Clinical studies have shown sex differences in onset, severity, and extra intestinal manifestation in IBD1,2,19. This is likely due to the hormonal change and studies have shown at puberty there is an increase in IBD diagnosis in females than males. We chose to use rats at the age of puberty to explore this effect in the indomethacin induced IBD rat model. We found that two injections 24 hours apart induced acute GI inflammation resulting in weight loss two days after injections in both male and female rats. IBD female rats showed a more significant loss in body weight, a significant increase in colon hypertrophy, and slightly higher disease activity score compared to IBD male rats. These results indicate that there are slight differences in the severity of the disease between males and females, which is similar to the observed clinical variations between males and females.
Mitochondrial function is important to maintaining epithelial barrier function11. To begin our exploration into mitochondrial function in our IBD model, we measured mitochondrial content as either citrate synthase activity or cardiolipin levels. Cardiolipin has been reported as the most reliable measure of mitochondrial content followed by citrate synthase27. What we have demonstrated here is that both measures show the same trend. Due to these similarities, we used citrate synthase activity to normalize to mitochondrial content. IBD male rats have a significant decrease in colon mitochondrial content compared to controls while females showed no change in mitochondrial content in the IBD rats. These results indicate that the males likely have improved mitophagy mechanisms compared to females.
Mitochondrial oxidative phosphorylation (OXPHOS) is important for generating ATP. ATP production is essential to maintaining epithelial barrier function. Electrons enter the electron transport chain through two different pathways. One pathway is by the oxidation of NADH by complex I. The other is the oxidation of succinate to fumarate by complex II. Both complex I and II will transfer electrons to a membrane bound electron acceptor ubiquinone to generate ubiquionol. Electrons are then transferred through complex III to cytochrome c. Cytochrome c will bind and transfer an electron to complex IV, which ultimately transfers electrons to oxygen to generate water. Complex I, III, and IV are proton pumps which pump protons from the matrix to the inner membrane space to generate a proton motive force that is utilized by ATP synthase to convert ADP to ATP. Defects in OXPHOS have been implicated in various diseases, especially those related to inflammation29. Respiratory control ratios (RCRs) are an indication of how efficiently the mitochondria are generating ATP. Female IBD rats showed a significant decrease in colonic mitochondrial RCR values. When normalized to citrate synthase, only the female IBD rats showed a decrease in intact colonic mitochondrial respiration. These results indicate that protons are leaking through the inner mitochondrial membrane by a pathway other than ATP synthase, resulting in lower ATP production and lower OXPHOS capacity. The loss of ATP in IBD female colon mitochondria will impair the ability of epithelial cells to maintain barrier function resulting in GI inflammation4.
When mitochondrial oxidative phosphorylation is decreased, mitochondrial ROS (mtROS) production is increased. This occurs through the production of superoxide, which will contribute to cellular oxidative stress and is linked to diseases and epithelial barrier dysfunction4,30. Both male and female IBD rats showed a significant increase in the production of mtROS compared to control regardless of substrate. This increase in mtROS will contribute to the gut dysbiosis, lower ATP production by the epithelial cells, and epithelial barrier dysfunction resulting in GI inflammation4. There are two significant locations for mtROS production from OXPHOS, complex I and complex III. Complex I will produce mtROS when the FMN site is reduced to FMNH2 leading to a high NADH/NAD+ ration, which increases the likelihood that electrons will be transferred to O2 instead of through the iron-sulfur centers31. Complex I and III will produce mtROS if there is an increase in the highly reactive semiquionone, which will quickly react with O2 to produce mtROS31. Any inhibition in the electron transport chain will result in an increase in the reduction of these intermediates that will increase the probability of mtROS production. In the IBD female colon mitochondria, we found that complex I and IV activities are decreased, which would lead to an increase in both FMH2 and the semiquinone, ultimately leading to the increase in mtROS.
