Anti-fatigue Effect And Mechanism Of Phlorizin On Exhaustive Exercise-induced Fatigue In Mice

Objective: The study was designed to investigate the anti-fatigue effect,and further to explore the possible mechanism of phlorizin(PHZ)on exhaustive swimming-induced fatigue in mice.Methods: The fatigue model of mice was established by exhaustive swimming,and SPF male C57BL/6J mice were divided into six groups by simple random-sampling method:control group,model group,model + PHZ 2.5 mg/kg dose group,model + PHZ 5 mg/kg dose group,model + PHZ 10 mg/kg dose group and model + PHZ 20 mg/kg dose group,respectively.Thereafter,wild type(WT)and Nrf2 deficient(Nrf2-/-)mice were randomly divided into four groups: WT-model group,WT-model + PHZ(10 mg/kg)group,Nrf2-/--model group,Nrf2-/--model + PHZ(10 mg/kg)group.Then the control group and model group were given the same volume-matched normal saline by continuous gavage for28 days.The exercise ability of mice in each group was tested by forced swimming with load and rotarod test.The binding ability of PHZ to Nrf2 was analyzed by molecular docking technique.Blood urea nitrogen(BUN),creatine kinase(CK),lactic acid(LA),liver glycogen(LG),skeletal muscle glycogen(MG),reactive oxygen species(ROS)and malondialdehyde(MDA)levels,as well as superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)activities were determined by enzyme linked immunosorbent assay.Furthermore,the expressions of nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase-1 NAD(P)H-quinone oxidoreductase-1 and the apoptosis releated protein expressions of Bcl2,Bax and caspase 3 were detected by western blot analysis.Results: Time of the forced swimming and time on rotard of mice were significantly reduced in model group than those of control group,while,these change of mice in PHZ treatment group were reversed than those of model group.PHZ also effectively decreased the levels of BUN,CK and LA,increased of the LG and skeletal MG of fatigue mice,as evidenced by enzyme linked immunosorbent assay.PHZ balanced the redox status through reducing generation of ROS,enhancing the activities of antioxidative enzymes.Furthermore,PHZ not only increased the ratio of Bcl2/Bax,but also decreased the level of cleaved-caspase 3.Notably,PHZ facilitated nucleus-Nrf2 translocated from cytoplasm to nucleus,and up-regulated its downstream antioxidant response element including heme oxygenase-1 and NADPH quinone oxidoreductase-1.Intriguingly,molecular docking results showed that PHZ directly bound to Nrf2,which suggested that the anti-fatigue effect of PHZ might through activating Nrf2.Thereafter,Of note,Nrf2-/-mice were used to explore the role of Nrf2 in the anti-fatigue effect in mice.The results showed that time of the forced swimming and time on rotard of Nrf2-/-mice were significantly decreased in model group than those of WT mice.Moreover,the levels of BUN,CK and LA were increased,the levels LG and MG were decreased in Nrf2-/-mice than those of WT mice.Meanwhile,ROS and MDA levels were enhanced,SOD,CAT and GSH-Px activities were decreased in Nrf2-/-mice than those of WT mice.However,the anti-fatigue effects of PHZ were substantially abolished in Nrf2-/-mice.Conclusion: PHZ exerts potent anti-fatigue effect on exhaustive swimming-induced fatigue in mice,which are mediated by Nrf2/ARE signaling pathway,thereby inhibits oxidative stress.