The Study On Heme Oxygenase-1 Regulating DNA Repair In Liver Damage Induced By Low Dose Of Deoxynivalenol

Objective:In this study,low-dose deoxynivalenol（DON）poisoning in vivo and in vitro were used to explore the effect of low-dose DON on pathological changes and antioxidant enzymes of liver in mice,and further explore the role of heme oxygenase-1（HO-1）in regulating oxidative enzymes and enhancing DNA repair under DON exposure.Methods:1.Low-dose DON exposure in vivo（1）After one week of adaptive feeding in SPF-level barrier system,C57BL/6J mice were randomly divided into 4 groups according to body weight,namely control group,DON group,DON+HO-1 overexpression group(hereinafter referred to as HO-1^OE group))and DON+HO-1 silence group(hereinafter referred to as HO-1^{sh RNA} group).Recombinant AAV8 virus that overexpressed and silenced HO-1were injected into mice in HO-1^OE group and HO-1^{sh RNA} group（iv,100μL/mouse）through the tail vein for 3 weeks,respectively.After 3 weeks,the control group was orally administered ultrapure water at 5μL/g per day,and the remaining three groups were orally administered DON（25μg/kg bw/day）per day.On the 30th and 90th days of gavage,half of the mice were randomly selected from each group and sacrificed,and biological samples were collected.（2）Liver tissue H&E staining was used to evaluate liver pathological changes.Visible light method was used to detect the activity of antioxidant enzymes（CAT,GSH）in liver tissue homogenate.Western blot test was used to detect the expressions of DNA repair proteins（ERCC1,XPC）in liver Expression.2.DON exposure in vitro（1）The CCK-8 test was used to detect cell viability.Hepa1-6 cells were treated with different DON concentrations（0,0.2,0.4,0.6,0.8,1.0,1.2 and 1.4μM）for 12 h,24 h,36 h and 48 h,respectively.According to the test results,the optimum dose and time were finally selected for the experiment in vitro.（2）Hepa 1-6 cell stable transfectants overexpressing HO-1(HO-1^OE Hepa 1-6cells)or silencing HO-1(HO-1^{sh RNA} Hepa 1-6 cells)was constructed by lentivirus transfection.（3）The cells were divided into control group,DON group,HO-1^OE Hepa 1-6group and HO-1^{sh RNA} Hepa 1-6 group.According to the CCK-8 test results,the cells were treated with 0.6μM DON for 24 h.（4）Visible light method was used to detect the level of intracellular antioxidant enzymes（SOD,GSH）;Western blot test was applied to detect the expression levels of XPC,ERCC1 and other proteins in cells.Results:1.Low-dose DON exposure in vivo（1）Histopathological changes of the mouse liver:the hepatic cells of the control mice were neatly arranged,the cell morphology was complete,and the nucleus was full.After intragastric administration of DON for 30 days,there was obvious infiltration of inflammatory cells around the central vein of the hepatic lobule in the DON group,and the hepatic cords were not arranged neatly.After intragastric administration of DON for 90 days,some hepatocytes were swollen,the nuclei were enlarged,and there were binuclear or multinuclear cells.The arrangement of the hepatic cords was very irregular,and the liver sinusoids were further reduced.The infiltration of inflammatory cells around the central vein of the hepatic lobule was not obvious,and the morphology of the hepatocytes was relatively intact and arranged neatly in the HO-1^OE group.In contrast,in the HO-1^{sh RNA}group,the infiltration of inflammatory cells around the central vein of the liver lobule was further aggravated,some hepatocytes were swollen and deformed,the nuclei were shrunk,and the cytoplasmic staining was deepened,showing typical eosinophilic features.