Korean J Physiol Pharmacol.  2017 Nov;21(6):579-589. 10.4196/kjpp.2017.21.6.579.

Genistein attenuates isoflurane-induced neurotoxicity and improves impaired spatial learning and memory by regulating cAMP/CREB and BDNF-TrkB-PI3K/Akt signaling

• Jiang T
• Wang XQ
• Ding C
• Du XL

• KMID: 2395252
• DOI: http://doi.org/10.4196/kjpp.2017.21.6.579

Abstract

Anesthetics are used extensively in surgeries and related procedures to prevent pain. However, there is some concern regarding neuronal degeneration and cognitive deficits arising from regular anesthetic exposure. Recent studies have indicated that brain-derived neurotrophic factor (BDNF) and cyclic AMP response element-binding protein (CREB) are involved in learning and memory processes. Genistein, a plant-derived isoflavone, has been shown to exhibit neuroprotective effects. The present study was performed to examine the protective effect of genistein against isoflurane-induced neurotoxicity in rats. Neonatal rats were exposed to isoflurane (0.75%, 6 hours) on postnatal day 7 (P7). Separate groups of rat pups were orally administered genistein at doses of 20, 40, or 80 mg/kg body weight from P3 to P15 and then exposed to isoflurane anesthesia on P7. Neuronal apoptosis was detected by TUNEL assay and FluoroJade B staining following isoflurane exposure. Genistein significantly reduced apoptosis in the hippocampus, reduced the expression of proapoptotic factors (Bad, Bax, and cleaved caspase-3), and increased the expression of Bcl-2 and Bcl-xL. RT-PCR analysis revealed enhanced BDNF and TrkB mRNA levels. Genistein effectively upregulated cAMP levels and phosphorylation of CREB and TrkB, leading to activation of cAMP/CREB-BDNF-TrkB signaling. PI3K/Akt signaling was also significantly activated. Genistein administration improved general behavior and enhanced learning and memory in the rats. These observations suggest that genistein exerts neuroprotective effects by suppressing isoflurane-induced neuronal apoptosis and by activating cAMP/CREB-BDNF-TrkB-PI3/Akt signaling.

Keyword

BDNF; CREB; Genistein; Isoflurane; Neurodegeneration; Phosphatidylinositol 3-kinase

MeSH Terms

Anesthesia
Anesthetics
Animals
Apoptosis
Body Weight
Brain-Derived Neurotrophic Factor
Cognition Disorders
Cyclic AMP Response Element-Binding Protein
Genistein*
Hippocampus
In Situ Nick-End Labeling
Isoflurane
Learning
Memory*
Neurons
Neuroprotective Agents
Phosphatidylinositol 3-Kinase
Phosphorylation
Rats
RNA, Messenger
Spatial Learning*
Anesthetics
Brain-Derived Neurotrophic Factor
Cyclic AMP Response Element-Binding Protein
Genistein
Isoflurane
Neuroprotective Agents
Phosphatidylinositol 3-Kinase
RNA, Messenger

Figure

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  Fig. 1 Genistein effectively reduced isoflurane-induced neuroapoptosis. The effect of genistein (20, 40 or 80 mg) on neuronal apoptosis following isoflurane exposure was assessed in the hippocampal regions by (A) TUNEL assay and (B) Fluoro-Jade B staining. Geinstein dose-dependently reduced isoflurane-induced apoptosis. The data are presented as mean±SD, n=6. aStatistically significant differences at p<0.05 vs respective control. b–fSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test.

• 

  Fig. 2 Genistein modulated the expression of proteins of the apoptotic cascade. (A) Western blotting analysis revealed that genistein significantly (p<0.05) reduced isoflurane-induced raised expressions of cleaved caspase-3, and pro-apoptotic proteins (Bad, and Bax), while geinstein markedly (p<0.05) up-regulated the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. (B) Represents relative expressions of proteins with control expressions set at 100%. The data are presented as mean±SD, n=6. aStatistically significant difference at p<0.05 vs respective control. b–eSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (Lane 1, Control; Lane 2, Isoflurane; Lane 3, Isoflurane+20 mg Genistein; Lane 4, Isoflurane+40 mg Genistein; Lane 5, Isoflurane +80 mg Genistein).

