GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion

doi:10.1038/srep20196

. 2016 Feb 3:6:20196.

doi: 10.1038/srep20196.

GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion

Xi Chen^{1

2

3}, Yuanling Liu^{1

2

3}, Jin Zhu^{1

2

3}, Shipeng Lei⁴, Yuan Dong⁵, Lingyu Li^{1

2

3}, Beibei Jiang^{1

2

3}, Li Tan^{1

2

3}, Jingxian Wu^{1

2

3}, Shanshan Yu^{1

2

3}, Yong Zhao^{1

2

3}

Affiliations

¹ Department of Pathology, Chongqing Medical University, 400016 Chongqing, People's Republic of China.
² Institute of Neuroscience, Chongqing Medical University, 400016 Chongqing, People's Republic of China.
³ Key Laboratory of Neurobiology, Chongqing Medical University, 400016 Chongqing, People's Republic of China.
⁴ Department of Respiratory Medicine, Jiangjin Center Hospital, 402260, Chongqing, People's Republic of China.
⁵ Department of Forensic Medicine, Chongqing Medical University, 400016 Chongqing, People's Republic of China.

PMID: 26838164
PMCID: PMC4738318
DOI: 10.1038/srep20196

GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion

Xi Chen et al. Sci Rep. 2016.

. 2016 Feb 3:6:20196.

doi: 10.1038/srep20196.

Authors

Affiliations

¹ Department of Pathology, Chongqing Medical University, 400016 Chongqing, People's Republic of China.
² Institute of Neuroscience, Chongqing Medical University, 400016 Chongqing, People's Republic of China.
³ Key Laboratory of Neurobiology, Chongqing Medical University, 400016 Chongqing, People's Republic of China.
⁴ Department of Respiratory Medicine, Jiangjin Center Hospital, 402260, Chongqing, People's Republic of China.
⁵ Department of Forensic Medicine, Chongqing Medical University, 400016 Chongqing, People's Republic of China.

PMID: 26838164
PMCID: PMC4738318
DOI: 10.1038/srep20196

Abstract

The NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a critical role in protecting against oxidative stress in brain ischemia and reperfusion injury. Glycogen synthase kinase 3β (GSK-3β) may play a critical role in regulating Nrf2 in a Kelch-like ECH-associated protein 1 (Keap1)-independent manner. However, the relationship between GSK-3β and Nrf2 in brain ischemia and reperfusion injury is not clear. In this study, we explored the mechanisms through which GSK-3β regulates Nrf2 and Nrf-2/ARE pathways in vitro and in vivo. We used oxygen and glucose deprivation/reoxygenation (OGD/R) in primary cultured cortical neurons and a middle cerebral artery occlusion-reperfusion (MCAO/R) rat model to mimic ischemic insult. In this study, GSK-3β siRNA and inhibitors (SB216763 and LiCl) were used to inhibit GSK-3β in vitro and in vivo. After inhibiting GSK-3β, expression of total and nuclear Nrf2, Nrf2-ARE binding activity, and expression of Nrf2/ARE pathway-driven genes HO-1 and NQO-1 increased. Overexpression of GSK-3β yielded opposite results. These results suggest that GSK-3β downregulates Nrf2 and the Nrf2/ARE pathway in brain ischemia and reperfusion injury. GSK-3β may be an endogenous antioxidant relevant protein, and may represent a new therapeutic target in treatment of ischemia and reperfusion injury.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

**Figure 1. P-GSK-3β (tyr216), total GSK-3β, β-catenin, and Nrf2 expression levels varied with reoxygenation time.**
After 1.5 h of oxygen and glucose deprivation (OGD), neurons were harvested after 0.5 h, 1 h, 4 h, and 6 h of reoxygenation. (A) Western blot analyses of p-GSK-3β (tyr216), total GSK-3β, β-catenin, and Nrf2. (**B–E**) Representative ratios of p-GSK-3β (tyr216), total GSK-3β, β-catenin, and Nrf2 to β-actin. P-GSK-3β (tyr216) underwent a short-term decrease after 0.5 h of reoxygenation and then initially reached the highest level after 1 h of reoxygenation. Total GSK-3β expression levels showed a similar trend. β-catenin and Nrf2 expression levels showed a reverse trend. Bars represent mean ± SEM (n = 4–6). **p < 0.01 vs. Normal, ^#p < 0.05 vs. 0.5 h reoxygenation, ^##p < 0.01 vs. 0.5 h reoxygenation.

