Bavachinin protects the liver in NAFLD by promoting regeneration via targeting PCNA

Introduction: Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease all over the world, and no drug is approved for the treatment of NAFLD. Bavachinin (BVC) is proven to possess liver-protecting effect against NAFLD, but its mechanism is still blurry. Objectives: With the use of Click Chemistry-Activity-Based Protein Profiling (CC-ABPP) technology, this study aims to identify the target of BVC, and investigate the mechanism by which BVC exerts its liverprotecting effect. Methods: The high fat diet induced hamster NAFLD model is introduced to investigate BVC's lipidlowering and liver-protecting effects. Then, a small molecular probe of BVC is designed and synthesized based on the CC-ABPP technology, and BVC's target is fished out. A series of experiments are performed to identify the target, including competitive inhibition assay, surface-plasmon resonance (SPR), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and coimmunoprecipitation (Co-IP). Afterward, the pro-regeneration effects of BVC are validated in vitro and in vivo through flow cytometry, immunofluorescence, and the terminal deoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL). Result: In the hamster NAFLD model, BVC shows lipid-lowing effect and improvement on the histology. PCNA is identified as the target of BVC with the method mentioned above, and BVC facilitates the interaction between PCNA and DNA polymerase delta. BVC promotes HepG2 cells proliferation which is inhibited by T2AA, an inhibitor suppresses the interaction between PCNA and DNA polymerase delta. In NAFLD Conclusion: This study suggests that, besides the anti-lipemic effect, BVC binds to the pocket of PCNA facilitating its interaction with DNA polymerase delta and pro-regeneration effect, thereby exerts the pro