Cloning Of Muscle Atrophy Gene Atrogin-1 And Regulation Of FoxO/atrogin-1 Signal Pathway Induced By Fasting And LPS Stimulation In Monopterus Albus

Atrogin-1 is a conserved ubiquitin ligase E3,which plays an important role in the early stage of skeletal muscle and myocardial atrophy and degeneration after hunger and inflammatory diseases.It is a major protein that adjusts the quality of skeletal muscle in mammals and fish.In this experiment,We characterised the Monopterus Albus atrogin-1gene,identifying two alternatively spliced atrogin-1 isoforms.The mRNA sequence of atrogin-1 x1 of Monopterus Albus is 2361 bp,including the coding sequence of 1074 bp,encoding 357 amino acids,the predicted mass is 41.8 KD,isoelectric point(Pi)is 8.77.The mRNA of atrogin-1 x2,another alternatively spliced atrogin-1 isoform,2353 bp,including the coding sequence of 894 bp,which encodes 298 amino acids with a predicted mass of 34.8kD,isoelectric point(Pi)is 9.26.Atrogin-1 x1 only 5 ’UTR is different from that of atrogin-1 x2.It is suggested that the transcription factors that regulate their expression may be different.It is predicted that the protein sequences have about 40 amino acids in the F-box conserved region.The expression of heart,hepatopancreas(liver),spleen,head and kidney,body kidney,foregut and posterior intestine in fed and healthy Monopterus Albus was quantitatively analyzed.RPL17,which can be stably expressed in all tissues,was used as internal reference gene to analyze the expression.The results showed that,unlike mammals,only in skeletal muscle and heart,atrogin-1 x1 and atrogin-1 x2 were widely expressed in all the examined Monopterus Albus tissues,and the relative expression of skeletal muscle is high in heart,spleen and skeletal muscle.The effects of starvation on atrogin-1 expression,FoxO gene and Akt gene expression in Monopterus Albus were divided into three groups: short-term,medium-term and long-term starvation.After 7 days of starvation,the expression of atrogin-1 x1 increased but not significantly,and the content of atrogin-1 x1 increased significantly at 3 months after starvation,but the expression of atrogin-1 x1 decreased after 7 months of starvation,which may be due to chronic starvation.The expression of atrogin-1 x2 was similar to that of atrogin-1 x1.The expression of Akt gene was significantly expressed in short-term,medium-term and long-term starvation.The expression of FoxO3 gene increased but was not significant after starvation.After 3 months of starvation,the level of FoxO3 increased significantly,but decreased after 7 months.MYOD1 gene is regulated by atrogin-1 gene expression,and the trend is consistent with it.In the LPS stimulation experimental group,30 ±5 g Monopterus Albus was injected with LPS into the abdominal cavity of 10mg/kg.In the PBS group,injection of PBS as control.Then,injection of LPS combined with cytokine inhibitor AX024 or tyrosine kinase inhibitor AG490,confirmed that LPS could cause muscle atrophy of Monopterus Albus byFoxO/atrogin-1 signaling pathway,and was regulated by cytokines and growth factors.