| 【Objective】To explore the changes of autophagy and differential expression of mi RNAs in skeletal muscle of septic rats,search for mi RNAs related to autophagy changes,and detect their impact on skeletal muscle autophagy.【Methods】(1)The male SPF-grade rats were divided into 6 groups: sham 24(sh24,n=3),48(sh48,n=3)and 72(sh72,n=3)hour groups,sepsis 24(sep24,n=5),48(sep48,n=7)and 72(sep72,n=10)hour groups,and the rats in the sepsis groups underwent cecal ligation and puncture.HE staining was used to detect the muscle fiber atrophy in rats’ tibialis anterior muscles,and western blot was used to detect the expression of autophagy proteins ULK 1 and LC3 B.The tibialis anterior muscles of the sh24 and sep24 groups were analyzed by mi RNA high-throughput sequencing,and then the sequencing results were validated by real-time fluorescence quantitative PCR,and target genes related to autophagy were predicted by target gene software.(2)L6 myoblasts were induced to differentiate into L6 myotubes after 6 days of cultivation by using 2% FBS medium.After differentiation,L6 myotubes were divided into 6 groups:control 24(con24),48(con48)and 72(con72)hours groups cultured with 10% fetal bovine serum + DMEM and sepsis 24(sep24),48(sep48)and 72(sep72)hours groups cultured with10% septic rat serum + DMEM.CCK-8 was used to detect cell viability,and western blot was used to detect the expression of autophagy proteins ULK 1 and LC3 B.(3)Mi RNA-429,which was proved to be differentially expressed through screening and predicted to be related to autophagy,was used for experiments.L6 myotubes were divided into 4groups:(1)control group(con),(2)sepsis group(sep),(3)sepsis + mi RNA-429 inhibitor group(sep+inhibitor),(4)sepsis + mi RNA-429 mimic group(sep+mimic).After transfection,except for the control group,the rests were stimulated with septic rat serum for 24 hours.Jimsa staining was used to observe the morphological changes of L6 myotubes.Western blot was used to detect the expression of autophagy proteins ULK 1 and LC3 B.【Results】(1)Compared with the sh24,sh48 and sh72 groups respectively,the diameter of the tibialis anterior muscles was shortened(P<0.05)and the expression of autophagy proteins ULK 1 and LC3 B was increased(P<0.05)in the sep24,sep48 and sep72 groups.The results of highthroughput sequencing of mi RNAs showed that 24 hours after CLP,there were significant differential changes in 14 mi RNAs in tibialis anterior muscles,of which 8 mi RNAs were increased and 6 mi RNAs were reduced.Among them,mi RNA-429 and mi RNA-141-3p showed the highest differential expression(P<0.001).The differential expression of mi RNAs between the sh24 and sep24 was further validated by PCR.Compared with the sh24 group,the expression levels of mi RNA-429 and mi RNA-141-3p in the sep24 group were significantly,they were 9.5and 4 times higher than those in the sh24 group,respectively(P<0.001).The prediction websites predicted that mi RNA-429 had a targeted relationship with the autophagy-related gene ULK 2.(2)Compared with the con24,con48 and con72 groups,the cell viability of the sep24,sep48 and sep72 group was decreased(P<0.05),and the expression levels of autophagy proteins ULK 1 and LC3 B were increased(P<0.05).(3)Compared with the con group,the expression levels of autophagy proteins ULK 1 and LC3 B in the sep group,sep+inhibitor group and sep+mimic group were significantly increased(P<0.05).Compared with the sep group,the expression levels of autophagy proteins ULK 1 and LC3 B in the sep+inhibitor group were significantly increased(P<0.05),while the levels of ULK1 and LC3 B in the sep+mimic group were reduced.【Conclusion】The autophagy of skeletal muscle in septic rats was enhanced.The expression of mi RNA-429 was increased,which could inhibit autophagy. |
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