苦参碱通过AMPK/ULK1/WIPI1信号轴诱导肝癌细胞自噬

doi:10.5246/jcps.2026.04.025

摘要/Abstract

摘要：

自噬是一种将细胞质成分运送至溶酶体进行降解的分解代谢过程。然而, 苦参碱诱导肝癌细胞自噬的机制尚不明确。本研究旨在探讨p53/AMPK通路与苦参碱诱导人肝癌细胞自噬之间的关联。首先通过免疫荧光和免疫印迹法验证PFT-α抑制剂联合RNA干扰抑制p53后, 苦参碱可显著增加LC3Ⅱ含量。在苦参碱处理过程中, p53沉默显著提升AMPK磷酸化水平, 而ULK1和WIPI-1表达略有下降。相反, 通过激动剂和拮抗剂靶向调控AMPK, 可正向调节苦参碱处理细胞中ULK1和WIPI-1的表达。敲低WIPI-1可抑制苦参碱诱导的凋亡并促进细胞增殖。此外, cDNA芯片技术全面解析了苦参碱治疗肝癌的体内分子机制。经实时PCR分析筛选并验证, 苦参碱处理后SRGN和BCL11A表达显著上调。这些发现揭示了苦参碱诱导自噬的复杂机制, 验证了p53/AMPK交叉调控通路的参与, 并鉴定出ULK1和WIPI-1等下游效应分子。此外, 对SRGN和BCL11A作为苦参碱调控基因的鉴定, 揭示了中药单体在肝细胞癌中发挥抗肿瘤作用的额外机制。通过对自噬相关通路及特定致癌靶点的双重调控, 苦参碱成为肝癌治疗中极具前景的多靶点药物, 其治疗潜力值得进一步深入研究。

关键词: 苦参碱, AMPK, ULK1, WIPI-1, 自噬

Abstract:

Autophagy is a highly conserved catabolic process that delivers cytoplasmic constituents to lysosomes for degradation. Despite its importance, the mechanisms underlying matrine-induced autophagy in hepatoma cells remain largely elusive. This study aimed to elucidate the role of the p53/AMPK signaling pathway in mediating matrine-induced autophagy in human hepatocellular carcinoma cells. Initially, inhibition of p53 using the chemical inactivator PFT-α or RNA interference (RNAi) in combination with matrine treatment led to a pronounced increase in LC3Ⅱ levels, as evidenced by immunofluorescence and immunoblot analyses. Concomitantly, p53 silencing markedly enhanced AMPK phosphorylation, whereas expression of ULK1 and WIPI-1 showed a modest decline. In contrast, pharmacological modulation of AMPK using specific agonists and antagonists positively regulated ULK1 and WIPI-1 expressions in matrine-treated cells. Notably, WIPI-1 knockdown attenuated matrine-induced apoptosis and facilitated cell proliferation. Furthermore, comprehensive cDNA array profiling explored the molecular mechanisms of matrine in vivo, revealing significant regulation of SRGN and BCL11A, which was subsequently validated by quantitative real-time PCR (qPCR). Collectively, these findings uncovered intricate mechanisms underlying matrine-induced autophagy, highlighting the pivotal cross-talk between p53 and AMPK and identifying key downstream effectors, including ULK1 and WIPI-1. Moreover, the identification of SRGN and BCL11A as matrine-responsive genes suggested additional pathways through which matrine exerted its anti-tumor activity. By concurrently modulating autophagy-related signaling and specific oncogenic targets, matrine emerged as a promising multi-target therapeutic agent for hepatocellular carcinoma, warranting further exploration of its clinical potential.

Key words: Matrine, AMPK, ULK1, WIPI-1, Autophagy

Supporting: /attached/file/20260509/20260509012911_370.pdf

谢步善, 周苗苗, 何星星. 苦参碱通过AMPK/ULK1/WIPI1信号轴诱导肝癌细胞自噬[J]. 中国药学(英文版), 2026, 35(4): 359-371.

Bushan Xie, Miaomiao Zhou, Xingxing He. Matrine induces autophagy in liver cancer cells via an AMPK/ULK1/WIPI1 signaling axis[J]. Journal of Chinese Pharmaceutical Sciences, 2026, 35(4): 359-371.

