Effects Of Repeated Exhaustion Exercise On Autophagy Of Skeletal Muscle In Mice And The Intervention Of Pyrroloquinoline Quinone (PQQ)

ObjectiveTo investigate the effects of two-week repetitive exhaustion exercise on oxidative stress and autophagy of skeletal muscle in mice and the intervention effect of pyrroloquinoline quinone,and to clarify the possible mechanism of pyrroloquinoline quinone on autophagy of skeletal muscles in exhaustive exercise mice,so as to provide a strong scientific basis for the development of safe and efficient pyrroquinolinone-related sports nutrition supplements.DesignForty male KM mice according to body weight were randomly divided into five groups:The quiet control group(NC group),exhausted exercise group(E group),Lowdose PQQ supplemented exhausted exercise group(LE group,5 mg/kg/d),Medium-dose PQQ supplemented exhausted exercise group(ME group,10 mg/kg/d)and High-dose PQQ supplemented exhausted exercise group(HE group,20 mg/kg/d),The mice in NC group and E group were given distilled water every day during 2 weeks of repeated exhaustive exercise intervention,while the mice in PQQ group were given different doses of PQQ every day.The mice in the exercise group were put into a round plastic bucket with a suitable amount of water and a suitable water temperature for swimming exercise until the mice became exhausted.The swimming exercise was intervened for 2 days(6days/week)to establish repeated exhaustive exercise model.Immediately after the last mouse exhaustive exercise intervention,the mice were anesthetized and their gastrocnemius muscles were collected.The swimming time of mice was recorded,the activity levels of oxidative stress indicators such as SOD,T-AOC and MDA were detected by biochemical kit,Real-time PCR was used to detect m RNA expression changes of autophagy markers such as Lc3,p62,Atg5,Atg12,Beclin1 and Bcl-2 in mice gastrocnemius muscle and Western blotting was used to detect the protein expression changes of autophagy markers such as Lc3-Ⅰ,Lc3-Ⅱ,p62,Atg5,Atg12,Beclin1,Bcl-2,Ulk1,p-Ulk1(Ser757),AMPK,p-AMPK,PI3 K,Akt,p-Akt,m TOR and p-m TOR in gastrectum muscle of mice.Result1 Compared with NC group,The MDA content in skeletal muscle of mice in E group was extremely significantly increased and SOD and T-AOC levels were extremely significantly decreased(p<0.01).Compared with group E,The MDA content in skeletal muscle of mice in the PQQ supplementation exercise group was extremely significantly reduced and SOD and T-AOC levels were extremely significantly increased(p<0.01),In addition,the decrease trend of MDA content in skeletal muscle and the increase trend of SOD content and T-AOC activity were more obvious in the ME group.2 Compared with the NC group,The m RNA expressions of Lc3 and The protein expression of Lc3-Ⅱ and The ratio of Lc3-Ⅱ/Lc3-Ⅰ in skeletal muscle of mice in E group were extremely significantly increased(p<0.01),And that The m RNA expressions and protein expression of p62 in skeletal muscle of mice in E group were extremely significantly decreased(p<0.01).Compared with the E group,The m RNA expressions of Lc3 and The protein expression of Lc3-Ⅱ and The ratio of Lc3-Ⅱ/Lc3-Ⅰ in skeletal muscle of mice in LE 、 ME and HE group were extremely significantly decreased(p<0.01),While The m RNA expressions of p62 were significantly elevated(p<0.05 or p<0.01),While The protein expression of p62 were extremely significantly increased(p<0.01),Among them,the m RNA expressions of Lc3 and p62 in skeletal muscle of mice in the ME group showed a more significant trend.3 Compared with the NC group,The m RNA and protein expressions of Atg5、Atg12in skeletal muscle of mice in E group were extremely significantly increased(p<0.01).Compared with the E group,The m RNA and protein expressions of Atg5 、 Atg12 in skeletal muscle of mice in LE、ME and HE group were