Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100193, China; Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Beijing 100193, China; Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing 100193, China; Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glyeolipid Metabolism Disorder Disease, State Administration of Traditional Chinese Medicine, Beijing 100193, China.
Beijing Increasepharm Safety and Efficacy Co., Ltd, Beijing, China.
J Adv Res. 2024 Jan;55:131-144. doi: 10.1016/j.jare.2023.02.007. Epub 2023 Feb 16.
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.
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 liver-protecting effect.
The high fat diet induced hamster NAFLD model is introduced to investigate BVC's lipid-lowering and liver-protecting effects. Then, a small molecular probe ofBVC is designed and synthesized based on theCC-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 co-immunoprecipitation (Co-IP). Afterward, the pro-regeneration effects of BVC are validated in vitro and in vivo through flow cytometry, immunofluorescence, and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).
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 hamsters, BVC enhances PCNA expression and liver regeneration, reduces hepatocyte apoptosis.
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 protective effect against HFD induced liver injury.
非酒精性脂肪性肝病（NAFLD）是全世界最常见的肝脏疾病，目前尚无药物被批准用于治疗 NAFLD。白桦脂酸（BVC）已被证明具有抗 NAFLD 的肝脏保护作用，但作用机制仍不清楚。
本研究采用点击化学-活性蛋白质谱（CC-ABPP）技术，旨在鉴定 BVC 的靶点，并探讨 BVC 发挥肝脏保护作用的机制。
采用高脂饮食诱导的金黄地鼠 NAFLD 模型，研究 BVC 的降脂和护肝作用。然后，根据 CC-ABPP 技术设计并合成了 BVC 的小分子探针，钓出 BVC 的靶标。通过一系列实验，包括竞争性抑制试验、表面等离子体共振（SPR）、细胞热转移试验（CETSA）、药物亲和反应靶标稳定性（DARTS）试验和免疫共沉淀（Co-IP），鉴定靶标。随后，通过流式细胞术、免疫荧光和末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）实验，在体外和体内验证了 BVC 的促再生作用。
在金黄地鼠 NAFLD 模型中，BVC 显示出降脂作用和改善组织学的作用。通过上述方法，鉴定出 PCNA 是 BVC 的靶标，BVC 促进 PCNA 与 DNA 聚合酶 delta 的相互作用。BVC 促进 HepG2 细胞增殖，而 T2AA 抑制剂抑制 PCNA 与 DNA 聚合酶 delta 的相互作用。在 NAFLD 金黄地鼠中，BVC 增强 PCNA 表达和肝再生，减少肝细胞凋亡。
本研究表明，除了降脂作用外，BVC 与 PCNA 的口袋结合，促进其与 DNA 聚合酶 delta 的相互作用，发挥促再生作用，从而对 HFD 诱导的肝损伤发挥保护作用。
