Astragaloside-IV promotes autophagy via the Akt/mTOR pathway to improve cellular lipid deposition

Abstract

The current study aimed to investigate the potential role of astragaloside IV (AS-IV) in improving cellular lipid deposition and its underlying mechanism. A fatty liver cell model was established by treating hepatoma cells with palmitic acid. AS-IV and SC79 were used for treatment. Oil Red O staining was applied to detect intracellular lipid deposition, and transmission electron microscopy was utilized to assess autophagosome formation. Immunofluorescence double staining was applied to determine microtubule-associated proteins 1A/1B light chain 3 (LC3) expression. Western blot analysis was performed to detect the expression of LC3, prostacyclin, Beclin-1, V-akt murine thymoma viral oncogene homolog (Akt), phosphorylated Akt, mTOR, and phosphorylated mTOR. Oil Red O staining revealed that AS-IV reduced intracellular lipid accumulation. Further, it increased autophagosome synthesis and the expression of autophagy proteins LC3 and Beclin-1 in the cells. It also reduced the phosphorylation levels of Akt and mTOR and the levels of prostacyclin. However, the effects of AS-IV decreased with SC79 treatment. In addition, LC3B + BODIPY493/503 fluorescence double staining showed that AS-IV reduced intracellular lipid deposition levels by enhancing autophagy. AS-IV can reduce lipid aggregation in fatty liver cells, which can be related to enhanced hepatocyte autophagy by inhibiting the Akt/mTOR signaling pathway.

Figure 1.

Screening of the effect of AS-IV at different concentrations on lipid concentrations in HepG2 cells. (A, B) Quantitative analysis of the cell viability rate. (C) Schematic representation of cell grouping and pretreatment. (D) Representative images of Oil Red O staining of intracellular lipid droplets in each group. (E) Quantitative analysis of Oil Red O staining. (F,G) Quantitative analysis of the TG and TC levels. Data were presented as mean ± standard deviations (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001, nsp > 0.05.

Figure 2.

Effect of AS-IV on the autophagy level in HepG2 cells. (A) Representative transmission electron microscopy images of HepG2 cells in each group. The red arrow shows mild pyknosis of the mitochondria. The blue arrow shows mild expansion of the rough endoplasmic reticulum, and the purple arrow shows autophagy. (B) Representative images of immunofluorescence staining of LC3 in each group. (C) Quantitative analysis of immunofluorescence staining. (D) Bands of autophagy-related indicators, including LC3II/I, Beclin-1, and p62. (E–G) Quantitative analysis of Western blotting findings. Data were presented as means ± standard deviations (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001, nsp > 0.05.

Figure 3.

Effect of AS-IV on the Akt/mTOR signaling pathway in HepG2 cells. (A) Western blot analysis of Akt, p-Akt, mTOR, and p-mTOR. (B–E) Quantitative analysis of Western blot analysis results. Data were presented as mean ± standard deviation (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001, nsp > 0.05.

Figure 4.

Effect of SC79 on Akt/mTOR induced by AS-IV. (A) Schematic representation of cell grouping and pretreatment. (B) Quantitative analysis of the cell viability rate. (C) Western blot analysis of Akt, p-Akt, mTOR, and p-mTOR. (D–G) Quantitative analysis of Western blot analysis results. Data were presented as mean ± standard deviation (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001, nsp > 0.05.

Figure 5.

The effect of SC79 on the regulation of autophagy by AS-IV. (A) Representative transmission electron microscopy images of HepG2 cells in each group. The red arrow shows mild pyknosis of mitochondria, the blue arrow shows mild expansion of rough endoplasmic reticulum, and the purple arrow shows autophagy. (B) Representative images of immunofluorescence staining of LC3 in each group. (C) Quantitative analysis of immunofluorescence staining. (D) Bands of autophagy-related indicators, including LC3II/I, Beclin-1, and p62. (E–G) Quantitative analysis of Western blotting findings. Data are presented as the means ± standard deviations (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001, nsp > 0.05.

Figure 6.

Effect of SC79 on AS-IV-regulated autophagy improvement of palmitic acid-induced lipid accumulation. (A) Representative images of Oil Red O staining of intracellular lipid droplets in each group. (B, C) Quantitative analysis of the TG and TC levels. (D) Quantitative analysis of Oil Red O staining. Data were presented as means ± standard deviations (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001, nsp > 0.05.

Figure 7.

Effect of SC79 on LC3 + BODIPY493/503 expression in HepG2 cells treated with AS-IV. (A) Representative images of immunofluorescence staining of LC3 and lipid droplets in each group. (B–D) Quantitative analysis of immunofluorescence staining findings. Data were presented as means ± standard deviations (n = 3). *P < .05, **P < .01, ***P < .001, ****P < .0001, nsp > 0.05.