Study On Injury Of Ammonium Chloride In Zebrafish Liver Cell And Grass Carp Liver Cell

Grass carp(Ctenopharyngodon idella)is one of the important freshwater aquaculture fish in China.With the promotion of intensive grass carp farming,the accumulation of ammonia nitrogen occure frequently.Exposured to high levels ammonia nitrogen inhibited the immune system and induced oxidative stress in grass carp.As the important detoxification organ in the animal body,the liver directly determines the health and growth of the farmed grass carp.At present,the research on the effect of ammonia nitrogen mainly focuses on the individual level such as fish body.In this study,zebrafish liver cell(ZFL)and grass carp liver cell(L8824)were used to explore the effect of ammonium chloride on hepatocyte growth and the damage mechanisms at the cellular level.1.In this study,ZFL cells were exposed to ammonium chloride to reveal the mechanisms of ammonium chloride-induced apoptosis of ZFL cells.The results showed that ammonium chloride exposure inhibited the growth of ZFL cells and significantly reduced cell viabilities.Cell morphology was observed by inverted microscope and fluorescence microscope,and it was found that ammonium chloride exposure resulted in early apoptosis,cell shedding and death of ZFL cells.At the same time,the exposure to ammonium chloride will activate the antioxidant system of ZFL cells,and the intracellular reactive oxygen species(ROS)level will decrease to a certain extent.The relative expression levels of endoplasmic reticulum stress-related genes atf6,hspa5,xbp1 s,ern1,chop,atf4b1,eif2s1 were increased continuously,and the maximum values appeared in the 10 mmol/L ammonium chloride exposure group.However,the expression level of eif2ak3 was increased first and then decreased,indicating that ammonium chloride induced endoplasmic reticulum stress(ERS)in ZFL cells.In sum,ammonium chloride exposure could increase the apoptosis rate of ZFL cells,and with the increase of ammonium chloride exposure concentration,the apoptosis rate of ZFL cells also increased.2.In this study,L8824 cells were exposed to ammonium chloride to evaluate the effect of ammonium chloride on hepatocyte and its mechanisms.The results showed that exposure to ammonium chloride led to a decrease in the viability of L8824 cells and an increase in the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST).Intracellular ROS and malondialdehyde(MDA)levels were increased,while mitochondrial membrane potential was decreased.It was suggested that ammonium chloride induced oxidative stress and L8824 cells suffered oxidative damage.The detection showed that the relative expression of ERS-related pathway genes perk,atf4 and ire-1α increased.Pre-incubation of L8824 cells with the ERS inhibitor 2-APB reduced ammonium chloride-induced calcium release from the endoplasmic reticulum.The apoptosis rate of L8824 cells was increased after exposure to ammonium chloride.In short,ammonium chloride could induce oxidative stress and ERS on L8824 cells,resulting in the apoptosis of L8824 cells.