Zebrafish Intestinal Vibrio Sp(Vibrio Sp) And Aeromonas Sp(Aeromonas Sp) Induce The Innate Immune Response Of Juveniles Through The NF-ΚB Signaling Pathway

The composition of the intestinal flora develops with the host from birth and undergoes complex interactions,dependent on the host’s genome,nutrition,and lifestyle.The intestinal flora is involved in the regulation of multiple host metabolic pathways,resulting in interacting host flora metabolism,signaling and immune inflammation.More and more studies have begun to pay attention to the interrelationships between intestinal microbial flora and various physiological functions such as host nutritional metabolism and immune defense.The important unknown functions of this huge microbial population are gradually being revealed.Among them,the mechanism of intestinal flora during host innate immunity has become a hot research area.The innate immune system acts as the second line of defense for microorganisms(including phagocytosis and induction of inflammatory responses).Innate immune cells includ macrophages,dendritic cells,neutrophils,and natural killer cells.Innate immune cells play a major role in recognizing microbial-associated molecular patterns(MAMPs),which are primarily recognized by pattern recognition receptors(PRR),which are expressed in all cells of a given type,including Toll-like receptors(Eg TLR1,TLR2,TLR4,TLR7,TLR8 and TLR9),Nod-like receptors(eg NOD1,NOD2,NLRP3 and NLRC4)and C-type lectin receptors(eg MR,DC-SIGN,Mincle,Dectin-1 and Dectin-2,Dectin-3,CL-LK and DCIR).The host coordinates signals from these pattern recognition receptors(PRRs)and initiates various cellular functions such as phagocytosis,autophagy,apoptosis,and activation of inflammasome.In this experiment,a sterile zebrafish model was used as the experimental material to investigate the effects of Vibrio sp and Aeromonas sp in the zebrafish intestinal bacteria on the innate immune function of juvenile fish.First,by 16SRNA sequencing analysis,the proportion of different strains in the zebrafish intestine was found,and the proportion of Proteobacteria was the highest.The abundance between females and males was 37.72%and 59.71%,respectively.Second,the abundance between females and males was 48.72%and 17.31%,respectively.At the genus level,the relative abundances of Aeromonas sp between females and males were 13.03%and 8.56%,and the relative abundances of Vibrio sp between females and males were 2.09%and 0.49%,respectively.Secondly,fluorescent staining of Vibrio sp and Aeromonas sp was performed to detect the colonization location and customization ability in zebrafish juveniles.It was found that Vibrio sp and Aeromonas sp were mainly colonized in the zebrafish juvenile intestines,and the colonization capacity of Vibrio sp and Aeromonas sp reached 4×10~3 CFU/larva on the seventh day,and four days later.It was that the number of intestinal flora increased on the 11th day,but then decreased.Finally,to investigate whether the innate immune system of zebrafish juveniles exposed to Vibrio sp or Aeromonas sp was affected,1×10~7 CFU/m L of Aeromonas sp and Vibrio sp were added to conventionally raised zebrafish and sterile zebrafish.After the juvenile fish,RNA was extracted from every 20 juveniles for q RT-PCR detection.The results showed that in the conventional feeding group exposed to Vibrio sp.,It was found that the expression of the proinflammatory cytokines tnfα,tnfβand il1βcan be increased by tlr4and tlr5 stimulation of the NF-κB subunit c-rel.In addition,in the conventional feeding group exposed to Aeromonas,it was found that the expression of the proinflammatory cytokines tnfα,tnfβand il1βcan be increased by nod1,nod2 stimulation of NF-κB subunit nfkb2.The aseptic zebrafish juvenile group exposed to Vibrio sp or Aeromonas sp did not cause a significant response to the innate immune system.The above results may infer the importance of fish intestinal microorganisms to the host’s innate immune response.The development of this study will provide strong evidence for elucidating the role of gut microbes in the host’s innate immune response,and it will also provide new research ideas for understanding the body’s inflammatory response.