Comparative Study On Oxidative Stress Level And Antioxidant Defense Mechanism In Different Tissues Of Daurian Ground Squirrel (spermophilus Dauricus) During Different Hibernation Periods

Oxidative stress occurs as a result of mechanical unloading,fasting,and ischemia-reperfusion in the non-hibernators?rats,mice,humans,etc.?.However,accompanied by various stress challenges such as“ischemia-reperfusion”,fasting,and mechanical unloading during“torpor-arousal”cycle,no oxidative damage is found in the tissues of hibernators after a prolonged period of hibernation.Thus,it is an interesting and thoughtful research to explore the unique mechanisms and countermeasures of hibernating animals against ischemia-reperfusion induced oxidative stress.The Nrf2?nuclear factor erythroid 2?NF-E2?-related factor 2?/Keap1?Kelch-like ECH-associated protein 1?pathway is a prominent regulator of cytoprotective responses to oxidative stress.Under basal conditions,Nrf2 is sequestered in the cytosol by a Keap1 homodimer,which facilitates the ubiquitination and proteasomal degradation of Nrf2.However,under conditions of oxidative stress,Nrf2 is released from Keap1,leading to phosphorylation and nuclear translocation.In the nucleus,phospho-Nrf2 heterodimerizes with small musculoaponeurotic fibrosarcoma?Maf?proteins and binds to the antioxidant response element?ARE?,thereby activating the expression of a battery of antioxidant genes that respond to oxidative stress.Therefore,this paper will use the hibernating mammalian?Spermophilus dauricus?as the research object to detect the changes of oxidative stress levels?hydrogen peroxide and malondialdehyde?,the protein expression and enzymatic activity of various antioxidant enzymes?superoxide dismutase,catalase and glutathione peroxidase?and total antioxidant capacity in different tissues and different types of skeletal muscle during different periods of hibernation.We also explored the molecular mechanisms involved in the regulation of antioxidant responses during hibernation by measuring the expression of the Nrf2/Keap1 pathway.In addition,we compare with the indicators of skeletal muscle atrophy,oxidative stress and antioxidant defense in non-hibernators?hindlimb unloading rats?and mammalian hibernators?hibernating Daurian ground squirrels?.This thesis consists three parts.The first part:the study on the oxidative stress level and antioxidant defense ability of different tissues in different periods of hibernation of Daurian ground squirrels.We investigated levels of oxidative stress,antioxidant capacity,and the underlying mechanism in heart,liver,brain,and kidney tissue as well as plasma during different periods of hibernation in Daurian ground squirrels?Spermophilus dauricus?.Hydrogen peroxide?H₂O₂?is a significant ROS that directly reflected the level of oxidative stress.In vivo,free radicals undergo lipid peroxidation to form MDA.As the final product of membrane lipid peroxidation and an index of oxidative damage of lipids,MDA indirectly reflected the level of oxidative stress.Our data showed that the levels of hydrogen peroxide significantly increased in the heart and brain during late torpor compared with levels during the summer active state.The content of malondialdehyde?MDA?was significantly lower during interbout arousal and early torpor state than that during summer active or pre-hibernation state,and MDA levels in the late torpor group brain were significantly higher than the levels in other states.Moreover,most changed indicators of oxidative stress recovered to summer active or pre-hibernation state levels in post-hibernation.Superoxide dismutase 2?SOD2?protein levels increased markedly in the heart throughout the torpor–arousal cycle.Catalase?CAT?expression remained at an elevated level in the liver during the hibernation cycle.Superoxide dismutase 1?SOD1?and glutathione peroxidase 1?GPx1?expression increased considerably in all tissues during the interbout arousal and early torpor states.In addition,the activities of the various antioxidant enzymes were higher in all tissues during interbout arousal and early torpor state than during late torpor state;however,GPx activity in plasma decreased significantly during the hibernation season.The expression of p-Nrf2decreased in all tissue types during interbout arousal group,but significantly increased during late torpor group,especially in liver tissue.These results suggest that increased reactive oxygen species during late torpor may activate the Nrf2/Keap1 antioxidant pathway and may contribute to the decreased ROS levels found during the interbout arousal and early torpor states,thereby protecting organisms from oxidative damage over the torpor-arousal cycle of hibernation.The second part:the study on the oxidative stress level and antioxidant defense ability of different type of skeletal muscles?slow-twitch muscle:soleus;fast-twitch muscle:extensor digitorum longus;mixed muscle:gastrocnemius?in different periods of hibernation of Daurian ground squirrels.The section study showed that H₂O₂ content of extensor digitorum longus and gastrocnemius significantly decreased at pre-hibernation and late torpor state compared with the summer active group.The content of MDA was significantly decreased during the torpor-arousal cycles in the extensor digitorum longus and gastrocnemius,and decreased at interbout arousal and early torpor states in the soleus compared with that in the SA group.However,MDA content increased in the soleus during the late torpor group compared with the interbout arousal and early torpor groups.Interestingly,most changed indicators recovered to summer or pre-hibernation levels in post-hibernation in the three skeletal muscles.Compared with the summer active group,GPx1 and CAT protein levels in the soleus and SOD2 in the gastrocnemius increased markedly throughout the hibernation season.In the extensor digitorum longus,SOD2 