Effects Of Moderate Load Exercise On Nrf2 Signaling Pathway Related Proteins In The Myocardium Of Aging Rats

Objective:The aging situation is getting worse nowadays,as a result,aging related neurodegenerative diseases and the decline of organ function caused by aging directly affect the quality of life and even life safety of the elderly.The decline of cardiovascular function as well as the cardiovascular system diseases which are induced by aging,such as myocardial infarction,coronary heart disease,angina,etc,have become the first killer that endangers the life and health of the elderly which is in parallel with cancer and diabetes.As one of the most important organs in the whole body,the heart bears the dynamic role of blood circulation.Cardiac myocytes are the source of cardiac power.The state of cardiac myocytes directly determines the magnitude of cardiac contractility.Modern sports medicine believes that exercise with appropriate load can delay the decline of biological activity of cardiomyocytes caused by oxidative aging to a certain extent,and play an important role in anti-aging.However,the mechanism of exercise against aging is unclear.The signal pathway of Nuclear factor 2-related factor 2（Nrf2）is an important antioxidant switch in vivo,which is responsible for the regulation of downstream antioxidant proteins and some antioxidant enzymes,as well as the anti-aging of cardiomyocytes.The purpose of this study is to explore the effect of moderate load exercise on Nrf2 signaling pathway of myocardial cells in aging rats induced by D-galactose and aluminum trichloride,and to elucidate the new mechanism of exercise against aging.Methods:60 male SD rats were randomly divided into three groups,and 20 rats were included in each group.They were normal control group（NC group）,aging control group（AC group）,aging exercise group（AE group）.NC group were fed normally.In AC group,the aging rat model was established by intraperitoneal injection of D-galactose[60mg/（kg·d）]and gavage of Al Cl₃[40mg/（kg·d）]with a concentration of 2mg/ml for 12 weeks.The aging model of rats in AE group was established firstly,and the aged rats were trained on treadmill for 12 weeks.3 days in a week were designed for the traning,namely,Monday,Wednesday and Friday.They rest at the rest of the time,and the running platform gradient is zero.The running speed was 10 m/min in the first week,15 m/min from the second week to the sixth week,20 m/min from the seventh week to the twelfth week,and the exercise time was 15 min/day from the first week,then increased by 5 min/day every week,until70 min/day in the twelfth week.The exercise time gradually increased from 15 min/day to 70 min/day.According to Bedford’s exercise load standard,the rats in the model training group took about 50ml of oxygen per minute and 60ml of maximum oxygen during exercise,which belonged to moderate load training.Within 24 hours after training,Morris water maze was used to evaluate the behavior,learning and memory function of rats.Within 24 hours after the water maze experiment,the rats were anesthetized.Five rats in each group were perfused with left ventricular aorta.After the experiment,the hearts were separated quickly.After 24 hours of fixation,the hearts were dehydrated,transparent and embedded.The expression of HO-1 and nqo-1 protein in myocardial tissue was detected by immunohistochemistry.After anesthesia,10 hearts in each group were quickly separated and frozen in liquid nitrogen for 12 hours,and then transferred to-80℃for preservation.The contents of HO-1,NQO-1,Bax and Bcl-2 were detected by Western blot,and the activities of superoxide dismutase（SOD）,glutathione（GSH）,and Ca²⁺-ATPase in myocardial cells were measured by corresponding kits.After anesthesia,5 hearts in each group were separated and fixed,dehydrated and embedded for he observation of myocardial morphology and structure,and Masson staining to observe the process of myocardial fibrosis.Results:1)Compared with NC group,the escape latency of AC group was 3 on the fourth and fifth day,the latency of AE group was significantly lower than that of AC group（P<0.01）.2)On the fourth and fifth day,the latency of AE group was significantly lower than that of AC group（P<0.01）.3)On the third day,compared with NC group,the duration of stay in target quadrant of AC group and AE group was significantly shorter（P<0.01）,P<0.05),the number of times of crossing the original platform was also significantly reduced（P<0.01,P<0.05）.4)Compared with the AC group,the retention time in the target quadrant in the AE group was significantly increased（P<0.01）.5)Compared with NC group,the activity of SOD,GSH and Ca²⁺-ATPase in AC group decreased significantly（P<0.01）.Compared with NC group,glutathione（GSH）activity decreased significantly in AE group（P<0.05）,but there was no difference in SOD activity in AE group.Compared with AC group,the activity of superoxide discrimination enzyme（SOD）in AE group was significantly increased（P<0.01）,the activity of glutathione（GSH）was slightly increased（P>0.05）,the activity of Ca²⁺-ATPase was significantly increased（P<0.05）;6)Wester blot showed that the expression of HO-1 protein in NC group was higher than that in AC group,and the difference was very significant（P<0.01）,the expression of HO-1 protein in AE group was higher than that in AC group,the difference was very significant（P<0.01）,and AE group was higher than NC group,the difference was very significant（P<0.01）;7)the expression of NQO-1protein in NC group was higher than that in AC group,the difference was significant（P<0.05）,the expression of NQO-1 protein in NC group was higher than that in AE group,the difference was not significant（P>0.05）,the expression of NQO-1protein in AE group was higher than that in AC group,the difference was not significant（P>0.05）.There was no significant difference（P>0.05）;8)the expression of Bax protein in NC group was slightly higher than that in AE group,and there was no significant difference（P>0.05）;9)the expression of Bcl-2protein in NC group was significantly higher than that in AC group and AE group,and the expression of Bcl-2 protein in AE group was significantly higher than that in AC group（P<0.01）（P<0.05）（P<0.05）;10)immunohistochemistry results showed that the expression of HO-1 protein in NC group was higher than that in AE group,and the structure had a very significant difference（P<0.01）.The expression of HO-1 protein in AE group was higher than that in AC group,and the difference was very significant（P<0.01）;There was no difference in Bax protein expression.Compared with NC group,Bcl-2 in AC group decreased significantly（P<0.05）;11)the expression of NQO-1 protein in NC group was higher than that in AE group,and the difference was very significant（P<0.05）;12)After HE staining,the arrangement of myocardial tissue in NC group was normal,the tissue structure was clear,there was no interstitial edema and inflammatory cell infiltration.In AC group,the myocardial injury was obvious,the interval of myofascicles was widened,the distribution of myocytes was uneven,the myocardial cells were scattered and inflammatory cells were infiltrated.The myocardial tissue of AE group was arranged in order,and some inflammatory cells were infiltrated occasionally;13)Masson staining showed that the myocardial tissue of AC group appeared aggregation fibrosis.The area of fibrosis was massive.In AE group,there were occasionally fibrotic myocardium,which was reticular and scattered around the myocardial cells.In NC group,there was no fibrotic myocardium,and the morphology of myocardium was normal.Conclusion:1)the same load treadmill exercise in 12 weeks can improve the anti-oxidation ability of aging rats Myocardium,the possible mechanism is to activate Nrf2 signal pathway,stimulate the expression of HO-1 and NQO-1protein in downstream;2)the same load exercise in 12 weeks can activate the activity of SOD and GSH,protect the myocardium;3)during 12 weeks,running on the same load can regulate the expression ratio of Bax protein and bcl-2 protein,resist myocardial apoptosis and promote the survival of cardiomyocytes.