| Background: Skeletal muscle is the largest organ of the human body and the main consumer of energy during exercise.Muscle function declines with age,but the pathophysiological reasons for this are not fully understood.Studies have shown that Rac1 plays an important role in a series of cellular physiological activities such as cell adhesion,proliferation and cytoskeleton regulation.It also participates in skeletal muscle regeneration and glucose metabolism through activation of downstream important effector kinases such as PAK1 and MAPK.However,whether exercise regulates the Rac1/PAK1/p38 MAPK signaling pathway and its related downstream factors in the aging body,as well as the intracellular mechanism that exercise mediates this pathway to promote protein synthesis and promote glucose uptake by skeletal muscle still need further research.Objectives: In this experiment,we conducted weight-bearing ladder training and treadmill exercise training on BALB/c naturally aging mice to explore the role and molecular mechanism of Rac1/PAK1/p38 MAPK signaling pathway in skeletal muscle regeneration and glucose metabolism,and further study the effects of exercise on the Rac1/PAK1/p38 MAPK signal pathway and its downstream factors MyoD,GLP-1R,etc.,in order to provide new therapeutic targets for exercise prevention and treatment of sarcopenia,type 2 diabetes and other related diseases.Methods: 25 healthy male BALB/c mice were raised to 52 weeks of age under standard conditions and randomly divided into 4 groups,including: tail-loaded ladder exercise training with high-fat diet group(HFD-R,n=6),tail-loaded ladder exercise training and treadmill exercise with high-fat diet group(HFD-RA,n=7),high-fat diet group(HFD-C,n=6),ordinary diet group(C,n=6).When formal training began,the ordinary food MD17111 was replaced by D12492 high-fat food.The HFD-R group received 8 weeks of tail-loaded ladder exercise training,and the HFD-RA group received 8 weeks of ladder exercise training and unloaded treadmill exercise training.During the experiment,the diet was monitored daily,the weight of the mice was weighed every Sunday,and the grip strength of the mice and the maximum load in the exercise group were measured every two weeks.24 hours after the last training intervention,the mice were weighed,blood was taken from the eyeballs,and the mice were sacrificed by cervical dislocation.The quadriceps tissue was taken on both sides.The Wes automatic protein expression quantitative analysis system was used to detect the protein levels of Rac1,PAK1,PAK2,p38 MAPK,Cdc42,p-PAK1,p-PAK2,p-38 MAPK,MyoD,MyoG,GLP-1R,GLUT4 in mouse quadriceps muscle tissue.Hematoxylin-eosin staining technique was used to detect the thickness of skeletal muscle fibers in mouse quadriceps tissue,and oil red staining technique was used to detect the distribution of lipid droplets in mouse inguinal fat(i WAT)tissue.After the experimental data was obtained,SPSS 21 and Graphpad prism 7 statistical software were used to analyze the results through one-way ANOVA and two-way ANOVA.Results:(1)After eight weeks,the body weight of four groups of aging mice decreased,the body weight of mice in the exercise group increased first and then decreased,but the body weight of mice in the non-exercise group gradually decreased,and the body weight of mice in HFD-R and HFD-RA group was always higher than that in HFD-C and C group.At the end of the eighth week,the body weight of HFD-R,HFD-RA and C groups was significantly lower than that before the experiment(P<0.05),and the mice in group C had the largest weight loss.The quadriceps femoris mass index in HFD-R,HFD-RA and HFD-C groups was slightly higher than that in C group,but there was no significant difference among them.(2)With the increase of age,the absolute grip strength of mice in each group was significantly decreased,and the grip strength of mice in the exercise group at week 4and 8 was significantly higher than that in the non-exercise group.At the end of the eighth week,the relative grip strength of HFD-RA mice was significantly higher than that of HFD-C and C group(P<0.05).(3)Rac1/PAK1/p38 MAPK signaling pathway related gene expression results in quadriceps femoris: Compared with HFD-C and C groups,Rac1 protein expression level in HFD-R and HFD-RA groups was significantly increased(P<0.001).There was no significant difference in the relative expression of Cdc42 protein among all groups.Compared with HFD-C group,the protein level of PAK1 in HFD-R and HFD-RA groups was significantly increased(P<0.05),and the protein level of PAK1 in HFD-R and HFD-RA groups was extremely significantly increased compared with C group(P<0.01).There was no significant difference in the protein level of p38 MAPK in the quadriceps among all groups.The phosphorylation level of p38 MAPK in HFD-R group was higher than that in the other three groups(P<0.001),while the protein level of p-p38 MAPK in HFD-RA and HFD-C groups was significantly higher than that in C group(P<0.001).(4)MyoD and MyoG protein expression results in quadriceps femoris: The protein levels of MyoD and MyoG in HFD-R and HFD-RA groups were significantly higher than those in HFD-C group(P<0.001).(5)GLUT4 and GLP-1R protein expression results in quadriceps femoris: The GLUT4 level in HFD-C group was slightly lower than that in C group,and the value in HFD-R and HFD-RA group was higher than that in HFD-C group(P<0.05).The level of GLP-1R protein in group C was significantly higher than that in group HFD-C(P<0.001),and the GLP-1R level in HFD-R and HFD-RA group was significantly higher than that in group HFD-C(P<0.001).Conclusions:(1)Tail-loaded ladder exercise training and combined exercise can maintain the body weight of aging mice with high-fat diet to a certain extent,and slow down the degradation of physical activity of aging body.(2)Tail-loaded ladder exercise training and combined exercise can improve the relative grip strength of aging mice with high-fat diet,which is beneficial to the maintenance of muscle strength and delay skeletal muscle atrophy.(3)Tail-loaded ladder exercise training and combined exercise can up-regulate the expression of MyoD and MyoG in high-fat diet aging mice through Rac1/PAK1/p38 MAPK signaling pathway,and promote skeletal muscle regeneration.(4)Tail-loaded ladder exercise training and combined exercise can regulate the gene expression of GLUT4 and GLP-1R in high-fat diet aging mice through the Rac1/PAK1/p38 MAPK signaling pathway,improving the glucose metabolism function of skeletal muscle and maintain glucose metabolism homeostasis. |
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