| Experiment 1:The Effect of Exercise Preconditioning on Oxidative Stress-Mediated Apoptosis in Cerebral Ischemia-Reperfusion RatsObjective:Investigate the impact of exercise preconditioning on oxidative stress,apoptosis,and anti-apoptosis during the acute phase of cerebral ischemiareperfusion in rats,offering a theoretical foundation and scientific proof for the use of exercise preconditioning as a preventive and therapeutic strategy against ischemic stroke.Methods:Using a random number table,allocate 120 male SD rats into four groups:Sham,Model,Exercise+Model(E+Model),and Exercise+Sham(E+Sham),with each group containing 30 rats.Subsequently,further categorize these groups into two subgroups based on two reperfusion time points,12 hours(n=12)and 24 hours(n=18).For three weeks,rats in the E+Model and E+Sham groups undergo exercise preconditioning for 30 minutes daily.Twenty-four hours following the final session of exercise preconditioning,establish a cerebral middle artery occlusion and reperfusion model in the rats from each group.At 12 hours and 24 hours postreperfusion,conduct neurological deficit evaluations using the Garcia score,determine the cerebral infarct size through TTC staining,examine ischemic side brain tissue for pathological changes with HE staining,analyze the expression levels of Bcl-2,Bax,and Caspase-3 proteins in ischemic side brain tissue via Western blot,assess rat ischemic side cortex apoptosis with TUNEL staining,and measure ROS and SOD levels in the infarcted brain tissue using ELISA.This approach aims to investigate the effects of exercise preconditioning on neurological deficits,acute phase cerebral infarct size and pathology,apoptosis,protein expression levels,and oxidative stress in rats experiencing cerebral ischemia-reperfusion.The goal is to demonstrate that exercise preconditioning can mitigate oxidative stress in the ischemic brain tissue of rats subjected to cerebral ischemia-reperfusion,reduce apoptosis,enhance anti-apoptotic mechanisms,decrease the size of cerebral infarcts and neurological deficits,thereby providing neuroprotection.Results:1.Garcia score indicated that 12h and 24h after cerebral ischemiareperfusion,significant neurological deficits were observed in the rats of all groups except the Sham group and E+Sham group.Moreover,the degree of neurological deficits in the E+Model group was smaller than that in the Model group at both time points(P<0.05).2.TTC staining indicated that 24 hours following cerebral ischemiareperfusion,distinct infarction areas appeared in all rat groups except for the Sham and E+Sham groups.Nonetheless,the cerebral infarct size in the E+Model group was less than that observed in the Model group(P<0.05).3.HE staining indicated that 12 and 24 hours post-cerebral ischemiareperfusion,significant pathological damage was observed in the brain tissues of both the Model and E+Model groups.However,the extent of pathological damage was less severe in the E+Model group compared to the Model group at both time intervals.4.TUNEL staining indicated that 12 and 24 hours after cerebral ischemia-reperfusion,numerous apoptotic cells were detected in both the Model and E+Model groups.However,the number of apoptotic cells in the E+Model group was lower than in the Model group at both observed times(P<0.05).5.Western Blot results indicated that after 12h and 24h of cerebral ischemia reperfusion,there were no significant changes in the expression of BAX,Bcl-2,and Caspase-3 in the brain tissue of the Sham group and E+Sham group.However,the expression of Bcl-2 in the E+Sham group was higher than that in the Sham group at both time points(P<0.05).The expression of BAX,Bcl-2,and Caspase-3 in the ischemic side brain tissue of the Model group and E+Model group was significantly higher compared to the Sham group and E+Sham group at both time points(P<0.05).Furthermore,the levels of BAX and Caspase-3 were significantly elevated in the Model group compared to the E+Model group(P<0.05),whereas Bcl-2 expression was notably reduced in the Model group relative to the E+Model group(P<0.05).6.Brain tissue ROS content results indicated that at 12 and 24 hours following cerebral ischemia-reperfusion,there were no significant changes in the ROS content in the brain tissue of the