A method and application of improving mitochondrial metabolic function
A technique for mitochondrial, metabolic functions, with applications in the fields of biochemistry and pharmacy
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[0092] 1. Preparation of AOX inhibitor and its inhibitory effect on AOX
[0093] The present inventors prepared LH-919-CoA. Using LH-919 acid as raw material, high-purity LH-919-CoA was prepared, and its inhibitory effect on AOX was tested. The synthesis method for preparing fatty acyl-CoA from free fatty acid is carried out according to literature (LiD.et.al., J.Am.Chem.Soc., 2001, 121, 9034-9042).
[0094] In the present invention, the inhibitory effect of the AOX inhibitor on AOX is tested in vitro and in animal body respectively.
[0095] The present inventors found for the first time that LH-919-CoA can significantly inhibit liver AOX activity in vitro. Firstly, the recombinantly expressed and highly purified rat liver AOX was used as the target protein to measure the inhibitory effect of LH-919-CoA on AOX. The results showed that LH-919-CoA could rapidly inhibit the activity of AOX, and this inhibitory effect was irreversible. Free LH-919 acid has no inhibitory effect...
Embodiment 1
[0155] Embodiment 1, preparation of AOX inhibitor and in vitro inhibition experiment
[0156] (1) Preparation of AOX inhibitor LH-919-CoA
[0157] The preparation method of LH-919-CoA was carried out according to the literature (Li D. et. al., J. Am. Chem. Soc., 2001, 121, 9034-9042). Concrete operation: LH-919 20mg (Sigma, St.Louis, MO), add 5mL anhydrous tetrahydrofuran (tetrahydrofuran, THF) (Acros, Geel, Belgium), add 0.1mmol triethylamine ( Triethylamine) (Sigma, St.Louis, MO), after mixing and stirring for 10 minutes, 0.1 mmol isobutyl chloroformate (isobutyl chloroformate) (Acros, Geel, Belgium) was added dropwise to the solution at 0°C, and stirred at room temperature for 1 h . Finally, dissolve 50 mg of coenzyme A sodium salt (Coenzyme A) (USB, Cleveland, OH) in distilled water, add 1 mol / L NaOH to adjust the pH to 8.0, add dropwise to the reaction solution under nitrogen protection, and continue stirring for 20 minutes. Slowly add 1M hydrochloric acid dropwise to ...
Embodiment 2
[0174] Example 2. Analysis of AOX inhibitor precursors inhibiting AOX in vivo in mice
[0175] (1) Experimental determination of the conversion of LH-919 into LH-919-CoA in animals
[0176] 6 Wistar rats of SPF grade, male, weighing 220-250g, after fasting for 12h, the 6 rats were divided into 2 groups, the control group and the drug group, 3 rats in each group, and the control group was given olive oil 0.2mL / In the drug group, LH-919 was dissolved in the corresponding volume of olive oil, LH-919 100 μg / kg was administered orally, and all rats were sacrificed 3 hours later. The liver was quickly dissected and removed, and immediately stored in liquid nitrogen.
[0177] The determination of fatty acyl-CoA content in subcellular units of liver cells was carried out by reference (Melde K. et. al., Biochem. J., 1991, 274, 395-400). The operation steps are as follows: Accurately weigh 0.3 g of liver tissue, cut it into pieces on an ice bath, suspend in 9 times the volume of homog...
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