Study On DNA Damage And Mechanism Of Staphylococcal Enterotoxin A In Hepatocytes

Staphylococcus aureus(S.aureus)is an important food-borne pathogenic bacteria,Staphylococcus aureus enterotoxin A(SEA)is one of the most common cause of the following symptoms,clinical symptoms,such as only a small amount of intake can cause vomiting and wound inflammation to cause rhinitis has a crucial influence,serious threat to public health.It is widely distributed and highly pathogenic,but the cytotoxicity of SEA has not been evaluated.Reactive oxygen species(ROS)have been shown to induce DNA damage in cells.(1)The changes of oxidative stress indexes in hepatocytes after SEA induction were evaluated.The results of cell activity assay showed that SEA could inhibit the growth of hepatocytes(human normal hepatocytes HL-7702 and rat hepatocytes BRL-3A),and the corresponding half lethal doses(600 and 800ng/m L)were obtained.The addition of SEA can significantly increase the ROS level in liver cells,and decrease the activity levels of SOD and GSH-Px in liver cells,and the migration of Nrf2 from cytoplasm to nucleus was observed by Western blot.The results showed that SEA could induce oxidative stress in hepatocytes and destroy the oxidative balance in hepatocytes,and there was a linear relationship between the changes of oxidative indexes and the dose of SEA.(2)The effects of SEA on?H2AX protein and its pathway in hepatocytes were investigated.Western blot method was used to observe the significant increase of target proteins(ATM,Chk2,H2AX,P53)and their phosphorylation in liver cells 24hours after SEA treatment,which was positively dependent on the concentration of SEA treatment,indicating that SEA can cause DNA double-strand damage in liver cells.The expression of DNA damage marker protein?H2AX was increased under SEA induced by immunofluorescence.Confocal imaging showed that SEA induced the formation of?H2AX\53BP1 aggregates in hepatocytes at 24 h.On this basis,by flow cytometry,it was found that the concentration of SEA in IC₅₀ could induce the cell cycle arrest in the G1 phase.(3)The DNA damage mediated by SEA through ROS in liver cells was investigated.The expression of 8-oxo G,a common ROS-mediated DNA damage protein,was detected.ELISA results showed that the expression of 8-oxo G increased significantly in a concentration-dependent manner,indicating that SEA is a ROS-mediated DNA damage.The antioxidant NAC was used to eliminate the effects of SEA on liver cell oxidation and DNA damage.The addition of NAC significantly alleviated the proliferation inhibition of hepatocytes by SEA.The expression of?H2AX protein decreased and tended to the normal value.The experiment proved that SEA mediated DNA damage of liver cells through ROS.In conclusion,SEA can inhibit the proliferation of liver cells and reduce cell activity.It can mediate DNA damage in liver cells by making them produce ROS.This study provides a theoretical basis for the future prevention and control of SEA.