Preliminary Study On The Toxic Effect Of AFB₁ And The Protective Effect Of NAC In Canine Liver Cells

Aflatoxin B1(AFB1)is the most toxic of AF,which can damage liver tissue seriously and even cause cancer.N-acetyl-1-cysteine(NAC)as a strong antioxidant has advantages in the treatment of liver diseases.In this study,primary hepatocytes of dogs were used as models to study the hepatocytotoxicity of AFB1 and the protective effect of NAC.At the logarithmic growth stage in vitro,the cultured hepatocytes were randomly divided into 7 groups:0,0.05,0.25,1.25,6.25,31.25 ?g/mLAFB1 and 6.25 ?g/mLAFB1+64 mmol/LNAC.Primary hepatocytes of dogs were treated for 36 hours,and the morphological and ultrastructural changes were observed by inverted microscope and electron microscope.Liver function indexes were measured by automatic biochemical analyzer.The effect of AFB1 on the oxidative and antioxidant capacity of hepatocytes was studied by enzyme linked immunosorbent assay(ELISA).Real-time fluorescence quantitative polynucleotide chain reaction(qRT-PCR)was used to detect the expression of apoptosis gene.The blank control group,6.25?g/mL AFB1 group and 6.25 ?g/mL AFB1+64 mmol/LNAC group were selected,and the apoptosis rate was determined by flow cytometry to study the protective effect of NAC on AFB1 hepatocytotoxicity.The results are showed as follows:1.AFB1 has inhibitory effect on cell activity,Liver cells treated for 36 hours showed a concentration dependence with the increase of toxin concentration and were used as follow-up test conditions.Liver cells treated with NAC of different concentrations showed the most significant protective effect when NAC concentration reached 64 mmol/L.2.Observation under an inverted microscope showed that the morphology and size of liver cells in the control group were different after 36 hours of culture,but the cell space was closely connected and the growth condition was good.With the increase of toxin concentration,the number of cells decreased obviously,the intercellular space increased,and the broken cells were distributed.Under electron microscope,the cell membrane and nuclear membrane of the control group were intact,and the distribution of cytoplasm and cytoplasm was uniform.With the increase of AFB1 concentration,cell damage was gradually obvious.Compared with 6.25 ?g/ml AFB1 cells in NAC group,the growth status of AFB1 cells was better.3.The results of cell apoptosis showed that the apoptosis rate of 6.25 ?g/mL AFB1 reached 12%,which was significantly higher than that of the control group(P<0.01).The apoptosis rate of NAC group was significantly higher than that of the control group and significantly lower than that of the AFB1 group(P<0.01).4.Liver function index test results showed that high concentrations of AFB1 group of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were significantly increased compared with control group,low concentration of AFB1 group ?-gamma glutamyl transpeptidase(?-GGT)and alkaline phosphatase(ALP)levels significantly increased compared with control group(P<0.01),NAC group compared with 6.25(?g/mL AFB1,ALT,AST and ALP were significantly lower.5.The results of oxidation and antioxidant indexes showed that the concentrations of 8-hydroxydeoxyguanosine(8-OHdG)and malondialdehyde(MDA)were significantly increased in the AFB1 treatment group compared with the control group.H2O2 increased significantly in high concentration AFB1(1.25-31.25 ?g/mL).Compared with the group of 6.25 ?g/mL AFB1,NAC was significantly reduced by 8-OHdG,MDA and H2O2.The activity of glutathione peroxidase(GSH-Px),catalase(CAT)and superoxide dismutase(SOD)decreased gradually with the increase of toxin concentration,and the activity of GSH-Px and CAT in NAC group increased significantly compared with that of 6.25?g/mLAFB1.6.The results of qRT-PCR showed that the expression levels of cleaved-caspase-3 and cleaved-caspase-9 mRNA were significantly increased compared with the control group,and the NAC group was significantly reduced compared with the 6.25 ?g/mL AFB1 group.In summary,AFB1 can inhibit primary liver cell activity in puppies,cause abnormalities in cell morphology,liver function indicators,cell oxidation and antioxidant function,and activate the expression of pro-apoptotic genes Caspase-3 and Caspase-9 mRNA,thus accelerating liver cell apoptosis.NAC can significantly improve antioxidant enzyme activity and reduce liver cell injury and apoptosis.To find effective clinical drugs for alleviating liver injury caused by AFB1,and provide a new research direction.