The Role Of Different Mitophagy Pathways In Anti-Tumor Effect Of Exercise

Background:Cancer remains the leading cause of death in the world’s population and a major obstacle to prolonging life expectancy.According to the latest WHO statistics,cancer is the first or second leading cause of death among people under the age of 70 in 112 of 183 countries.China has the largest proportion of cancer cases and deaths in the world,among which patients with liver cancer account for half of the world’s total.As an anti-cancer and anti-cancer lifestyle,exercise has been proved to be able to be used in all stages of the occurrence and development of cancer.The anticancer mechanisms of exercise include improvement of metabolic disorders,chronic systemic inflammation,impaired immune function,mitochondrial function.As the energy factory in cells and the center of cell signal transduction,mitochondria play a vital role in a variety of physiological activities.Therefore,maintaining mitochondrial homeostasis is a prerequisite for cellular metabolism.Mitochondrial proliferation and biogenesis maintain the quantity,morphology and timely update of mitochondria,while mitophagy maintains mitochondrial quality by clearing damaged mitochondria.Studies have revealed that there are three pathways of mitophagy,which are ubiquitin-mediated,receptor-mediated and lipid-mediated,to guide the degradation of mitochondrial components under different physiological or pathological conditions.Diethylnitrosamine has a stable liver carcinogenesis,and is often used to induce hepatocellular carcinoma(HCC)in mice or rat.Mitophagy can promote and inhibit the development of tumors.Different studies have shown that exercise can promote and inhibit mitophagy.Whether or through which pathway exercise regulates mitophagy and thus affects the occurrence and development of liver tumors are not clear and need to be further explored.Objective:To investigate the effects of different kinds of exercise on the occurrence and development of DEN-induced liver tumors,and to compare the antitumor effects of different kinds of exercise and their differences from the perspective of different pathways of mitophagy.Methods:Forty-eight three-week-old C57BL/6 male mice weighing 1015 g were selected.They were randomly divided into four groups:NC group,the control group,n=12;DEN group,namely,the DEN injection control group,n=12;MET group,namely,DEN injection+moderate intensity endurance exercise group,n=12;HIIT group,DEN injection+high intensity intermittent exercise group,n=12.When the mice in the NC group were 3 weeks old,25mg/kg normal saline was intraperitoneally injected once a week.DEN,MET and HIIT mice were intraperitoneally injected with 25mg/kg DEN once every week at the age of 3 weeks.They were injected for 10 weeks in total.At 13 weeks of age,the mice in the MET group started moderate intensity endurance exercise with intensity of 65%-75%VO2 max,treadmill speed of 12 m/min,and continued to exercise for 40 min/day,five times a week.At the age of 13 weeks,HIIT group mice began high-intensity intermittent exercise with the intensity of 85%-90%VO2 max at a speed of 25 m/min for 2 min and an interval of 2 min,which was repeated for 10 groups every day,five times a week.The daily exercise paths of mice in MET and HIIT groups were approximately the same.Body weights were measured weekly and body composition was measured at 27 weeks.At the age of 27 weeks,the mice in each group were anesthetized with isoflurane,and blood was taken from the heart cavity and the adjacent liver tissues were taken out.The contents of tumor markers such as DCP,AFP-L3,GPC3,AFP and AST in serum of mice were detected by ELISA.The mRNA expression levels of PINK1,NBR1,OPTN,FUNDC1,BNIP3L,BNIP3,PHB2,P62,Parkin,NDP52 and other genes related to mitophagy pathway in the liver were detected by real-time quantitative PCR.The expressions of Parkin,FUNDC1,PUMA,BNIP3,BNIP3L,PINK1,P62,LC3B and other mitophagy-related proteins in the liver were measured by Western Blotting.Results:(1)The incidence of tumor in NC group was 0%,that in DEN group was 45%,that in MET group was 64%,and that in HIIT group was 17%.The results showed that the incidence of tumor was the lowest in the highintensity intermittent exercise group,and the incidence of tumor was increased by moderate-intensity exercise.(2)Compared with NC group,the weight of mice in DEN group was significantly lower(p<0.05),and the weight of mice in HIIT group was significantly lower than that in DEN group(p<0.05).The percentage of lean body mass in DEN group was significantly higher than that in NC group(p<0.01),but the percentage of body fat was lower than that in NC group(p<0.01).There was no significant difference in lean body weight and body fat rate between HIIT group and other groups.(3)The content of serum DCP in DEN group was significantly lower than that in NC group(p<0.01).The levels of serum AFP-L3 in DEN group(p<0.01)and MET group(p<0.01)were significantly higher than those in HIIT group.Compared with DEN group,the serum AST content in HIIT group decreased significantly(p<0.05).(4)Compared with DEN group,the level of PINK 1 mRNA in MET group was significantly higher(p<0.01).Compared with DEN group,the level of mitophagy receptor protein P62 mRNA in MET group was significantly higher(p<0.05).There was no significant difference in the mrna levels of Parkin,NDP52 and NBR1 in liver between groups.There was no significant difference in the expression levels of PINK1,Parkin and P62 proteins among the groups.(5)Compared with DEN group,the expression of LC3B protein in HIIT group increased significantly(p<0.05).There was no significant difference in PUMA mRNA level between groups.The level of BNIP3L(NIX)mRNA in MET group was significantly higher than that in DEN group(p<0.01).Compared with NC group,the expression of PUMA protein in the liver of DEN group decreased significantly(p<0.05).The expression of BNIP3L protein in HIIT group was significantly higher than that in DEN group(p<0.05).(6)Compared with DEN group,the level of FUNDC1 mRNA in MET group increased significantly(p<0.01).Compared with MET group,the level of FUNDC1 mRNA in mice in HIIT group decreased significantly(p<0.05).There was no significant difference in the level of BNIP3 mRNA in liver between groups.There was no significant difference in the expression levels of BNIP3 and FUNDC1 proteins among the groups.Conclusion:(1)Compared with moderate intensity of endurance exercise,highintensity intermittent exercise can significantly reduce the incidence of DEN-induced liver tumors.This indicated that high intensity intermittent exercise had good anti-tumor effect.(2)High-intensity intermittent exercise significantly reduced serum liver injury markers AFP-L3 and AST in mice,indicating that high-intensity intermittent exercise could better prevent DEN-induced liver injury.(3)High-intensity intermittent exercise improves the expression of mitophagy proteins LC3B and BNIP3L,indicating that high-intensity intermittent exercise tends to reduce the incidence of DEN-induced liver tumors through the BNIP3L receptor-mediated mitophagy pathway.