**Figure 2. GSK-3 β regulation of Nrf2 in neurons under normal conditions.**
(A) Western blot analysis of GSK-3β and p-GSK-3β (tyr216). (**B,C**) Representative ratios of GSK-3β and p-GSK-3β (tyr216) to β-actin. GSK-3β and p-GSK-3β (tyr216) expression significantly decreased in the siRNA (GSK-3β siRNA) and inhibitor [SB(SB216763) and LiCl] groups compared with the normal group. (D) Western blot analysis of Nrf2 and nuclear Nrf2. (**E,F**) Representative ratios of Nrf2 and nuclear Nrf2 to β-actin. There was no significant difference in the expression levels of Nrf2 and nuclear Nrf2 in siRNA, inhibitor, and GSK-3β groups compared with the normal group. Con siRNA indicates control siRNA. Bars represent mean ± SEM (n = 4–6) *p < 0.05 vs. Normal.

**Figure 3. GSK-3β regulates Nrf2 in neurons after oxygen and glucose deprivation/reoxygenation (OGD/R).**
Cells were subjected to OGD for 1.5 h followed by 1 h of reoxygenation, after which cells were harvested. (A) Western blot analyses of GSK-3β and p-GSK-3β (tyr216). (**B,C**) Representative ratios of GSK-3β and p-GSK-3β (tyr216) to β-actin. GSK-3β and p-GSK-3β (tyr216) expression significantly decreased in the siRNA + OGD/R and inhibitor +OGD/R groups compared with the OGD/R group. (D) Western blot analysis of Nrf2 and nuclear Nrf2. (**E,F**) Representative ratios of Nrf2 and nuclear Nrf2 to β-actin. Expression levels of Nrf2 and nuclear Nrf2 significantly increased in the siRNA + OGD/R and inhibitor + OGD/R groups. Nrf2 and nuclear Nrf2 expression levels significantly decreased in the GSK-3β + OGD/R group. Bars represent mean ± SEM (n = 4–6). ^#p < 0.05 vs. OGD/R, ^##P < 0.01 vs. OGD/R.

**Figure 4. Quantitative RT-PCR analysis of GSK-3β and Nrf2 mRNA levels in neurons.**
(**A,B**) GSK-3β and Nrf2 mRNA levels analyzed by quantitative RT-PCR from neurons in Fig. 2. (**C,D**) GSK-3β and Nrf2 mRNA levels analyzed by quantitative RT-PCR from neurons in Fig. 3. Bars represent mean ± SEM (n = 4–6). *p < 0.05 vs. Normal. **p < 0.01 vs. Normal, ^#p < 0.05 vs. OGD/R, ^##p < 0.01 vs. OGD/R. OGD/R = oxygen and glucose deprivation/reoxygenation.

**Figure 5. GSK-3β regulates Nrf2-ARE binding in neurons after glucose deprivation/reoxygenation (OGD/R).**
(A) Electrophoretic Mobility Shift Assay (EMSA) analysis of Nrf2-ARE binding. (B) Semiquantitative analysis of Nrf2-ARE binding. CK, 100x, (+) and (−) indicate controls. Bars represent mean ± SEM (n = 4–6). ^##p < 0.01 vs. OGD/R.

**Figure 6. GSK-3β regulates Nrf2/ARE-driven genes in neurons after oxygen and glucose deprivation/reoxygenation (OGD/R).**
Protein and RNA were collected after OGD for 1.5 h and reoxygenation for 1 h. (A) Western blot analysis of HO-1 and NQO1. (**B,C**) Representative ratios of HO-1 and NQO1 to β-actin. (**D,E**) Representative HO-1 and NQO1 mRNA levels analyzed by quantitative RT-PCR. The expression levels of HO-1 and NQO1 significantly increased in the siRNA (GSK-3β siRNA) + OGD/R and inhibitors + OGD/R groups. Reverse results were obtained in the GSK-3β + OGD/R group. Results from quantitative RT-PCR were consistent with those from western blot analysis. Bars represent mean ± SEM (n = 4–6). ^#p < 0.05 vs. OGD/R, ^##p < 0.01 vs. OGD/R.

