Effect And Mechanism Of Ochratoxin A On Mouse Primary Hepatocytes

Mycotoxins have always been a major problem of crop pollution.Among them,aflatoxin and ochratoxin are common.A great deal of previous researches have been on aflatoxin,but in terms of food pollution,the impact of ochratoxin should be taken into consideration.OTA is very stable and is a colorless,odorless crystalline compound with strong kidney toxicity,liver toxicity,teratogenicity,mutagenicity,and can cause cancer.However,this process is not very clear,and the mechanism of action of OTA-damaged cells needs to be investigated.In order to clarify the specific mechanism of OTA’s effect on liver cells,this paper took primary mouse hepar cells and mouse hepar cancer cells as research objects to clarify the damage of OTA on mouse hepar cells,as well as to study the biological mechanism of OTA and the specific mode of liver cell death.The first part is about how OTA damage the mice primary hepar cells.The experimental animals we selected were male C57 BL / 6 mice 28 days after birth,and the primary liver cells were isolated in vivo using a modified two-part separation method.The Half lethal dose of OTA to the cell detected was 21.5μmol/L.In order for the research subjects to be in good condition during the research process,the toxin concentration of 0,1.25,2.5,5,10 μmol/L toxin was selected for the experiment.First,after extracting the adherent cells,the cells were be disposed of the above toxin concentration and the cell proliferation activity was observed within 120 h.Secondly,the apoptosis and lactate dehydrogenase(LDH)leakage were detected after 48 h of treatment with the above toxin concentration.The results indicate that high-dose toxin suppress proliferation.The greater the toxin concentration,the higher the LDH leakage rate.Flow cytometry and laser confocal can be used to clearly see the effect of the toxin on mouse hepatocytes.Early apoptosis appears in toxin-treated mouse hepar cells and may turn to late apoptosis.These phenomena all indicated that the toxin had obvious damage to the primary liver cells of mice.The second part mainly discusses the damage effect of OTA on mouse hepar cells and the reasons that causes this effect,as well as the way that OTA causes the death of mouse liver cells.Treat primary cells with OTA toxin doses of 0,1.25,2.5,5,10μmol/L,and then detect the level of reactive oxygen species(ROS),mitochondrial function(ATP level)and DNA damage status(8-hydroxyl)in liver cells Deoxyguanosine levels).The results showed that ROS levels in primary liver cells treated with OTA increased significantly,ATP levels decreased significantly,and8-hydroxydeoxyguanosine levels increased significantly.On the other hand,we are investigating the death method through which OTA causes liver cell death.Considering that there may be apoptosis,necroptosis,pyroptosis.We selected a more stable mouse liver cancer cell line Hepa1-6,which can effectively compare the differences between the various death modes caused by toxins.The quantity of Caspase 3,Caspase 9 and PARP were analyzed by Werstern blot.The expression of RIP1,RIP3,MLKL,p-MLKL,Caspase8 are related to cell necrosis apoptosis,and the follow expression of GSDMD and GSDME related to death.The results showed that the quantity of Caspase 3,Csapase 9,and PARP protein in the OTA treatment group were significantly increased,and the cell death caused by OTA may be apoptosis;the OTA treatment group also had RIP1,RIP3,MLKL,p-MLKL,Caspase8 Significantly increased,indicating that cell necrosis apoptosis may also be a way for OTA to cause liver cell death;however,the expression levels of GSDMD and GSDME related to cell pyrolysis have not increased,indicating that cell pyrolysis may not participate in OTA-induced Liver cell damage.