Effects Of Different Doses Of Hydrogen On Oxidative Stress And Intestinal Flora In Rats Undergoing Intensive Exercise

Objective:To observe the effects of long-term hydrogen supplementation on exercise ability,oxidative damage,antioxidant activity and intestinal barrier in rats undergoing intensive exercise,and to clarify its improvement effect and optimal supplementation dose.Furthermore,the mechanism of hydrogen in preventing and treating exercise-induced oxidative injury was further studied from the perspectives of Keapl/Nrf2/ARE signaling pathway and intestinal flora,so as to provide theoretical reference for the application of hydrogen in exercise-induced oxidative injuryMethods:Forty male SD rats(200±20g)were randomly divided into five groups:quiet group(N=8,C group),exercise group(N=8,E group),low-dose hydrogen intervention exercise group(N=8,H2-? group),medium-dose hydrogen intervention exercise group(N=8,H2-? group),high-dose hydrogen intervention exercise group(N=8,H2-? group).In group E,H2-?,H2-? and H2-?,rats in group H2-?,H2-? and H 2-?were given four weeks of intensive exercise.Immediately after each exercise,rats in group H2-?,H2-? and H2-? were put into hydrogen culture chambers with concentrations of 0.5±0.1%,0.9±0.1%and 1.5±0.1%respectively for one hour.During the experiment,rats were weighed every week.After 4 weeks of training,cardiac apex blood was collected and hind limb gastrocnemius and colon were taken.The levels of ROS in skeletal muscle were measured;the indexes of serum motor function(CK,LDH),oxidative stress(MDA,3-NT),antioxidant(T-AOC,SOD,CAT),intestinal barrier(D-lactic acid,LPS)were measured after serum was separated;16S rDNA sequencing and tight junction protein were performed in feces and colon of rats in exercise group,quiet group and exercise group,respectively.The expression levels of ccludin and ZO-1 mRNA were determined.Results:(1)Compared with group C,except for the second week of exercise,the weight of rats in group E decreased significantly(P<0.05);there was no significant difference in the weight of rats in H2-? group,H2-? group and H2-? group.(2)Compared with group C,serum CK and LDH levels in group E increased,but there was no statistical difference;serum CK levels in group H2-? decreased significantly(P<0.05),and serum LDH levels in group H2-? decreased significantly(P<0.05).(3)Compared with group C,ROS and serum MDA levels of skeletal muscle in group E increased significantly(P<0.05),while ROS levels of skeletal muscle in group H2-?,H2-? and H2-? had no significant difference,while serum MDA levels in group H2-?,H2-? and H2-? increased significantly(P<0.05);compared with group E,ROS levels of skeletal muscle in group H2-? decreased significantly(P<0.001).(4)Compared with group C,serum T-AOC,CAT and SOD levels in group E increased significantly(P<0.05),serum T-AOC levels in group H2-? increased significantly(P<0.05),serum SOD levels in group H2-? increased significantly(P<0.05),and serum T-AOC,CAT and SOD levels in group H2-? had no significant difference;compared with group E,serum CAT levels in group H2-? decreased significantly(P<0.01).(5)Compared with group C,the serum D-lactic acid levels in group E and H2-? increased significantly(P<0.05),but there was no significant change in serum D-lactic acid levels in group H2-?and H2-?;compared with group C,the serum LPS levels in group E,H2-?,H2-? and H2-? increased,but there was no significant difference.(6)Compared with group C,there was no significant difference in the expression of ZO-1 and Occludin in intestinal epithelium in group E.The expression of ZO-1 and Occludin in group H2 increased significantly(P<0.01),and the expression of ZO-1 and Occludin in group H2 also increased significantly(P<0.01).(7)Compared with group C,the expression of Nrf2 protein increased in group E,and the expression of Nrf2 protein increased in group H2,but the expression of Nrf2 protein did not increase significantly in group H.Compared with group E,the expression of Nrf2 protein decreased and the expression of Nrf2 protein decreased in group H2.(8)Compared with group C,the diversity and abundance of intestinal flora in group E and H2 had no significant difference;the abundance of beneficial bacteria Ruminococcaceae,Romboutsia,Ruminococcaceae_UCG-014 and Peptostreptococceae in group E decreased significantly(P<0.05),and the abundance of beneficial bacteria Lachnospiracea in group H2 increased significantly(P<0.05),while the proportions of beneficial bacteria such as Ruminococceae,Rucocceae_UCG-014 increased.There was no significant difference.Conclusions:(1)During the four-week intensive exercise,the weight and exercise ability of rats decreased with the increase of exercise intensity and exercise time.Supplementary hydrogen can alleviate weight loss,promote the recovery of exercise fatigue and improve exercise function.(2)High intensity exercise around the body caused oxidative stress injury,resulting in a significant increase in the level of antioxidant enzymes;long-term hydrogen supplementation can effectively eliminate free radicals and improve the exercise-induced oxidative stress injury in rats.(3)Intensive exercise can increase the permeability of intestinal epithelial cells.Hydrogen can increase the expression of tight junction protein in intestinal epithelium and enhance the intestinal barrier function.(4)Compared with different doses,medium-dose hydrogen has the best effect in regulating the imbalance of redox system caused by intensive exercise and promoting the recovery of exercise fatigue and intestinal barrier.(5)Hydrogen may alleviate exercise-induced oxidative stress injury by scavenging excessive free radicals and increasing the abundance of beneficial bacteria,improving the structure of bacteria and promoting the restoration of microecological balance of intestinal flora.