Regulation And Mechanism Of Intelectin And Seaweed Polysaccharide On Macrophage Phagocytosis Of Megalobrama Amblycephala

It has been shown that Megalobrama amblycephala intelectin(MaINTL)can enhance the phagocytosis of M.amblycephala macrophages,but the regulatory mechanism was not clear.In this study,recombinant M.amblycephala intelectin(rMaINTL)was obtained by prokaryotic expression.Digital expression profiling was performed after rMaINTL injection into juvenile M.amblycephala,and GO enrichment and KEGG pathway analysis were performed to determine the expression of phagocytosis-related signals.rMaINTL-treated macrophages were examined by fluorescence microscopy and transmission electron microscopy to determine the morphology,cytoskeleton and internal structure of macrophages.Expression of phagocytosis-related signaling molecules at the protein level and molecular level was detected by Western blotting and qRT-PCR after rMaINTL treatment of cells,and the expression of signaling molecules,actin changes and changes in phagocytosis function were detected after inhibitor blockade.The seaweed polysaccharides that could bind to MaINTL were screened by sugar binding assay,and the effect of seaweed polysaccharides on MaINTL expression was examined by qRT-PCR and Western blot.The regulation of macrophage phagocytosis by seaweed polysaccharide was detected by incubation of macrophages with seaweed polysaccharide.The aim of this study was to elucidate the molecular and cellular mechanisms of rMaINTL-mediated macrophage phagocytosis and the effect of seaweed polysaccharides on macrophage phagocytosis.The results were as follows:(1)Analysis of differential gene enrichment in juvenile fish kidney showed that after rMaINTL injection into juvenile fish,21.59 M of raw transcriptome data were measured in both control and experimental groups,with 97.67% and 97.76% sequencing quality greater than 20%in control and experimental groups,respectively,and 93.48% and 94.21% matching rate to the M.amblycephala database,respectively.The overall analysis with DEseq2 showed a total of 201 differential genes,of which 121 were up-regulated and 80 were down-regulated.In GO enrichment analysis differential genes were mainly enriched in biological processes and molecular functions,the most significant enrichment in biological processes was in cellular processes and metabolic processes,the most significant enrichment in cellular components in cellular anatomical entities,protein-containing complexes,and the most significant enrichment in molecular functions in catalytic activity,binding activity and transmembrane activity.the results of KEGG pathway analysis showed that differential genes were enriched in phagosomes,actin cytoskeleton regulation,PI3K-Akt signaling pathway,antigen processing and presentation,and C-type lectin receptor signaling pathways were significantly enriched,with key signals related to phagocytosis including CDC42 and Rac1,and elevated expression of immune-related genes CXCL11,CCR4,and CD74.The results indicated that rMaINTL injection into juvenile fish could promote the expression of phagocytosis and immune-related genes.(2)The cytoskeleton of macrophages changed significantly after rMaINTL treatment,and the cell surface area became larger,the pseudopods extended,and the internal structure of the cells also changed,the intracellular endoplasmic reticulum expanded and phagosomes and lipid droplets appeared,and the ratio of F-actin/G-actin increased after rMaINTL incubation.The above results indicated that rMaINTL incubation of macrophages could promote the morphological and structural changes of macrophages,and the phagocytic and killing functions of macrophages were enhanced after the morphological and structural changes.(3)The expression of phagocytosis-related signaling molecules CDC42,PCDC42/Rac1,WASP,and Arp2 were upregulated after rMaINTL treatment,and the ratio of macrophage cytoskeleton protein F-actin/G-actin was elevated,and the phagocytic function of macrophages was also enhanced.The expressions of WASP and Arp2 were down-regulated after CDC42 inhibitor blockade,and the expressions of CDC42 did not change significantly after WASP inhibitor blockade,and the expressions of WASP and Arp2 decreased.the ratio of F-actin/G-actin decreased,the phagocytosis of macrophages was decreased after CDC42 inhibitor and WASP treatment.(4)It was screened by sugar binding assay that kelp polysaccharide and rockweed polysaccharide could bind to MaINTL,and the expression of INTL was significantly elevated at both protein level and mRNA level after incubation of macrophages with rockweed polysaccharide and kelp polysaccharide.The macrophage cytoskeleton was changed,macrophages extended pseudopods,and macrophage phagocytosis was enhanced after incubation of macrophages with the two kelp polysaccharides.