| ObjectiveDimethylformamide(N,N-dimethylformamide,DMF)is a colorless and slightly ammonia liquid,which can be soluble in water and most organic solvents.DMF is widely used in medicine,polyacrylonitrile silk,polyurethane synthetic leather and other fields.Although DMF has incomparable advantages compared with other organic solvents,DMF exposure can lead to severe liver damage without effective therapeutic medicines.Along with the frequent reports of DMF poisoning cases,DMF poisoning has been becoming an ignorable public health issue in China.Oxidative stress and Kupffer cells(KCs)mediated inflammation are involved in the development and progression of many types of liver diseases.Previous studies have shown that the C YP2E1-mediated active metabolite of DMF,methyl isocyanate(MIC),can form adducts with glutathione(GSH)leading to the depletion of GSH in liver cells.At the same time,DMF will induce the production of reactive oxygen species(ROS)during the metabolism of CYP2E1.All these can cause oxidative stress.However,whether oxidative stress plays causal roles in c DMF-induced liver injury remains to be revealed.KCs accounts for about 90%of the total number of fixed macrophages in the body and nearly one-third of the non-parenchymal cells in the liver.Therefore,KCs play a key role in maintaining the homeostasis of liver tissues and the body.Activated KCs can release a variety of inflammatory mediators,and thus enhancing liver injury.The role of KCs in hepatotoxicity of DMF has not been reported.This study aims to explore the roles of oxidative stress in DMF-induced liver injury in mice by investigating the time-course of oxidative stress-related parameters and the protective roles of N-acetyl cysteine(NAC)and Nrf2 agonist sulforaphane(SF).In addition,we investigated the roles of GdCl3,which could eliminate the Kupffer cells in liver,against DMF-induced acute liver injury.Methods1 The determination of the 50%lethal dose(LD50)of DMF in male ICR mice.Specific Pathogen-free male ICR mice(18～22 g)were randomly divided into five groups with 10 in each group.The mice were orally treated with 4.50,5,49,6.71,8.19,10.00 g/kg bw DMF and observed for 7 days for behavior and death.The LD50 was calculated by using Bliss method.2 Establishment of acute liver injury model induced by DMF in miceSPF ICR mice(18～22 g)were randomly divided into four groups with 10 in each group:control group,DMF(24 h)group,DMF(48 h)group and DMF(72 h)group.All mice in the DMF group were fasted for eight hours and then were gavaged with 3 g/kg bw DMF.The mice were sacrified at according time points.Serum was collected to measure the activity of ALT and AST.Liver tissues were isolated and the degree of liver injury was evaluated by HE staining.3 Measurement of the hepatic malondialdehyde(MDA),glutathione(GSH)and 4-Hydroxynonenal(4-HNE)levelsThe contents of MDA and GSH in liver were determined by using commercial assy kits.Hepatic 4-HNE in liver tissues were measured using western blotting.4 Determination of protein expression level and mRNA levelTotal liver protein was extracted using RIPA buffer and protein levels of Nrf-2,etc,were determined by western blotting.The mRNA levels of Nrf-2 and its downstream genes were determined by qRT-PCR.5 The effects of NAC on acute DMF-intoxicated miceSPF ICR mice(18～22 g)were randomly divided into six groups(n=10):Control group,DMF group,and NAC intervention group with different doses(62.5 mg/kg bw,125 mg/kg bw,250 mg/kg bw,and 500 mg/kg bw).Mice in each intervention group were intraperitoneally injected with different doses of NAC for 5 days and then exposed to 3 g/kg bw DMF by gavage;the mice in the control group were given the same volume of distilled water.Mice in each group were sacrified 48 h after DMF exposure and the serum was collected for the determination of the activity of ALT and AST.Liver was isolated for HE staining and the determination of MDA and GSH levels.6 The effects of SF on acute DMF-intoxicated miceSPF ICR mice(18～22 g)were randomly divided into four groups(n=10):control group,DMF group,SF group and DMF+SF group.Mice in SF group and DMF+SF group were intraperitoneally injected with SF(30 mg/kg bw)for 5 days,and then exposed to DMF(3 g/kg bw)by oral gavage,while the mice in control group were treated with