Complexity of Compensatory Effects in Nrf1 Knockdown: Linking Undeveloped Anxiety-Like Behavior to Prevented Mitochondrial Dysfunction and Oxidative Stress

Abstract

Anxiety-related disorders are complex illnesses that underlying molecular mechanisms need to be understood. Mitochondria stand as an important link between energy metabolism, oxidative stress, and anxiety. The nuclear factor, erythroid-derived 2,-like 1(Nrf1) is a member of the cap "n" collar subfamily of basic region leucine zipper transcription factors and plays the major role in regulating the adaptive response to oxidants and electrophiles within the cell. Here, we injected small interfering RNA (siRNA) targeting Nrf1 in dorsal third ventricle of adult male albino Wistar rats and subsequently examined the effect of this silencing on anxiety-related behavior. We also evaluated apoptotic markers and mitochondrial biogenesis factors, along with electron transport chain activity in three brain regions: hippocampus, amygdala, and prefrontal cortex. Our data revealed that in the group that received Nrf1-siRNA, anxiety-related behavior did not show any significant changes compared to the control group. Caspase-3 did not increase in Nrf1-siRNA-injected rats even though Bax/Bcl2 ratio markedly elevated in Nrf1-knockdown rats in all three mentioned regions compared to control rats. Also, Nrf1 silencing of complex I and II-III did not alter, generally. In addition, Nrf1-knockdown affected mitochondrial biogenesis markers. The level of peroxisome proliferator-activated receptor gamma coactivator-1α and cytochrome-c increased, which indicates a possible role for mitochondrial biogenesis in anxiety.

Keywords: Anxiety; Electron transport chain; Nrf1; PGC-1α; siRNA.

Fig. 1

siRNA-mediated down regulation of Nrf1 in 3 areas of rat’s brain. Evaluation of protein level (Western blotting) after direct in vivo injection of scrambled siRNA (control) and Nrf1-siRNA in hippocampus (a), prefrontal (b), and amygdala (c). Sixty μg proteins were separated on SDS-PAGE, Western blotted, probed with anti-Nrf1 antibody, and reprobed with anti-β-actin antibody (one representative Western blot was shown). The densities of corresponding bands were measured and the ratio to β-actin bands was reported. Three rats were used for each 4 and 8 h groups, and experiment was repeated 3 times independently. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control group

Fig. 2

Effect of Nrf1-siRNA administration on rat anxiety-like behavior in the EPM. The animals received scrambled siRNA or Nrf1-siRNA into D3V. Mean ± SEM of a percentage of open arm time, b percentage of open arm entries, c enclosed arm enteries, d time spend in the open arm, e time spend in the close arm, and f time spend in the central area of EPM (n = 8 in each group)

Fig. 3

Bax/Bcl2 ratio and caspase-3 cleavage in hippocampus, amygdala, and prefrontal cortex after siRNA injection are shown by Western blotting. Sixty μg proteins were separated on SDS-PAGE, Western blotted, probed with anti-Bax, -Bcl2 and -caspase-3 antibodies, and reprobed with anti-β-actin antibody. The ratio to the β-actin bands was reported. Each point shows the mean ± SEM. ***P < 0.001 compared to the control group

Fig. 4

Western blot analysis of Nrf2/β-actin ratio in hippocampus, amygdala, and prefrontal cortex after Nrf1-siRNA injection. Method was as described in Fig. 1 legend. Each point shows the mean ± SEM. *P < 0.05 and ***P < 0.001 compared to the control group

Fig. 5

PGC-1α, cytochrome-c, NRF-1, and TFAM level in hippocampus, amygdala, and prefrontal after siRNA injection are shown by Western blotting. Method was as described in Fig. 1 legend. Each point shows the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the control group