Effect of acute treadmill exercise and voluntary freewheel running on cytokine and apoptotic protein expression in intestinal lymphocytes of older female C57BL/6 mice
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Background: Colorectal cancer (CRC) is the second leading cause of Canadian cancer mortality. Inflammation is a fundamental risk factor in the aetiology of sporadic intestinal carcinoma. Reducing the frequency or duration of gastrointestinal inflammation may decrease CRC risk. Over 200 population studies demonstrate reduced odds of developing CRC among physically active persons. Preliminary data suggests that regular exercise may slow CRC pathogenesis by decreasing and increasing intestinal expression of pro- and anti-inflammatory cytokines, respectively. This research was designed to further our understanding of how exercise influences the colonic cytokine milieu, even in the presence of immunoscenescent changes. Objectives: The objective of the first experiment (Study #1) was to compare cytokine and apoptotic protein expression in intestinal lymphocytes (IL) at baseline and in response to acute exercise-induced oxidant stress in both young and older C57BL/6 female mice. A second objective (Study #2) was to examine the effect of exercise training on the expression of pro- and anti-inflammatory cytokines and pro- and anti-apoptotic proteins in IL of older C57BL/6 female mice under ‘resting’ conditions. The final objective (Study #3) was to compare the effect of acute exercise-induced stress on IL cytokine and apoptotic protein expression in trained versus untrained older C57BL/6 mice. Methods: Immediately following sacrifice, plasma was collected from the mice and stored (-80ºC) until corticosterone and 8-iso-PGF2α assessment by enzyme immunoassay. Soleus and plantaris skeletal muscles were excised and frozen in liquid nitrogen (-80ºC) until spectrophotometric assessment of cytochrome c oxidase (CO) activity. Finally, the entire mouse intestinal compartment was removed and IL lysates were prepared for flow cytometric analysis of percent apoptosis (% Annexin V+ IL) and for western blot analysis of pro-inflammatory (TNF-α, IL-1β), pleiotropic (IL-6) and anti-inflammatory (IL-10) cytokine, and pro-(caspase-3, -7) and anti-(Bcl-2) apoptotic protein expression. Results: Findings from Study #1 indicate that, in mice, acute exercise increases caspase-3 (IMM and 2Hr groups vs. SED; p<0.05) and TNF-α (IMM vs. SED and 2Hr groups; p<0.001), and decreases Bcl-2 (IMM and 2Hr groups vs. SED; p<0.01) expression in intestinal lymphocytes. Furthermore, IL expression of Bcl-2 was lower (p<0.001) and % Annexin V+ IL was higher (p<0.05) in the older vs. young mice. The results from Study #2 indicate that trained older mice had lower (p<0.05) expression of TNF-α and caspase-7 in IL, and lower (p<0.05) concentration of 8-iso-PGF2α in plasma compared to sedentary untrained controls. Finally, Study #3 shows that older trained mice display increased expression of pro-(TNF-α) and anti-(IL-10) inflammatory cytokines and pro-apoptotic (caspase-3, caspase-7) proteins, and decreased expression of anti-apoptotic (Bcl-2) protein in IL after acute exercise challenge compared to older untrained controls. In both Study #1 & #3, the treadmill protocol induced stress: plasma corticosterone and 8-iso-PGF2α were higher in mice sampled immediately after acute exercise relative to the no acute exercise (sedentary) condition. This exercise effect did not differ by age (Study #1) or by training (Study #3) condition. In addition, Study #2 & Study #3 showed elevations in cytochrome c oxidase activity following long-term training. Conclusion: Collectively, these results suggest that, in C57BL/6 female mice, IL expression of pro-apoptotic proteins and pro-inflammatory cytokines does not differ by age (young vs. older animals) in response to a single intense exercise bout. However, older mice display lower expression of ‘protective’ anti-apoptotic proteins and a higher percentage of early apoptotic IL compared to young mice. Additionally, long-term exercise may protect the bowel from inflammation by reducing inflammatory cytokine and apoptotic protein expression under ‘resting’ (no stress) conditions. Finally, long-term training preserves the IL cytokine and apoptotic protein responses in older mice to a magnitude similar to that previously described in young mice. Alternatively, older untrained mice display reduced responsiveness to acute treadmill exercise, suggestive of immunosenescence.