Related Experiment Video
Updated: Jul 27, 2025
Establishment of Hepatocarcinoma in BALB/c-nu Mice and Investigation of the Therapeutic Effect of the Sanleng Jiashen Formula
Published on: January 26, 2024
Amelioratory effects of astragaloside IV on hepatocarcinogenesis via Nrf2-mediated pSmad3C/3L transformation
Yong Fang Gong1, Shu Hou2, Jia-Cheng Xu2
1Department of Pharmacology, School of Basic Medicine, Anhui Medical University, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, 230032, 81 Meishan Road, Hefei, China; School of Nursing, Anhui Medical University, No.15, feicui Road, Economic and Technological Development Zone, Hefei, China.
Background:
Phosphorylated Smad3 isoforms are reversible and antagonistic, and the tumour-suppressive pSmad3C can shift to an oncogenic pSmad3L signal. In addition, Nrf2 has a two-way regulatory effect on tumours, protecting normal cells from carcinogens and promoting tumour cell survival in chemotherapeutics. Accordingly, we hypothesised that the transformation of pSmad3C/3L is the basis for Nrf2 to produce both pro- and/or anti-tumourigenic effects in hepatocarcinogenesis. Astragaloside IV (AS-IV), the major component of Astragalus membranaceus, exerts anti-fibrogenic and carcinogenic actions. Lately, AS-IV administration could delay the occurrence of primary liver cancer by persistently inhibiting the fibrogenesis and regulating pSmad3C/3 L and Nrf2/HO-1 pathways synchronously. However, effect of AS-IV on hepatocarcinogenesis implicated in the bidirectional cross-talking of pSmad3C/3 L and Nrf2/HO-1 signalling, especially which one contributes palpably than the other still remains unclear.
Purpose:
This study aims to settle the above questions by using in vivo (pSmad3C+/- and Nrf2-/- mice) and in vitro (plasmid- or lentivirus- transfected HepG2 cells) models of HCC.
Study Design And Methods:
The correlation of Nrf2 to pSmad3C/pSmad3L in HepG2 cells was analysed by Co-immunoprecipitation and dual-luciferase reporter assay. Pathological changes of Nrf2, pSmad3C, and pSmad3L in human HCC patients, pSmad3C+/- mice, and Nrf2-/- mice were gauged by immunohistochemical, haematoxylin and eosin staining, Masson, and immunofluorescence assays. Finally, western blot and qPCR were used to verify the bidirectional cross-talking of pSmad3C/3L and Nrf2/HO-1 signalling protein and mRNA in vivo and in vitro models of HCC.
Results:
Histopathological manifestations and biochemical indicators revealed that pSmad3C+/- could abate the ameliorative effects of AS-IV on fibrogenic/carcinogenic mice with Nrf2/HO-1 deactivation and pSmad3C/p21 transform to pSmad3L/PAI-1//c-Myc. As expected, cell experiments confirmed that upregulating pSmad3C boosts the inhibitory activity of AS-IV on phenotypes (cell proliferation, migration and invasion), followed by a shift of pSmad3L to pSmad3C and activation of Nrf2/HO-1. Synchronously, experiments in Nrf2-/- mice and lentivirus-carried Nrf2shRNA cell echoed the results of pSmad3C knockdown. Complementarily, Nrf2 overexpression resulted in the opposite result. Furthermore, Nrf2/HO-1 contributes to AS-IV's anti-HCC effect palpably compared with pSmad3C/3L.
Conclusion:
These studies highlight that harnessing the bidirectional crosstalk pSmad3C/3 L and Nrf2/HO-1, especially Nrf2/HO-1 signalling, acts more effectively in AS-IV's anti-hepatocarcinogenesis, which may provide an important theoretical foundation for the use of AS-IV against HCC.
Insights
Astragaloside IV (AS-IV) shows promise in combating liver cancer by modulating the pSmad3C/3L and Nrf2/HO-1 pathways. Targeting the Nrf2/HO-1 signaling pathway offers a more effective strategy for AS-IV
Area of Science:
- Hepatocellular carcinoma (HCC) research
- Molecular signaling pathways in cancer
- Natural product pharmacology
Background:
- Phosphorylated Smad3 (pSmad3) isoforms (pSmad3C/3L) and Nrf2 exhibit dual roles in hepatocarcinogenesis.
- Astragaloside IV (AS-IV) delays liver cancer by regulating pSmad3C/3L and Nrf2/HO-1 pathways.
- The precise contribution of each pathway to AS-IV's anti-cancer effects remains unclear.
Purpose of the Study:
- To elucidate the role of pSmad3C/3L and Nrf2/HO-1 crosstalk in AS-IV's anti-hepatocarcinogenesis effects.
- To determine which pathway, pSmad3C/3L or Nrf2/HO-1, contributes more significantly to AS-IV's efficacy.
- To validate findings using in vivo (pSmad3C+/-, Nrf2-/- mice) and in vitro (HepG2 cells) models.
Main Methods:
- Co-immunoprecipitation and dual-luciferase reporter assays to analyze Nrf2-pSmad3C/pSmad3L correlation.
- Immunohistochemistry, H&E, Masson, and immunofluorescence assays to assess pathological changes.
- Western blot and qPCR to verify signaling pathway crosstalk in vivo and in vitro.
Main Results:
- pSmad3C+/- mice showed reduced AS-IV efficacy with Nrf2/HO-1 deactivation and a shift to pSmad3L.
- Upregulating pSmad3C enhanced AS-IV's anti-cancer activity, activating Nrf2/HO-1.
- Nrf2/HO-1 signaling demonstrated a more significant contribution to AS-IV's anti-HCC effects compared to pSmad3C/3L.
Conclusions:
- AS-IV exerts anti-hepatocarcinogenesis effects by modulating the bidirectional crosstalk of pSmad3C/3L and Nrf2/HO-1 pathways.
- Targeting the Nrf2/HO-1 signaling pathway is a more effective strategy for AS-IV's anti-liver cancer action.
- This study provides a theoretical basis for utilizing AS-IV in treating HCC.