**Figure 7. P-GSK-3β (tyr216), total GSK-3β, β-catenin, and Nrf2 expression levels varied with reperfusion time in the cerebral cortex of rats.**
Rats were subjected to 1 h of middle cerebral artery occlusion (MCAO), followed by 1 h, 6 h, or 24 h of reperfusion. (A) Western blot analysis of p-GSK-3β (tyr216), total GSK-3β, β-catenin, and Nrf2. (**B–E**) Representative ratios of p-GSK-3β (tyr216), total GSK-3β, β-catenin, and Nrf2 to β-actin. P-GSK-3β (tyr216) underwent a short-term decrease after 1 h of reperfusion and then initially reached the highest level after 6 h of reperfusion. β-catenin and Nrf2 expression levels showed a reverse trend. Bars represent mean ± SEM (n = 4–6). *p < 0.05 vs. Normal, ^#p < 0.05 vs. MCAO/R 1 h, ^##p < 0.01 vs. MCAO/R 1 h.

**Figure 8. GSK-3β regulation of Nrf2 in the cerebral cortex of rats in normal conditions.**
(A) Western blot analyses of GSK-3β, p-GSK-3β (tyr216), Nrf2, and nuclear Nrf2. (**B–E**) Representative ratios of GSK-3β, p-GSK-3β (tyr216), Nrf2, and nuclear Nrf2 to β-actin. GSK-3β and p-GSK-3β (tyr216) expression significantly decreased in the siRNA (GSK-3β siRNA) and inhibitor [SB(SB216763) and LiCl] groups compared with the normal group. There was no significant difference in the expression levels of Nrf2 and nuclear Nrf2 in the siRNA and inhibitor groups compared with the normal group. Bars represent mean ± SEM (n = 4–6). *p < 0.05 vs. Normal, **p < 0.01 vs. Normal.

**Figure 9. GSK-3β regulates Nrf2 in the cerebral cortex of rats after middle cerebral artery occlusion-reperfusion (MCAO/R).**
Rats were subjected to MCAO for 1 h followed by 6 h of reperfusion. (A) Western blot analysis of GSK-3β, p-GSK-3β (tyr216), Nrf2, and nuclear Nrf2. (**B–E**) Representative ratios of GSK-3β, p-GSK-3β (tyr216), Nrf2, and nuclear Nrf2 to β-actin. GSK-3β and p-GSK-3β (tyr216) expression significantly decreased in the siRNA + MCAO/R and inhibitors + MCAO/R groups compared with the MCAO/R group. Expression levels of Nrf2 and nuclear Nrf2 significantly increased in the siRNA + MCAO/R and inhibitors + MCAO/R groups. Bars represent mean ± SEM (n = 4–6). ^##P < 0.01 vs. MCAO/R.

**Figure 10. Quantitative RT-PCR analysis of GSK-3β and Nrf2 mRNA levels in the cerebral cortex of rats.**
(A,B) GSK-3β and Nrf2 mRNA levels analyzed by quantitative RT-PCR from the cerebral cortex in Fig. 8. (**C,D**) GSK-3β and Nrf2 mRNA levels analyzed by quantitative RT-PCR from the cerebral cortex in Fig. 9. Bars represent mean ± SEM (n = 4–6). *p < 0.05 vs. Normal, ^#p < 0.05 vs. MCAO/R. MCAO/R = middle cerebral artery occlusion-reperfusion.

**Figure 11. GSK-3β regulates Nrf2-ARE binding activity in the cerebral cortex of rats after middle cerebral artery occlusion-reperfusion (MCAO/R).**
(A) Electrophoretic Mobility Shift Assay (EMSA) analysis of Nrf2-ARE binding. (B) Semiquantitative analysis of Nrf2-ARE binding. CK, 100x, (+) and (−) indicate different controls. Bars represent mean ± SEM (n = 4–6). ^##p < 0.01 vs. MCAO/R.

