such as SREBP-1c, fatty acid synthase (FAS), and acetyl-CoA carboxylase α (ACC), were significantly down-regulated by betulin ( Figure 4 B). Furthermore, consistent with previous observation...
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