Expression of apoptosis, autophagy and necroptosis effectors in cells of rat hippocampus after excessive F- consumption

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Abstract

The work examined the expression of apoptosis, autophagy and necroptosis markers in hippocampal cells of rats after long-term consumption of excessive F- doses at the transcriptional and translational levels. Male Wistar rats were divided into 4 groups receiving 0.4 (control), 5, 20 and 50 mg/l F- (as NaF) for 12 months. The changes in contents of effectors of mitochondrial (Bcl-2, Bax, Caspase-9, Caspase-3) and receptor (Caspase-8, Fas) pathways of apoptosis, mediators (Ulk-1, Beclin-1) and modulators (AMPK, Ark, mTOR) of autophagy, as well as that of necroptosis (RIP and MLKL) were assessed by immunoblotting, the gene expression (Bcl2, Bax, Casp3, Ulk1, Beclin1, Prkaa1, Akt, and mTor) – by real-time PCR. In the hippocampus of F – exposed animals, the expression ratio of Bcl2/Bax genes and Bcl-2/Bax proteins decreased, caspase-9 and caspase-3 were activated, but the level of caspase-8 and membrane Fas receptor remained stable. Long-term F- consumption had no effect on the content of autophagy initiator Ulk-1 and protein kinases AMPK, Akt and mTOR, but resulted in inhibition of key autophagy mediator Beclin-1. The expression level of necroptosis RIP and MLKL effectors in the hippocampal cells of rats received excessive F- did not change as well. Thus, long-term F- exposure was accompanied by activation of apoptosis, mainly through the mitochondrial pathway, at the background of autophagy suppression.

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About the authors

O. V. Nadei

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences

Email: nagalak@mail.ru
Russian Federation, Saint-Petersburg

N. I. Agalakova

Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences

Author for correspondence.
Email: nagalak@mail.ru
Russian Federation, Saint-Petersburg

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2. Fig. 1. Changes in the expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in the rat hippocampus after exposure to F- at the level of transcription and translation. (a) – Bcl2 and Bax gene expression levels in hippocampal cells normalized to the expression of the reference gene pair Eef1a1+Ppia (mean values ​​± SE, n = 7–9). (b) – Representative immunoblots for one rat from each group. (c) – Mean ± SE content of Bcl-2 and Bax proteins in hippocampal cells calculated in relation to the optical density of the reference protein GAPDH (n = 7). (d) – Ratio of Bcl-2 and Bax gene expression. (e) – Ratio of Bcl-2 and Bax protein levels. * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control group (Con).

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3. Fig. 2. Level of caspase-9 in the hippocampus of rats fed excess F-. (a) Representative examples of immunoblots of native caspase-9 and active caspase-9 for one rat from each experimental group. (b) Mean ± SE content of the two forms of caspase-9 normalized to GAPDH (n = 5). * p < 0.05, *** p < 0.001 compared to control.

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4. Fig. 3. Stimulation of caspase-3 in the hippocampus of rats fed excess F-. (a) Casp3 mRNA levels in hippocampal cells of rats from all experimental groups (mean ± SE, n = 8–10). (b) Representative examples of immunoblots of native caspase-3 and active caspase-3 for one rat from each experimental group. (c) Mean ± SE content of the two forms of caspase-3 normalized to GAPDH. (d) Changes in the ratio of procaspase-3 to active caspase-3 levels (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control.

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5. Fig. 4. Protein expression of two forms of caspase-8 and Fas receptors in rat hippocampal cells after long-term exposure to F-. (a) Example of immunoblots for one animal from each group. (b) Average caspase-8 and Fas protein content ± SE in cells calculated relative to the content of the reference protein GAPDH (n = 4).

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6. Fig. 5. Expression of autophagy mediators in the hippocampus of F-exposed rats. (a) Representative immunoblots and mean ± SE ULK-1 protein levels in hippocampal cells of rats treated with different doses of F- (n = 7). (b) Changes in Beclin1 gene expression in the hippocampus of animals treated with excess F- at the transcriptional level (n = 6). (c) Changes in Beclin-1 protein content in the hippocampus of animals after long-term F- consumption (n = 6). Representative immunoblots for one animal from each group and mean ± SE values ​​of native and phosphorylated (Ser30) Beclin-1 forms are shown. * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.0001 compared to control.

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7. Fig. 6. Expression of autophagy modulators AMPK, Akt and mTOR in the hippocampus of F-excess-treated rats at the transcriptional and translational levels. (a) Expression levels of Prkaa1, Akt and mTOR genes in rat hippocampal cells. (b) Representative immunoblots for one animal from each group. (c) Mean ± SE values ​​of AMPK, Akt and mTOR protein levels (n = 5).

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8. Fig. 7. RIP and MLKL protein levels in the hippocampus of rats treated with different doses of F-. (a) Representative immunoblots for one animal from each group. (b) Mean ± SE values ​​of RIP and MLKL protein content in hippocampal cells (n = 5).

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