Nitric Oxide Synthase(NOS)Functions In Gut Microbiotic Homeostasis Of Shrimp

A large number of microorganisms exists in the gastrointestinal tract of animals,and this number in the human gastrointestinal tract has even exceeded the total number of human cells.The intestinal flora and host are interdepedent and mutually restrictive,forming a unique intestinal microbial system during long-term evolution.This system plays important role in assisting the host to digest and absorb nutrients,participating in the host metabolic process,providing substrate and energy for host metabolism,promoting the development of tissues and organs such as the intestine,and providing protection for the host by forming a bacterial membrane barrier.At the same time,there are some pathogens in intestinal tract which can damage the intestinal epithelial barrier,disrupt the intestinal flora balance,and make the host sick or even die.Therefore,maintaining the homeostasis of the gut microbiota is particularly important in resisting pathogenic bacteria.However,due to the diversity of intestinal bacteria and the multiple cross-effects of intestinal immune regulation,how can the host tolerate these bacteria and maintain homeostasis of the microbiota has not been fully revealed.In previous studies,ROS and antibacterial peptides have been considered as the major regulators of homeostasis in the intestine.However,recent studies have found that the metabolites of intestinal symbiotic bacteria can down-regulate the production of nitric oxide(NO),which indicate that NO may also participate in the regulation of intestinal flora.NO is a small molecule that can be diffused freely in body.Most of NO is synthesized by nitric oxide synthase(NOS).NO can act as a signaling molecule to activate multiple pathways.High concentrations of NO can also cause nitrogen stress in cells,which is biologically toxic.However,how NOS/NO regulates gut microbiotic homeostasis is unclear.In this research,we used Marsupenaeus japonicus as the experimental material to study the function of a nitric oxide synthase(NOS)in the regulation of gut microbiotic homeotasis of shrimp.Challenged by the pathogen Vibrio anguillarum,the expression of Nos in shrimp gastrointestinal tract was significantly up-regulated at 3 and 6 h,respectively.In order to study the function of NOS,we knocked down the expression of Nos by RNA interference and found that the NO concentration in stomach and intestine decreased,and the number of bacteria increased 5.3 and 4.2 times,respectively.In addition,after treating with the NOS inhibitor L-NMMA,the NO concentration of the gastrointestinal tract decreased,and the number of bacteria increased significantly along with the increase of the inhibitor concentration.We also analyzed the expression of several antibacterial peptides in shrimp after RNAi of Nos or the inhibitor injection following bacterial challenge,and found that the experssion of Alf-el,Crusl-1,CrusI-2 and Crusl-5 was suppressed.In further research,we found that the phosphorylation of extracellular regulated protein kinases(ERK)and the nuclear translocation of the transcription factor Dorsal were declined in intestine of L-NMMA-treated shrimp.The above results suggest that NOS plays an important role in regulation of microbiotic homeostasis in gastrointestinal tract of M.japonicus via NO production to induce ERK phosphorylation and Dorsal nuclear translocation to promote the expression of AMPs against gut pathogens.