Effects Of Acute Nitrite Stress On Endoplasmic Reticulum Stress Perk Pathway Of Grass Carp Ctenopharyngodon Idella

Grass carp（Ctenopharyngodon idella）is one of the important fish in China,and it has ranked the first in the total freshwater aquaculture fish production in the country for many years.Nitrite,which is a major intermediate in the ecosystem nitrogen cycle,can pollute aquaculture water,excessive nitrite in water would damage,even kill the aquatic animals.The endoplasmic reticulum stress（ERS）is a common stress response,and the PERK pathway of unfolded protein reaction plays an important role in protecting cells against environmental stress.To provide the basis for the control and prevention of nitrite in the culture environment,our study investigated the effects of acute nitrite exposure on endoplasmic reticulum stress,antioxidant,blood biochemistry of grass carp in vivo and cell viability,apoptosis,[Ca²⁺]_i signaling of cells in vitro.The healthy grass carp（1 year old,37.21±0.30 g）were exposed to nitrite（0mg/L,8 mg/L,13 mg/L,25 mg/L,50 mg/L）for 0 h,12 h,24 h,48 h,and 96 h respectively,then the gill,liver,muscle and blood samples were collected.The expressions of grp78,perk,eif-2α,atf4,chop,bax,and bcl-2 genes in the liver and muscle were detected,gill and liver tissue were used for the histological observation,the physiological and biochemical indexes and antioxidant enzyme activities in the serum were detected.Grass carp isolated liver cells（L8824）were exposed to the nitrite solution for the in vitro experiments,the cells were collected to detect the viability,the expressions of grp78,perk,eif-2α,atf4,chop,bax,bcl-2,apoptosis,cells morphology changes and intracellular[Ca²⁺]_i signaling.After exposure of grass carp isolated kidney cells（CIK）to nitrite,the cell viability,apoptosis,cell morphology,and related genes expression were also analyzed.The main results were as follows:1.In the liver of the control group,the liver cells were arranged neatly,and the nucleus was located at the center.However,after exposed to 50 mg/L nitrite for 96 h,the structure of grass carp liver was impaired seriously,it show lots of vacuoles,and the position of nucleus shifted obviously.In the gill of control group,the silkworms and sputum were arranged neatly and closely,and after 96 h of exposure to 50 mg/L nitrite,the sputum was disorderly arranged and the capillaries were dilated and enlarged.After acute exposure to nitrite,the activities of serum catalase（CAT）were significantly up-regulated in 8 mg/L group and then decreased with the increase of concentration,while the activities of superoxide dismutase（SOD）decreased gradually in the 8 mg/L,13 mg/L,25 mg/L,and 50 mg/L groups as the time prolonged.After exposed to nitrite for 12 h,24 h,48 h and 96 h,the activitives of alanine aminotransferase（ALT）in the serum were significantly increased in high concentration group.After exposed for 12 h,24 h and 48 h,the activities of alkaline phosphatase（AKP）,cholesterol（CHO）increased significantly.The activities of aspartate aminotransferase（AST）decreased significantly,while glucose（GLU）,albumin（Alb）,total protein（TP）and triglyceride（TG）shows no significant change in48 h.Genes in the PERK pathway have tissue expression differences in grass carp,compared with the liver,the expressions of perk and atf4 were lower in muscle and higher in other tissues;the expressions of eif-2αwere lower in muscle and kidney,but higher in other tissues;and the levels of chop were higher in intestine and gill,lower in other tissues.After 96 h of acute nitrite exposure,the expression of ERS marker gene grp78 in the liver was significantly up-regulated in the treatment group,the expressions of perk,eif-2α,atf4 and chop were increased firstly and then decreased gradually,the expression of pro-apoptotic gene bax was significantly increased while the expression of bcl-2 was decreased,which can inhibit the apoptosis,thus the ratio of bax/bcl-2 was significantly up-regulated.In the muscle of grass carp,the expressions of perk,eif-2α,atf4 and chop were increased significantly at 24 h in the 8mg/L group,then decreased gradually,the expression of pro-apoptotic gene bax were increased in muscle firstly and then decreased,the expression of bcl-2 decreased significantly.2.After acute nitrite exposure,the activities of L8824 cells were significantly decreased,the apoptosis increased significantly after 12 h and 24 h,and the cells morphology were changed significantly in 100 mg/L group.The intracellular Ca²⁺fluorescence intensity increased with a time and concentration-dependent manner,but the Ca²⁺fluorescence intensity were decreased after added the Ca²⁺release channel inhibitor 2-APB.The expression of ERS marker gene grp78 was significantly increased,the expressions of perk and eif-2αwere significantly increased in the 10mg/L group,and the expression of atf4 shown no significant change.And the expression of chop were significantly increased in the 10 mg/L and 100 mg/L,the expression of bax was significantly up-regulated in the 10 mg/L,while the expression of bcl-2 was significantly down-regulated in the 2.5 mg/L,compared with the control group,it’s lower in the 10 mg/L and 100 mg/L.After the addition of PERK pathway inhibitor GSK2656157,the apoptosis of L8824 cells was significantly reduced compared to 10 mg/L nitrite exposure.3.After 24 h of nitrite exposure,the cells viability of CIK cells were decreased significantly with increasing concentration.However,the expressions of perk,eif-2αand atf4 did not changed significantly,while the expression of chop was significantly increased in 50 mg/L,100 mg/L,250 mg/L,the expression of bax was significantly increased while the bcl-2 was significantly decreased,and ratio of apoptosis gene bax/bcl-2 was increased,after 24 h of nitrite exposure,cells apoptosis were significantly increased,and it shown signs of apoptosis in the morphology observation of treatment group.In summary,acute nitrite exposure induced endoplasmic reticulum stress in grass carp,activated the PERK pathway of unfolded protein reaction,it also damaged the gill and liver tissue of grass carp,and reduced the antioxidant performance,interfered with the stability of serum biochemical indicators in grass carp.Meanwhile,the acute nitrite stress reduced the cell viability,resulting in the increase of apoptosis,the Ca²⁺efflux in the endoplasmic reticulum.