图/表 4

Figure 1. p53 negatively regulates matrine-induced autophagy in hepatoma cells. (a) Upper panel: Immunoblotting analysis confirmed that RNAi inhibited p53 (p53 siRNA). Grayscale analysis of expression of the p53/β-actin ratio (*P < 0.05). Middle panel: Western blotting analysis of LC3 expression in hepatoma cells treated with 0.8 mg/mL matrine for 48 h, with or without 30 μM PFT-α. β-Actin served as a loading control. Grayscale analysis of expression of the LC3-II/β-actin ratio. Significant differences between matrine treatment and combined treatment with matrine and PFT-α (*P < 0.05). Lower panel: LC3-II expression in p53 siRNA-transfected cells treated with matrine, compared to scrambled siRNA control. Grayscale analysis of expression of the LC3-II/GAPDH ratio. The combined treatment of matrine and p53 siRNA resulted in significantly greater effects than matrine plus control siRNA (*P < 0.05). (b) Immunofluorescence analysis of LC3 puncta formation (red) in p53-knockdown cells treated with matrine, counterstained with DAPI (blue) for nuclei.

Figure 2. AMPK positively regulates ULK1 and WIPI1 in matrine-treated HepG2 cells. (a) Western blotting analysis of p-AMPKα (Thr172), AMPK, WIPI1, and ULK1 expression in HepG2 cells transfected with p53 siRNA or scrambled RNA (control) for 48 h prior to matrine treatment. Grayscale analysis of expression of the p-AMPK/AMPK, WIPI1/GAPDH and ULK1/GAPDH ratio. The combined treatment of matrine and p53 siRNA resulted in significantly greater effects than matrine plus control siRNA (nsIndicates no significant difference between groups (P > 0.05), *P < 0.05). (b) Immunoblot analysis of AMPK signaling components (AMPK, WIPI1, ULK1, p-ULK1 (Thr757)) in HepG2 cells co-treated with matrine and either AMPK activator (0.1 mmol/L AICAR) (Left panel) or inhibitor (10 µmol/L Compound C) (Right panel). GAPDH served as a loading control. Grayscale analysis of expression of the AMPK/GAPDH, ULK1/GAPDH, p-ULK1/GAPDH and WIPI1/GAPDH ratio. Significant differences between matrine treatment and combined treatment with matrine and AICAR (Left panel) or Compound C (Right panel) (*P < 0.05). (c) Left panel: CCK-8 assay showing relative cell viability (mean ± SEM, n = 3) in HepG2 cells treated with matrine alone or in combination with AICAR (P < 0.05 vs. matrine treatment). Right panel: CCK-8 assay demonstrating relative cell viability (mean ± SEM, n = 3) in HepG2 cells treated with matrine alone or in combination with Compound C (*P < 0.05 vs. matrine treatment).

Figure 3. WIPI1 knockdown suppresses apoptosis and promotes proliferation in matrine-treated HepG2 cells. (a) qPCR analysis confirming WIPI1 knockdown efficiency in three experimental groups of HepG2 cells transfected with three pairs of WIPI1 siRNA; (b) Western blotting analysis verifying WIPI1 protein knockdown, with grayscale quantification presented as mean ± SEM (n = 3 independent experiments); (c) Flow cytometric analysis of apoptosis using Annexin V/PI staining showing reduced apoptosis in WIPI1 siRNA-transfected cells treated with 0.8 mg/mL matrine compared to control siRNA (mean ± SEM, n = 3); (d) Cell viability assessed by CCK-8 assay demonstrated increased cell proliferation in WIPI1 siRNA-transfected cells treated with 0.8 mg/mL matrine (mean ± SEM, n = 3). Untreated cells and control siRNA-transfected cells served as negative controls. nsIndicates not significant (P ≥ 0.05), *P < 0.05, **P < 0.01.

Figure 4. A cDNA array explores the molecular mechanism of matrine treatment of liver cancer. (a) Changes of gene expression induced by matrine in vivo (at least 2-fold change); (b) Real-time-PCR analysis of the expression of SRGN, BCL11A after exposure to matrine in xenograft, as compared with control (*P < 0.05).

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