in the interbout arousal and post-hibernation groups,CAT and GPx1 protein in the early torpor and late torpor groups significantly increased.In addition,the activities of the various antioxidant enzymes were higher in all tissues during interbout arousal and early torpor state than during late torpor,and CAT activity during the hibernation season increased significantly in three muscles.Nrf2 and p-Nrf2 protein levels in the soleus and extensor digitorum longus significantly elevated during the hibernation periods,and increased during the pre-hibernation,interbout arousal and early torpor state in the gastrocnemius.These results suggest that activated of Nrf2/Keap1 antioxidant pathway over the hibernation time course may contribute to the increased of protein levels of downstream antioxidant enzymes and the decreased MDA levels found during the hibernation bouts,thereby protecting organisms from oxidative damage over the torpor-arousal cycle of hibernation.This section suggested that the Nrf2-mediated up-regulation of multiple antioxidant enzymes may be a key preventative defense against oxidative stress induced by the ischemia-reperfusion and disuse in the skeletal muscle in hibernating Daurian ground squirrels?S.dauricus?.The third part:a comparative study on the relationship among the oxidative stress level,antioxidant defense ability and muscle atrophy in different type of skeletal muscles in hindlimb unloading rats and hibernating Daurian ground squirrels.Compared to control rats,our data showed that the muscle wet weight,the muscle-to-body mass ratio and the fiber cross sectional area were significantly reduced in soleus and extensor digitorum longus muscle after 14-day hindlimb unloading?HU?;However,in the hibernating Daurian ground squirrels,no significant changes of the muscular atrophy index in soleus and extensor digitorum longus were observed between hibernation and summer active group.The results of oxidative stress level showed that H₂O₂ content significantly increased in the soleus and extensor digitorum longus of HU rats,and the content of MDA was significantly increased in the soleus of HU rats;but both are not increased in hibernating ground squirrels.SOD2,CAT,and GPx1 expression dramatically decreased in the soleus of HU rats,and GPx1 in the extensor digitorum longus of HU rats decreased,however,SOD1 in the soleus of HU rats significantly increased.While they were maintained and even increased in soleus and extensor digitorum longus of hibernating ground squirrels.For example,CAT and GPx1 protein level of the hibernation group significantly increased in the soleus and extensor digitorum longus.Nrf2 and p-Nrf2 protein levels maintained in the soleus and decreased in the extensor digitorum longus of HU rats,while the general elevation of Nrf2 protein in the skeletal muscles during the hibernation ground squirrels.These results suggest that under disuse conditions increased ROS don't activate the Nrf2/Keap1 antioxidant pathway and upregulated the antioxidant enzymes and may cause oxidative stress and induced muscle atrophy in the rats;however,elevated the Nrf2/Keap1 pathway and antioxidant enzymes in the ground squirrels during hibernation prevent oxidative stress and muscle atrophy.Overall,the oxidative stress and Nrf2-medatied antioxidant defense appears to differ in slow and fast muscles in non-hibernators and hibernators under disuse conditions,which may be an important reason for the different degrees of muscle atrophy.In summary,we studied the oxidative stress level,antioxidant defense ability and its regulation mechanism in different tissues during different periods of hibernation in Daurian ground squirrels?S.dauricus?.Firstly,the level of ROS in the late torpor in different tissues increased in certain extent,but most changed indicators recovered to summer or pre-hibernation levels in post-hibernation,indicating that the hibernating Daurian ground squirrels maintained redox homeostasis under multiple stress conditions.Secondly,elevated H₂O₂ content in the heart and brain tissue during late torpor was observed,implying that these tissues are more susceptible to the effects of oxidative stress.Thirdly,the changing patterns of the antioxidant enzymes were tissue specific.For example,CAT expression markedly increased in the liver throughout the entire hibernation season,but there was no significant change in brain tissue;GPx1 and CAT protein expression were significantly increased in the soleus muscle during the entire hibernation season,but there was no significant change in gastrocnemius muscle.Fourthly,the skeletal muscle atrophy,oxidative stress level and antioxidant regulation of hibernating squirrels and hindlimb unloading rats under disused conditions were comparative study.The results showed that muscle atrophy of the soleus and extensor digitorum longus in the hindlimb unloading rats was obvious,and the levels of ROS level were significantly increased,but the expression of various antioxidant enzymes was significantly decreased.These results indicate that disuse induces the increased of oxidative stress and decreased of antioxidant defense capacity,which may be one of the important reasons for inducing disuse muscle atrophy.The skeletal muscles of the hibernating squirrel were no obvious atrophy.The level of oxidative stress remained stable,and multiple antioxidant enzymes were highly expressed.These results indicate that the enhanced antioxidant defense ability of hibernating ground squirrels in low-metabolism and disuse state may be an important mechanism to prevent disuse muscle atrophy caused by hibernation inactivity during hibernation.The current study not only provide a new perspective for us to understand the adaptation mechanism of hibernating squirrels to special ecological environment,but also provide theoretical basis and new ideas on therapies for free radical-related diseases.