الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
The globally increasing burden of IR exposure highlights the importance of pursuing research to develop new radioprotectors to protect individuals from the IR-induced MAP. IR induces cellular stress that regulates pro-apoptotic members of the Bcl-2 family of proteins including BAX, which forms the apoptotic pore and result in MOMP. HtrA2 is one of the released apoptogenic factors. It induces apoptosis through caspase-dependent and independent mechanism. Caspase 9 is an initiator caspase that cleaves and activates downstream effector caspases that directly mediate cascade of events leading to the well-known apoptosis phenotypic changes.
Ectoine is a compatible solute, that is biosynthesized by microorganisms under extreme conditions including radiation. It is a hydroxyl radical scavenger, and it can protect organs and cells against ultraviolet-A radiation damage, IR-induced DNA single strand breaks, inflammation, and oxidative stress. Thus, we aimed to evaluate the effect of ectoine on the expression level of BAX, HtrA2, and caspase 9, that are considered as vital markers of MAP in irradiated mice as a possible mechanism of radioprotection.
We assessed the locomotor level of activity as an indicator for the physical and mental status of mice. Our results demonstrated that mice in the ectoine treated group had a comparable activity level relative to the control group, while those in the irradiated group showed a highly significantly decreased activity level. Interestingly, ectoine elevated the locomotor activity in the combined treatment group.
We evaluated the body weight of mice, liver weight and the liver index as a sign of toxicity and morbidity. Our results demonstrated that γ-irradiation caused severe body weight loss at day 16 post-treatment. Also, the liver weight was high during the first week, then it was gradually decreasing over the follow up period. This increase could be due the IR-induced accumulation of fatty hepatocytes. Our results demonstrated that treating mice with 200 mg/kg of ectoine alone resulted in an increased body and liver weight at the first week post-treatment. In the combined treatment group, ectoine insignificantly increased the body weight at day 16 post-treatment. We also reported that it significantly reduced the liver weight and the liver index relative to the irradiated group. Hence, ectoine has the capability to improve IR-induced morbidity.
200 mg/kg of ectoine effectively enhanced the survival of irradiated mice and improved the IR-induced death rate from 58.33% in the radiation treated group to 22.22% in the combined treatment group, two hours post-irradiation.
We studied the effect of 6 Gy γ-irradiation on the expression level of BAX, HtrA2, and caspase 9 to indicate its ability to induce cell death through MAP. IR resulted in a 14.08, 3.75, and 3.72-fold increase in expression level of BAX, HtrA2 and caspase 9, respectively at day 3 post-treatment. Then, the gene expression of BAX, HtrA2 and caspase 9 were down-regulated at day 30, due to cell recovery.
Summary, Conclusion and Recommendations
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Our results demonstrated a statistically significant moderate positive correlation between the expression of BAX and HtrA2, which could be due to the effect of apoptosis-induced HtrA2. There was a moderate positive correlation between the expression of BAX and caspase 9. Also, there was a low positive correlation between HtrA2 and caspase 9 that is due to the ability of HtrA2 to induce caspase-dependent MAP.
200 mg/kg of ectoine resulted in a 3.23, 2.29 and 2.86-fold increase in expression level of BAX, HtrA2 and caspase 9, respectively at day 3 post-treatment. However, their expression levels were gradually decreased, to 0.16, 0.19, and 0.05-fold, respectively at day 30 post-treatment. Although ectione caused increased MAP genes expression, it resulted in high survival rates among other experimental groups. Consequently, this pro-apoptotic effect of ectoine was only at the gene expression level, as BAX, HtrA2 and caspase 9’s active protein structure requires further activation steps.
We studied the effect of 200 mg/kg of ectoine and 6 Gy γ-irradiation on the expression level of BAX, HtrA2, and caspase 9 to indicate the ability of ectoine to inhibit the MAP. In the combined treatment group, ectoine effectively reduced the expression levels of the three genes relative to the irradiated group during the first week post-treatment. However, after the first week, BAX and HtrA2 showed elevated levels of expression at day 16 and 30, while caspase 9 had reduced levels relative to the irradiated group. At day 16 and 30, although BAX’s expression was up-regulated, caspase 9 was down-regulated. Also, elevated levels of HtrA2 were not reciprocated by elevated levels of caspase 9.
We employed univariate and multivariate analysis of the genes’ expression to describe its effect on the survival and DNA fragmentation. Our results demonstrated only caspase 9’s up-regulation was associated with death and DNA fragmentation. Hence, caspase 9 was a strong prognostic factor, as the increased expression was associated with increased risk, which in turn affected the survival, the DNA fragmentation and the likelihood of death.
We further studied DNA fragmentation as a biochemical hallmark of apoptosis, where DNA laddering was prevalently characteristic to the irradiated group, specially at day 7 post-treatment.
We determined the association between treatment type and both DNA fragmentation and death. There was a significant association between them, where IR increased the risk of DNA fragmentation and death relative to the control group at 5.86 and 1.93, respectively. Ectoine in the combined treatment group efficiently reduced the hazard ratios.
All in all, our study supports the notion that ectoine has a radioprotective effect. Such that it alleviated the IR-induced fatigue, morbidity, and mortality. It mostly indirectly affects the molecular markers of MAP, of which caspase 9 was a strong independent prognostic factor for DNA fragmentation and death. Ectoine is a promising inhibitor of the MAP. However, more elaborate research needs to be done due to the implication of other molecules in the pathway and to identify them and the magnitude of their effect.
Summary, Conclusion and Recommendations
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• Conclusions:
from this study, we conclude the following:
1. IR decreased the locomotor activity and resulted in an IR-induced fatigue-like behavior, while ectoine improved this effect.
2. IR caused ARS resembled in severe body weight loss at day 16 post-treatment, and increased liver weight and liver index, while ectoine reversed the irradiation influence.
3. 200 mg/kg of ectoine effectively improved the IR-induced death rate and enhanced the survival of irradiated mice.
4. 200 mg/kg of ectoine can inhibit the effect of IR-induced MAP, where in the combined treatment group, ectoine effectively reduced their expression levels during the first week post-treatment.
5. Caspase 9 is a strong prognostic factor of DNA fragmentation and death.
6. 200 mg/kg of ectoine reduced the radiation’s elevated hazard ratio.
• Recommendations: from this study, we recommend the following: 200 mg/kg of ectoine could be used as a potent radioprotector in future clinical and/or non-clinical situations.
from this study we recommend the following as possible ways to further continue with our study: 1. Studying the locomotor activity using an automated system for monitoring mice individually in their respective housing cages to avoid observational bias and social isolation, and to provide longer periods of monitoring.
2. The pro-apoptotic effect of ectoine needs to be further studied at the protein level.
3. Ectoine’s pharmacodynamics needs to be further studied to verify whether other molecules are involved in the signaling mechanism and to identify them.
4. Ectoine’s pharmacokinetics requires more elaborate studies.