Controllable oxidative stress and tissue specificity in major tissues during the torpor-arousal cycle in hibernating Daurian ground squirrels

Abstract

Mammalian hibernators experience repeated hypoxic ischaemia and reperfusion during the torpor-arousal cycle. We investigated levels of oxidative stress, antioxidant capacity, and the underlying mechanism in heart, liver, brain and kidney tissue as well as plasma during different periods of hibernation in Daurian ground squirrels (Spermophilus dauricus). Our data showed that the levels of hydrogen peroxide significantly increased in the heart and brain during late torpor (LT) compared with levels during the summer active (SA) state. The content of malondialdehyde (MDA) was significantly lower during interbout arousal (IBA) and early torpor (ET) than that during SA or pre-hibernation (PRE), and MDA levels in the LT brain were significantly higher than the levels in other states. Superoxide dismutase 2 protein levels increased markedly in the heart throughout the entire torpor-arousal cycle. Catalase expression remained at an elevated level in the liver during the hibernation cycle. Superoxide dismutase 1 and glutathione peroxidase 1 (GPx1) expression increased considerably in all tissues during the IBA and ET states. In addition, the activities of the various antioxidant enzymes were higher in all tissues during IBA and ET than during LT; however, GPx activity in plasma decreased significantly during the hibernation season. The expression of p-Nrf2 decreased in all tissue types during IBA, but significantly increased during LT, especially in liver tissue. Interestingly, most changed indicators recovered to SA or PRE levels in post-hibernation (POST). These results suggest that increased reactive oxygen species during LT may activate the Nrf2/Keap1 antioxidant pathway and may contribute to the decreased MDA levels found during the IBA and ET states, thereby protecting organisms from oxidative damage over the torpor-arousal cycle of hibernation. This is the first report on the remarkable controllability of oxidative stress and tissue specificity in major oxidative tissues of a hibernator.

Keywords: ROS; antioxidant defence; antioxidant enzymes; hibernation; oxidative stress.

Figure 1.

Effects of hibernation on body weight, organ wet weight and organ-to-body-mass ratio in organs of S. dauricus over the course of a torpor–arousal cycle. (a) Changes in organ wet weight of heart, liver, brain and kidney in different periods (n = 8, one-way ANOVA). (b) Changes in organ-to-body-mass ratio in heart, liver, brain and kidney in different periods (n = 8, one-way ANOVA). SA, summer active; PRE, pre-hibernation; IBA, interbout arousal; ET, early torpor; LT, late torpor; POST, post-hibernation. Data are means ± s.e; *p < 0.05, compared with SA; **p < 0.01 compared with SA; ***p < 0.001 compared with SA; ^#p < 0.05, compared with PRE; ^##p < 0.01, compared with PRE; ^###p < 0.001, compared with PRE; ^&&p < 0.01, compared with IBA; ⁺⁺p < 0.01, compared with ET; ⁺⁺⁺p < 0.001, compared with ET; ^{^}p < 0.05, compared with LT; ^{^^}p < 0.01, compared with LT; ^{^^^}p < 0.001, compared with LT.

Figure 2.

Levels of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) in tissue and plasma samples. (a) Changes in H₂O₂ levels in heart, liver, brain, kidney and plasma during different periods (n = 8, one-way ANOVA). (b) Changes in MDA levels in heart, liver, brain, kidney and plasma during different periods (n = 8, one-way ANOVA). SA, summer active; PRE, pre-hibernation; IBA, interbout arousal; ET, early torpor; LT, late torpor; POST, post-hibernation. Data are means ± s.e; *p < 0.05, compared with SA; **p < 0.01, compared with SA; ^#p < 0.05, compared with PRE; ^##p < 0.01, compared with PRE; ^&p < 0.05, compared with IBA; ^&&p < 0.01, compared with IBA; ⁺p < 0.05, compared with ET; ⁺⁺p < 0.01, compared with ET; ⁺⁺⁺p < 0.001, compared with ET; ^{^}p < 0.05, compared with LT; ^{^^}p < 0.01, compared with LT.

Figure 3.

