AS-IV Attenuates Oxidative Stress-Induced Apoptosis in Zebrafish via Modulation of the AKT/NRF2/HO-1/Caspase-3 Signaling Axis

Abstract

As the primary active component of Astragalus membranaceus, Astragaloside IV (AS-IV) is widely recognized in pharmacological research for its multifaceted therapeutic potential, particularly its antioxidative, immunostimulatory, and cardioprotective properties. Oxidative stress is an important mechanism in the induction of many diseases. The present study investigates the antioxidative mechanism of Astragaloside IV in zebrafish, using menaquinone exposure to induce oxidative stress conditions. The findings revealed that AS-IV effectively attenuated oxidative stress-induced mortality and morphological abnormalities in zebrafish. AS-IV exhibited a concentration-dependent protective effect against developmental abnormalities, with progressive reduction in pericardial effusion, body curvature, and growth retardation observed at higher doses. Moreover, AS-IV treatment not only effectively reduced reactive oxygen species (ROS) accumulation and attenuated oxidative DNA damage but also significantly decreased apoptosis in the cardiac region of zebrafish embryos under oxidative stress conditions. Western blot analysis revealed that AS-IV treatment significantly reduced the protein levels of both Cleaved Caspase-3 and γ-H2AX, indicating its ability to inhibit DNA damage-induced apoptosis. AS-IV mediates its antioxidant defense mechanisms through the activation of the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway, inducing the significant upregulation of cytoprotective enzymes. This molecular mechanism underlies the observed phenotypic improvements in oxidative stress-related damage. Upstream analysis demonstrated that AS-IV activates NRF2 primarily through protein kinase B (AKT/PKB) pathway modulation, independent of KEAP1 regulation. Comprehensive mechanistic analysis reveals that Astragaloside IV mitigates oxidative stress-induced apoptosis in zebrafish through coordinated regulation of the AKT/NRF2/HO-1/Caspase-3 signaling axis.

Keywords: AS-IV; NRF2; network pharmacology; oxidative stress; zebrafish.

Figure 1

Developmental abnormalities in zebrafish phenotypes due to menaquinone. (A) The phenotype of developmental abnormalities in zebrafish caused by menaquinone; (B) effects of menaquinone on zebrafish mortality; (C) effects of 72 hpf menaquinone on zebrafish deformity rates; (D) effects of menaquinone on zebrafish reactive oxygen species accumulation. (PE: pericardium edema; YR: yolk retention; SBD: swim bladder deficiency; CBS: curved body shape). Note: ** p < 0.01 versus the CTL group. In this and the subsequent figures, CTL signifies vehicle control (0.1% DMSO).

Figure 2

Antioxidant effect of AS-IV. (A) AS-IV inhibits increased mortality in an oxidative stress model; (B) AS-IV reduces deformity rate in zebrafish; (C) AS-IV inhibits the area of pericardial edema; (D) fluorescence intensity statistics of DCFH-DA reactive oxygen content assay; (E) AS-IV inhibits ROS accumulation in zebrafish. Note: * p < 0.05 or ** p < 0.01 versus the CTL group. # p < 0.05 or ## p < 0.01 versus the MEN group.

Figure 3

AS-IV treatment of oxidative stress-induced cardiac injury. (A) Decrease in heart rate in AS-IV-treated oxidative stress model; (B) electrocardiogram of AS-IV intervention oxidative stress model. Note: ** p < 0.01 versus the CTL group. # p < 0.05 or ## p < 0.01 versus the MEN group.

Figure 4

AS-IV antioxidant mechanisms network pharmacology analysis. (A) Venn diagram of AS-IV effective targets and oxidative stress targets; (B) results of PPI network analysis of AS-IV interfering with oxidative stress targets; (C) GO enrichment analysis (the top 20 terms of BP, CC, and MF enrichment analysis were shown in orange, blue, and blue bars, respectively); (D) bubble plot of antioxidant pathway enrichment analysis of 93 targets (Top 15 results); (E) AS-IV antioxidant major gene KEGG genes mapping.

Figure 5

Experimental validation of the antioxidant molecular mechanism of AS-IV. (A) Inhibition of zebrafish apoptosis by AS-IV detected by using AO staining (arrowheads point to apoptotic cells); (B) assay of γH2AX and cleaved caspase-3 content via Western blot analysis showed that AS-IV inhibited oxidative stress-induced DNA damage and cell apoptosis; (C) Western blot analysis results showed that AS-IV increased NRF2 accumulation and AKT/PKB phosphorylation without affecting KEAP1 levels; (D) results of the relative protein level. * p < 0.05 or ** p < 0.01 versus the CTL group. # p < 0.05 or ## p < 0.01 versus the MEN group.

Figure 6

Expression of antioxidant enzymes of AS-IV. (A–D) The results of qRT-PCR assay showed that AS-IV activated the expression of antioxidant genes HO-1, Gpx-1, CAT, and SOD-2. * p < 0.05 or ** p < 0.01 versus the CTL group. ## p < 0.01 versus the MEN group.

Figure 7

The molecular mechanism through which AS-IV suppresses cell apoptosis triggered by ROS involves the mediation of NRF2 accumulation.