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Application of a polypeptide in the preparation of medicines for treating or preventing metabolic syndrome

A metabolic syndrome and drug technology, applied in metabolic diseases, drug combinations, pharmaceutical formulations, etc., can solve the problem of lack of TRB3 protein inhibitors and achieve significant curative effect

Active Publication Date: 2017-02-22
胡卓伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a polypeptide that can specifically bind to TRB3 and its derivatives in the preparation of drugs for the treatment and / or prevention of metabolic syndrome in view of the current lack of effective use of TRB3 protein inhibitors Applications

Method used

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  • Application of a polypeptide in the preparation of medicines for treating or preventing metabolic syndrome
  • Application of a polypeptide in the preparation of medicines for treating or preventing metabolic syndrome
  • Application of a polypeptide in the preparation of medicines for treating or preventing metabolic syndrome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 demonstrates that free fatty acids increase the expression level of TRB3 protein in mouse hepatocytes and block autophagic flux by immunoblotting.

[0055] The free fatty acid preparation method is as follows:

[0056] Weigh 106 μl of oleic acid (317.37ml / mol) and 0.0427g of palmitic acid (256.42g / mol) in 5ml of 0.1M NaOH solution and heat at 70°C until completely dissolved. In addition, incubate at 50°C with 10% BSA. Add 50 μl of 100 mM FFA solution to 950 ul of 10% BSA solution, incubate in a water bath at 55° C. for 10 minutes and then vortex for 10 seconds to prepare a 5 mM FFA / 10% BSA stock solution. Store at -20°C after filtration with a 0.45 μm syringe filter. When in use, take it out at 50°C to dissolve, and lower it to 37°C for use. and serum-free DMEM at a ratio of 1:9 to prepare 0.5mM fatty acid sodium high-fat medium.

[0057] Oleic acid and palmitic acid were purchased from Sigma Aldrich, USA, and DMEM was purchased from Invitrogen, USA.

[00...

Embodiment 2

[0070] Example 2 The combination of P62 protein and TRB3 protein was verified by laser confocal method.

[0071] Confocal laser detection was performed on the AML-12 cells treated with free fatty acids for 48 hours in Example 1, and the specific operations were as follows:

[0072] (1) Spread the cells in the logarithmic growth phase on a 12-well plate pre-placed with round slides (treated with polylysine), and culture overnight until the cells adhere to the wall.

[0073] (2) The next day, the above culture medium was discarded.

[0074] (3) Wash with PBS 5 times.

[0075] (4) Incubate overnight at 4°C with TRB3 and P62 primary antibodies.

[0076] (5) Wash 5 times with PBS the next day.

[0077] (6) Use specific secondary antibody (purchased from Beijing Zhongshan Jinqiao Co., Ltd.), incubate at 37 degrees for half an hour, wash 5 times with PBS

[0078] (7) Take the round glass slide out of the well plate, and seal it with DAPI (purchased from Beijing Zhongshan Jinqiao ...

Embodiment 3

[0081] Example 3 Co-immunoprecipitation verified that Pep2-A1, Pep2-A2 and Pep2-A3 can block the binding of P62 protein and TRB3 protein.

[0082] Co-immunoprecipitation reagents are as follows:

[0083] Lysis solution A: 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl 2 ·6H 2O, 0.0742g EDTA, 10mL glycerin, 10mL 10% NP40, add deionized water to 150mL, adjust the pH value to 7.6 with HCl, adjust the volume to 191mL, mix well, filter through a 0.45μm membrane filter, and store at 4°C.

[0084] Lysis Solution B: 200μL 2M β-glycerophosphate, 4mL 2.5M NaF, 2mL 100mM NaVO 3 , 2mL 100mMPMSF, 200μL 1M DTT, 200μL each of 1mg / mL Leu, Pep, and Apr, the total volume is 9mL. The mother liquor was stored at -20°C. Before use, thaw the mother liquor of each component in liquid B, add it to liquid A according to the above composition ratio and mix well.

[0085] Protein A / G Plus-Agarose was purchased from Santa Cruz, USA.

[0086] The specific operation steps are as follows:

[0087] (1)...

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PUM

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Abstract

The invention discloses an application of a polypeptide specifically bingd to TRB3 or a derivative of the polypeptide in the preparation of medicines for treating and / or preventing metabolic syndrome. The amino acid sequence of the polypeptide is represented by anyone of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and the derivative of the polypeptide is a chimeric peptide formed by connecting the polypeptide with a cell penetrating peptide and / or a medicinal composition formed by cooperating the polypeptide with a medicinal assistant. The polypeptide or the polypeptide derivative can specifically bind to the TRB3 in order to block out interaction between the TRB3 and a P62 protein, and the peptide derivatives Pep2-A1, Pep2-A2 and Pep3-A3 can be used to treat high fat diet induced mouse metabolic syndrome.

Description

technical field [0001] The present invention relates to the application of a polypeptide in the preparation of medicines for treating and / or preventing metabolic syndrome. Background technique [0002] Metabolic syndrome and its complications are complex metabolic disorders with high morbidity and mortality, which seriously threaten the health of the global population. Metabolic syndrome is associated with type II diabetes, nonalcoholic fatty liver, insulin resistance, obesity or overweight, atherosclerosis, hypertension, hyperinsulinemia, impaired glucose tolerance, diabetic cardiomyopathy, dyslipidemia, and tumor development . Among them, at least 2.8 million people die each year from obesity or overweight. The intake of high-calorie diet can induce multi-organ inflammatory response and disrupt the dynamic balance of cell metabolism, which is the primary inducing factor of metabolic syndrome. [0003] TRB3 (Tribbles Homologue 3) is one of the members of the Tribbles hom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/10A61K38/16A61P1/16A61P3/10A61P9/00A61P13/12A61P3/00
Inventor 胡卓伟余娇娇付小明
Owner 胡卓伟
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