Screening Of TFEB-Mediated Autophagy Agonists And Their Role In Lipid Degradation Of Hepatocytes

When cells are in a state of stress,they will provide necessary materials and energy through autophagy by degrading their own protein self-aggregation and damaged organelles and other non-essential components,thus maintaining homeostasis and metabolic balance of internal environment.The dysfunction of autophagy is closely related to the occurrence of many diseases such as abnormal lipid metabolism,inflammation,tumor and nervous system disorders.Transcription factor TFEB is one of the main intracellular precursors to autophagy.TFEB regulates the expression of autophagy-related genes.TFEB is activated and phosphorylated by the m TOR protein complex 1（m TORC1）located on the surface of the lysosome membrane.Phosphorylated TFEB is transferred from the cytoplasm to the nucleus.Therefore,targeting TFEB to screen compounds that regulate autophagy provides theoretical basis for drug development of autophagy-lysosomal related diseases.In this study,two He La cell lines with stably expressed TFEB-GFP and GFP-LC3were used to screen out drugs that can initiate autophagy by TFEB from a library of small molecular compounds and investigate their mechanisms of action.The main experimental results include:（1）Two small molecule compounds,Nebivolol HCl and Bazedoxifene HCl,were found to induce TFEB to enter the nucleus by screening stably expressing TFEB-GFP cell lines.Nucleus and cytoplasm separation experiments showed that both compounds could induce nuclear localization of endogenous TFEB.（2）Western blot analysis showed that there was no significant change in p-AMPK protein level in He La cells treated by Nebivolol HCl and Bazedoxifene HCl.The results showed that Nebivolol HCl and Bazedoxifene HCl induced TFEB activation independently of AMPK signaling pathway.In addition,the protein level of p-P70S6K increased after Nebivolol HCl treatment,and decreased after Bazedoxifene HCl treatment.The results showed that Nebivolol HCl activated TFEB independently of m TORC1 signaling pathway,but Bazedoxifene HCl activated TFEB through m TORC1 signaling pathway.（3）BAPTA-AM was used to detect the effect of Ca2+on TFEB activation.It was found that Nebivolol HCl and Bazedoxifene HCl could induce TFEB activation,but TFEB activation was independent of Ca²⁺.（4）Stably expressed GFP-LC3 He La cells were treated with Nebivolol HCl and Bazedoxifene HCl,respectively,resulting in increased fluorescence spot distribution.Meanwhile,Western blot showed that LC3-II and free-GFP proteins were increased.In addition,the co-treatment of these two small molecule compounds with Baf A1 resulted in further accumulation of LC3-II protein level,suggesting that Nebivolol HCl and Bazedoxifene HCl promoted autophagy.（5）Acridine orange（AO）staining experiment showed that both Nebivolol HCl and Bazedoxifene HCl could increase the number of lysosomes.Western blot results showed that LAMP2,CTSB and CTSD protein levels increased after treatment with these two small molecule compounds.In conclusion,both Nebivolol HCl and Bazedoxifene HCl can enhance the activity of autophagic lysosomal pathway.（6）The effect of two small molecular compounds on lipid droplet metabolism was detected by inducing lipid droplet experiment in vitro.The results showed that Nebivolol HCl could promote lipid droplet clearance in Hep G2 cells,but Bazedoxifene HCl could not.In summary,Nebivolol HCl and Bazedoxifene HCl were identified in this study as small molecular compounds that activate TFEB to initiate autophagy,both of which can enhance lysosome function.Activation of TFEB by Bazedoxifene HCl depends on the m TORC1 signaling pathway.Nebivolol HCl can promote the removal of lipid droplets from liver cells.These results provide theoretical basis for the research of autophagy activator and drug development of lipid metabolism diseases.