Nrf2 pathway activation promotes the expression of genes related to glutathione metabolism in alcohol-exposed astrocytes

Abstract

Introduction: Oxidative and antioxidant pathways play essential roles in the development of alcohol-induced brain injury. The Nrf2 pathway is an endogenous antioxidant response pathway, but there has been little research on the role of Nrf2 in alcohol-related diseases. Thus, we examined the effects of alcohol and an Nrf2 agonist (TBHQ) on astrocyte function, mRNA expression, and metabolite content to further explore the protective mechanisms of Nrf2 agonists in astrocytes following alcohol exposure.

Methods: CTX TNA2 astrocytes were cultured with alcohol and TBHQ and then subjected to transcriptome sequencing, LC-MS/MS analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and malondialdehyde (MDA) and superoxide dismutase (SOD) activity assays.

Results: Alcohol exposure significantly increased malondialdehyde (MDA) levels while decreasing superoxide dismutase (SOD) levels in astrocytes. Treatment with TBHQ effectively reversed these effects, demonstrating its protective role against oxidative stress induced by alcohol. Transcriptome sequencing and qRT-PCR analysis revealed that TBHQ specifically upregulates genes involved in glutathione metabolism, including a notable increase in the expression of the glutathione S-transferase A5 (GSTA5) gene, which was suppressed by alcohol exposure. Additionally, metabolomic analysis showed that TBHQ regulates key components of ether lipid metabolism in alcohol-exposed astrocytes, with significant reductions in the levels of lysophosphatidylcholine (18:0) (LysoPC (18:0)) and 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine, both of which are critical markers in the ether lipid metabolic pathway.

Discussion: The findings underscore the role of TBHQ as an Nrf2 agonist in mitigating alcohol-induced oxidative damage in astrocytes by modulating glutathione metabolism and ether lipid metabolism. The regulation of GSTA5 gene expression emerges as a key mechanism through which Nrf2 agonists confer neuroprotection against oxidative stress and lipid oxidation. These insights pave the way for potential therapeutic strategies targeting the Nrf2 pathway to protect astrocytes from alcohol-induced damage.

Keywords: Alcohol; Astrocyte; LC-MS/MS; Nrf2 pathway; Transcriptome sequencing.

Figure 1. Alcohol exposure increases MDA levels and decreases SOD levels in astrocytes, and these changes were reversed by TBHQ.

(A) MDA levels in astrocytes after alcohol and TBHQ intervention (n = 5). (B) SOD levels in astrocytes after alcohol and TBHQ intervention. (n = 5). *P < 0.05; **P < 0.01.

Figure 2. Alcohol exposure regulates metabolism-related gene expression in astrocytes.

(A) KEGG annotation analysis of DEGs between the control and alcohol groups. (B) KEGG pathway analysis of DEGs between the control and alcohol groups.

Figure 3. Heat map of the 49 DEGs between the alcohol and TBHQ groups (n = 4).

Figure 4. TBHQ modulates the expression of genes involved in the metabolism of glutathione in astrocytes exposed to alcohol.

(A) KEGG annotation analysis of DEGs between the alcohol and TBHQ groups. (B) KEGG pathway analysis of DEGs between the alcohol and TBHQ groups.

Figure 5. Alcohol exposure decreases GSTA5 expression in astrocytes, which was reversed by TBHQ.

(A) Venn diagram of six overlapping DEGs from the control and alcohol groups and the alcohol and TBHQ groups. (B) Effects of alcohol and TBHQ on GSTA5 transcript levels in astrocytes (n = 3). (C) Heat map of the six overlapping DEGs (n = 4). (D) KEGG enrichment analysis of the six overlapping DEGs. *P < 0.05, **P < 0.01.

Figure 6. OPLS-DA score plots for all detected metabolites in astrocytes treated with alcohol and TBHQ.

(A) OPLS-DA model in the cationic mode. (B) OPLS-DA model in the anionic mode.

Figure 7. Heatmap of differential metabolites between the TBHQ and alcohol groups (n = 6).

*P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8. Statistical analysis of differential metabolites in astrocytes treated with alcohol and TBHQ.

(A) Classification of differential metabolites and their proportion between the TBHQ and alcohol groups. (B) KEGG enrichment analysis of 38 different metabolites between the TBHQ and alcohol groups. *P < 0.05, **P < 0.01, ***P < 0.001.