Sham group and E+Sham group.The ROS content in the ischemic side brain tissue of the Model group and E+Model group was significantly higher compared to the Sham group and E+Sham group at both time points(P<0.05).Furthermore,the ROS content in the E+Model group was significantly lower than that in the Model group at both time points(P<0.05).7.Brain tissue SOD content results indicated that at 12 and 24 hours following cerebral ischemia-reperfusion,there were no significant changes in the SOD content in the brain tissue of the Sham group and E+Sham group.The SOD content in the ischemic side brain tissue of the Model group and E+Model group was significantly lower compared to the Sham group and E+Sham group at both time points(P<0.05).Additionally,the SOD content in the E+Model group was significantly higher than that in the Model group at both time points(P<0.05).Conclusions:1.Exercise preconditioning is capable of mitigating acute cerebral ischemia-reperfusion damage,enhancing neurological function,diminishing the size of cerebral infarcts,and providing neuroprotection.;2.Exercise preconditioning can alleviate acute oxidative stress and cell apoptosis during cerebral ischemia-reperfusion,promote anti-apoptotic mechanisms,and provide theoretical basis for its application in the prevention and treatment of ischemic brain diseases.Experiment 2:The Effect of Exercise Preconditioning on Mitochondrial Energy Metabolism and ATP Generation in Cerebral IschemiaReperfusion Rats.Objective:Investigating the impact of exercise preconditioning on energy metabolism during the acute phase of cerebral ischemia-reperfusion in rats’brain tissue,including mitochondrial energy metabolism,oxidative phosphorylation,and glycolysis pathways,to provide a theoretical basis and scientific evidence for exercise preconditioning as a preventive and therapeutic measure for ischemic stroke.Methods:Using a random number table,allocate 120 male SD rats into four groups:Sham,Model,Exercise+Model(E+Model),and Exercise+Sham(E+Sham),with each group containing 30 rats.Subsequently,further categorize these groups into two subgroups based on two reperfusion time points,12 hours(n=12)and 24 hours(n=18).For three weeks,rats in the E+Model and E+Sham groups undergo exercise preconditioning for 30 minutes daily.Twenty-four hours following the final session of exercise preconditioning,establish a cerebral middle artery occlusion and reperfusion model in the rats from each group.At 12h and 24h after reperfusion,Western blot was used to detect the protein expression of COXIV,TIGAR,and PFKFB3 in the ischemic brain tissue;q-PCR was used to detect the mRNA expression of COXIV,TIGAR,and PFKFB3 in the ischemic brain tissue;and assay kits were used to detect ATP content and mitochondrial respiratory chain complex V(F1-F0-ATP)activity in the ischemic brain tissue.The effects of exercise preconditioning on mitochondrial energy production,oxidative phosphorylation,and glycolysis-related protein and mRNA expression,ATP content,and ATP synthase mitochondrial respiratory chain complex V activity in the ischemic brain tissue of rats were observed to elucidate how exercise can enhance mitochondrial energy production and oxidative phosphorylation pathways,reduce glycolysis pathways,increase ATP content in the ischemic brain tissue,improve neuronal energy metabolism,and protect neurons.Results:1.Western Blot analyses indicate that post-cerebral ischemia reperfusion at 12 and 24 hours,COXIV protein levels in the Model group were notably lower than in the Sham,E+Sham,and E+Model groups(P<0.05).At these time points,COXIV protein expression in the E+Model group did not show significant differences compared to the Sham group and was reduced compared to the E+Sham group(P<0.05).The E+Sham group exhibited significantly higher COXIV protein levels than the other three groups at both time intervals(P<0.05).The TIGAR protein levels in the Model group were significantly elevated compared to those in the Sham and E+Sham groups at both time points(P<0.05),yet they were lower than those observed in the E+Model group(P<0.05).At the same time points,the expression levels of TIGAR protein in the E+Model group surpassed those in all other groups significantly(P<0.05).PFKFB3 protein expression