Study On Oxidative Stress And Autophagy Induced By Ochratoxin A In HEK293T Cells

PurposeTo study on the relationship between oxidative stress and autophagy process induced by ochratoxin A(OTA)in renal cells(HEK293T cells).MethodsUsing HEK293 T cells as the experimental object.Cells were exposed to ochratoxin A(OTA)and cell activity were detected by CCK-8 kit.The expression level of ROS was detected by reactive oxygen detection kit.The expression of key proteins(p-ULK1,p-Beclin1,p-mTOR,LC3-B)in autophagy pathway and oxidative stress related proteins(ATM/LKB1/AMPK pathway and TRAP1,VDAC1 and LONP1)were detected by Western and blot.Results 1.After 24 hours of OTA treatment,the cell growth and cell survival rate were significantly increased in 1 μM,3 μM and 5 μM groups when compared with control group(without any treatment)and the survival rate respectively were 15.1%,121.7% and 121.3%.But the 7 μM,8 μM,9μM and 10 μM groups were inhibited obviously.The survival rate of the7 μM group was about 89.6%,the survival rate of the 8 μM group is about 49.4%,the 9 μM group is 10.5% and the 10 μM group is 5.7%.2.Western blot detection of autophagy related proteins showed that the expression of p-ULK1,p-Beclin1 and LC3 B proteins in OTA treatment groups were significantly higher than that of the control group(without any treatment),the results showed that OTA induced autophagy of HEK293 T cells and the autophagy level of 8 μM group was higher than the 7 μM group.At the same time,the expression of TRAP1,VDAC1 and LONP1 proteins in OTA treatment group were lower than that of control group.The result indicated that OTA inhibited the expression of TRAP1,VDAC1 and LONP1 proteins.3.The reactive oxygen detection kit showed that OTA induced increasing of ROS level in HEK293 T cells.The fluorescence intensity in 8 μM group was 138.33.the The fluorescence intensity in 7 μM group was 37.70 and The fluorescence intensity in control group(without any treatment)was 1.36.4.Compared with the control group(without any treatment),the expression of p-ATM,p-LKB1 and p-AMPK proteins increased significantly in 8 μM group.But the 7 μM group had no significant changes in expression of p-ATM,p-LKB1 and p-AMPK proteins.These results indicated that 8 μM may induce activation of ATM/LKB1/AMPK signaling pathway 5.Compared with the control group(without any treatment),the expression of p-mTOR protein 7 μM group did not change significantly.The expression of p-mTOR protein in 8 μM group was significantly lower than that in control group.Conclusion 1.HEK293 T cells treated with 7 μM ochratoxin A(OTA)and 8 μM ochratoxin A(OTA)for 24 hours both induce oxidative stress and autophagy.HEK293 T cells treated with 8 μM ochratoxin A(OTA)for 24 hours may activate ATM/LKB1/AMPK signaling pathway.2.HEK293 T cells treated with 7 μM ochratoxin A(OTA)and 8 μM ochratoxin A(OTA)for 24 hours both inhibited the expression of TRAP1,VDAC1 and LONP1 proteins.