The Effects And Mechanisms Of HO-1-mediated Autophagy Against Hepatotoxicity Of Deoxynivalenol

Deoxynivalenol（DON）is one of the most prevalent mycotoxins around the world.Ingested DON is mostly metabolized by liver,however,when DON intake amounts exceed the metabolic capacity of liver,it can cause acute and chronic toxicity on liver and other organs.According to the newest study,the maximum human daily DON intake in China is 12.7 g/kg bw based on wheat flour consumption,and the intake of DON from wheat sources accounts for 56%to 100%of the total DON intake.However,the effects of chronic relatively low dose of DON exposure on liver remain unclear.Therefore,studying chronic relatively low dose of DON exposure on liver can not only provide a basis for the health hazard assessment of human daily DON intake,but also provide references for the improvement of food safety related policies.The Heme oxygenase（HO）family are the rate-limiting enzymes during heme catabolism,through which biliverdin,free iron ion and carbon monoxide（CO）produce.HO-1 plays an important role in the process of oxygen transfer between cells and tissues,peroxides metabolism and xenobiotics biodegradation.It has been shown that HO-1 could alleviate liver inflammation,acute liver failure,and liver ischemia-reperfusion injury by mediating autophagy.In addition,relevant studies indicated that autophagy mediated by HO-1 was closely related to the production of CO during heme metabolism.However,whether HO-1 can act as a molecular target for liver protection by regulating autophagy remains unclear.Therefore,it is of great public health importance to explore the protective effect and potential mechanism of HO-1 on the liver,which could provide a new idea for the prevention and treatment of human DON exposure.Overall,the present study contained three parts:（1）we first gave mice 25μg/kg bw/day DON via oral gavage for 30 and 90 days to explore the impacts of such dose of DON on liver,thus to provide a basis for the health hazard assessment of human daily DON intake;（2）then,in vitro experiments were carried out to study the influences of DON on cells as well as the effects of HO-1/CO through regulating cell autophagy;（3）finally,we established liver-specific over-expression or silence mice model by virus-transfection and then gave mice 25μg/kg bw/day DON via oral gavage for 30 and 90days to investigate the effects of HO-1-mediated cell autophagy on liver during DON treatment in vivo,thus to provide a new idea for the prevention and treatment of human DON exposure.Section 1:The liver toxicity of DON in vivoObjectiveTo investigate the impacts of 25μg/kg bw DON administration for 30 and 90 days on liver.Methods（1）40 8-week old male C57BL6/J mice were housed in SPF animal facilities and were randomly assigned to Control group and DON group（20 mice per group）.Mice were then sacrificed at 30 days（10 mice）and 90 days（10 mice）of DON administration and samples were collected.（2）Liver histological examination,serum liver function indexes and three pro-inflammatory cytokines,IL-1β,IL-6 and TNF-αwere detected.Results（1）The alterations of development of miceCompared with Control group,body weight of mice in DON group was not significantly changed（P<0.05）.The liver organ coefficient of mice in DON group was significantly increased compared with Control group（P<0.05）.（2）The alterations of liver histopathologyAfter 30-day DON administration,lymphocyte infiltration occurred around the central lobular vein in livers of mice.Compared with Control group,the Ishak score of the liver was significantly increased（5.33±0.58 vs.0.67±0.29,P<0.001）.Compared with the30-day DON group,inflammatory infiltration around the central lobular vein in the 90-day DON group was aggravated,and the liver Ishak score was further increased（8.00±1.00 vs.5.33±0.58,P<0.001）.