The Role Of PI3K/AKT Signaling Pathway In Acrylonitrile-induced Apoptosis Of Rat Hepatocytes

Objective: To establish a liver injury model of male SD rats induced by acrylonitrile(ACN),observe and study the manifestations of liver injury in rats,and explore the role of oxidative stress,PI3K/AKT signaling pathway in the apoptosis of rat liver cells induced by acrylonitrile,so as to provide a reliable basis for further study on the hepatotoxicity mechanism of ACN.Methods: 60 adult healthy male SD rats were randomly divided into 5 groups,12 in each group,respectively: control group(corn oil),low ACN group(11.5 mg/kg),medium ACN group(23.0 mg/kg),high ACN group(46.0 mg/kg),N-acetylcysteine(NAC)group.The gavage volume was 5.0 mL/kg once a day for 6 days a week for a total of 28 days.In the NAC group,300.0 mg/kg NAC was given for 30 min before high-dose ACN was given.The rats were anesthetized with ether 24 hours after the end of the last exposure,and sacrificed after heart blood collection.(1)Liver tissue was extracted from the rats,and the wet weight of the liver was weighed to calculate the liver organ coefficient.(2)Prepared HE pathological sections of liver tissue and observed the pathological changes of rat liver tissue structure under the microscope;(3)Centrifuge plasma and measure the levels of liver function-related enzymes alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in rat serum.(4)Apoptosis of rat liver cells was detected by TUNEL staining.(5)Rat liver tissue homogenate was prepared and catalase(CAT),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),total antioxidant capacity(T-AOC)and glutathione(GSH)and malondialdehyde(MDA)were detected.(6)The relative expression levels of PI3 K and AKT mRNA related to the PI3K/AKT signaling pathway and the mRNA levels of Bad,Bcl-2,Cyt c and Caspase-3 related to the mitochondrial apoptosis pathway in rat liver tissues were detected by qRT-PCR.(7)The relative expression levels of PI3 K,p-PI3 K,AKT and p-AKT related proteins in the PI3K/AKT signaling pathway and the relative expression levels of mitochondrial apoptosis related proteins Bad,p-Bad,Bcl-2,Bax,Caspase-9,Cyt c and Caspase-3 were detected by Western Blot.Results:(1)Liver wet weight and organ coefficients of rats: liver wet weight and organ coefficients in the low ACN group and organ coefficients in the high ACN group were higher than those in the control group(P<0.05).(2)Liver tissue structure changes in rats: in the control group,there were hepatic lobule structure,normal hepatocyte morphology,uniform size,round nucleus,neat hepatic cords,and obvious hepatic sinus;in the low ACN group,no significant abnormal changes were observed under the microscope;in the medium ACN group,the cytoplasm was slightly loose,the hepatocyte morphology was damaged,and the boundaries of the hepatic cords were blurred;in the high ACN group,the hepatic lobule structure was not clear,the hepatic cells were swollen,the nuclear vacuoles were changed,the fat droplets were increased,and the hepatic sinus was congested;in the NAC group,the hepatocytes increased in size,the cytoplasm was loose,and the hepatic cords were arranged in a neat way,the hepatocytes were less damaged compared with the high ACN group.(3)Serum enzymes related to liver function in rats: compared with the control group,ALT activity in the low and the high ACN group was increased,and AST activity in the medium and the high ACN group was increased(P<0.05);the ALT activity in the NAC group was decreased compared with the high ACN group(P<0.05).(4)TUNEL staining: compared with the control group,the number of positive cells in the low and medium ACN groups was significantly increased(P<0.05).(5)Oxidative stress index of rat liver: compared with the control group,CAT activity decreased,while SOD and GSH-Px activity increased in the low ACN group(P<0.05);in the medium ACN group,CAT activity decreased and MDA content increased(P<0.05);in the high ACN group,CAT activity decreased,SOD activity increased,GSH content decreased,and MDA content increased(P<0.05).The MDA content in the NAC group was decreased compared with thehigh ACN group(P<0.05).(6)qRT-PCR: compared with the control group,the relative expressions of Bad and Caspase-3 mRNA of the liver of rats in the low ACN group were increased,while the relative expression of Bcl-2 mRNA was decreased(P<0.05);in the medium ACN group,the relative expressions of PI3 K and Bcl-2 mRNA were decreased,while the relative expression of Cyt c mRNA was increased(P<0.05);the relative expressions of PI3 K and Bcl-2 mRNA in the high ACN group were decreased,while the relative expression of Bad mRNA was increased(P<0.05);there was no significant difference in the relative expression level of AKT mRNA among the ACN exposed groups(P>0.05).The relative expression of Bad mRNA in the NAC group was decreased compared with the high ACN group(P<0.05).(7)Western Blot results: compared with the control group,the pro-Caspase-3 protein relative expression was decreased,while the cleaved-Caspase-3 protein relative expression was increased in the low ACN group(P<0.05);in the medium ACN group,the relative expressions of PI3 K,p-Bad,pro-Caspase-3 protein were decreased,while the relative expressions of Bad,Bax,Caspase-9 and Cyt c protein were increased,and the ratio of Bcl-2/Bax was decreased(P<0.05);in the high ACN group,the relative expressions of p-PI3 K,p-AKT,p-Bad,Bcl-2,pro-Caspase-3 were decreased,the relative expressions of Bad,Bax,Caspase-9,Cyt c,cleaved-Caspase-3 were increased,and the ratio of Bcl-2/Bax was decreased(P<0.05);there was no significant difference in the relative expression level of AKT protein among ACN exposed groups(P>0.05).Compared with the high ACN group,the relative expressions of PI3 K,p-Bad,pro-Caspase-3 proteins were increased,and the relative expressions of Bax,Caspase-9,and cleaved-Caspase-3 proteins were decreased in the NAC group(P<0.05).Conclusion:(1)ACN can change the structure of rat liver tissue,affect the activity of serum liver function-related enzymes,and cause liver damage.(2)ACN-induced oxidative stress,inhibition of PI3K/AKT signaling pathway and activation of mitochondrial apoptotic pathway are the possible mechanisms of ACN-induced hepatocyte apoptosis in rats.