Mitochondrial Proteins Unveil The Mechanism By Which Physical Exercise Ameliorates Memory, Learning And Motor Activity in Hypoxic Ischemic Encephalopathy Rat Model Part 2

2.3. Mitochondrial Proteins in the Nuclei of the Hippocampus

The mitochondrial apoptosis indicators in the nuclear portion, cytochrome c, apoptosis-inducing factor (AIF), and cleaved caspase-3, as well as Smac/Diablo and OPA1, increased significantly in HIE, as demonstrated by Western blot semi-quantification (Figure 3). 

Cytochrome is an important neurochemical that facilitates communication between neurons and helps people maintain good memory. Many studies have shown that cytochromes have a huge impact on the function of the human brain and are indispensable in learning, memory, cognition, and other aspects.

First of all, cytochromes can improve people's attention and concentration, which is one of the important factors in improving memory. The role of cytochromes becomes more apparent when we need to remember certain information and we need to concentrate on it with full concentration. It can coordinate the activity of neurons and enhance the transmission of information between different areas, thereby helping us learn and remember more effectively.

Secondly, cytochromes can also promote connectivity between brain neurons and further enhance the efficiency of information transmission. The presence of cytochromes modulates synaptic transmission and strengthens synaptic connections. This process promotes interactions and information transfer between neurons, making the brain more flexible and efficient.

Finally, cytochromes can also help people better process and store memory information. Research shows that cytochromes play a very important role in the process of memory formation and preservation. It can strengthen the connections and interactions between neurons, thereby helping us better store and recall information and improve the effectiveness and durability of memory.

To sum up, cytochromes have a very important impact on brain function and memory. We should focus on maintaining and promoting the production of cytochrome, maintaining brain health, and improving memory through a healthy diet, moderate exercise, and good sleep. It can be seen that we need to improve memory, and Cistanche deserticola can significantly improve memory, because Cistanche deserticola has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidation and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche deserticola can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning, and thinking speed, and may also prevent the development of cognitive dysfunction and neurodegenerative diseases.

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Cytochrome c (Figure 3B), cleaved caspase-3 (Figure 3C), and Smac/Diablo (Figure 3D) were all expressed differently in HIE with and without exercise but there was no statistically cally significant difference when compared to SHAM, NT. Nonetheless, nuclear AIF and OPA1 (Figure 3A, E) showed statistically significant.

2.4. Mitochondrial Proteins in the Cytoplasm of the Cerebral Cortex

The mitochondrial apoptosis indicators in the cytoplasmic portion, apoptosis-inducing factor (AIF), cytochrome c, cleaved caspase-3, as well as Smac/Diablo and OPA1, increased significantly in HIE, as demonstrated by Western blot semi-quantification (Figure 4). 

AIF (Figure 4A), Cytochrome c (Figure 4B), and Smac/Diablo (Figure 4D) were all expressed differently in HIE with and without exercise but there was no statistically significant difference when compared to SHAM, NT. Despite this, cleaved caspase-3 (Figure 4C) and OPA1 (Figure 4E) showed statistically significant changes.

2.5. Mitochondrial Proteins in the Nuclei of the Cerebral Cortex

The mitochondrial apoptosis indicators in the nuclear portion, cytochrome c, apoptosis-inducing factor (AIF), and cleaved caspase-3, as well as Smac/Diablo and OPA1, increased significantly in HIE, as demonstrated by Western blot semi-quantification (Figure 5), AIF (Figure 5A), Cytochrome c (Figure 5B), cleaved caspase-3 (Figure 5C), and OPA1 (Figure 5E) were all expressed differently in HIE and exercise and were statistically significant compared to SHAM, NT. 

Changes in nuclear Smac/Diablo (Figure 5D), on the other hand, were not statistically significant.

Taken together, these findings support the proposition that exercise improves motor function, learning, and memory recovery by suppressing hippocampal and cortical mitochondrial apoptotic protein expression in the brains of HIE rats.

2.6. Immunofluorescence Analysis of Proteins in the Motor Cortex

Figure 6 shows representative immunofluorescence images from the motor cortex of the cerebral cortex region stained for AlF, Cytochrome C, Cleaved caspase-3, Smac, and OPAlThe quantitative analysis of the mean fluorescent intensity (MFI) of the proteins are also summarized in Figures 6 and 7. $wimming exercise caused a significant reduction in the expression of the proteins. images and the mean fluorescent intensity (MFI) graphs.

Figure 6. Immunofluorescence images and mean fluorescent intensities of the proteins in the motor cortex. (A) Representative immunofluorescence images and MFI of AIF in the motor cortex of each group. AIF molecules are green. AIF molecules were analyzed in the motor cortex. 

