| Objective:To investigate whether exogenous supplementation of nicotinamide mononucleotide(NMN)produces a protective effect on myocardial oxidative stress injury in rats undergoing acute high-intensity exercise,and to provide a reference for the study on the prevention of exercise-induced myocardial injury by NMN.Methods:Fifty-six 7-week-old male SD rats were randomly divided into five groups:quiet control group(C,n=8),exercise control group(CE,n=12),low-dose NMN pretreatment exercise group(LNE,n=12),medium-dose NMN pretreatment exercise group(MNE,n=12),and high-dose NMN pretreatment exercise group(HNE,n=12).After the acclimatization feeding,the NMN supplementation group was gavaged with three different concentrations of NMN solution according to rat body weight:low(100 mg/kg),medium(300 mg/kg)and high(500 mg/kg),and the C and CE groups were gavaged with an equal volume of saline once daily for 4 weeks.After 1 h of the last gavage,rats in all exercise groups(CE,LNE,MNE,and HNE groups)were subjected to acute high-intensity exercise for 1 h at a speed of 27 m/min on a running platform with an inclination of 10%.After 1 h of exercise,4 rats in each group were randomly selected to continue exercise until exhaustion,and the time of exhaustion was recorded;the rest of the rats stopped exercise immediately.The rats were sampled2 h after the end of acute high-intensity exercise.Light microscope observation of HE-stained sections of rat myocardial tissue,transmission electron microscope observation of electron microscopic sections of rat myocardial tissue,ELISA determination of myocardial injury markers c Tn I,CK-MB,NT-pro BNP levels and inflammatory factors IL-6,TNF-α,IL-4,IL-10 levels in rat serum,Western blot detection of apoptosis proteins Bax,Caspase-3 expression levels,reflecting the extent of myocardial injury in rats;ROS level in myocardial tissue by DHE staining,MDA content of lipid peroxidation products and activities of antioxidants SOD,GPx and CAT by the kit,reflecting the extent of oxidative stress injury in rat myocardium;NAD~+and NADH levels and NAD~+/NADH ratio in myocardial tissue by the kit,reflecting NMN entered the heart and enhanced the cellular NAD~+level;Western blot detected the protein expression levels of Sirt1,Nrf2 and HO-1 in rat myocardium,reflecting the effect of NMN supplementation on this signaling pathway related to oxidative stress response;Graphpad Prism 8.0.1 software was used for statistical analysis and plotting of data.Results:(1)Effect of NMN on body weight of rats.All exogenous supplementation of NMN at different concentrations had no significant effect on the body weight of healthy rats.(2)The effect of NMN on the time to exhaustion in rats.Compared with the CE group,the mean values of exhaustion exercise time of rats in MNE and HNE groups were prolonged by 9.39%,16.97%and 17.27%,respectively,among which,the exhaustion exercise time of rats in MNE and HNE groups was significantly prolonged(P<0.05),and the exhaustion time of rats in LNE group tended to increase,but there was no significant difference(P>0.05).The results indicated that NMN could prolong the time of exhaustion and improve the exercise ability of rats,and the effect was better in medium and high doses.(3)Effects of NMN on myocardial injury in rats with acute high-intensity exercise.(1)The levels of myocardial injury markers c Tn I,CK-MB and NT-pro BNP were significantly increased(P<0.01)in the CE group compared with the C group;the levels of c Tn I,CK-MB and NT-pro BNP were significantly decreased(P<0.05)in the LNE,MNE and HNE groups compared with the CE group.(2)Compared with group C,myocardial fibers in the CE group were disordered in arrangement,with swollen and blurred cell contours and inflammatory cell infiltration;compared with the CE group,morphological and structural changes were smaller in the LNE,MNE and HNE groups,and the effect was particularly pronounced in the MNE and HNE groups.(3)Compared with group C,the levels of pro-inflammatory factors IL-6 and TNF-αwere significantly increased(P<0.01)and the levels of anti-inflammatory factors IL-4 and IL-10 were significantly decreased(P<0.05)in the CE group;compared with the CE group,the levels of TNF-αwere significantly decreased(P<0.05)in the MNE and HNE groups,and the levels of IL-10 were significantly increased(P<0.05)in the HNE group.(4)The protein expression levels of myocardial Bax and Caspase-3 were significantly increased(P<0.01)in the CE group compared with the C group;the myocardial Caspase-3 levels were significantly decreased(P<0.05)in the LNE,MNE and HNE groups compared with the CE group,and the myocardial Bax levels were significantly decreased(P<0.05)in the MNE and HNE groups.The results suggest that NMN supplementation can produce protective effects on acute high-intensity exercise-induced myocardial injury,with better effects at medium and high doses.