in the Model group was significantly elevated compared to the other groups at both time points(P<0.05),and in the E+Model group,it was lower than in the Model group but higher than in the Sham and E+Sham groups at both intervals(P<0.05).2.q-PCR results show that 12h and 24h after reperfusion,the expression of COXIV,TIGAR,and PFKFB3 mRNA in the ischemic side brain tissue of the Model group and E+Model group undergo significant changes compared to the Sham group and E+Sham group(P<0.05).At both observed intervals,COXIV mRNA levels in the Model group were markedly lower than in the Sham,E+Sham,and E+Model groups(P<0.05).Specifically,at 12 hours,COXIV mRNA expression in the E+Model group was below that of the Sham and E+Sham groups(P<0.05),and at 24 hours,it surpassed that of the Sham group yet remained below the E+Sham group(P<0.05).At the 12hours,there was no significant variance in TIGAR mRNA levels between the Model group and both the Sham and E+Sham groups(P>0.05);however,these levels were beneath those seen in the E+Model group(P<0.05).By the 24-hour mark,the expression was solely below that of the E+Model group(P<0.05).Across both the 12h and 24h intervals,the TIGAR mRNA expression in the E+Model group exceeded that of the other three groups(P<0.05).PFKFB3 mRNA expression in the Model group was significantly greater than in the other three groups at both times(P<0.05),and in the E+Model group,it was lower than in the Model group but exceeded that in the Sham and E+Sham groups at both intervals(P<0.05).3.The results of ATP content in brain tissue show that there is no significant change in ATP content in the brain tissue of the Sham group and E+Sham group at 12h and 24h.12h and 24h after reperfusion,the ATP content in the ischemic side brain tissue of the Model group and E+Model group is significantly lower than that in the Sham group and E+Sham group(P<0.05).At both time points,the ATP content in the E+Model group is higher than the Model group(P<0.05).4.Assessments of the activity of Mitochondrial respiratory chain complex V(F1-F0-ATP)indicate that there are no significant differences between the Sham and E+Sham groups at both 12 and 24 hours.At these time points,complex V activity in the ischemic brain tissue of the Model group was significantly reduced compared to the Sham and E+Sham groups(P<0.05).While the E+Model group experienced a decrease in complex V activity at 12 hours,this reduction was not statistically significant compared to the Sham and E+Sham groups(P>0.05).Moreover,at both intervals,complex V activity in the E+Model group was higher than in the Model group(P<0.05).Conclusions:Exercise preconditioning can enhance the activity of mitochondrial respiratory chain complex in brain tissue of rats with cerebral ischemiareperfusion,promote oxidative phosphorylation pathway,reduce glycolysis pathway,improve metabolic conditions,and increase energy production.Experiment 3:The Impact of Exercise Preconditioning on Mitochondrial Metabolic Reprogramming in Cerebral Ischemia-Reperfusion Rats.Objective:To explore how exercise preconditioning affects the NAMPT/AMPK pathway,oxidative phosphorylation,glycolysis pathway,and its products in the acute phase brain tissue of cerebral ischemia-reperfusion rats,aiming to provide theoretical and scientific basis for the application of exercise preconditioning in the prevention and treatment of ischemic stroke.Methods:120 male SD rats were adaptively fed under the same conditions for one week,and then randomly divided into 5 groups using a random number table,namely,the Sham group,the Model group,the Exercise+Model(E+Model)group,and the Exercise+Sham(E+Sham)group,with 30 rats in each group.After reperfusion,the rats were divided into 2 subgroups at 12h(n=12)and 24h(n=18)after reperfusion.The E+Model group and E+Model group were given 30 minutes of exercise preconditioning every day for 5 days a week for a total of 3 weeks.After the last exercise preconditioning,the rat models of middle cerebral artery ischemia-reperfusion were established.At 12h and 24h after reperfusion,Western blot was used to detect the expression of pAMPK,AMPK,and NAMPT proteins in the ischemic brain tissue;q-PCR was used to detect the expression of AMPK and NAMPT mRNA in the ischemic brain tissue;and