Application of polypeptide in preparation of medicines for treating or preventing metabolic syndrome
A technology for metabolic syndrome and drugs, applied in metabolic diseases, drug combinations, pharmaceutical formulations, etc., can solve problems such as lack of TRB3 protein inhibitors, and achieve significant curative effects
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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 50μl of 100mM FFA solution to 950ul of 10%BSA solution, incubate in a water bath at 55°C for 10 minutes and then vortex for 10 seconds to prepare a 5mM 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.
[0058] The ...
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, discard the above culture medium.
[0074] (3) Wash 5 times with PBS.
[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 slide out of the well plate, and seal it with DAPI (purchased from Beijing Zhongshan Jinqiao Co., Ltd.) c...
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, 10mL10%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: 200 μL 2M β-glycerol phosphate, 4 mL 2.5M NaF, 2 mL 100 mM NaVO 3 , 2 mL of 100 mM PMSF, 200 μL of 1M DTT, 200 μL of 1 mg / mL Leu, Pep, and Apr each, with a total volume of 9 mL. 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 fo...
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