Changes in levels of SOD1, SOD2, CAT and GPx1 proteins in different tissues of S. dauricus over a torpor–arousal cycle. (a) Representative immunoblots of SOD1, SOD2, CAT and GPx1 in heart tissue during six hibernation periods. (b) Relative SOD1, SOD2, CAT and GPx1 protein expression in heart tissue. (c) Representative immunoblots of SOD1, SOD2, CAT and GPx1 in liver tissue during six hibernation periods. (d) Relative SOD1, SOD2, CAT and GPx1 protein expression in liver tissue. (e) Representative immunoblots of SOD1, SOD2, CAT and GPx1 in brain tissue during six hibernation periods. (f) Relative SOD1, SOD2, CAT and GPx1 protein expression in brain tissue. (g) Representative immunoblots of SOD1, SOD2, CAT and GPx1 in kidney tissue during six hibernation periods. (h) Relative SOD1, SOD2, CAT and GPx1 protein expression in kidney tissue. SA, summer active; PRE, pre-hibernation; IBA, interbout arousal; ET, early torpor; LT, late torpor; POST, post-hibernation. Values are means ± s.e., n = 8; *p < 0.05, compared with SA; **p < 0.01, compared with SA; ***p < 0.001, compared with SA; ^#p < 0.05, compared with PRE; ^##p < 0.01, compared with PRE; ^###p < 0.001, compared with PRE; ^&p < 0.05, compared with IBA; ^&&p < 0.01, compared with IBA; ⁺p < 0.05, compared with ET; ⁺⁺p < 0.01, compared with ET; ^{^}p < 0.05, compared with LT; ^{^^}p < 0.01, compared with LT.

Figure 4.

Changes in activities of SOD1, SOD2, CAT, GPx1 and TAC in different tissues of S. dauricus over the course of a torpor–arousal cycle. (a) Activities of SOD1, SOD2, CAT, GPx and TAC in heart tissue during six hibernation periods. (b) Activities of SOD1, SOD2, CAT, GPx and TAC in liver tissue during six hibernation periods. (c) Activities of SOD1, SOD2, CAT, GPx and TAC in brain tissue during six hibernation periods. (d) Activities of SOD1, SOD2, CAT, GPx and TAC in kidney tissue during six hibernation periods. (e) Activities of SOD1, SOD2, CAT, GPx and TAC in plasma during six hibernation periods. SA, summer active; PRE, pre-hibernation; IBA, interbout arousal; ET, early torpor; LT, late torpor; POST, post-hibernation. Values are means ± s.e., n = 8; *p < 0.05, compared with SA; **p < 0.01, compared with SA; ***p < 0.001, compared with SA; ^#p < 0.05, compared with PRE; ^##p < 0.01, compared with PRE; ^###p < 0.001, compared with PRE; ^&p < 0.05, compared with IBA; ^&&p < 0.01, compared with IBA; ^&&&p < 0.001, compared with IBA; ⁺p < 0.05, compared with ET; ⁺⁺p < 0.01, compared with ET; ⁺⁺⁺p < 0.001, compared with ET; ^{^}p < 0.05, compared with LT.

Figure 5.

Changes in levels of Nrf2, p-Nrf2 and Keap1 proteins in various tissues during different periods. (a) Representative immunoblots of Nrf2, p-Nrf2 and Keap1 in heart tissue during six hibernation periods. (b) Relative Nrf2, p-Nrf2 and Keap1 protein expression in heart tissue. (c) Representative immunoblots of Nrf2, p-Nrf2 and Keap1 in liver tissue during six hibernation periods. (d) Relative Nrf2, p-Nrf2 and Keap1 protein expression in liver tissue. (e) Representative immunoblots of Nrf2, p-Nrf2 and Keap1 in brain tissue during six hibernation periods. (f) Relative Nrf2, p-Nrf2 and Keap1 protein expression in brain tissue. (g) Representative immunoblots of Nrf2, p-Nrf2 and Keap1 in kidney tissue during six hibernation periods. (h) Relative Nrf2, p-Nrf2 and Keap1 protein expression in kidney tissue. SA, summer active; PRE, pre-hibernation; IBA, interbout arousal; ET, early torpor; LT, late torpor; POST, post-hibernation. Values are means ± s.e., n = 8; *p < 0.05, compared with SA; **p < 0.01, compared with SA; ^#p < 0.05, compared with PRE; ^##p < 0.01, compared with PRE; ^&p < 0.05, compared with IBA; ^&&&p < 0.001, compared with IBA; ^{^}p < 0.05, compared with LT.