Baicalein Inhibits Cerebral Ischemia-Reperfusion Injury through SIRT6-Mediated FOXA2 Deacetylation to Promote SLC7A11 Expression

Baicalein upregulated SIRT6 expression to inhibit apoptosis and partial role of ferroptosis, thereby alleviating I/R injury in mice. MCAO/R mice were treated with baicalein. A, Timeline for baicalein treatment. B, The infarct size was measured using TTC staining. C, Neurological deficit scores were used to evaluate the nerve injuries. D, E, The levels of MDA, GSH, Fe²⁺ were detected using kits. F, Expression of GXP4, SLC7A11, and ACSL4 was determined using Western blotting. G, H, SIRT6 mRNA and protein expression were ascertained by qRT-PCR and Western blotting. I, After 7 d of baicalein treatment, the expression of apoptosis-related proteins was ascertained by Western blotting. n = 9. *p < 0.05, **p < 0.01, ***p < 0.001.

Baicalein inhibited ferroptosis in OGD/R-induced HT22 cells by upregulating SIRT6 expression. OGD/R-treated HT22 cells were treated with baicalein. A, Cell viability was evaluated using a CCK-8 assay. B, The cell death rate was determined by flow cytometry. C–E, Lipid ROS, MDA, GSH, and Fe²⁺ levels were measured using kits. F, GXP4, SLC7A11, and ACSL4 expression levels were determined using by western blotting. G, H, Expression of SIRT6 mRNA and protein was substantiated by qRT-PCR and Western blotting. Data from each study were tested three times. *p < 0.05, **p < 0.01, ***p < 0.001.

Baicalein alleviated OGD/R-induced HT22 cells injury through SIRT6-mediated partial role of ferroptosis. OGD/R-induced HT22 cells were treated with baicalein and transferred with sh-SIRT6. A, B, Expression of SIRT6 mRNA and protein was confirmed by qRT-PCR and Western blotting. C, Cell viability was determined using the CCK-8 assay. D, The cell death rate was ascertained using flow cytometry. E–G, Lipid ROS, MDA, GSH, and Fe²⁺ levels were measured using kits. H, Expression of GXP4, SLC7A11, and ACSL4 was determined using Western blotting. Data from each study were tested thrice. *p < 0.05, **p < 0.01, ***p < 0.001.

Baicalein promoted FOXA2 deacetylation by upregulating SIRT6. A, B, FOXA2 mRNA and protein expressions were substantiated by qRT-PCR assay and Western blotting. C–E, FOXA2 expression and acetylation was substantiated by IP assay. F, The regulatory relationship of FOXA2 with SLC7A11 was ascertained by Co-IP assay. G, SIRT6 and FOXA2 were colocated in the nucleus. H, FOXA2 expression and acetylation was ascertained by IP assay. The data of each study were tested thrice. *p < 0.05, **p < 0.01, ***p < 0.001.

SIRT6 inhibited the transcriptional regulation of SLC7A11 by FOXA2. OGD/R-induced HT22 cells were treated with baicalein and transfected with the pcDNA3.1-SIRT6. A, The potential binding site of FOXA2 on SLC7A11 was analyzed using the StarBase database. B–D, Dual-luciferase reporter and ChIP assays were used to substantiate the relationship between FOXA2 and SLC7A11. E, F, SIRT6 mRNA and protein expression were substantiated by qRT-PCR and Western blot analysis (WB). Data from each study were tested thrice. *p < 0.05, **p < 0.01, ***p < 0.001.

SIRT6 regulated OGD/R-induced HT22 cell partial role of ferroptosis through FOXA2. OGD/R-induced HT22 cells were treated with baicalein and transfected with pcDNA3.1-FOXA2 or pcDNA3.1-SIRT6. A, B, FOXA2 mRNA and protein expression were substantiated by qRT-PCR and WB. C, Cell viability was determined using the CCK-8 assay. D, The cell death rate was ascertained using flow cytometry. E–G, Lipid ROS, MDA, GSH, and Fe²⁺ levels were measured using kits. H, Expression of GXP4, SLC7A11, and ACSL4 was determined by Western blotting. Data from each study were tested thrice. *p < 0.05, **p < 0.01, ***p < 0.001.

Baicalein inhibited partial role of ferroptosis by upregulating SIRT6 to alleviate I/R injury in mice. The MCAO/R mice were injected with baicalein and sh-SIRT6. A, The infarct size was measured using TTC staining. B, Neurological deficit scores were used to evaluate the nerve injuries. C, D, SIRT6 mRNA and protein expression were substantiated by qRT-PCR and Western blotting. E, F, The levels of MDA, GSH, Fe²⁺ were detected using kits. G, The expression of GXP4, SLC7A11, ACSL4, and FOXA2 was determined by Western blotting. n = 9. *p < 0.05, **p < 0.01, ***p < 0.001.