Improvement And Mechanism Of Different Exercises On Myocardial Injury In Mice With High Fat Diet Induced NAFLD

Objective: Research Objective: Studies have shown that NAFLD is a metabolic syndrome manifested in the liver,which covers a wide range in clinical practice.NAFLD can not only lead to liver injury,but also increase the risk of extrahepatic complications,especially cardiovascular diseases.It can also lead to myocardial cell injury due to lipid toxicity and autophagy disorders caused by lipid deposition,thus affecting the damage of cardiac structure and function.Previous studies have shown that exercise can reduce the risk of cardiovascular diseases and induce myocardial protection.However,the improvement effect and molecular mechanism of appropriate intensity exercise on myocardial injury in NAFLD induced by high fat diet alone are not completely clear.Therefore,this paper aims to explore the specific mechanism of different exercise modes regulating glycolipid metabolism and mediating autophagy to promote myocardial protection in NAFLD.To further reveal the mechanism of exercise to improve metabolic cardiomyopathy,and to provide a theoretical basis for the formulation of exercise prescription in patients with NAFLD.Methods: Forty 3-week-old male C57BL/6J mice were randomly divided into blank Control group(Control group,n=10)and NAFLD model group(HFD group,n=30).Blank group was fed standard ordinary diet,while model group was fed high fat diet(fat 60%,protein 20%,carbohydrate 20%).At the end of the 18 th week,with the average body weight of HFD group exceeding 20% of the body weight of Control group as the cut-off point,4mice in HFD group whose body weight did not meet the above requirements were excluded,and then 2 mice in each group were randomly selected for liver oil red O staining to observe the liver steatosis and judge whether the modeling of mice in HFD group was successful.After successful modeling,the remaining mice in HFD group were randomly divided into model group(HFD group),moderate intensity exercise group(HFD+MICT group)and high intensity intermittent exercise group(HFD+HIIT group),with 8 mice in each group,and HFD+MICT group and HFD+HIIT group received 8 weeks of different exercise interventions.The body weight of the experimental mice was recorded once a week.After the intervention,glucose tolerance test and insulin resistance index(HOMA-IR)of the mice were tested by intraperitoneal injection.Then samples of serum,myocardial tissue and abdominal adipose tissue were collected,and the contents of TC,TG,HDL-C and LDL-C in serum were detected by ELISA kit.Myocardial fibrosis was observed by Masson staining,ultrastructure of myocardial cells was observed by transmission electron microscopy,and expression changes of mitochondrial autophagy,glucose transport,lipid metabolism and other related proteins in myocardial tissue were detected by Western blot.Research results:1.Changes of body weight,glucose sensitivity and lipid protein in each group1)Body weight: compared with Control group,body weight of mice in HFD group was significantly increased(p<0.001);After exercise intervention,the body weight of HFD+MICT and HFD+HIIT groups was significantly lower than that of HFD group(p<0.001).The body weight of mice in HFD group after the experiment was significantly higher than that before the experiment(p<0.01),and the body weight of mice in HFD+MICT group and HFD+HIIT group after exercise showed a decreasing trend compared with that before the intervention,without statistical significance(p>0.05).2)Heart index and fat index: After the experiment,compared with Control group,heart index in HFD group was significantly decreased and abdominal fat index was significantly increased(p<0.01);Compared with HFD group,heart index was significantly increased and abdominal fat index was significantly decreased in HFD+MICT and HFD+HIIT groups(p<0.05).3)Fasting blood glucose,insulin resistance index and intraperitoneal injection glucose tolerance: After the experiment,there was no significant difference in the fasting blood glucose level between the four groups.Compared with Control group,insulin resistance index(HOMA-IR)and AUC level of intraperitoneal glucose tolerance test in HFD group were significantly increased(p<0.01).After exercise intervention,HOMA-IR of mice in HFD+MICT and HFD+HIIT groups was significantly decreased compared with HFD group(p<0.01).The AUC area of intraperitoneal injection glucose tolerance test in HFD+MICT group was significantly decreased(p<0.05),and the AUC area of HFD+HIIT group had a downward trend compared with that in HFD group,but there was no significant difference(p>0.05).4)Contents of TC,TG,HDL-C,LDL-C and LDL/HDL ratio in serum: After the experiment,compared with Control group,the levels of TC and LDL/HDL in serum of HFD group were significantly increased(p<0.01),the contents of TG and LDL-C were significantly increased(p<0.05),and the content of HDL-C was significantly decreased(p<0.01).Compared with HFD group,the contents of TG and LDL-C in serum of mice in HFD+MICT and HFD+HIIT groups were significantly decreased(p<0.05),and LDL/HDL was significantly decreased(p<0.05).TC content and LDL/HDL content in HFD+HIIT group were significantly decreased(p<0.01),and HDL-C content was significantly increased(p<0.05),but the contents of TC and HDL-C in HFD+MICT group were decreased or increased,but there was no significant difference(p>0.05).These results suggest that long-term high-fat diet can increase the weight and abdominal fat of NAFLD mice,promote the formation of IR in vivo,decrease the glucose tolerance and sensitivity in vivo,and cause different lipid protein abnormalities,increasing the risk of cardiovascular diseases.It can significantly reduce body weight,improve IR,glucose tolerance and dyslipidemia in NAFLD mice.2.Changes of myocardial morphology and structure in each group1)Masson staining: Compared with Control group,the content of