（3）The alterations of serum liver function indexesAfter 30-day DON administration,the serum ALT activity was significantly increased compared with Control mice（27.87±2.95 IU/L vs.19.30±3.20 IU/L,P<0.05）.After90-day DON administration,the serum ALT activity was increased compared with the30-day DON group mice（33.53±2.95 IU/L vs.27.87±2.95,P<0.001）.There were no significant differences in serum AST activity（P>0.05）.（4）The alterations of serum pro-inflammatory cytokinesAfter 30-day and 90-day DON administration,compared with Control group,the serum levels of IL-6,IL-1βand TNF-αwere significantly increased（P<0.05）.Conclusion25μg/kg bw DON administration for 30 days and 90 days could induce low-grade inflammatory reactions in liver and increase serum ALT activity.The liver damages caused by 90-day DON administration were more serious than that at 30-day DON administration,which presented a time-dependent effect.Section 2:The toxicity of DON on Hepa 1-6 cells and the effectsand mechanisms of HO-1-mediated autophagy in vitroObjectiveTo explore the impacts of DON treatment on Hepa 1-6 cells and the protective effects and mechanisms of HO-1 on cells by regulating autophagy.Methods（1）Determination of the dose and treatment duration of DONCell viabilities after DON treatment were determined by LDH assays.（2）Impacts of DON on Hepa 1-6 cellsHepa 1-6 cells were treated with 30 n M DON for 0,1,3,6,9,and 12 h,respectively.Cell viabilities were detected by LDH assays,cell apoptosis rates were detected by flow cytometry and expressions of apoptosis-related proteins were detected by Western blot.DAPGreen staining and transmission electron microscopy were used to observe autophagic vesicles,expressions of autophagy-related proteins were detected by Western blot.（3）The protective effects of HO-1 on cells HO-1-overexpressed Hepa 1-6 cells(HO-1^OE Hepa 1-6 cells)and HO-1-silenced Hepa 1-6 cells(HO-1^{sh RNA} Hepa 1-6 cells)were treated with DON.Cell viabilities,cell apoptosis and autophagy were measured.（4）The mechanism of HO-1 against DONHO-1^OE Hepa 1-6 cells and HO-1^{sh RNA} Hepa 1-6 cells were first pretreated with 100μM CORM-A1 for 30 minutes,and then HO-1^OE Hepa 1-6 cells and HO-1^{sh RNA} Hepa 1-6 cells were treated with DON.Cell viability,cell apoptosis and autophagy were measured at each time point.Results（1）The impacts of DON on cell viability showed typical time effect and dose effect.After being treated for 12 hours,the cell viability of Hepa 1-6 cells was significantly decreased and was above 80%（P<0.01）.In order to ensure the stability of detections in cells,30n M was chosen as DON treatment dose,and 0,1,3,6,9 and 12 hours were selected as DON treatment time.（2）Apoptosis in Hepa 1-6 cells was significantly increased at 6,9,and 12 h（P<0.05）;autophagy in Hepa 1-6 cells was significantly increased at 1 h and 3 h（P<0.05）.（3）The cell viability of HO-1^OE Hepa 1-6 cells was significantly higher than that of Hepa1-6 cells after DON treatment（P<0.05）;autophagy was significantly prolonged and apoptosis was significantly delayed（P<0.05）;the cell viability of HO-1^{sh RNA} Hepa 1-6cells was significantly lower than that of Hepa 1-6 cells（P<0.001）,autophagy was inhibited and apoptosis occurred earlier（P<0.05）.（4）After CO pre-treatment,autophagy in HO-1^OE Hepa 1-6 cells and HO-1^{sh RNA} Hepa 1-6 cells was further prolonged and apoptosis was inhibited（P<0.05）.ConclusionDON treatment could first induce autophagy and then trigger apoptosis in cells.HO-1could help prolong autophagy through CO and delay the onset of apoptosis.Section 3:The effects of HO-1-mediated autophagy againstdeoxynivalenol-induced liver toxicityObjectiveTo study the effects of HO-1-mediated cell autophagy in vivo by liver specific HO-1overexpression and silence mouse models.Methods（1）40 8-week old male C57BL6/J mice were housed in SPF animal facilities and were randomly assigned to HO-1^OE+DON group and HO-1^{sh RNA}+DON group（20 mice per group）.Mice were then sacrificed at 30 days（10 mice）and 90 days（10 mice）of DON administration and samples were collected.（2）Liver histological examination,serum liver ALT and AST activity and three pro-inflammatory cytokines,IL-1β,IL-6 and TNF-αwere detected.