distilled water of the equal volume.All mice were sacrificed 48 h after DMF exposure.The blood was collected for the determination of the activities of ALT and AST.Liver tissue was isolated for H&E staining.The contents of MDA and GSH in liver tissues were determined by commercial kits.The protein levels of Nrf-2 and its target proteins were determined by western blotting,while mRNA levels of Nrf-2 and its related genes were determined by qRT-PCR.7 The effects of GdCl3,an inhibitor of Kupffer cells,on acute DMF-intoxicated miceSPF ICR mice(18～22 g)were randomly divided into four groups with 10 in each group:Control group,DMF group,GdCl3 group and DMF+GdCl3 group.Mice in GdCl3 group and DMF+GdCl3 group were intraperitoneally injected with GdCl3(10 mg/kg bw)24 h before and after exposure,and then were gavaged with 3 mg/kg bw DMF;while mice in control group were given distilled water of the same volume.Mice in each group were sacrificed 48 h after DMF exposure and serum was collected for the determination of the activity of ALT and AST.Liver was isolated for HE staining and the determination of MDA and GSH levels.Results1 The LD50 of DMF exposed orally in miceDMF-exposed mice showed symptoms such as decreased activity,decline of body weight,etc.1 mouse died in the 4.5 g/kg bw group;2 died in the dose group of 5.49 g/kg bw;8 died in the dose group of 6.71 g/kg bw;9 died in the 8.19 g/kg bw roup;10 died in the 10.00 g/kg bw group.The LD50 was 6.0583g/k bw(95%CI:5.4319-6.7061 g/kg bw).2 Effects of DMF acute exposure on liver injury in ICR male miceCompared with the control group,the body weight of mice in the DMF-exposed mice were decreased significantly at the three time points(24 h,48 h and 72 h)(P<0.01);the liver weight and the ratio of liver weight to body weight,and the serum aminotransferase actiities were increased significantly at 48 h and 72 h time points(P<0.01).Pathological examination by HE staining showed that the necrotic and enlarged hepatocytes with a great amount of infiltrated proinflammatory cells in DMF-exposed mice liver at 48 h and 72 h time points.3 Changes of oxidative stress index in liver of DMF-exposed miceCompared with the control group,the content of MDA in the liver tissues in DMF group mice were increased significantly at 24 h and 48 h time points(P<0.05),while the content of GSH and the ratio of GSH/GSSG in the liver tissues in DMF-treated mcie showed a decreasing trend(P<0.05).The expression level of 4-HNE adducts in mouse liver was increased significantly at the 48h and 72 h groups(P<0.05).4 Effects of DMF exposure on the protein and mRNA levels of Nrf-2 in miceCompared with the control group mice,the protein levels of Nrf-2 were increased signfiantly at 24 h,48 h and 72 h time points(P<0.01),while the protein level of Keap-1 appeared to be decreased(P<0.01);the protein levels of NQO-1 were signfiantly increased at 48 h and 72 h time points compared with the control value(P<0.01),while the HO-1 protein expression were decreased(P<0.01);SOD expression level were decreased at 24 h and 48 h(P<0.05),and the expression level of y-GCS did not change significantly(P>0.05).Compared with the control group,the mRNA level of Nrf2 was not affected by DMF,while the mRNA levels of NQO-1,SOD,GCLc,and GCLm were significantly increased at 24 h time point(P<0.01);the mRNA level of NQO-1 was increased significantly at 48 h time point(P<0.01),while the mRNA level of SOD and GCLm were decreased at 48 h time point(P<0.01);the mRNA level of NQO-1,SOD,GCLc and GCLm kept unchanged at 72 h time point(P>0.05).5 Effects of NAC on acute liver injury induced by DMF in mice5.1 Effects of NAC intervention on body weight,liver weight and liver index of miceCompared with the Control group mice,the body weight was signfiantly decreased in DMF group mice(P<0.01),while the liver weight and the liver index were signfianty increased(P<0.01).Compared with the DMF model group mice,the liver weight and body weight were all not significantly altered in NAC-cotreated mice.