myocardial collagen fiber in HFD group was significantly increased(p<0.01);However,after exercise intervention,the content of myocardial collagen fiber in HFD+MICT and HFD+HIIT groups was significantly decreased compared with that in HFD group(p<0.05).2)Transmission electron microscopy: Compared with the Control group,the myocardial structure of HFD group was abnormal,the myofibrillar arrangement was disorganized,broken,the light and dark bands were blurred or even disappeared,the myocardial morphology was uneven,the mitochondrial swelling and the ridge were blurred.After exercise intervention,compared with HFD group,the myocardial fiber arrangement in HFD+MICT and HFD+HIIT groups was improved,more clear light and dark bands could be seen,the size and density of lipid droplets were significantly reduced,mitochondrial structure was restored to normal,and the improvement was more obvious in HFD+MICT group.These studies on tissue structure showed that exercise could effectively improve myocardial fibrosis,abnormal muscle fiber arrangement and structure induced by long-term high-fat diet,and effectively promote the recovery of mitochondrial structure,among which the effect of moderate intensity aerobic exercise was better than that of high-intensity intermittent exercise.3.Western blot analysis1)Lipid metabolism: Compared with Control group,the expression levels of ACC and PPARγ protein in myocardial tissue of HFD group were significantly increased(p<0.05),and the expression of PPARα protein was significantly decreased(p<0.05).Compared with HFD group,the expression level of ACC protein in myocardial of mice in HFD+MICT group was significantly decreased(p<0.05),the expression level of PPARγ protein in HFD+HIIT group was significantly decreased(p<0.05),and the expression level of ACC protein was significantly decreased(p<0.01).The expression of PPARα protein in the two exercise groups showed an increasing trend but no statistical significance(p>0.05).These results indicate that long-term high-fat diet can cause the disturbance of myocardial lipid metabolism,and exercise intervention can promote the myocardial lipid oxidation and the expression of lipid synthetic protein to achieve a relatively balanced state,in which moderate intensity exercise and high intensity intermittent exercise have better effects.2)Glucose transport: compared with Control group,the ratio of P-AMPK/AMPK in myocardium of HFD group was significantly decreased(p<0.05),and GLUT4 protein expression level was significantly decreased(p<0.01).Compared with HFD group,P-AMPK/AMPK in myocardium of mice in HFD+MICT group was significantly up-regulated(p<0.01),GLUT4 protein expression was significantly up-regulated(p<0.05),and P-AMPK/AMPK in myocardium of mice in HFD+HIIT group was significantly up-regulated(p<0.05).These results suggest that glucose transport in the myocardium of NAFLD mice is impaired,which inhibits the effective uptake of glucose by the cardiomyocytes,and exercise can restore normal glucose metabolism in the myocardium by improving related glucose transport pathways.3)Autophagy: Compared with Control group,Parkin protein level in HFD group was significantly down-regulated(p<0.01),but PINK1 protein level had no significant change.Compared with HFD group,Parkin protein level in myocardial of mice in HFD+MICT group was significantly up-regulated(p<0.05),while Parkin protein level in HFD+HIIT group showed an up-regulated trend,but there was no significant difference.Since mitochondria can be cleared through the general autophagy pathway,we detected general autophagy related indicators,and the results showed that compared with the Control group,the expression of Beclin1,an autophagy related protein in myocardial tissue of HFD group had no significant difference.However,LCII/LCI ratio was significantly up-regulated(p<0.01),protein expression level of p62 was significantly up-regulated(p<0.05),and protein expression of LAMP1 was significantly decreased(p<0.01).After exercise intervention,compared with HFD group,LCII/LCI in HFD+MICT group was significantly decreased(p<0.01),and LAMP1 protein expression was significantly up-regulated(p<0.05).LCII/LCI in myocardium of HFD+HIIT group was significantly decreased(p<0.05),and p62 protein expression level was significantly increased(p<0.05).These results indicate that high-fat diet can inhibit mitochondrial autophagy and autophagy flux in mouse myocardial cells,which may be due to blocking lysosome degradation of contents rather than blocking the initiation of autophagy.Therefore,we speculate that there may be dysfunctional mitochondrial accumulation in the heart,but the expression level of PGC-1α protein is significantly decreased(p<0.01).The expression of PGC-1α protein in the myocardium of mice in the two groups showed an increased trend after exercise,indicating that the increase of mitochondria in the myocardium of mice in the high-fat diet group may be due to the impaired ability to remove damaged mitochondria.Conclusion: Both moderate intensity aerobic exercise and high intensity intermittent aerobic exercise for 8 weeks can effectively improve the myocardial structure damage caused by mitochondrial dysfunction in mouse cardiomyocytes induced by high fat diet.The mechanism may be through improving lipid metabolism disorder,reducing insulin resistance in NAFLD mice,enhancing myocardial tissue sensitivity to glucose,and up-regulating mitochondrial autophagy.Restore autophagy flow,so as to improve the normal autophagy function of myocardial cells,effectively maintain the homeostasis of myocardial cells,and promote the recovery of myocardial structure and function in mice with non-alcoholic fatty liver disease.Among them,the effect of high-intensity intermittent aerobic exercise was better in improving dyslipidemia,and the effect of moderate intensity aerobic exercise was more significant in improving glucose metabolism and mitochondrial function of cardiomyocytes.