Effects Of Different Loads Of Swimming On Skeletal Muscle Oxidative Stress, Autophagy In Ovariectomized Rats

Objective:Sarcopenia is a loss of muscle mass and function that reduces mobility and quality of life in older adults and can lead to falls related injuries.This symptom is associated with increased oxidative stress in skeletal muscles due to aging,increased oxygen free radicals in the body,accumulation of lipid peroxides,and lipid peroxidation and protein damage in cells.At the same time,as a highly conserved self-protection mechanism of eukaryotic cells in evolution,autophagy plays an important role in the realization of intracellular material recycling and the maintenance of cell homeostasis,and participates in the growth,development,senescence and death of skeletal muscle cells and many other physiological processes.Studies have shown that oxidative stress can activate autophagy and participate in the process of skeletal muscle atrophy.But other studies have shown that oxidative stress can inhibit autophagy.After menopause,due to the degeneration of ovarian function and the decrease of estrogen secretion,skeletal muscle mass and function are damaged,leading to the function loss.The relationship between this and oxidative stress and autophagy is still uncertain at present.Regular exercise has a beneficial effect on the human body.However,it is unclear whether the improvement in skeletal muscle shape and function in ovariectomized rats is related to oxidative stress and autophagy levels.This study used ovariectomized rats,simulating postmenopausal women,through different load swimming,analyzing effects of exercise training on contraction of rat skeletal muscle,oxidative stress level and autophagy,to explore the relationship and mechanism of the occurrence of the disease of the rats skeletal muscle.The results will provide theoretical basis for postmenopausal women rehabilitation exercise.Methods:A total of 50 clean female Sprague-Dawley rats at 8 weeks were randomly divided into 5 groups: Sham group,OVX group,ovaries removal 60min/per time exercise group(OVX60ET),90min/per time exercise group(OVX90ET),and 120min/per time exercise group(OVX120ET).After a week adaptation,50 rats underwent bilateral ovariectomy.After 2 weeks of surgery and postoperative recovery,adaptive swimming exercises were conducted.After 1 week of adaptive swimming,the rats in the 3 exercise groups underwent 60 min,90min and 120 min swimming training every day,6 days a week,for a total of 8 weeks.The sham group and the ovariectomized sedentary group did not engage in any form of exercise,and all animals were anesthetized after 24 hours at the last swim.Serum estrogen levels and uterine weight were measured to assess the effect of ovariectomy.The peak and amplitude of single contraction and tentanic contraction,fatigue index of skeletal muscle were measured to evaluate the function of gastrocnemius muscle.The level of SOD,GSH-Px and MDA in gastrocnemius muscle was measured to evaluate the level of oxidative stress.The change degree of gastrocnemius sarcopenia was evaluated by hematoxylin staining.Western blot was used to detect the expression levels of IGF-1,AMPK,PGC-1?,Beclin 1,ULK1,Atg7,LC3,mTOR and P62 in gastrocnemius muscle,and to evaluate the changes in functions and autophagy.The levels of LC3-?and P62 were evaluated by immunohistochemistry.All the results were analyzed by SPSS software,and the values were expressed as mean ± standard error.One-way ANOVA was used for inter-group comparison,and p<0.05 was considered statistically significant.By analyzing the changes of indicators,the effects of different loads of swimming exercise training on skeletal muscle function and autophagy in ovariectomized rats were investigated.Results: 1.After 8 weeks,the weight and gastrocnemius of the rats in the OVX group were significantly higher than those in the sham group(P<0.01)and those in OVX 60 ET and OVX90 ET groups increased as well(P<0.05).However,there was no significant difference between the weight of exercise rats in OVX120 ET and that in the sham group.Among the ovariectomized rats,the weight in OVX120 ET group gained much less.In terms of uterine weight and serum estrogen,the uterine weight and serum estrogen levels of the four ovariectomized groups were significantly lower than those of the sham group after 8 weeks(P<0.01).After 8 weeks of swimming exercise,the serum estrogen level of rats in OVX90 ET group was significantly higher than that in the OVX group(P<0.05).2.Histological changes of gastrocnemius muscle in ovariectomized rats.After 8 weeks of ovariectomy,the skeletal muscle cells in the sedentary group increased in diameter and nuclear arrangement.After 8 weeks of swimming,the muscle diameter of the rats in OVX60 ET and OVX90 ET decreased,and the nuclei of the rats in the OVX90 ET were arranged neatly in lines.In OVX120 ET,the cell diameter decreased significantly,and the number of nuclei decreased.3.Changes of muscle contractile function in skeletal muscle of ovariectomized rats.Compared with sham group,after 8 weeks peak amplitude of the single and tetanic contraction decreased significantly(P< 0.01)in OVX group and OVX120 ET group,and the fatigue index decreased in both groups(P<0.01)and(P<0.05)respectively..After 8 weeks of training,the amplitude and peak tension of single contraction and tentanic contraction of rats in OVX90 ET were significantly increased(P<0.01)compared with those in OVX group,the fatigue index increased significantly(P<0.05)as well,while the amplitude of single contraction and peaktension of rats in OVX60 ET were significantly different(P<0.05).4.Compared with the sham group,after 8 weeks SOD level in the gastrocnemius of OVX group decreased significantly(P<0.01),and MDA increased significantly(P<0.05),while MDA level increased significantly in the OVX129ET(P<0.01).After 8 weeks swimming,SOD increased significantly(P<0.01)and MDA level decreased significantly(P<0.05)in OVX90 ET compared with those of OVX.There was no significant difference in SOD and MDA levels between OVX60 ET,OVX120ETand the OVX group.Compared with the sham group,the content of GSH-Px in gastrocnemius muscle of the OVX and OVX120 ET significantly reduced(P<0.01 and P<0.05,respectively).GSH-Px level of rats in OVX90 ET was significantly higher than that in the OVX group(P<0.05).5.Changes of proteins in ovariectomized rats.After 8 weeks,compared with the sham group,the protein in other four groups,except P62,others proteins significantly reduced IGF-1(P<0.05),mTOR,ULK1,Beclin1,Atg7 and LC3(P<0.01).AMPK in OVX and OVX120 ET increased significantly than that in sham group.On the contrary,PGC-1? in OVX and OVX120 ET decreased significantly.After 8 weeks of swimming,mTOR(P<0.05),ULK1 and LC3 levels in OVX60 ET group were much lower than those in the sham group(P<0.01).Compared with OVX group,ULK1,LC3 in OVX90 ET and OVX120 ET increased significantly(P<0.05),PGC-1? in OVX90 ET increased as well(P<0.05).mTOR and Beclin 1 increased much higher(P<0.01)than those in OVX.furthermore,.Atg7 in three exercise groups increased significantly(P<0.01)compared with OVX,and P62 showed countery change(P<0.05)Conclusion: 1.The contractibility of gastrocnemius in OVX sedentary group was decreased,the level of oxidative stress was increased,and the autophagy flux was inhibited 2.The autophagy of ovariectomized rats was activated by aerobic exercise,which was load dependent.3.90 min swimming exercise can improve the antioxidant capacity of skeletal muscle,activate autophagy,and improve the decline of skeletal muscle function caused by estrogen deficiency in ovariectomized rats.4.120 min swimming exercise increased oxidative stress in ovariectomized rats,which induced to excessive activation of autophagy,and exacerbated the further decline in skeletal muscle function.