The Chinese herbal formula Free and Easy Wanderer ameliorates oxidative stress through KEAP1-NRF2/HO-1 pathway

Abstract

Posttraumatic stress disorder (PTSD) gains a lot of attention due to high prevalence and strong psychological upset, but the etiology remains undefined and effective treatment is quite limited. Growing studies demonstrated the involvement of oxidative stress in various psychiatry diseases, suggesting anti-oxidation therapy might be a strategy for PTSD treatment. Free and Easy Wanderer (FAEW) is a poly-herbal drug clinically used in China for hundreds of years in the treatment of psychiatric disorder. We hypothesized that FAEW exerts clinical effects through the activity against oxidative stress with fluoxetine as antidepressant control drug. Our results revealed that FAEW significantly reduced both endogenous and H₂O₂-induced exogenous ROS levels in the human glioblastoma T98G and neuroblastoma SH-SY5Y cell lines. Transcriptome-wide microarray analysis indicated NRF2/HO-1 as the common target of FAEW and fluoxetine. Western blotting assay proved that the two drugs promoted NRF2 release from KEAP1 in the cytoplasm and translocation to the nuclei in a KEAP1-dependent manner, the expression of the protein HO-1 increased accordingly, suggesting the participation of KEAP1-NRF2/HO-1 pathway. The chemical constituents of FAEW (i.e. paeoniflorin, baicalin) bound to KEAP1 in silico, which hence might be the effective substances of FAEW. In conclusion, FAEW counteracted H₂O₂-induced oxidative stress through KEAP1-NRF2/HO-1 pathway.

Figure 1

Cytotoxicity of FAEW (1) and fluoxetine (2) determined by the resazurin assay in the human glioblastoma T98G (A) and human neuroblastoma SH-SY5Y (B) cells. Shown are mean values ± SD of three independent experiments.

Figure 2

Inhibition of reactive oxygen species by FAEW (1) or fluoxetine (2) in the human glioblastoma T98G (A) and human neuroblastoma SH-SY5Y (B) cells. Shown are mean values ± SD of three independent experiments. ^★ p < 0.05, ^★★ p < 0.01, compared with control; ^▲ p < 0.05, ^▲▲ p < 0.01, compared with H₂O₂.

Figure 3

Gene expression profiling of T98G cells upon treatment of FAEW or fluoxetine. (A and B) Pathway analyses: Top cellular pathways affected by FAEW and fluoxetine examined by mRNA microarray hybridization. (A) Shows the comparison between FAEW and fluoxetine. (B) Shows the comparison between H₂O₂, H₂O₂ and FAEW, H₂O₂ and fluoxetine. P-values were calculated using right-tailed Fisher’s exact test. (C) Deregulated genes under the influence of the common upstream regulator NFE2L2 (NRF2) inhibited by FAEW (left) and fluoxetine (right).

Figure 4

NRF2, HO-1, CAT and KEAP1 protein expression affected by FAEW or fluoxetine in human glioblastoma T98G cells and human neuroblastoma SH-SY5Y cells. (A–D) Show NRF2, HO-1, CAT and KEAP1 protein expression affected by FAEW or fluoxetine in human glioblastoma T98G cells with different induction times for H₂O₂ (A), 10 min; (B), 6 h; (C), 12 h; (D), 24 h, respectively. TBP was used as loading control for nuclear proteins and β-actin was used as loading control for total protein and cytoplasmic proteins. (E and F) show cytoplasmic and nuclear NRF2, cytoplasmic KEAP1 and total HO-1 protein expression in human glioblastoma T98G (E) and human neuroblastoma SH-SY5Y (F) cells with different concentrations of FAEW and fluoxetine for 12 h. Lamin A/C was used as loading control for nuclear protein, β-actin was used as loading control for total protein and cytoplasmic protein. All the blots were cropped according to their locations in the membrane in order to fit the size, see the original figures in the supplementary information. Shown are mean values ± SD of three independent experiments. ^a p < 0.01, ^b p < 0.05.

Figure 5

Visualization of molecular docking of chemical compounds isolated from FAEW binding to KEAP1.