（2）Changes in liver antioxidant enzymes in mice:compared with that in the control group,mice in the DON group had a decrease in GSH content and increased CAT activity on the 30th day of DON gavage,but there was no statistical difference（P>0.05）.On 90th day,there was no significant change in GSH content and CAT activity in the liver（P>0.05）.After HO-1 overexpression,there was no significant change in CAT activity and GSH content in the HO-1^OE group compared with the DON group on the 30th day and 90th day of DON gavage（P>0.05）.After HO-1 was silenced,on the 30th day,the CAT activity of the HO-1^{sh RNA} group was significantly lower than that of the DON group（P<0.05）and HO-1^OE group（P<0.01）,and the GSH content was significantly lower than that of the HO-1^OE group（P<0.05）;on the90th day,the GSH content of the HO-1^{sh RNA}group was significantly lower than that of the DON group（P<0.01）and HO-1^OE group（P<0.05）.（3）The expression of key proteins of DNA repair in mouse liver:compared with that in the control group,on the 30th day,the expression of ERCC1 protein in the DON group was significantly decreased（P<0.05）,and the expression of XPC protein was also decreased,but the difference was not statistically significant（P>0.05）;on the 90th day,the expressions of ERCC1 and XPC proteins in the DON group were not statistically different from those in the control group（P>0.05）.After HO-1 overexpression,on the 30th day,the expressions of ERCC1 and XPC proteins in HO-1^OE group were significantly higher than those in DON group（P<0.05）;on the 90th day,the DNA repair proteins in HO-1^OE group were not significantly changed than that in the DON group（P>0.05）.After HO-1 silence,on the 30th day,the ERCC1 protein in the HO-1^{sh RNA} group was significantly lower than that in the DON group（P<0.05）and HO-1^OE group（P<0.001）,and the XPC protein was significantly lower than that in the HO-1^OE group（P<0.05）;On the 90th day,the expressions of ERCC1 and XPC proteins in the HO-1^{sh RNA} group were both lower than those in the HO-1^OE group,but there were no statistical difference（P>0.05）.2.DON exposure in vitro（1）Changes in cellular antioxidant enzymes:after 0.6μM DON exposure for 24h,the SOD activity and GSH content of cells in the DON group were decreased compared with that in the control group,but there were no statistical differences（P>0.05）.The GSH content of cells in the HO-1^OE Hepa 1-6 group was significantly higher than that in the DON group（P<0.01）,while the SOD activity of cells in the HO-1^{sh RNA} Hepa 1-6 group was significantly lower than those in the control group（P<0.01）and HO-1^OE Hepa 1-6 group（P<0.01）,and the GSH content was significantly lower that in the HO-1^OE Hepa 1-6 group（P<0.01）.（2）Expression of key proteins for DNA repair in cells:after 0.6μM DON intervention,the expression of XPC protein in DON group cells was significantly decreasced compared with that in the control group（P<0.05）,and the expression of ERCC1 protein was also decreased,but there was no statistical difference（P>0.05）.The expressions of XPC and ERCC1 proteins in the HO-1^OE Hepa 1-6 group were significantly higher than those in the DON group（P<0.05）,and the expression of XPC protein and ERCC1 protein in the HO-1^{sh RNA} Hepa 1-6 group was significantly lower than that in the HO-1^OE Hepa 1-6 group（P<0.01）.Conclusions:1.In vivo studies,it has shown that short-term exposure（30 days）of 25μg/kg bw/day DON can cause slight inflammatory damage to the liver of mice,reduce GSH content and expression of ERCC1 protein;and liver damage in mice had become more serious,and the expressions of antioxidant enzymes and DNA repair proteins in the liver were disturbed under long-term exposure（90 days）to 25μg/kg bw/day DON.2.It has shown that the activity of cells was inhibited,and the expressions of antioxidant enzymes（SOD,GSH）and DNA repair proteins（XPC,ERCC1）were reduced under 0.6μM DON exposure in vitro studies.3.HO-1 can alleviate liver damage caused by 25μg/kg bw/day DON by regulating the levels of antioxidant enzymes（CAT,GSH）and DNA repair,but under the long-term DON exposure,the body did not have enough time for repair or the expression of DNA repair showed“exhaustion expression”phenomenon,which indirectly indicated that the role of HO-1 may be weakened with the increased exposure time.At the same time,the study in vitro has shown that HO-1 can not only help maintain the level of CAT,but also maintain the level of SOD and the ability of“chain scavenging”,thereby indirectly inhibiting the decline in DNA repair capacity.