Resveratrol attenuates the CoCl2-induced hypoxia damage by regulation of lysine β-hydroxybutyrylation in PC12 cells

Abstract

Background: Stroke is a cerebrovascular disease that is the main cause of death and disability worldwide. Hypoxia is a major factor that causes neuronal damage and even cellular death. However, the mechanism and therapeutic drugs for hypoxia are not completely understood.

Methods: In this study, PC12 cells (a rat adrenal pheochromocytoma cell line) were exposed to Cobalt chloride (CoCl₂) to induce hypoxia. Using this cell model, the impacts of hypoxia on cell viability, proliferation, reactive oxygen species (ROS), and the levels of lysine β-hydroxybutyrylation (Kbhb) and the inflammatory signaling factor P65 were examined. In addition, we explored the ability of resveratrol (RES) to alleviate CoCl₂-induced hypoxia damage.

Results: RES attenuated CoCl₂-induced decreases of cell viability and cell proliferation and increase of ROS production in PC12 cells. CoCl₂ downregulated Kbhb in PC12 cells, but RES alleviated this effect. In addition, upregulated Kbhb by 3-hydroxybutyric acid sodium could partially recover the CoCl₂-induced hypoxia damage to PC12 cells, including cell viability, cell proliferation, oxidative stress, and the protein level of the inflammatory signaling factor P65.

Conclusion: Our results indicate that RES protects against CoCl₂-induced hypoxia damage in PC12 cells by modulating Kbhb, a novel post-translational modification.

Keywords: CoCl2; Hypoxia; Lysine β-hydroxybutyrylation; PC12 cells; Reactive oxygen species; Resveratrol.

Fig. 1

Effect of CoCl₂ on the viability of PC12 cells. (a-c) PC12 cells were exposed to 5 µM, 50 µM, or 100 µM CoCl₂ for assessment of cell viability at 24 h (a), 48 h (b) and 72 h (c) using the CCK-8 assay. n = 3. The data conformed to normal distribution analyzed by Shapiro-Wilk test. The data are presented as the mean ± SD. Differences between groups were assessed by one-way ANOVA. **p < 0.01 and ***p < 0.001

Fig. 2

Effect of CoCl₂ combined with resveratrol (RES) on the viability of PC12 cells. PC12 cells were exposed to 100 µM CoCl₂, 50 μm RES, and 100 µM CoCl₂ plus 50 μm RES for 48 h. (a) Morphological characterization of PC12 cells was performed using an optical microscope (the red arrows for abnormal morphology); scale bars in the figures indicate 100 μm. (b) The percentage of abnormal cells in different groups have been quantified. (c) Cell viability was examined by CCK-8 assay. The data are presented as the mean ± SD. n = 3. The data conformed to normal distribution analyzed by Shapiro-Wilk test. Differences between groups were assessed by one-way ANOVA. *p < 0.05, **p < 0.01

Fig. 3

Effect of CoCl₂ combined with RES on PC12 cell proliferation. (a) EdU staining (red) and Hoechst staining (blue) of PC12 cells treated with 100 µM CoCl₂ combined with 50 µM RES. Scale bar: 100 μm. (b) Statistical result of CoCl₂ combined with RES on PC12 cell proliferation. EdU positive cells (%) were calculated as the ratio of EdU staining cells to Hoechst staining cells and multiplied by 100 (%). The data are presented as the mean ± SD. n = 4. The data conformed to normal distribution analyzed by Shapiro-Wilk test. Differences between groups were assessed by one-way ANOVA. ns: not significant, **p < 0.01, ***p < 0.001

Fig. 4

Effect of RES on CoCl₂-induced reactive oxygen species (ROS) production in PC12 cells. PC12 cells were exposed to 100 µM CoCl₂, 50 µM RES, and 100 µM CoCl₂ plus 50 µM RES for 48 h. ROS production was measured with the ROS fluorescent dye DCFH-DA. The data are presented as the mean ± SD. n = 6. The data conformed to normal distribution analyzed by Shapiro-Wilk test. Differences between groups were assessed by one-way ANOVA. ns: not significant, **p < 0.01, ***p < 0.001

Fig. 5

Effect of CoCl₂, RES, and CoCl₂ plus RES on the level of Kbhb in PC12 cells. (a) PC12 cells were exposed to CoCl₂ (5, 50 and 100 µM) for 48 h, and the level of Kbhb was analyzed via western blotting. (b) PC12 cells were exposed to 50 µM RES, 100 µM CoCl₂, and 50 µM RES plus 100 µM CoCl₂ for 48 h, and the level of Kbhb was analyzed via western blotting. The data are presented as the mean ± SD. n = 3. The data conformed to normal distribution analyzed by Shapiro-Wilk test. Differences between groups were assessed by one-way ANOVA. *p < 0.05, **p < 0.01

Fig. 6

Effect of 3-hydroxybutyric acid sodium (3-HBA·Na) on the level of Kbhb and viability of PC12 cells. PC12 cells were exposed to 100 µM CoCl₂, 20 mM 3-HBA·Na, and 20 mM 3-HBA·Na plus 100 µM CoCl₂ for 48 h. (a and b) The level of Kbhb was examined by western blotting. (c) Cell viability was examined via a CCK-8 assay. The data are presented as the mean ± SD. n = 3 or 6. The data conformed to normal distribution analyzed by Shapiro-Wilk test. Differences between groups were assessed by one-way ANOVA. **p < 0.01, ***p < 0.001

Fig. 7

Effect of 3-hydroxybutyric acid sodium (3-HBA·Na) on the proliferation of PC12 cells. EdU staining (red) and Hoechst staining (blue) of PC12 cells treated with 100 µM CoCl₂, 20 mM 3-HBA·Na, and 20 mM 3-HBA·Na plus 100 µM CoCl₂. Scale bar: 100 μm. (b) Statistical result of 3-HBA·Na combined with RES on PC12 cell proliferation. The data are presented as the mean ± SD. n = 3. The data conformed to normal distribution analyzed by Shapiro-Wilk test. Differences between groups were assessed by one-way ANOVA. ns: not significant, *p < 0.05

Fig. 8

Effect of 3-hydroxybutyric acid sodium (3-HBA·Na) on the expression of P65. PC12 cells were exposed to 100 µM CoCl₂, 20 mM 3-HBA·Na, and 20 mM 3-HBA·Na plus 100 µM CoCl₂ for 48 h. The protein level of P65 in the nuclear fraction was analyzed via western blotting (a and b)