Department of Infectious Disease, The Affiliated Hospital of Guizhou Medical University, Guiyang 550000, China.
Department of Vascular Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang 550000, China.
Phytomedicine. 2024 Dec;135:156047. doi: 10.1016/j.phymed.2024.156047. Epub 2024 Sep 13.
Hepatic fibrosis is a pathological process in a variety of acute or chronic liver injuries. Catalpol (CAT), an iridoid glycoside found in Rehmannia glutinosa, has several pharmacological properties, including anti-inflammatory, antidiabetic and anti-fibrotic effects. Nevertheless, there is currently no report on whether CAT regulates the aerobic glycolysis of hepatic stellate cells (HSCs) to inhibit liver fibrosis.
This study aimed to investigate the protective effects of CAT on hepatic fibrosis and elucidate its underlying mechanisms.
To explore whether CAT improved liver fibrosis in vivo and in vitro, hepatic fibrosis was induced to mice by intraperitoneally injecting carbon tetrachloride (CCl). Additionally, LX-2 cells were stimulated with transforming growth factor-β (TGF-β) to simulate fibrosis in vitro. Serum markers of liver injury were examined by using an automatic biochemical analyzer. Histopathological staining, Immunofluorescence (IF) staining, Western blot (WB) analysis, co-immunoprecipitation (Co-IP), drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), etc. were employed to identify the targeting between CAT and EphA2 and detect the expression of aerobic glycolysis related proteins, fiber markers and signaling pathways that are responsible for CAT's anti-fibrotic effects of CAT.
Results showed that CAT significantly inhibited hepatic injury, fibrogenesis and inflammation in mice treated with CCl. This was demonstrated by the enhancement of fibrosis markers, liver function indices, and histopathology. In addition, CAT significantly inhibited the activation of HSCs in TGF-β-induced LX-2 cells, as indicated by decreased proliferation, migration, and expression of collagen I and a-SMA. The study results also suggested that CAT may exert anti-fibrotic effects by inhibiting glycolysis in activated HSCs and in CCl-treated mice. Mechanistically, CAT directly targets Ephrin type-A receptor 2 (EphA2) to reduce binding with focal adhesion kinases (FAK) and significantly inhibits the FAK/Src pathway. In addition, the pharmacological inhibition of EphA2 cannot further increase the therapeutic effects of CAT on liver fibrosis in vivo and in vitro.
The study findings generally demonstrated that CAT presented a novel therapeutic method to treat hepatic fibrosis; this method which inhibits the aerobic glycolysis of activated HSCs through the EphA2/FAK/Src signaling pathway.
肝纤维化是多种急性或慢性肝损伤的病理过程。在地黄中发现的环烯醚萜苷（CAT）具有多种药理作用，包括抗炎、抗糖尿病和抗纤维化作用。然而，目前尚无报道称 CAT 是否调节肝星状细胞（HSCs）的有氧糖酵解以抑制肝纤维化。
本研究旨在探讨 CAT 对肝纤维化的保护作用及其机制。
为了探讨 CAT 是否改善体内和体外肝纤维化，通过腹腔注射四氯化碳（CCl）诱导小鼠肝纤维化。此外，用转化生长因子-β（TGF-β）刺激 LX-2 细胞体外模拟纤维化。使用自动生化分析仪检测血清肝损伤标志物。采用组织病理学染色、免疫荧光（IF）染色、Western blot（WB）分析、免疫共沉淀（Co-IP）、药物亲和力反应靶稳定性（DARTS）、细胞热转移测定（CETSA）等方法鉴定 CAT 与 EphA2 之间的靶向关系，并检测与 CAT 抗纤维化作用相关的有氧糖酵解相关蛋白、纤维标志物和信号通路的表达。
结果表明，CAT 显著抑制 CCl 处理小鼠的肝损伤、纤维化和炎症。这表现为纤维化标志物、肝功能指数和组织病理学的增强。此外，CAT 显著抑制 TGF-β诱导的 LX-2 细胞中 HSCs 的激活，表现为增殖、迁移和胶原 I 和 a-SMA 的表达减少。研究结果还表明，CAT 可能通过抑制激活的 HSCs 和 CCl 处理的小鼠中的糖酵解来发挥抗纤维化作用。在机制上，CAT 直接靶向 Ephrin 型受体 A2（EphA2）以减少与粘着斑激酶（FAK）的结合，并显著抑制 FAK/Src 通路。此外，EphA2 的药理学抑制不能进一步增加 CAT 对体内和体外肝纤维化的治疗效果。
本研究结果表明，CAT 通过 EphA2/FAK/Src 信号通路抑制激活的 HSCs 的有氧糖酵解，为治疗肝纤维化提供了一种新的治疗方法。