• 

  Fig. 3 Genistein effectively upregulated CREB signalling. (A) Expression analysis by Western blotting revealed up-regulated CREB signaling in the hippocampal tissues on geinstein supplementation. Genistein significantly (p<0.05) increased isoflurane suppressed CREB phosphorylation and CaMKIV expression. (B) Represents relative expressions of proteins with control expressions set at 100%.The data are presented as mean±SD, n=6. aStatistically significant differences at p<0.05 vs respective control. b–eSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (Lane 1, Control; Lane 2, Isoflurane; Lane 3, Isoflurane + 20 mg Genistein; Lane 4, Isoflurane + 40 mg Genistein; Lane 5, Isoflurane + 80 mg Genistein).

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  Fig. 4 Genistein increased hippocampal cAMP levels. cAMP levels in the hippocampal tissues following isoflurane exposure was assessed by ELISA. Genistein was found to cause significant (p<0.05) increase in the cAMP levels in a dose-dependent manner. The data are presented as mean±SD, n=6. aStatistically significant difference at p<0.05 vs respective control. b–eSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test.

• 

  Fig. 5 Genistein up-regulated BDNF –TrkB signaling. The BDNF and TrkB expressions in the hippocampal tissues were assessed both at mRNA and as well protein levels. Genistein dose-dependently increased the expression of BDNF and TrkB at both (A and B) mRNA and (C and D) protein levels. (C and D) Geinstein increased phosphorylation of TrkB. (B and D) Represents relative expressions of proteins with control expressions set at 100%. the data are presented as mean±SD, n=6. aStatistically significant difference at p<0.05 vs respective control. b–fSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (Lane 1, Control; Lane 2, Isoflurane; Lane 3, Isoflurane+20 mg Genistein; Lane 4, Isoflurane + 40 mg Genistein; Lane 5, Isoflurane+80 mg Genistein).

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  Fig. 6 Genistein modulated the expression of PI3K/Akt pathway proteins. (A) Western blot analysis of the PI3K pathway proteins revealed that genistein upregulated the expression of Akt, GSK-3β, phospho-Akt and phospho- GSK-3β and mTORc1 while suppressed PTEN, signifying activation of PI3K/Akt signaling cascade. (B) The data are presented as mean±SD, n=6. aStatistically significant difference at p<0.05 vs respective control. b–fSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test (Lane 1, Control; Lane 2, Isoflurane; Lane 3, Isoflurane+20 mg Genistein; Lane 4, Isoflurane+40 mg Genistein; Lane 5, Isoflurane+80 mg Genistein).

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  Fig. 7 Geinstein improved the general behavior of P35 rats following isoflurane exposure at P7. (A) The behavior of the rats in a novel environment was assessed by open-field test. Geinstein treatment improved the movement of the rats in comparison with isoflurane-alone exposed rats. (B) Genistein enhanced the freezing responses of P35 rats in context and cued fear conditioning tests. The data are presented as mean±SD, n=6. aStatistically significant difference at p<0.05 vs respective control. b–fSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test.

• 

  Fig. 8 Geinstein effectively improved learning and memory of rats in the Morris water maze tests. (A) The escape latency of the rats during training sessions reveals that geinstein treatment reduced the latency time. (B) Genistein treatment enhanced the performance of the rats in identifying the submerged platform at a shorter latency time demonstrating effectively improved spatial navigation in cued and place trials. In probe trials geinstein-treated rats spent longer time looking out for the platform in the target quadrant in comparison with isoflurane-alone exposed rats, indicating improved memory retention on geinstein treatment. The data are presented as means±SD, n=6. aStatistically significant at p<0.05 vs respective control. b–fSignificant differences (p<0.05) between mean values as determined by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test.

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