Astragaloside IV represses hepatocellular carcinoma progression by modulating HMGB1-ferroptosis axis

Abstract

Astragaloside IV (AST-IV), as one of the main functional components of Astragalus membranaceus, has physiological functions such as regulating metabolism and anti-tumor. However, the role of AST-IV on hepatocellular carcinoma (HCC) was still poorly understood. In this study, our work explored whether AST-IV could induce ferroptosis and repress HCC tumorigenesis. Results indicated that AST-IV could repress the tumor progression (viability, migration) of HCC in vitro. Besides, AST-IV induced the ferroptosis (Fe²⁺, malondialdehyde, lipid peroxidation) in HCC cells. Molecular docking and microscale thermophoresis indicated that high mobility group protein B1 (HMGB1) acted as the target of AST-IV. AST-IV could repress the HMGB1 expression and HMGB1 reversed the role of AST-IV on HCC cells' ferroptosis. In vivo, AST-IV administration repressed the tumor progression. In conclusion, AST-IV represses HCC progression by modulating HMGB1-ferroptosis axis, which provides a novel insight for HCC.

Keywords: Astragaloside IV; Ferroptosis; HMGB1; Hepatocellular carcinoma.

Fig. 1

AST-IV repressed the tumor progression of HCC in vitro. (A) The chemical structural formula of AST-IV. (B) The human HCC cell line (Huh7, MHCC97H) and normal human hepatic cells (THLE-3) were administrated by continuous concentration of AST-IV (0-200 µmol/L). The viability was tested by CCK-8. (C) Transwell assay was performed to test the migration of HCC cells. AST-IV (0 µmol/L, 25 µmol/L, 50 µmol/L) was administrated to cells in vitro. (D, E, F) EdU analysis was performed to test the proliferative ability of HCC cells with AST-IV concentration-dependent treatment. ** p < 0.01; * p < 0.05

Fig. 2

AST-IV accelerated the ferroptosis of HCC cells. (A) The iron (Fe²⁺) concentration accumulation was tested in HCC cells (Huh7, MHCC97H) treated with AST-IV (0 µmol/L, 25 µmol/L, 50 µmol/L). (B) The content of MDA was determined in HCC cells (Huh7, MHCC97H) treated with AST-IV. (C) The GSH was determined by the commercial kits. (D) The TEM was determined for the ultrastructure of the mitochondria in HCC cells. (E, F) The cellular lipid peroxidation level was tested by BODIPYTM 581/591 C11 on flow cytometer. ** p < 0.01; * p < 0.05

Fig. 3

HMGB1 acted as the target of AST-IV. (A) RNA-Seq was performed in the AST-IV treated HCC cells (Huh-7). (B) Gene set enrichment analysis (GSEA) shows the ferroptosis characteristic in AST-IV treated HCC cells (Huh-7) compared with control. (C) The molecule docking was performed to test the possible interaction between HMGB1 and the AST-IV bioactive compound. (D) Microscale thermophoresis (MST) assay was performed to test the interaction of HMGB1-AST-IV

Fig. 4

AST-IV targeted HMGB1 to trigger HCC ferroptosis. (A) Immunofluorescence analysis was performed to test the HMGB1 level in Huh7 cells with overexpression of HMGB1 (HMGB1) and AST-IV administration (50 µmol/L). (B) The iron (Fe²⁺) concentration accumulation was tested in HCC cells (Huh7) treated with overexpression of HMGB1 (HMGB1) and AST-IV (50 µmol/L). (C) The content of MDA was determined in HCC cells (Huh7) treated with HMGB1 and AST-IV. (D) The GSH was determined by the commercial kits. (E, F) The cellular lipid peroxidation level was tested by BODIPYTM 581/591 C11 on flow cytometer. ** p < 0.01; * p < 0.05

Fig. 5

AST-IV repressed the HCC tumor growth in vivo. (A) The in vivo mice assay was performed with Huh-7 cells subcutaneous tumor. Mice were treated with AST-IV intraperitoneal injection. (B) The volume and (C) weight were calculated in the AST-IV treated mice or control group. (D) Immumohistochemical (IHC) staining revealed the expression of HMGB1, ki67, and GPX4. * p < 0.05

Fig. 6

AST-IV represses HCC progression by modulating HMGB1-ferroptosis axis, which provides a novel insight for HCC