**Figure 12. GSK-3β regulates Nrf2/ARE-driven genes in the cerebral cortex of rats after middle cerebral artery occlusion-reperfusion (MCAO/R).**
Protein and RNA were collected after MCAO for 1 h and reperfusion for 6 h. (A) Western blot analysis of HO-1 and NQO1. (**B,C**) Representative ratios of HO-1 and NQO1 to β-actin. (**D,E**) Representative HO-1 and NQO1 mRNA levels analyzed by quantitative RT-PCR. Expression levels of HO-1 and NQO1 significantly increased in the siRNA and inhibitor groups. Results from quantitative RT-PCR were consistent with those from western blot analysis. Bars represent mean ± SEM (n = 4–6). ^#p < 0.05 vs. MCAO/R, ^##P < 0.01 vs. MCAO/R.

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References

1. Doyle K. P. & Simon R. P. Mechanisms of ischemic brain damage. Neuropharmacology. 55, 310–318 (2008). - PMC - PubMed
1. Danilov C. A. et al.. Sulforaphane protects astrocytes against oxidative stress and delayed death caused by oxygen and glucose deprivation. Glia. 57, 645–656 (2009). - PMC - PubMed
1. Gu J. et al.. Icariside II enhances Nrf2 nuclear translocation to upregulate phase II detoxifying enzyme expression coupled with the ERK, Akt and JNK signaling path-ways. Molecules. 16, 9234–9244 (2011). - PMC - PubMed
1. Hwang Y. P. & Jeong H. G. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells. Toxicol. Appl. Pharmacol. 242(1), 18–28 (2010). - PubMed
1. Calkins M. J. et al.. The Nrf2/ARE pathway as a potential therapeutic target in neurodegener-ative disease. Antioxid. Redox. Signal. 11, 497–508 (2009). - PMC - PubMed

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[1] Doyle K. P. & Simon R. P. Mechanisms of ischemic brain damage. Neuropharmacology. 55, 310–318 (2008). - PMC - PubMed

[2] Doyle K. P. & Simon R. P. Mechanisms of ischemic brain damage. Neuropharmacology. 55, 310–318 (2008). - PMC - PubMed

[3] Danilov C. A. et al.. Sulforaphane protects astrocytes against oxidative stress and delayed death caused by oxygen and glucose deprivation. Glia. 57, 645–656 (2009). - PMC - PubMed

[4] Danilov C. A. et al.. Sulforaphane protects astrocytes against oxidative stress and delayed death caused by oxygen and glucose deprivation. Glia. 57, 645–656 (2009). - PMC - PubMed

[5] Gu J. et al.. Icariside II enhances Nrf2 nuclear translocation to upregulate phase II detoxifying enzyme expression coupled with the ERK, Akt and JNK signaling path-ways. Molecules. 16, 9234–9244 (2011). - PMC - PubMed

[6] Gu J. et al.. Icariside II enhances Nrf2 nuclear translocation to upregulate phase II detoxifying enzyme expression coupled with the ERK, Akt and JNK signaling path-ways. Molecules. 16, 9234–9244 (2011). - PMC - PubMed

[7] Hwang Y. P. & Jeong H. G. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells. Toxicol. Appl. Pharmacol. 242(1), 18–28 (2010). - PubMed

[8] Hwang Y. P. & Jeong H. G. Ginsenoside Rb1 protects against 6-hydroxydopamine-induced oxidative stress by increasing heme oxygenase-1 expression through an estrogen receptor-related PI3K/Akt/Nrf2-dependent pathway in human dopaminergic cells. Toxicol. Appl. Pharmacol. 242(1), 18–28 (2010). - PubMed

[9] Calkins M. J. et al.. The Nrf2/ARE pathway as a potential therapeutic target in neurodegener-ative disease. Antioxid. Redox. Signal. 11, 497–508 (2009). - PMC - PubMed

[10] Calkins M. J. et al.. The Nrf2/ARE pathway as a potential therapeutic target in neurodegener-ative disease. Antioxid. Redox. Signal. 11, 497–508 (2009). - PMC - PubMed

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GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion

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GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion

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