Research On The Mechanism Of Probiotics Supplementation To Improve Muscle Inflammation In Overtraining Rats

Objective:Purpose of the study was to investigate whether the supplementation of mixed probiotics can protect gut barrier function by regulating intestinal flora homeostasis in overtraining rats,so as to alleviate skeletal muscle inflammation,oxidative stress,cell apoptosis,and investigating the underlying signaling pathway mechanism.Methods:Thirty-two male rats were randomly allocated to four groups(n=8):control(SC),exercise(EC),probiotics(SP)and exercise with probiotics(EP).SP and EP groups received mixed probiotics(at a dose of 4.0×10~9 CFU/day in distilled water,for 8 weeks);SC and EC groups received maltodextrin(1ml/day)by oral gavage during the experiment.After 1h of gavage,the rats in EC and EP groups were trained for 8 weeks of increasing load running platform,6 days a week.After the last training,fasting for 20-24 hours,blood was taken from the abdominal aorta to separate the serum,the soleus muscle and ileocecal faeces were frozen at-80℃ for testing indexes.(1)CK,LDH and BUN indexes in rat serum were measured by an automatic biochemical analyzer;MYO in rat serum indexes were measured by automatic immunoanalyzer.(2)16S rDNA amplicon sequencing was employed to determine the microbial diversity and composition in rat feces.(3)Serum D-LAC and DAO levels were detected by ELISA;serum and skeletal muscle levels of LPS,IL-6,IL-Iβ,TNF-αwere detected by ELISA.(4)The contents of T-AOC,SOD,MDA,GPx and CAT in serum and skeletal muscle of rats were measured by colorimetry.(5)Western blots were used to determine the relative protein expression levels of skeletal muscle proteins TLR4,MyD88,p-NF-κB/NF-κB,Bax/Bcl-2,cleaved caspase-9/procaspase-9 and cleaved caspase-3/procaspase-3.Results:(1)Compared with the SC group,the body weight of the EC group decreased significantly after the 2nd week of exercise intervention(p<0.05).Compared with SP group,the weight of EP group decreased significantly after the first week of exercise intervention(p<0.01).(2)Exercise intervention had a major effect.Compared with the SC group,CK,BUN,MYO values in the serum of the EC group were significantly increased(p<0.05).Compared with SP group,the serum LDH,BUN values of EP group were also significantly increased(p<0.05).(3)Long-term endurance exercise intervention improved the intestinal microbial diversity of rats.Compared with SC,the abundance adaptability of Lactobacillaceae(p=0.015,T-test)in EC group increased,but the abundance of Bacteroidetes (gram-negative bacteria)(p=0.038,T-test)and other pathogenic bacteria increased,which damaged the intestinal microbial homeostasis.Compared with the SC group,the probiotics colonized the intestinal tract in the SP group,the abundance of Actinobacteria(LDA score>4,LEfSe)increased.Compared with EC group,the level of beneficial strains such as Bifidobacteriaceae(q<0.05,MetaStat)in EP group were significantly increased.(4)Compared with the SC group,the serum levels of D-LAC and DAO in the EC group were significantly increased(p<0.05).Compared with EC group,the content of serum D-LAC in EP group was significantly decreased(p<0.05)and the content of DAO was decreased,but there was no significant difference(p>0.05).(5)Compared with the SC group,the serum and skeletal muscle LPS content of EC group was significantly increased(p<0.01).Compared with EC group,the content of serum and skeletal muscle LPS in EP group was significantly decreased(p<0.05).(6)Compared with the SC group,serum and skeletal muscle IL-6,IL-Iβand TNF-αlevels in the EC group were significantly increased(p<0.05).Compared with EC group,serum and skeletal muscle IL-Iβand TNF-αlevels in EP group were significantly decreased(p<0.05),serum IL-6 levels were significantly decreased (p<0.05),and skeletal muscle IL-6 levels were decreased,but there was no significant difference(p>0.05).(7)Compared with the SC group,relative protein expressions of skeletal muscle total protein TLR4,MyD88 and p-NF-κB/NF-κB in EC group were significantly increased(p<0.01).Compared with EC group,the relative protein expression levels of skeletal muscle protein TLR4,MyD88 and p-NF-κB/NF-κB in EP group were significantly decreased(p<0.05).(8)Compared with the SC group,MDA levels in serum and skeletal muscle of the EC group were significantly increased(p<0.01),and serum T-AOC,SOD,GPx and CAT levels were significantly decreased(P<0.05).Compared with EC group,MDA content in serum and skeletal muscle of EP group was significantly decreased (p<0.05),T-AOC and GPx content in serum and skeletal muscle were significantly increased(p<0.05),SOD and CAT in serum and skeletal muscle were increased,but there was no significant difference(p>0.05).(9)Compared with the SC group,the relative protein expression levels of skeletal muscle apoptotic protein Bax/Bcl-2,cleaved caspase-9/procaspase-9 and cleaved caspase-3/procaspase-3 in the EC group were significantly increased(p<0.05). Compared with EC group,the relative protein expression levels of skeletal muscle apoptotic protein Bax/Bcl-2,cleaved caspase-9/procaspase-9 and cleaved caspase-3/procaspase-3 in the EP group were decreased,but there was no significant difference(p>0.05).Conclusion:Probiotics alleviated overtraining-induced muscle inflammation and oxidative stress damage through modulating intestinal flora homeostasis and the TLR4/MyD88/NF-κB signaling pathway in rats.