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Lithium protects against paraquat neurotoxicity by NRF2 activation and miR-34a inhibition in SH-SY5Y cells.

Alural B, Ozerdem A, Allmer J, Genc K, Genc S - Front Cell Neurosci (2015)

Bottom Line: Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a.Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity.Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

View Article: PubMed Central - PubMed

Affiliation: Izmir Biomedicine and Genome Center, Dokuz Eylul University Izmir, Turkey ; Department of Neuroscience, Health Science Institute, Dokuz Eylul University Izmir, Turkey.

ABSTRACT
Lithium is a mood stabilizing agent commonly used for the treatment of bipolar disorder. Here, we investigated the potential neuroprotective effect of lithium against paraquat toxicity and its underlying mechanisms in vitro. SH-SY5Y human neuroblastoma cells were treated with paraquat (PQ) 0.5 mM concentration after lithium pretreatment to test lithium's capability in preventing cell toxicity. Cell death was evaluated by LDH, WST-8, and tryphan blue assays. Apoptosis was analyzed using DNA fragmentation, Annexin V immunostaining, Sub G1 cell cycle analysis, and caspase-3 activity assays. BCL2, BAX, and NRF2 protein expression were evaluated by Western-blotting and the BDNF protein level was determined with ELISA. mRNA levels of BCL2, BAX, BDNF, and NRF2 target genes (HO-1, GCS, NQO1), as well as miR-34a expression were analyzed by qPCR assay. Functional experiments were done via transfection with NRF2 siRNA and miR-34a mimic. Lithium treatment prevented paraquat induced cell death and apoptosis. Lithium treated cells showed increased anti-apoptotic protein BCL2 and decreased pro-apoptotic protein BAX expression. Lithium exerted a neurotrophic effect by increasing BDNF protein expression. It also diminished reactive oxygen species production and activated the redox sensitive transcription factor NRF2 and increased its target genes expression. Knockdown of NRF2 abolished neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium. Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a. Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity. Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

No MeSH data available.


Related in: MedlinePlus

NRF2 siRNA transfection decreases NRF2 and target genes mRNA and NRF2 protein expression in SH-SY5Y cells. Cells were transfected with NRF2 siRNA or non-target siRNA for 24 h. (A,B) NRF2 protein levels were measured by Western blot, (C) mRNA levels of NRF2 were evaluated with qPCR. Transfection of cells with NRF2 siRNA decreased NRF2 mRNA and protein expression. Knocking-down NRF2 expression reduced the mRNA expression of (D)HO-1 and (E)NQO1(F)GCS and (G)BCL-2 determined by qPCR. The data are presented as mean ± S.E, n = 5. (*p < 0.05 compared to non-target siRNA transfected cells).
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Figure 6: NRF2 siRNA transfection decreases NRF2 and target genes mRNA and NRF2 protein expression in SH-SY5Y cells. Cells were transfected with NRF2 siRNA or non-target siRNA for 24 h. (A,B) NRF2 protein levels were measured by Western blot, (C) mRNA levels of NRF2 were evaluated with qPCR. Transfection of cells with NRF2 siRNA decreased NRF2 mRNA and protein expression. Knocking-down NRF2 expression reduced the mRNA expression of (D)HO-1 and (E)NQO1(F)GCS and (G)BCL-2 determined by qPCR. The data are presented as mean ± S.E, n = 5. (*p < 0.05 compared to non-target siRNA transfected cells).

Mentions: To confirm the repression by NRF2 specific siRNA, we evaluated NRF2 mRNA expression by qPCR and NRF2 protein expression by Western blotting. Transfection of cells with NRF2 siRNA decreased the NRF2 expression at both mRNA and protein levels (Figures 6A–C). In addition, NRF2 inhibition significantly reduced the mRNA expression of NRF2-regulated genes, HO-1, NQO1, GCS, and BCL-2 compared to cells transfected with non-targeting siRNA (Figures 6D–G).


Lithium protects against paraquat neurotoxicity by NRF2 activation and miR-34a inhibition in SH-SY5Y cells.

