Quercetin Attenuates Acetaldehyde-Induced Cytotoxicity via the Heme Oxygenase-1-Dependent Antioxidant Mechanism in Hepatocytes

Abstract

It is still unclear whether or how quercetin influences the toxic events induced by acetaldehyde in hepatocytes, though quercetin has been reported to mitigate alcohol-induced mouse liver injury. In this study, we evaluated the modulating effect of quercetin on the cytotoxicity induced by acetaldehyde in mouse hepatoma Hepa1c1c7 cells, the frequently used cellular hepatocyte model. The pretreatment with quercetin significantly inhibited the cytotoxicity induced by acetaldehyde. The treatment with quercetin itself had an ability to enhance the total ALDH activity, as well as the ALDH1A1 and ALDH3A1 gene expressions. The acetaldehyde treatment significantly enhanced the intracellular reactive oxygen species (ROS) level, whereas the quercetin pretreatment dose-dependently inhibited it. Accordingly, the treatment with quercetin itself significantly up-regulated the representative intracellular antioxidant-related gene expressions, including heme oxygenase-1 (HO-1), glutamate-cysteine ligase, catalytic subunit (GCLC), and cystine/glutamate exchanger (xCT), that coincided with the enhancement of the total intracellular glutathione (GSH) level. Tin protoporphyrin IX (SNPP), a typical HO-1 inhibitor, restored the quercetin-induced reduction in the intracellular ROS level, whereas buthionine sulphoximine, a representative GSH biosynthesis inhibitor, did not. SNPP also cancelled the quercetin-induced cytoprotection against acetaldehyde. These results suggest that the low-molecular-weight antioxidants produced by the HO-1 enzymatic reaction are mainly attributable to quercetin-induced cytoprotection.

Keywords: acetaldehyde; aldehyde dehydrogenase; glutathione; heme oxygenase-1; quercetin.

Figure 1

Quercetin protected Hepa1c1c7 cells from the cytotoxicity induced by acetaldehyde. After the 24 h pretreatment with quercetin (Que), acetaldehyde (AA, 10 mM) was treated with the cells for 3 h, then an MTT assay was carried out. All values are presented as the means ± SD of three separate experiments and subjected to one-way ANOVA using SPSS 26.0 software, followed by Tukey’s HSD. The different letters over the bars correspond to significant differences between treatments in each condition (p < 0.05).

Figure 2

Quercetin modulated the ALDH gene expression and the ALDH activity in Hepa1c1c7 cells. (A) After the 6 h treatment with quercetin (Que), the total RNA was subjected to an RT-PCR analysis for each gene. (B) After the 24 h treatment with quercetin, the ALDH activity was evaluated. All values are presented as the means ± SD of three separate experiments and subjected to a Student’s t-test by using SPSS 26.0 software (**, p < 0.01; *, p < 0.05 vs. control).

Figure 3

Quercetin inhibited the intracellular reactive oxygen species (ROS) accumulation induced by acetaldehyde. After the 24 h pretreatment with quercetin (Que), 10 mM of acetaldehyde (AA) was treated with the cells for 3 h. The cells were subjected to a dichlorofluorescin diacetate (DCFH-DA) assay using an image-based cytometer. All values are presented as the means ± SD of three separate experiments and subjected to one-way ANOVA using SPSS 26.0 software, followed by Tukey’s HSD. The different letters over the bars correspond to significant differences between treatments in each condition (p < 0.05).

Figure 4

Quercetin enhanced the expression of the antioxidant-producing enzymes and the total glutathione level. (A) After the 6 h treatment with quercetin (Que), the total RNA was subjected to an RT-PCR analysis for each gene. (B) After the 24 h treatment with quercetin, the intracellular GSH level was evaluated. (C) After the 24 h treatment with quercetin (10 μM), the protein expression level of HO-1 was determined by western blotting. All values are presented as the means ± SD of three separate experiments and subjected to a Student’s t-test by using SPSS 26.0 software (**, p < 0.01; *, p < 0.05 vs. control).

Figure 5

The HO-1 inhibitor, tin protoporphyrin IX (SNPP) (A), and the glutathione biosynthesis inhibitor, buthionine sulphoximine (BSO) (B), modulated the quercetin-induced inhibition of ROS accumulation. After the 1 h pretreatment with SNPP (10 μM) or BSO (0.5 μM), quercetin (Que) was treated with the cells for 24 h. After the 3 h treatment with 10 mM of acetaldehyde (AA), the cells were subjected to the DCFH-DA assay. All values are presented as the means ± SD of three separate experiments and subjected to one-way ANOVA using SPSS 26.0 software, followed by Tukey’s HSD. The different letters over the bars correspond to significant differences between treatments in each condition (p < 0.05).

Figure 6

The HO-1 inhibitor cancelled the quercetin-induced cytoprotection against acetaldehyde. After the 1 h pretreatment with SNPP (10 μM), quercetin was treated with the cells for 24 h. After the 3 h treatment with 10 mM of acetaldehyde, the cells were subjected to an MTT assay. All values are presented as the means ± SD of three separate experiments and subjected to one-way ANOVA using SPSS 26.0 software, followed by Tukey’s HSD. The different letters over the bars correspond to significant differences between treatments in each condition (p < 0.05).