Although mtROS may be produced there is a balance between antioxidants and mtROS. However, when the balance between antioxidants and ROS species is disrupted, oxidative stress can occur28. Assessment of the antioxidant status is correlated to the extent of oxidative stress. Redox homeostasis is regulated either enzymatically or non-enzymatically. Superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase and glutathione levels are common components that are altered in disease states28. When we explored these antioxidant mechanisms, the only one that showed a significant difference was catalase. Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen32. In colons from male IBD rats, we found that catalase was significantly decreased. The amplex red assay measures the presence of H2O2. Therefore, since the male IBD rats have a decrease in catalase activity there will be an increase in H2O2.
Based on these results, using a mitochondrial-targeted therapy is a feasible option for treatment. MitoTEMPO is a mitochondria-targeted superoxide dismutase mimetic that possesses superoxide and alkyl radical scavenging properties33.1 This compound combines the antioxidant piperidine nitroxide TEMPO with the lipophilic cation triphenylphosphonium, which allows it to pass through lipid bilayers and accumulate in mitochondria33. Mitochondrial targeting of superoxide scavenging via mitoTEMPO has been examined for potential therapeutic benefit to a variety of mitochondrial dysfunctions arising from excessive reactive oxygen species34–36. IBD disease activity was improved with mitoTEMPO treatment in female rats but not males. As expected mitoTEMPO decreased mtROS in the colon of IBD females rats and it also improved the activity of complex I and IV. However, mitoTEMPO increased mtROS in males. This is likely due to mitoTEMPO converting superoxide to H2O2. However, the males have lower catalase activity leading to an increase in the levels of H2O2. A mitochondrial-targeted catalase mimetic will potentially be a better treatment option for the males but creating a catalase mimetic can be challenging. However, a study in 2016 designed a nanosystem based on Tempol and a biocompatible B-cyclodextrin-derived material23. The Tempol serves as a SOD mimetic, while the nanocontainer is mainly composed of a hydrogen peroxide-eliminating material that functions as a catalase-mimicker23. A treatment like this may work better for our male IBD colon mitochondrial function and will be explored in the future. Another feasible option is the supplementation with MCFAs. MCFAs have been shown to lower mtROS in other disease models26. In this study, the males produce less mtROS with MCFA than other mitochondrial substrates. We will also explore other mitochondrial-targeted therapies in the future.
Interestingly, our findings also highlight sex differences in colon mitochondrial function. There have been several studies exploring the effect of sex hormones on mitochondrial function37. Estrogen acts through the estrogen receptor β (ERβ) to increase mitochondrial DNA (mtDNA)–encoded transcription of cytochrome oxidase subunits I, III, and IV; increase respiration capacity; elevate antioxidant activity; and inhibit apoptosis22. Studies have shown that an increase in the androgen receptor (AR) negatively regulates the expression, assembly, integrity, and function of individual mitochondrial complexes resulting in decreased mitochondrial OXPHOS21. The animals used in this study are at the age of puberty for rats. Therefore, sex hormones are on the rise, which will increase the chances of observing sex differences. When comparing control males and females, we observed differences in colon mitochondrial function. Control males showed a significant decrease in complex I-driven respiration, complex II-driven respiration and MCFA oxidation compared to control females. Control males also showed a significant decrease in complex II, III, and IV activity compared to control females. This highlights the importance of identifying sex differences in mitochondrial function in disease models. In future studies, we will alter the sex hormone status in our IBD animals to further determine the effects of sex hormones on mitochondrial function.
In conclusion, we have shown that female SD rats have an increase in disease severity compared to males. We have also demonstrated that there are differences in colon mitochondrial function between male and female SD rats. This may contribute to the differences in disease severity. While both male and female rats have increases in mtROS production, the cause for the increase is different between males and females. In females, complex I and IV have decreased activity resulting in the potential for increased mtROS. In males, catalase activity is decrease which will result in an increase of H2O2. Targeting mitochondria with the superoxide mimetic (mitoTEMPO) improves the disease activity in females, decreases mtROS, and improves complex I and IV activity. However, it did not prove to be a viable treatment option for males.