extremely significantly decreased(p<0.01),Among them,the m RNA and protein expression of Atg12 in skeletal muscle of mice in the ME group decreased more significantly.4 Compared with the NC group,The m RNA and protein expressions of Beclin1 in skeletal muscle of mice in E group were extremely significantly decreased(p<0.01),While The m RNA and protein expressions of Bcl-2 were extremely significantly increased(p<0.01).Compared with the E group,The m RNA and protein expressions of Beclin1 in skeletal muscle of mice in LE、ME and HE group were extremely significantly decreased(p<0.01),While The m RNA expressions of Bcl-2 were significantly increased(p<0.05 or p<0.01),While The protein expressions of Bcl-2 were extremely significantly increased(p<0.01),Among them,the m RNA and protein expressions of Beclin1 and the m RNA expressions of Bcl-2 in skeletal muscle of mice in the ME group showed a more significant trend.5 Compared with the NC group,The protein expressions of p-AMPK and the ratio of p-AMPK/AMPK in skeletal muscle of mice in E group were extremely significantly increased(p<0.01).Compared with the E group,The protein expressions of p-AMPK and the ratio of p-AMPK/AMPK in skeletal muscle of mice in E group were significantly increased(p<0.05 or p<0.01),Among them,the protein expressions of p-AMPK and the ratio of p-AMPK/AMPK in skeletal muscle of mice in the ME group showed a more significant trend.6 Compared with the NC group,The protein expressions of p-m TOR and the ratio of p-m TOR/m TOR in skeletal muscle of mice in E group were extremely significantly decreased(p<0.01).Compared with the E group,The protein expressions of p-m TOR and the ratio of p-m TOR/m TOR in skeletal muscle of mice in LE、ME and HE group were extremely significantly increased(p<0.01),Among them,the protein expressions of p-m TOR and the ratio of p-m TOR/m TOR in skeletal muscle of mice in the ME group showed a more significant trend.7 Compared with the NC group,The protein expressions of p-Ulk1(Ser757)and the ratio of p-Ulk1(Ser757)/Ulk1 in skeletal muscle of mice in E group were extremely significantly decreased(p<0.01).Compared with the E group,The protein expressions of p-Ulk1(Ser757)and the ratio of p-Ulk1(Ser757)/Ulk1 in skeletal muscle of mice in LE、ME and HE group were extremely significantly increased(p<0.01),Among them,the protein expressions of p-Ulk1(Ser757)and the ratio of p-Ulk1(Ser757)/ Ulk1 in skeletal muscle of mice in the ME group showed a more significant trend.8 Compared with the NC group,The protein expressions of PI3K、p-Akt and the ratio of p-Akt/Akt in skeletal muscle of mice in E group were extremely significantly decreased(p<0.01).Compared with the E group,The protein expressions of PI3K、p-Akt and the ratio of p-Akt/Akt in skeletal muscle of mice in LE、ME and HE group were extremely significantly increased(p<0.01),Among them,the protein expressions of PI3 K 、 p-Akt and the ratio of p-Akt/Akt in skeletal muscle of mice in the ME group showed a more significant trend.Conclusion1 The oxidative stress and autophagy in skeletal muscle of mice were induced by 2weeks of repetitive exhaustive exercise.Repetitive exhaustive exercise may be involved in inducing autophagy activation of skeletal muscle in mice through AMPK/m TOR/Ulk1 and PI3K/Akt/m TOR signaling pathways.2 Pyrroquinoline quinone supplementation can reduce oxidative stress and autophagy induced by repeated exhaustive exercise in skeletal muscle of mice.The mechanism of inhibiting autophagy may be through the PI3K/Akt/m TOR signaling pathway3 Low-dose,Medium-dose and High-dose of pyrroquinoline quinone supplementation can reduce oxidative stress and autophagy level of skeletal muscle in mice to different degrees.There is no dose-effect relationship between different doses,and the effect of pyrroquinoline quinone at Medium-dose is better.