Results are presented as the Mean ± SEM of 3 rats from each group. (** p < 0.001 and *** p < 0.0001) compared with the SHAM, NT group; (## p < 0.001 compared with the HIE, NT group; each group, n = 3). Scale bar, 100 µm. (B) Representative immunofluorescence images and MFI of Cytochrome C in the motor cortex of each group. Cytochrome C molecules (green) were analyzed in the motor cortex. 

Data are presented as the Mean ± SEM of 3 rats from each group. (** p < 0.001 and *** p < 0.0001) compared with the SHAM, NT group; (## p < 0.001 compared with the HIE, NT group); each group, n = 3. Scale bar, 100 µm. (C) Representative immunofluorescence images and MFI of Cleaved caspase-3 in the motor cortex of each group. Cleaved caspase-3 molecules (green) were analyzed in the motor cortex. 

Results are presented as the Mean ± SEM of 3 rats from each group. (* p < 0.05 and *** p < 0.0001) compared with the SHAM, NT group); each group, n = 3. Scale bar, 100 µm. (D) Representative immunofluorescence images and MFI of SMAC in the motor cortex of each group. 

SMAC molecules (green) were analyzed in the motor cortex. Results are presented as the Mean ± SEM of 3 rats of each group. (** p < 0.001 and *** p < 0.0001) compared with the SHAM, NT group); each group, n = 3. Scale bar, 100 µm. (E) Representative immunofluorescence images and MFI of OPA1 in the motor cortex of each group. OPA1 molecules (green) were analyzed in the motor cortex. 

Individual data are presented as the Mean ± SEM from 3 rats in each group. (*** p < 0.0001) compared with the SHAM-NT group; (## p < 0.001 compared with the HIE, NT group); each group, n = 3. Scale bar, 100 µm.

3. Discussion

Because mitochondria are essential for energy production within brain cells [23,24], we investigated the effect of swimming exercise on mitochondrial apoptotic and dynamic signals in HIE. 

To accomplish this, we used an exercise routine and assessed five mitochondrial function regulators (AIF, Cytochrome c, Cleaved caspase-3, Smac/Diablo, and OPA1). Our results demonstrated that exercise training affects these proteins regardless of the health state (that is, in HIE rat models or sham groups). 

Swimming exercise lowered the expression of mitochondrial apoptosis-related proteins; AIF, cytochrome c, and cleaved caspase-3 in the cytosol and nuclei of the hippocampus, and cortex, which is consistent with the statement made by Moore et al. [25] that physical activity impacts every aspect of the mitochondrion. 

Furthermore, the four weeks of exercise lowered the expression levels of the fission marker Smac/Diablo and the cristae remodel-ing protein OPA1, which was concomitant with enhanced motor performance, learning, and memory retention. 

In the hippocampus and cerebral cortex of HIE and sedentary rats, the levels of mitochondrial apoptotic indicators; cytochrome c, AIF, and cleaved caspase-3 increased significantly. In comparison to SHAM, NT rats, when HIE and normal rats were subjected to swimming exercise, the levels of AIF, cytochrome c, and cleaved caspase-3 decreased significantly, an observation in tandem with [24,26] findings that exercise restores mitochondria function in neurodegenerative disorders. 

With AIF pointed out as a major contributor to neuron loss in the immature brain following hypoxia-ischemia and a hypomorphic mutation causing decreased AIF expression reported to protect against neonatal hypoxic ischemia [27], we observe here that swimming exercise has the same effect in the HIE rat brain by suppressing the protein's cytosolic and nuclear translocation. 

The antiapoptotic impact of exercise training corresponds to improvements in motor function, learning, and memory, implying that exercise training circumvents mitochondrial malfunction and apoptosis. 

This explains the neuronal protective mechanisms reported after exercise training that promote neurogenesis and myelin repair in the penumbra following stroke [28], as these are high energy-demanding processes that necessitate stable functional mitochondria. 

Given exercise's ability to modulate mitochondrial protein stabilization, and enhance motor function, learning, and memory, it would be remarkable to determine whether the exercise-related metabolite, lactate, that crosses the blood-brain barrier [29] contributes significantly to exercise's beneficial effects of suppressing apoptosis and promoting motor function, learning, and memory in HIE. 

Swimming exercise significantly improved motor activity, memory, and learning in rats with HIE by reducing mitochondrial apoptosis through the Cyto.C/Cleaved Caspase-3 and AIF signaling pathways. 

Aside from that, swimming exercise intervention has been shown to stabilize mitochondrial cristae and membrane potential in HIE rats, as evidenced by Smac/Diablo and OPA1 reversal in these animals, which is consistent with [30,31] findings emphasizing the importance of OPA1 in mitochondrial cristae stabilization. 

With the underlying neurobiological mechanisms of exercise-induced neuroplasticity still mostly elusive [8], our findings provide an intriguing and plausible platform for future research on identifying additional molecular pathways that could be modulated to effectively manage HIE and the resulting impairments.

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