Alural B, Ozerdem A, Allmer J, Genc K, Genc S - Front Cell Neurosci (2015)

NRF2 siRNA transfection decreases NRF2 and target genes mRNA and NRF2 protein expression in SH-SY5Y cells. Cells were transfected with NRF2 siRNA or non-target siRNA for 24 h. (A,B) NRF2 protein levels were measured by Western blot, (C) mRNA levels of NRF2 were evaluated with qPCR. Transfection of cells with NRF2 siRNA decreased NRF2 mRNA and protein expression. Knocking-down NRF2 expression reduced the mRNA expression of (D)HO-1 and (E)NQO1(F)GCS and (G)BCL-2 determined by qPCR. The data are presented as mean ± S.E, n = 5. (*p < 0.05 compared to non-target siRNA transfected cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446540&req=5

Figure 6: NRF2 siRNA transfection decreases NRF2 and target genes mRNA and NRF2 protein expression in SH-SY5Y cells. Cells were transfected with NRF2 siRNA or non-target siRNA for 24 h. (A,B) NRF2 protein levels were measured by Western blot, (C) mRNA levels of NRF2 were evaluated with qPCR. Transfection of cells with NRF2 siRNA decreased NRF2 mRNA and protein expression. Knocking-down NRF2 expression reduced the mRNA expression of (D)HO-1 and (E)NQO1(F)GCS and (G)BCL-2 determined by qPCR. The data are presented as mean ± S.E, n = 5. (*p < 0.05 compared to non-target siRNA transfected cells).
Mentions: To confirm the repression by NRF2 specific siRNA, we evaluated NRF2 mRNA expression by qPCR and NRF2 protein expression by Western blotting. Transfection of cells with NRF2 siRNA decreased the NRF2 expression at both mRNA and protein levels (Figures 6A–C). In addition, NRF2 inhibition significantly reduced the mRNA expression of NRF2-regulated genes, HO-1, NQO1, GCS, and BCL-2 compared to cells transfected with non-targeting siRNA (Figures 6D–G).

Bottom Line: Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a.Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity.Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

View Article: PubMed Central - PubMed

Affiliation: Izmir Biomedicine and Genome Center, Dokuz Eylul University Izmir, Turkey ; Department of Neuroscience, Health Science Institute, Dokuz Eylul University Izmir, Turkey.

ABSTRACT
Lithium is a mood stabilizing agent commonly used for the treatment of bipolar disorder. Here, we investigated the potential neuroprotective effect of lithium against paraquat toxicity and its underlying mechanisms in vitro. SH-SY5Y human neuroblastoma cells were treated with paraquat (PQ) 0.5 mM concentration after lithium pretreatment to test lithium's capability in preventing cell toxicity. Cell death was evaluated by LDH, WST-8, and tryphan blue assays. Apoptosis was analyzed using DNA fragmentation, Annexin V immunostaining, Sub G1 cell cycle analysis, and caspase-3 activity assays. BCL2, BAX, and NRF2 protein expression were evaluated by Western-blotting and the BDNF protein level was determined with ELISA. mRNA levels of BCL2, BAX, BDNF, and NRF2 target genes (HO-1, GCS, NQO1), as well as miR-34a expression were analyzed by qPCR assay. Functional experiments were done via transfection with NRF2 siRNA and miR-34a mimic. Lithium treatment prevented paraquat induced cell death and apoptosis. Lithium treated cells showed increased anti-apoptotic protein BCL2 and decreased pro-apoptotic protein BAX expression. Lithium exerted a neurotrophic effect by increasing BDNF protein expression. It also diminished reactive oxygen species production and activated the redox sensitive transcription factor NRF2 and increased its target genes expression. Knockdown of NRF2 abolished neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium. Furthermore, lithium significantly decreased both basal and PQ-induced expression of miR-34a. Transfection of miR-34a specific mimic reversed neuroprotective, anti-apoptotic, and anti-oxidant effects of lithium against PQ-toxicity. Our results revealed two novel mechanisms of lithium neuroprotection, namely NRF2 activation and miR-34a suppression.

No MeSH data available.


Related in: MedlinePlus