A butyrolactone derivative and autophagy inhibitor
3BDO is a butyrolactone derivative and inhibitor of autophagy.1,2 It increases phosphorylation of the mammalian target of rapamycin (mTOR) substrates eIF4E-binding protein 1 (EIF4EBP1) and RPS6KB1/p70S6K1 in human umbilical vein endothelial cells (HUVECs) when used at a concentration of 60 μM.1 3BDO (60 μM) also prevents rapamycin-induced MAP1LC3B puncta formation, a marker of autophagy, in HUVECs. It inhibits apoptosis, senescence, and increases in integrin β4 levels induced by serum- and FGF2-deprivation in HUVECs when used at a concentration of 40 μg/ml.3 3BDO (80 mg/kg per day) reduces cortical and hippocampal amyloid plaque burden, inhibits autophagy in the brain, and rescues learning and memory deficits in the AβPP/PS1 transgenic mouse model of Alzheimer's disease.2
1.Ge, D., Han, L., Huang, S., et al.Identification of a novel MTOR activator and discovery of a competing endogenous RNA regulating autophagy in vascular endothelial cellsAutophagy10(6)957-971(2014) 2.Wei, L., Yang, H., Xie, Z., et al.A butyrolactone derivative 3BDO alleviates memory deficits and reduces amyloid-β deposition in an AβPP/PS1 transgenic mouse modelJ. Alzheimers Dis.30(3)531-543(2012) 3.Wang, W., Liu, X., Zhang, Y., et al.Both senescence and apoptosis induced by deprivation of growth factors were inhibited by a novel butyrolactone derivative through depressing integrin β4 in vascular endothelial cellsEndothelium14(6)325-332(2007)
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Kinase experiment: |
Total protein is obtained from HUVECs by using of IP lysis buffer after treatment with rapamycin (10 μM), 3BDO (60 μM) or both for 6 h. After centrifuging at 4°C, the supernatant is collected and incubated with protein A/G agarose beads and TIA1 antibody or normal mouse IgG as a control at 4°C overnight. The beads are washed 3 times with IP lysis buffer and then eluted with 4×SDS loading buffer. Ser phosphorylation is detected by western blot assay with Ser phosphorylation antibody[1]. |
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Cell experiment: |
HUVECs are isolated from umbilical cords and cultured in M199 medium with 20% (v/v) fetal bovine serum and 10 IU/mL fibroblast growth factor 2 (FGF2) in a humidified incubator at 37°C with 5% CO2. Cells up to passage 10 are used for experiments. When HUVECs are grown to 80% confluency, HUVECs are treated with DMSO or 60 μM 3BDO for 24 h, then total RNA is extracted[1]. |
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Animal experiment: |
Male apoE-/- mice (8 weeks old) are used in this study. ApoE-/- mice are fed an atherogenic diet (containing 21% fat and 0.15% cholesterol). To avoid the potential confounding effects of variation among batches of diet, a single batch is reserved and used throughout the experiment. Mice at 20 weeks older are divided into 3 groups for treatment (n=8 mice/group) for 8 weeks: control (DMSO), low-dose 3BDO (50 mg/kg/d; 3BDO-L) and high-dose 3BDO (100 mg/kg/d; 3BDO-H). The body weight of mice is measured every week during 3BDO injection. Blood samples are taken from the inferior vena cava, and animals are killed by exsanguination[2]. |
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参考文献: [1]. Ge D, et al. Identification of a novel MTOR activator and discovery of a competing endogenous RNA regulating autophagy in vascular endothelial cells. Autophagy. 2014 Jun;10(6):957-71. |
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