(P>0.05).5.2 Effects of NAC on serum transaminase activity in miceCompared with the control group,the serum ALT and AST activity in the model group was significantly increased(P<0.01).Compared with the model group,there was no significant difference in serum ALT and AST activity in the intervention groups(P>0.05).5.3 Histological examinationDMF induced obvious damage to the liver,while no improvement shown in the intervention groups by the results of the H&E staining.5.4 Effects of NAC intervention on oxidative stress related indicators in miceCompared with the control group group,the MDA level in DMF was signfiantly increased(P<0.01),while the GSH level was significantly decreased(P<0.01).Compared with the DMF group,there was no changes in the levels of hepatic MDA and GSH in the intervention groups(P>0.05).6 Effects of SF on acute liver damage induced by DMF in mice6.1 Effects of SF on body weight,liver weight and liver index of miceCompared with the control group,the body weight of mice in the model group was decreased significantly(P<0.01),while the liver weight and liver index were increased significantly(P<0.01).Compared with the model group,the body weight,liver weight,and liver index in SF-cotreated mice were not significantly affected(P>0.05).6.2 Effect of SF on the serum transaminase activity in DMF-exposed miceCompared with the control group,the serum ALT and AST activity in the model group mice was significantly increased(P<0.01).There was no significant difference in serum ALT and AST activity between SF+DMF group and model group(P>0.05).6.3 Pathological examination of liver in DMF-exposed mice treated with and without SFThe results of H&E staining showed no significant improvement in liver damage in the SF intervention group when compared with the model group.6.4 Effects of SF on oxidative stress related indicators in miceCompared with the control group,MDA content in the liver of the model group was significantly increased,while the GSH level was significantly decreased(P<0.05).Compared with the model group,MDA content in the SF intervention group was significantly decreased,GSH level was significantly increased(P<0.05).6.5 Effect of SF on the expression level of Nrf-2 and related proteinsCompared with Control group mice,the protein levels of Nrf-2 and NQO-1 in DMF group mice liver were significantly increased(P<0.01),while the Nrf-2 and NQO-1 in SF group mice liver were also significantly increased(P<0.01).Compared with the DMF model group,the Nrf-2 protein level in SF/DMF group mice liver were further increased(P<0.05),while the protein levels of NQO-1 and HO-1 were slightly increased(P>0.05)7 Effects of DMF exposure on the protein levels of p65,p-p65,and iNOS in mice liverCompared with the Control group mice,the protein levels of NF-κB p65 were signfiantly increased at 24 h and 48 h time points(P<0.05),the protein levels of p-p65 and iNOS were significantly increased at 24 h,48 h,and 72 h time points(P<0.05).8 Effects of GdCl3,an inhibitor of Kupffer cells,on acute DMF-exposed mice8.1 Effects of GdCl3 on body weight,liver weight and viscera coefficient of miceCompared with the Control group mice,the body weight was signfiantly decreased in DMF group mice(P<0.01),while the liver weight and the liver index were signfianty increased(P<0.01).Compared with the DMF model group mice,no changes were observed in the body weight,liver weight,and liver index in GdCl3/DMF group mice.8.2 Effects of GdCl3 on serum transaminase activity in miceCompared with the control group,the serum ALT and AST activity of mice in DMF model group were significantly increased(P<0.01).Compared with the model group,the serum ALT and AST activity of mice in the GdCl3 intervention group were significantly decreased(P<0.05).8.3 Histopathological examinationCompared with mice in the control group,liver sections of mice in DMF group showed obvious cell swelling,necrosis,and inflammatory cell infiltration.Compared with mice in DMF model group,liver injury was greatly improved by GdCl3 treatment.Conclusion1.The LD50 of DMF for male ICR mice was about 6.0583 g/kg bw(95%CI:5.4319-6.7061 g/kg bw).2.DMF exposure could result in oxidative stress in mice liver,but antioxidants SF and NAC could not prevent DMF-induced liver damage,which suggest that ROS is not the primary mechanism for DMF-induced acute liver injury.3.GdCl3 could significantly inhibit DMF acute liver injury,indicating that Kupffer cells mediated liver inflammation may drive DMF-induced acute liver injury. |
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