Astragaloside IV Suppresses Hepatic Proliferation in Regenerating Rat Liver after 70% Partial Hepatectomy via Down-Regulation of Cell Cycle Pathway and DNA Replication

Abstract

Astragaloside IV (AS-IV) is one of the major bio-active ingredients of huang qi which is the dried root of Astragalus membranaceus (a traditional Chinese medicinal plant). The pharmacological effects of AS-IV, including anti-oxidative, anti-cancer, and anti-diabetic effects have been actively studied, however, the effects of AS-IV on liver regeneration have not yet been fully described. Thus, the aim of this study was to explore the effects of AS-IV on regenerating liver after 70% partial hepatectomy (PHx) in rats. Differentially expressed mRNAs, proliferative marker and growth factors were analyzed. AS-IV (10 mg/kg) was administrated orally 2 h before surgery. We found 20 core genes showed effects of AS-IV, many of which were involved with functions related to DNA replication during cell division. AS-IV down-regulates MAPK signaling, PI3/Akt signaling, and cell cycle pathway. Hepatocyte growth factor (HGF) and cyclin D1 expression were also decreased by AS-IV administration. Transforming growth factor β1 (TGFβ1, growth regulation signal) was slightly increased. In short, AS-IV down-regulated proliferative signals and genes related to DNA replication. In conclusion, AS-IV showed anti-proliferative activity in regenerating liver tissue after 70% PHx.

Keywords: 70% partial hepatectomy; Astragali Radix; Astragalus membranaceus; astragaloside IV; huang qi; liver; liver regeneration; proliferation; rat.

Figure 1

Chemical structure of astragaloside IV. (C₄₁H₆₈O₁₄, molecular weight: 794.97 g/mol).

Figure 2

DEGs and gene network. (a) heatmap with hierarchical clustering of 2-fold changed DEGs, (b) expression pattern for 2-fold changed DEGs, (c) heatmap with hierarchical clustering of 5-fold changed DEGs, (d) expression pattern for 5-fold changed DEGs, (e) Gene network of 5-fold changed DEGs. 20 DEGs (in blue dotted line) and 12 DEGs (in red dotted line) were clustered. Markedly, DEGs within the blue dotted line showed strong relation. 14 other DEGs (not marked) were linked with only one or two genes. The green color in the figure indicates decreased expression; the red color in the figure indicates increased expression.

Figure 3

Results of KEGG pathway mapping. 2-fold changed DEGs were analyzed using the KEGG pathway database. 24, 22, and 22 DEGs were included in cell cycle, MAPK signaling, and PI3K-Akt signaling pathways, respectively. AS-IV showed down-regulatory effects on these pathways. Only a few DEGs were up-regulated in these three pathways. MAPK signaling and PI3K-Akt signaling pathways were upstream of the cell cycle pathway. The red and blue color in the figure indicate up-regulation and down-regulation of genes, respectively. Intensity of color is proportional to fold change.

Figure 4

Immunostaining for HGF. HGF-immuno-positive reactions were a brown color after immunostaining. HGF peaked at 12 h after PHx. The AS-IV group showed a low expression of HGF. Hematoxylin was used as a counter stain. (Scale bar indicates 100 μm, CV: central vein, n = 3, mean ± standard deviation, *: p < 0.05).

Figure 5

Immunostaining for cyclin D1. Cyclin D1 expression in regenerating liver tissues rapidly increased 12 h after PHx, peaking at 12 h after PHx then decreasing by 24 h. The AS-IV group showed a low expression of cyclin D1. Hematoxylin was used as a counter stain. (Scale bar indicates 100 μm, CV: central vein, n = 3, mean ± standard deviation, **: p < 0.01).

Figure 6

Relative expressions of cyclin D1 and TGF β1. Expression of cyclin D1 and TGFβ1 were analyzed by Western blot analysis. 12 h after PHx, cyclin D1 expression decreased in the experimental group and decreased further by 24 h while showing increased TGFβ1 expression. Relative protein expressions are presented as a ratio of the β-actin loading control. (mean ± standard deviation, n = 3, **: p < 0.01).

Figure 7

Administration of AS-IV and 70% PHx. Rats were received 10mg/kg of AS-IV diluted in D.W. (experimental group) or D.W. (control group) 2 h before 70% PHx and were sacrificed at 12 h or 24 h after PHx.

Figure 8

Graphical abstract of study. AS-IV suppressed hepatic proliferation of regenerating liver tissues after 70% PHx.