(4)The effect of NMN on myocardial oxidative stress levels in rats with acute high-intensity exercise.Compared with group C,the level of ROS in myocardial tissue of rats in CE group was significantly increased(P<0.01);compared with group CE,the mean values of LNE,MNE and HNE were reduced by 19.49%,32.63%and35.66%,respectively,all of which were significant(P<0.05).Myocardial MDA levels were significantly increased(P<0.01)in the CE group compared with the C group;their mean values were reduced by 18.88%,30.45%and 33.47%in the LNE,MNE and HNE groups,respectively,compared with the CE group,all of which were significant(P<0.05).Compared with group C,there was no significant change(P>0.05)in the activity of antioxidant enzymes SOD,CAT and GPx in myocardial tissue of rats in group CE;however,compared with group CE,there was an increase in the activity of antioxidant enzymes in myocardium of rats in groups LNE,MNE and HNE,with mean values of SOD activity increasing by 12.27%,20.41%and23.55%,CAT activity increasing by 12.83%,20.58%and 21.9%,and GPx activity increasing by 12.27%,18.41%and 18.23%,respectively,in which there was no significant difference(P>0.05)between the three antioxidant enzymes in the LNE group and the CE group,while the myocardial antioxidant enzymes in the MNE and HNE groups were significantly higher(P<0.05)than those in the CE group.The results showed that NMN could reduce myocardial oxidative stress injury in acute high-intensity exercise rats on the one hand,and improve their myocardial antioxidant capacity on the other hand,thus playing a protective role against myocardial oxidative stress injury induced by acute high-intensity exercise,among which,medium and high doses were more effective.(5)Effects of NMN on NAD~+and NADH levels and NAD~+/NADH ratio in rats with acute high-intensity exercise.Compared with group C,the myocardial NAD~+level of rats in CE group was significantly decreased(P<0.05);compared with group CE,the mean myocardial NAD~+level of rats in LNE,MNE and HNE groups was increased by 16.83%,25.66%and 30.67%,respectively,in which,the myocardial NAD~+level of rats in MNE and HNE groups was significantly increased(P<0.05).In contrast to NAD~+levels,myocardial NADH levels were significantly higher(P<0.01)in rats in the CE group compared with the C group;compared with the CE group,the mean myocardial NADH values were reduced by 13.65%,20.08%and 20.07%in the LNE,MNE and HNE groups,respectively,with significant reductions(P<0.05)in the MNE and HNE groups.Compared with group C,the myocardial NAD~+/NADH level was significantly lower(P<0.01)in the CE group;compared with the CE group,the mean values of myocardial NAD~+/NADH ratio were increased by 36.01%,59.92%and 65.00%in the LNE,MNE and HNE groups,respectively,with a significant increase(P<0.01)in the MNE and HNE groups.The results indicated that NMN supplementation could increase the myocardial NAD~+level,reduce the accumulation of NADH caused by high-intensity exercise,and increase the NAD~+/NADH ratio in rats with acute high-intensity exercise,thus ensuring the normal redox reaction and activation of downstream signaling pathways,and the effect was better at medium and high doses.(6)Effect of NMN on the oxidative stress pathway Sirt1/Nrf2/HO-1 in rats subjected to acute high-intensity exercise.Immunohistochemistry method can be obtained that the myocardial Sirt1 level was significantly decreased(P<0.05)in the CE group compared with the C group;its expression was significantly increased(P<0.01)in the MNE and HNE groups compared with the CE group;and its expression was significantly increased(P<0.05)in the HNE group compared with the LNE group.Western blot experiments showed that myocardial Sirt1 protein expression was significantly lower(P<0.05)in the CE group compared with the C group;myocardial Sirt1 protein expression was significantly higher(P<0.01)in the MNE and HNE groups compared with the CE group;myocardial Sirt1 protein expression level was significantly higher(P<0.05)in the HNE group compared with the LNE group.Compared with the CE group,the nuclear Nrf2 levels in cardiomyocytes and HO-1 levels in cardiomyocytes were significantly higher(P<0.05)in both the MNE and HNE groups of rats.The results indicated that NMN supplementation could activate the Sirt1/Nrf2/HO-1 signaling pathway in rats with acute high-intensity exercise,in which,medium and high doses,especially high doses,were more effective.Conclusions:Exogenous supplementation of NMN for 4 weeks was able to reduce ROS and MDA levels and increase the activities of antioxidant enzymes SOD,CAT and GPx in rats with acute high-intensity exercise,and attenuate myocardial oxidative stress,thus producing protective effects against cell swelling,inflammation,apoptosis and other related myocardial damage and improving exercise capacity in rats.The mechanism may be related to the increase of NAD~+level and the activation of Sirt1/Nrf2/HO-1 signaling pathway.Medium and high doses of NMN supplementation are more effective. |
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