| Apoptosis is vital to the development of biological beings, which is regulated by a series of genes. It is a type of programmed cell death, which is the most important way to clear dead cells that could be triggered by internal or external apoptotic stimulants. Apoptosis is in charge of clearing damaged or aged cells, which makes it important to development and maintenance of homeostasis. Besides, apoptosis is also closely related to the occurrence and development of many diseases. For example, neurodegenerative diseases such as Alzheimer’s disease or Huntington’s diseases are related to apoptosis. Besides, cell death caused by toxins as well as viral infections is also somehow related to apoptosis. In addition, cancer is also related to apoptosis, in that the occurrence of cancer is mainly due to the inhibition of apoptosis of cancer cells.There are three main pathways of apoptosis. The first and main one is called mitochondria-dependent apoptosis, of which the regulating mechanism is relatively clear. Mitochondria-dependent pathway is usually activated when cells are damaged internally or exposed to stressors. Pro-apoptotic factors such as cytochrome c, SMAC, Endo G and AIF are then released from mitochondria, activating caspases, and eventually causing cell death. Besides mitochondria-dependent apoptosis, the rest two apoptotic pathways are death receptor-dependent apoptosis and ER-dependent pathway. The three pathways mentioned above are not isolated. On the contrary, they could form a more complicated network by interacting with each other under certain circumstances.Besides apoptotic pathways, anti-apopototic pathways also play vital roles in the maintenance of cellular homeostasis. Researchers recently found a new pathway that possibly had anti-apoptotic effect, which is Parkin-NEMO-NF-κB-OPAl. The above pathway is also a type of mitochondria-dependent pathway. It was shown in a recent study that Parkin linked NF-κB to apoptosis via ubiquitination. In other words, drug stimulant could protect cells from apoptosis via Parkin-NEMO-NF-κB-OPAl pathway.Objective:The purpose of the present study was to examine the time-dependent changes of key genes and proteins of Parkin-mediated anti-apoptotic pathway caused by acute exhaustive exercise and subsequent recovery period, in the hope of investigating the relevance of Parkin-mediated anti-apoptotic pathway and apoptosis of skeletal muscle cells, exploring the underlying mechanism of Parkin-mediated anti-apoptotic pathway, and pondering the effect of acute exhaustive exercise on apoptosis of skeletal muscle cells as well as its tentative mechanism.Method:56 four-week-old male C57/BL6 mice were randomly divided into seven groups, which are:control (C), post exercise (EO),2h post exercise (E2),6h post exercise (E6),12h post exercise (E12),24h post exercise (E24) and 48h post exercise (E48). After one week adaptive feeding, the mice were allowed to perform acute exhaustive swimming in a tank with 50cm depth and water temperature of 30±2℃. The exercise was terminated when the mice sank and could not come back to water surface within 10s. The mice of Group Es were sacrificed with cervical dislocation. The gastrocnemius muscles of both sides were quickly removed, and mRNA and protein levels were measured with RT-PCR and Western Blot, respectively. Mitochondria membrane potential was also measured with JC-1 assay kit in order to reflect cell apoptosis level.Results:After acute exhaustive swimming, the mRNA expression of Parkin decreased significantly at E6, E24 and E48 (p<0.05) and E12 (p＜0.01). The mRNA expression of IκBα increased significantly at EO and E12 (p＜0.01), E2 and E6 (p<0.05). The mRNA expressions of NEMO, P50, P65 and OPA1 were not significantly different from group C. After acute exhaustive swimming, the protein level of Parkin increased significantly at EO (p＜0.01), E2 and E6 (p＜0.05). The protein level of P-IκB increased significantly at EO (p＜0.05) and E2 (P＜0.01). On the contrary, the protein level of IκBα decreased at E6, E12, E24 and E48 (p<0.05). The ratio of P-IκB and IκBα significantly decreased (p＜0.05) after acute exhaustive swimming exercise. The protein expression of OPA1 increased significantly at EO, E6 and E12 (p<0.05). After acute exhaustive swimming, the mRNA expression of Cyt C decreased significantly at EO (p＜0.05), E12, E24 and E48 (p＜0.01). The mRNA expression of CASP3 decreased significantly at E0, E2, E6, E12, E48 (p<0.05) and E24 (p<0.01). The mRNA expression of CASP6 decreased significantly at E0, E2, E12 and E48 (p<0.01). The mRNA expression of CASP9 decreased significantly at E0, E2, E12, E24 and E48 (p<0.01). The result of JC-1 assay kit suggested that after acute exhaustive swimming, the mitochondria membrane potential of the skeletal muscle of mice decreased significantly at E0, E2, E6, E12, E24 and E48 (p<0.05).Conclusion:1) Apoptosis was observed after acute exhaustive swimming exercise, and lasted for over 48 hours.2) Acute exhaustive swimming could effectively decrease the sensitivity of apoptotic factors in the skeletal muscle, such as Caspase 3, Caspase 6, Caspase 9 and Cyt C.3) Acute exhaustive swimming exercise immediately initiated Parkin-mediated anti-apoptotic pathway. However, the changes of the key genes did not necessarily fully follow the sequence of the pathway. Besides, the changes of mRNA expressions and protein levels were not entirely consistent with each other. |
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