Liver And Kidney IRE1 Pathway And Apoptosis In Grass Carp (Ctenopharyngodon Idellus) Induced By Nitrite Acute Exposure

With the development of intensive high-density aquaculture,a large number of nitrogen-containing organic matters deposited in the water,resulting in the increasing concentration of nitrite in the water.Oxidative stress,unfolded,misfolded and mutant protein in the fishes could be induced by high concentration of nitrite exposure.It could break the homeostasis of endoplasmic reticulum(ER),leading to endoplasmic reticulum stress(ERS)and mass mortality of fishes.ER could have an unfolded protein response to protect and restore the homeostasis to deal with the environmental stress.If ERS was happening continually,cell apoptosis would be induced.In this study,the damage of liver,liver cells and kidney cells induced by sodium nitrite were detected from the perspective of ER and cell apoptosis to provide a reliable scientific basis for scientific culture of grass carp.The grass carp were exposed to 0 mg/L,8 mg/L,13 mg/L,25 mg/L and 50 mg/L sodium nitrite for 0 h,12 h,24 h,48 h and 96 h,respectively.Three replicates were set up in each experimental group.After the exposure,the livers of grass carp were collected to detect the biochemistry indexes,antioxidant enzymes activities and the relative expressions of cell apoptosis related genes and IRE1 pathway related genes.The gills and liver of grass carp were collected to observed the histological changes.The grass carp hepatocytes were exposed to 0 mg/L,20 mg/L and 50 mg/L sodium nitrite for 0 h,12 h and 24 h,respectively.Cells were collected to assay the cell viabilities,apoptosis rates,the changes of calcium ions and the relatively expressions of apoptosis related genes and ER related genes.After adding IRE1α inhibitor STF-083010 to the cells,the relatively expressions of genes,apoptosis rate and the changes of calcium ions in the cells were detected.Grass carp kidney cells were exposed to 0 mg/L,25 mg/L and 50 mg/L sodium nitrite for 0 h,12 h and 24 h.Cells were collected to measure the cell viabilities,the relatively expressions of genes and the apoptosis rate.The experimental results were as follows:1.In the control group,the gill tissue and gill filaments of grass carp were well-organized and closely arranged.After exposure to nitrite at 50 mg/L for 96 h,the basal epithelium of grass carp gill patches became thicker and the secretion of mucous cells increased.The gill lobules adhered to each other.Mucous cells infiltrated between the gill lobules in large numbers,and the gill lobules proliferated and distorted.In the hepatocyte control group,the overall morphology of hepatocytes and the location of nuclei were obvious.However,after nitrite exposure of grass carp hepatocytes at 50 mg/L for 96 h,the hepatocytes showed obvious fat vacuolation.The content of hepatic glycogen decreased significantly in the high concentration group exposed to sodium nitrite.The alkaline phosphatase activity of grass carp liver increased significantly at 12 h and 24 h of sodium nitrite exposure,but decreased significantly after 48 h and 96 h of sodium nitrite exposure.The activity of SOD increased significantly after 12 h of sodium nitrite exposure.With the prolongation of exposure time,the activity of SOD increased significantly.The photographic ratio decreased significantly.The expressions of grp78,ire1α and xbp1 s increased significantly after exposure to sodium nitrite compared with control.The expression of grp78 reached the maximum after 96 hours of nitrite exposure,while ire1 a and xbp1 s reached the maximum after 12 h of exposure.2.In the experiments of acute sodium nitrite exposure to L8824 cells,the apoptotic rate of the treated group increased significantly,the expressiosn of jnk,bax,caspase9,caspase3,ire1α,xbp1 s and grp78 increased significantly,the expression of bcl-2 decreased significantly,and the concentration of calcium ion in the cytoplasm increased significantly.Compared with sodium nitrite treatment group,the apoptotic rate of STF-083010 treatment group decreased significantly,ire1α,xbp1 s,grp78,jnk,bax,caspase3 expressions decreased significantly,bcl-2 expression increased significantly,and calcium concentration in cytoplasm of 2-APB and STF-083010 treatment groups decreased significantly.3.In the experiments of CIK cells exposed to sodium nitrite,the viabilities of cells decreased significantly.AO staining results showed that the fluorescence intensity of nucleus and cytoplasm increased,the apoptotic rate increased significantly,and the expressions of jnk,bax,caspase9,caspase3,ire1α,xbp1 s and grp78 mRNA increased significantly after exposure to high concentration of sodium nitrite.ire1α,xbp1 s and grp78 mRNA expressions increased significantly when exposed to low concentration of sodium nitrite,and decreased significantly with the prolongation of time.In summary,acute nitrite exposure induced stress response in grass carp,resulting in the damage to liver and gill tissues,significant changes in liver biochemical indicators and antioxidant enzymes activities.The expressions of IRE1 pathway and apoptosis-related genes increased significantly in liver,liver cells and kidney cells.After the acute nitrite exposures,the viabilities of L8824 and CIK cells decreased significantly,and the apoptosis rates and the concentrations of calcium in hepatocytes increased significantly.IRE1 pathway might play a role in the apoptosis of grass carp hepatocytes induced by the exposure of sodium nitrite.