Naringin Activates AMPK/SIRT1/PGC-1α Signal Axis To Reduce Exercise Injury In Mice Under Low Temperature

ObjectiveThe current study investigated the protective effect and possible mechanism of naringin on exercise ability and muscle injury in mice under low temperature.To provides new experimental evidence for alleviating muscle injury and improving exercise ability under low temperature.As well as,providing scientific basis for the development of naringin-rich food resources.MethodsForty male C57BL/6J mice were randomly divided into four groups,normal temperature quiet control group(Ctrl),hypothermia exercise group(LE),50 mg/kg naringin+hypothermia exercise group(50NG group),and 100 mg/kg naringin+hypothermia exercise group(100NG group).Gavage intervention lasted for 4 weeks.Mice were trained with adaptive running twice a week.The weekly weight change of mice was monitored.The running to exhaustion time of mice in low temperature environment(4±1)℃ was recorded.Biochemical indices were determined by ELISA,including fatigue and damage indicators:lactic acid(LA),lactate dehydrogenase(LDH),creatine kinase(CK)and urea nitrogen(BUN)in serum;energy metabolism indicators:adenosine triphosphate(ATP)in muscle tissue;oxidative stress:serum malondialdehyde(MDA),muscle tissue superoxide dismutase(SOD).The morphology and the ultrastructure of skeletal muscle tissue were observed by HE staining and transmission electron microscopy,respectively.The expression of AMPK/SIRT1/PGC-1α signal axis was detected by Western blot.The mRNA expression levels of mitochondrial biogenesis(NRF1,TFAM)and antioxidantion-related genes(NRF2,SOD1,HO-1 and GCLC)in PGC-1αdownstream were detected by RT-qPCR.SPSS 25.0 software was applied for statistical analysis.Analysis of variance was used to evaluate the differences among multiple groups.The test level α=0.05.Results1.There was no significant difference in body weight among groups before and after NG intervention.Compared with LE group,100NG group prolonged the running exhaustion time of mice under low temperature,and the difference was statistically significant(P<0.05).2.The exercise-induced fatigue and injury indexes of mice in low temperature environment were decreased by NG.Compared with Ctrl group,serum LA,LDH,CK and BUN in LE group were increased,and the differences were statistically significant(P<0.05).NG reduced the levels of LA,LDH,CK and BUN,100NG group had the most obvious effect,and the differences were statistically significant(P<0.05).3.Exercise induced oxidative stress in mice at low temperature was reduced by NG.Compared with Ctrl group,oxidative stress level of mice in LE group was increased,MDA level in serum was increased,and SOD activity in gastrocnemius and soleus muscle was decreased,the differences were statistically significant(P<0.05).Compared with LE group,NG(100 mg/kg)supplementation decreased MDA level in serum,and increased SOD activity in gastrocnemius and soleus muscle(P<0.05).4.The muscle energy metabolism of mice under low temperature was improved by NG.Compared with Ctrl group,ATPase in gastrocnemius and soleus muscle tissues in LE group was decreased,and the difference was statistically significant(P<0.05).Compared with LE group,100NG group up-regulated ATPase activity in gastrocnemius and soleus muscle tissues(P<0.05).5.The injuries of muscle and myofibrils in mice after exhaustive exercise at low temperature could be protected by NG.HE staining and transmission electron microscopy showed that NG improved the disordered arrangement of muscle fibers,broken connections and rupture muscle cells caused by low temperature exhaustion,as well as,reduced myofibrils arrangement disorder,sparse and fracture.6.Naringin activated AMPK/SIRT1/PGC-1α signaling axis in muscle of mice with low temperature exhaustion.WB experiment showed that compared with Ctrl group,pAMPK/AMPK,SIRT1 and PGC-1α expression levels in gastrocnemius and soleus muscle of mice in LE group were decreased,and the differences were statistically significant(P<0.05).Compared with LE group,NG increased the expression levels of pAMPK/AMPK,SIRT1 and PGC-lα in mice with low temperature exhaustion,and the differences were statistically significant(P<0.05).7.The mRNA expression of mitochondrial biogenic factors were up-regulated by Naringin.Compared with Ctrl group,mRNA expressions of NRF1 and TFAM factors in gastrocnemius and soleus muscle of mice in LE group were inhibited,and the differences were statistically significant(P<0.05).Compared with LE group,naringin up-regulated NRF1 and TFAM mRNA expressions in gastrocnemius muscle and soleus muscle of mice with low temperature exhaustion,the effects of 100NG group intervention were obvious,and the differences were statistically significant(P<0.05).8.The mRNA expression of antioxidant stress-related factors were up-regulated by Naringin.Compared with Ctrl group,mRNA expressions of NRF2,SOD1,HO-1 and GCLC factors in gastrocnemius and soleus muscle of mice in LE group were inhibited,and the differences were statistically significant(P<0.05).Compared with LE group,naringin up-regulated the mRNA expression levels of NRF2,SOD1,HO-1 and GCLC in gastrocnemius and soleus muscle of mice with low temperature exhaustion,the improvement effects of 100NG group were obvious,and the differences were statistically significant(P<0.05).ConclusionNaringin can improve the antioxidant levels and muscle energy metabolism of exercise mice under low temperature,improve the exercise endurance of mice,and reduce exercise injuries,possibly associated with the activation of muscle AMPK/SIRT1/PGC-1α-NRF1/NRF2 signaling pathway.