（3）TUNEL staining and Western blot were used to detect apoptosis.Transmission electron microscopy and DAPGreen staining were used to observe autophagic vesicles in hepatocytes and Western blot was used to detect expressions of autophagy-related proteins in liver（including Control and DON groups）.Results（1）The alterations of development of miceCompare with Control group,body weights of mice in HO-1^OE+DON and HO-1^{sh RNA}+DON groups were not significantly changed（P>0.05）.The liver organ coefficient was not significantly changed between HO-1^OE+DON group and Control group（P>0.05）,the liver organ coefficient in HO-1^{sh RNA}+DON group was significantly higher than that in Control group（P<0.01）.（2）The alterations of liver histopathologyCompared with Control group,lymphocytes infiltration around the central vein in liver lobule was reduced in HO-1^OE+DON group,and the liver Ishak scores were significantly lower than that of the DON group（30-day:0.33±0.58 vs.5.33±0.58,90-day:1.67±0.58 vs.8.00±1.00,P<0.001）;in HO-1^{sh RNA}+DON group,lymphocyte infiltration around the central vein in liver lobule was aggravated.The liver Ishak scores were significantly higher than that of the DON group（30-day:7.66±1.15 vs.5.33±0.58,90-day:11.67±1.53 vs.8.00±1.00,P<0.001）.（3）The alterations of serum liver function indexesAfter DON administration,the serum ALT activities in HO-1^OE+DON group were significantly decreased compared with DON group（30-day:22.95±3.90 IU/L vs.31.00±2.95 IU/L,90-day:19.62±1.89 IU/L vs.32.28±4.39 IU/L,P<0.05）,the serum ALT activities in HO-1^{sh RNA}+DON group were significantly increased compared with DON group（30-day:38.46±2.87 IU/L vs.31.00±2.95 IU/L,90-day:36.23±2.73 IU/L vs.32.28±4.39 IU/L,P<0.001）.（4）The alterations of serum pro-inflammatory cytokinesThe serum IL-6,IL-1βand TNF-αlevels in HO-1^OE+DON group were lower than those in DON group（P<0.05）;the serum IL-6,IL-1βand TNF-αlevels in HO-1^{sh RNA}+DON group were significantly higher than those in DON group（P<0.05）.（5）Autophagy in liverCompared with Control group,the number of autophagy vesicles in hepatocytes in DON group was increased（30-day:3.5±1.0 vs.0.2±0.4,90-day:4.5±1.0 vs.0.4±0.3,P<0.001）and the expressions of Beclin-1,ATG5,ATG12 as well as the ratio of LC3B-Ⅱ/LC3B-Ⅰwere significantly increased（P<0.05）,p62 was significantly decreased（P<0.05）.Compared with DON group,the number of autophagy vesicles in hepatocytes in HO-1^OE+DON group was significantly increased（30-day:8.0±1.4 vs.3.5±1.0,90-day:8.7±1.6 vs.4.5±1.0,P<0.001）,the expressions of Beclin-1,ATG5,ATG12 as well as the ratio of LC3B-Ⅱ/LC3B-Ⅰwere significantly increased（P<0.05）,p62 was significantly decreased（P<0.05）.Compared with DON group,the number of autophagy vesicles in hepatocytes in HO-1^{sh RNA}+DON group was significantly decreased（30-day:0.3±0.5 vs.3.5±1.0,90-day:0.2±0.4 vs.4.5±1.0,P<0.001）,the expressions of autophagy-related proteins were not significantly changed（P>0.05）.（6）Apoptosis in liverCompared with Control group,apoptosis rate of hepatocytes in DON group was significantly increased（30-day:1.90±0.52%vs.0.62±0.24%,90-day:3.14±0.74%vs.0.68±0.20%,P<0.05）and the expressions of Cleaved caspase-3 and Cleaved caspase-7 were up-regulated（P<0.05）.Compared with DON group,apoptosis rate of hepatocytes in HO-1^OE+DON group was significantly decreased（30-day:1.04±0.36%vs.1.90±0.52%,90-day:1.48±0.41%vs.3.14±0.74%,P<0.05）and the expressions of Cleaved caspase-3 and Cleaved caspase-7 were significantly decreased（P<0.05）.Compared with DON group,apoptosis rate of hepatocytes in HO-1^{sh RNA}+DON group was significantly increased（30-day:4.77±0.42%vs.1.90±0.52%,90-day:5.30±1.39%vs.3.14±0.74%,P<0.05）,and the expressions of Cleaved caspase-3 and Cleaved caspase-7 were significantly up-regulated（P<0.05）.Conclusion25μg/kg bw DON administration could induce autophagy and apoptosis in hepatocytes of mice.HO-1-mediated autophagy could alleviate DON-induced liver damages.