assay kits were used to detect glucose,pyruvate,lactate,and NADPH/NADP+in the ischemic brain tissue.The changes in energy-related proteins,oxidative phosphorylation,and glycolysis products in the brain ischemia-reperfusion rats after exercise preconditioning were observed to elucidate the metabolic reprogramming of exercise preconditioning in improving brain ischemia-reperfusion injury and reducing the accumulation of glycolysis products such as lactate to protect nerve cells.Results:1.Western Blot results showed that there was no significant change in the expression of AMPK protein in each group(P>0.05);at both time points,the expression of p-AMPK protein in the Model group and E+Model group was significantly increased compared to the Sham group and E+Sham group(P<0.05),with a more significant increase in the E+Model group compared to the Model group(P<0.05);there was no significant change in the expression of p-AMPK protein compared between the Sham group and E+Sham group(P>0.05);Following reperfusion at 12 hours and 24 hours,there was a significant reduction in NAMPT protein expression in the Model group when compared to the other groups(P>0.05);conversely,in the E+Model group,NAMPT protein expression was significantly elevated in comparison to the other groups(P>0.05);there was no significant change in the expression of p-AMPK protein compared between the Sham group and E+Sham group(P>0.05).2.q-PCR results showed that after 12h and 24h of reperfusion,there was no significant change in AMPK mRNA expression in each group(P<0.05);at both time points,NAMPT mRNA expression in the Model group was lower than in the other three groups(P<0.05),and the NAMPT mRNA expression in the E+Model group was not significantly different from the Sham and E+Sham groups at the 12h time point(P>0.05),but was higher than the Sham group at the 24h time point(P<0.05).3.The results of brain tissue glucose content showed that there was no significant change in glucose content in the brain tissue of the Sham and E+Sham groups at 12h and 24h.Following 12 hours and 24 hours of reperfusion,the glucose levels in the ischemic brain tissue were significantly elevated in both the Model and E+Model groups when compared to the Sham and E+Sham groups(P<0.05).Additionally,at these time points,the glucose levels in the E+Model group were found to be lower than those in the Model group(P<0.05).4.The results of brain tissue pyruvic acid content showed that there was no significant change in pyruvic acid content in the brain tissue of the Sham and E+Sham groups at 12h and 24h.The pyruvic acid levels in the ischemic brain tissue were significantly reduced in the Model and E+Model groups compared to the Sham and E+Sham groups after 12 hours and 24 hours of reperfusion(P<0.05).Furthermore,at these same time intervals,the ketone body levels in the E+Model group exceeded those in the Model group(P<0.05).5.The results of brain tissue lactic acid content showed that there was no significant change in lactate content in the brain tissue of the Sham and E+Sham groups at 12h and 24h.Following 12 hours and 24 hours of reperfusion,the lactic acid content in the ischemic brain tissue of both the Model and E+Model groups was notably reduced when compared to the Sham and E+Sham groups(P<0.05).Moreover,at each of these time points,the lactic acid content was greater in the E+Model group than in the Model group(P<0.05).6.The results of brain tissue NADPH/NADP+content showed that there was no significant change in NADPH/NADP+content in the brain tissue of the Sham and E+Sham groups at 12h and 24h.After 12h and 24h of reperfusion,the NADPH/NADP+content in the ischemic brain tissue of the Model and E+Model groups was significantly lower compared to the Sham and E+Sham groups(P<0.05),and at both time points,the NADPH/NADP+content in the E+Model group was higher than in the Model group(P<0.05).Conclusions:Exercise preconditioning can activate the NAMPT/AMPK pathway to stimulate energy production,improve metabolic reprogramming of brain tissue in cerebral ischemia-reperfusion rats,promote oxidative phosphorylation and the pentose phosphate pathway,reduce glycolysis and its products accumulation,and protect neurons. |
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