Mechanism Of PHB Improving Immunity Of Carassius Auratus Gibelio Based On Combined Analysis Of MRNA And MicroRNA

Gibel carp(Carassius auratus gibelio)is one of the important freshwater aquaculture species in China,which provides abundant high quality protein sources and huge economic benefits.However,with the increase of breeding density and the gradual improvement of intensive farming,the pollution of aquaculture water is becoming more and more serious,resulting in various diseases of gibel carp.Studies have confirmed that poly-β-hydroxybutyrate(PHB),as a kind of novel probiotics,plays an important role in improving the immunity and disease resistance of aquatic animals.This study mainly analyzed the sequencing results of transcriptome mRNA and miRNA in the spleen of gibel carp after adding PHB to the diet,and the effects of β-HB on the antioxidant enzyme activity of spleen leukocytes and the expression levels of key genes in JAK/STAT and Th1/Th2 cell differentiation pathways at the cellular level.The results are as follows.(1)Compared with the control group,7005 differentially expressed mRNAs and 41 differentially expressed miRNAs were screened in the PHB group.Among them,3095 mRNAs expression levels were significantly up-regulated and 3910 genes were significantly down-regulated;41 differentially expressed miRNAs included 22 known miRNAs(10known miRNAs were up-regulated and 12 known miRNAs were down-regulated)and 19 novel miRNAs(8 novel miRNAs were up-regulated and 11 novel miRNAs were down-regulated).(2)The 22 known miRNAs differentially expressed mainly target 241 immune-related genes,including STAT1,JAK1,NF-κB1,and these miRNAs are involved in 20immune-related pathways,such as B cell receptor signaling pathway,Th1 and Th2 cell differentiation,and JAK/STAT signaling pathway.GO enrichment and KEGG pathway analysis showed that these target genes were mainly enriched in B cell receptor signaling pathway,Th17 cell differentiation,Th1 and Th2 cell differentiation,JAK/STAT signaling pathway and chemokine signaling pathway.(3)Combined miRNA-mRNA analysis most differentially expressed miRNAs negatively regulate mRNAs expression,and the number of target genes for each differentially expressed miRNA varies from 1 to 22.The q RT-PCR results of differentially expressed miRNAs(DE-miRNAs)and their target genes were basically consistent with those obtained by high-throughput sequencing.(4)Based on the fact that PHB is water-insoluble and degraded naturally into β-HB in the intestinal tract of animals,the immune enhancement mechanism of β-HB was investigated by using primary cultured leukocytes of spleen as a model.The results showed that β-HB could reduce intracellular NO concentration and cytotoxicity,increase leukocyte antioxidant enzyme activity and reduce MDA content,reduce leukocyte oxidative damage,and have good protective effect on oxidative damage of cells.Furthermore,the function of miR-1971-x in PHB regulation of leukocyte antioxidant activity was analyzed by enzyme activity determination and RT-q PCR.The results showed that miR-1971-x was involved in the regulation of antioxidant capacity of immune cells by PHB,and miR-1971-x negatively regulated the expression of target genes and downstream genes in JAK / STAT signaling pathway and Th1 and Th2 cell differentiation pathways.