Study On The Target Signaling Pathway Of Inherent Immune Deficiency In Zebrafish Exposed To Low Dose Of Trichlorocarban

Trichlorocarban(TCC)is a common antibacterial agent,which has been widely added to daily life products such as soap,shower gel,hand sanitizer and so on.It has been used for nearly 50 years in the world.However,there are a large number of literature reports that with its frequent use,sewage enters the environment,which poses a serious threat to environmental quality and human health,and is harmful to health Objects have carcinogenic and teratogenic effects.At present,a large number of studies on TCC are focused on the metabolism and genetics of organisms.Although some studies have found that TCC has toxic effects on the growth,development and immunity of organisms,the in-depth mechanism is still unclear.In order to further clarify the mechanism of innate immune toxicity,zebrafish were used as model organism to determine the appropriate exposure concentrations of 125,175 and 225 ?g/L.zebrafish embryos were exposed to6-120 HPF acute exposure to try to explain the molecular mechanism of innate immune deficiency induced by TCC.(1)It was observed that TCC exposure resulted in high mortality,delayed hatching rate and increased malformation rate of zebrafish embryos.For example,the body length shortens,the heart rate slows down,the ear sac area reduces,the egg yolk absorption delay and the traveling sac closes and so on.At the same time,TCC affected the formation and normal morphology of inferior intestinal vein,internode vessel and posterior caudal artery in zebrafish juveniles.Especially at the concentration of 225 ?g/L,the area of inferior intestinal vein of zebrafish juveniles decreased,branches decreased or even ruptured,which indicated that TCC had significant developmental toxicity to zebrafish embryos.(2)The results showed that TCC could inhibit the number of neutrophils,macrophages and myeloid cells in zebrafish juveniles.According to the tail cutting experiment,TCC significantly affected the function of innate immune cells in zebrafish juveniles,and with the increase of concentration,the number of innate immune cells gathered to the wound decreased.The subsequent Ao staining also showed that most of the apoptotic cells were located in the tail hematopoietic tissue where a large number of innate immune cells accumulated in the early stage of zebrafish.(3)After exposure to 120 HPF,the zebrafish juveniles in the treatment group and the control group were subjected to full transcriptome sequencing and bioinformatics analysis.The results showed that the main effects of TCC on zebrafish juveniles were redox,immune response and other biological processes.The main enrichment pathways are Nrf2 signal pathway,complement coagulation signal pathway and NF-? B signal pathway.The results of thermographic clustering showed that TCC significantly activated TLR4 signaling pathway,which is an early innate immune signaling pathway in zebrafish.At the same time,TCC significantly inhibited antioxidant stress pathway,and the complement genes related to immune cells were also significantly changed.(4)After exposure to TCC for 120 HPF,the contents of malondialdehyde(MDA),superoxide dismutase(SOD)and catalase(CHT)in zebrafish juveniles were detected.Compared with the control group,the contents of MDA and CHT increased significantly,while the content of SOD decreased significantly.A large amount of reactive oxygen species(ROS)were found in the head,heart and swimming sac of zebrafish.By verifying the expression patterns of Nrf2 anti oxidative stress pathway related genes(Nrf2,Keap1,etc.)and anti / Pro apoptotic genes(Bax,BCL,etc.),it was found that TCC significantly inhibited zebrafish anti oxidative stress pathway and triggered abnormal expression of apoptotic genes.(5)The abnormal expression of TLR4 signaling pathway marker genes(TLR4,My D88,etc.)and the activation of downstream inflammatory factors(TNF-?,IL-6,etc.)in zebrafish juveniles exposed to TCC at 120 HPF,as well as the changes of TLR4,My D88 and NF-? B at protein level,proved that TCC activated TLR4 signaling pathway.The inhibition of TLR4 signaling pathway by ca-4948 rescued the decreasing trend of immune cells induced by TCC,which proved that TCC targeted TLR4 signaling pathway.Subsequent docking results showed that TCC could bind to TLR4 receptor hydrophobically and form hydrogen bond.