Rapamycin and 3-Methyladenine Influence the Apoptosis, Senescence, and Adipogenesis of Human Adipose-Derived Stem Cells by Promoting and Inhibiting Autophagy: An In Vitro and In Vivo Study

Objective: We aimed to clarify the changes in Apoptosis, proliferation, senescence, and adipogenesis after promoting and inhibiting Autophagy in adipose-derived stem cells (ADSCs) by rapamycin and 3-methyladenine in vitro and in vivo.

Methods: After rapamycin and 3-methyladenine pretreatment, ADSC Autophagy was detected by immunofluorescence for LC3, RT-PCR for ATG genes, and western blotting (WB) for the LC3 II/I and p62 proteins. TUNEL staining, PCR of Bax, and WB of Caspase-3 were preformed to assess ADSC Apoptosis. The adipogenesis of ADSCs was evaluated by Oil red O staining and PCR of PPAR-γ. CCK8 assays were conducted to detect proliferation. Senescence was tested by Sa-β-gal staining and PCR of the P16/ 19/21 genes. Moreover, the mass and volume retention rate were determined, and perilipin and CD31 staining were performed in vivo.

Results: Rapamycin and 3-methyladenine pretreatment increased and decreased Autophagy of ADSCs, respectively, under normal and oxygen-glucose deprivation conditions. Apoptosis and senescence of ADSCs were decreased, and adipogenesis was increased along with the upregulation of Autophagy. However, the proliferation of ADSCs was inhibited after either rapamycin or 3-methyladenine pretreatment. In vivo, the volume and mass retention rate and the angiogenesis of the grafts were also improved after rapamycin pretreatment.

Conclusions: Rapamycin pretreatment reduced Apoptosis, delayed senescence, and promoted adipogenesis of ADSCs. These effects were inhibited by 3-methyladenine, indicating that the changes may be mediated by Autophagy. Moreover, the survival rate and angiogenesis of the grafts were increased after upregulation of ADSC Autophagy in vivo, which may help improve the efficiency of clinical fat transplantation.

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