Effects Of Short-term Fasting On The Oxidative Stress And Autophagy In Intestinal Of Siniperca Chuatsi

Siniperca chuatsi has a relatively high market valuable fish with high economic value.It is widely distributed in rivers and lakes in China,and has excellent traits such as fast growth,delicate meat,delicious taste and high nutrient content.Starvation stress is one of the problems often encountered in the process of fish growth.Hunger can cause oxidative stress in the fish body.First,the rise of reactive oxygen species ROS,attacking protein molecules,fatty acid molecules and nucleic acids in cells,and then the body starts antioxidant enzyme systems.It produces SOD,CAT,GST,etc.to scavenge ROS.When the antioxidant system cannot clear ROS,the body begins to activate autophagy.The primary sensor of cellular responses consists of keap1 and the transcription factor Nrf2,that is essential for maintaining cell homeostasis.This study investigated the effect of starvation on oxidative stress,antioxidant system and autophagy in the intestinal of Siniperca chuatsi.The main result as blow:1.Cloning and sequence analysis of Keap1 gene.By extracting the genomic DNA of Siniperca chuatsi,the full-length of Keap1 gene was cloned by RACE technology(MK654680).The Keap1 gene is 5026 bp in length,and the CDS region was 1878 bp,which encoding 625 amino acids.The function domains of keap1 gene was analyzed with pfam online tools.There was a highly conserved DNA binding domain in the N N-terminal of KEAP1 protein,which consist of a BTB domain(48aa-150aa),a BACK domain(155aa-257aa)and six Kelch domains.Phylogenetic analysis revealed the keap1 of Siniperca chuatsi close to Larimichthys crocea keap1 genes,consisting with phylogenetic relationships of the two species,which indicated the name of keap1 gene cloning from Siniperca chuatsi was correct.2.The effect of short-term starvation on oxidative stress in the intestines of Siniperca chuatsi.we treated the adult Siniperca chuatsi a series day(0,3,7 days)of starvation,and resumed feeding for 3 days.We selected seven factors related to oxidative stress: ROS,CAT,GPX,GSH,GST,MDA,SOD,and assayed for starvation at different times using an active kit to determine their content.At 0d,3d and 7d,the ROS content in the intestine increased significantly with the hunger time(P < 0.05),and the ROS content decreased significantly after 3 days of recovery(P < 0.05).The activities of GSH,GPX and CAT in starvation for 3 days were observed.There was a significant increase(P < 0.05);the activities of GST and SOD decreased(P < 0.05).GST,CAT,GPX and SOD activities were significantly decreased after 7 days of starvation(P < 0.05).SOD was significantly increased(P < 0.05),GST,GSH and GPX were significantly decreased(P < 0.05),and the changes of CAT and GPX were not significant.We also selected the expression of Keap1,Nrf2,GPX,GR and Kappa1 in oxidative stress-related genes,which showed that the expression of Keap1 gene increased gradually with the increase of starvation time(P > 0.05),and the expression of Keap1 gene increased significantly(P < 0.05).The expression of Nrf2 gene increased at 3d after starvation(P > 0.05),and increased significantly at 7d after starvation(P < 0.05),and increased significantly at 3d after recovery(P < 0.01).The expression of GPX gene showed a trend of decreasing first.After the increase,the larval decreased to the lowest point on 7d,and the recovery of feeding for 3d was significantly higher than that of hunger for 7d and 3d(P < 0.05).The expression of GR gene decreased,and the recovery of feeding for 3d and starvation for 3d and 7d were more than starvation.There was a significant decrease(P < 0.05);the expression of Kappa1 gene showed an increasing trend,and the difference of 0d,3d,and 7d hunger was not significant(P > 0.05),and the recovery of feeding for 3d was significantly higher than that of starvation 0d and 3d(P < 0.05).3.The effect of short-term starvation on autophagy in the intestines of Siniperca chuatsi.Transmission electron microscopy was used to observe the intestinal cells of different hunger-time points.The results showed that: autopsy bodies were almost undetectable after starvation 0d,autophagy bodies began to appear on starvation for 3 days,and autophagy bodies were significantly increased after 7 days of starvation.The autophagosome began to decrease when fed for 3 days.The relative expression levels of autophagy-related genes mTOR,S6K1,Atg4 b,Beclin1,GabarapL1 and LC3 were detected by real-time fluorescence quantification at 0d,3d,7d and 3d after feeding.The results showed that the expression of mTOR gene was first.The trend of lowering after rising increased to the highest in 3d after starvation,and decreased significantly after 7d of hunger and 3d of recovery(P < 0.05).The expression of S6K1 gene showed a downward trend,starvation 3d was slightly lower than 0d,starvation 7d and recovery feeding 3d was significantly lower than starvation 0d and starvation 3d(P < 0.05);Atg4b gene expression first decreased and then increased,in starvation The 7d decreased to the lowest(P < 0.05),and the recovery of feeding for 3d was significantly higher than that of starvation for 0d,3d and 7d(P < 0.05).The expression of Beclin1 and GabarapL1 genes at 3d and 7d was more than 3d,0d.There was a significant increase(P < 0.05).There was no significant difference in the expression level of LC3 gene at 0d,3d,7d and 3 days after recovery.