| Objective:To explore the role and mechanism of endoplasmic reticulum(ER)stress in N,N-dimethylformamide(DMF)exposure induced liver injury in mice,an in vivo model of liver injury through 28-day oral administration of DMF in mice was constructed to investigate whether ER stress-associated apoptosis is involved in DMF-induced liver injury.This study can also provide experimental basis for the prevention and treatment of DMF-induced liver injury.Methods:1.Sixty male C57BL/6 mice(20-24 g)and sixty female mice(18-22 g)were randomly assigned to control group(n=10),DMF1d group(n=10),DMF3d group(n=10),DMF7d group(n=10),DMF14d group(n=10)and DMF28d group(n=10).In DMF exposure groups,DMF were administrated by gavage with at 150 mg/kg of bodyweight for consecutive 28 days.Five exposure groups were sacrificed on the 1St day,3rd day,7th day,14th day,28th day,respectively.Blood samples from eyes were collected after euthanasia.The serum levels of ALT and AST were measured by blood biochemical analyzer.N-methylcarbmoylated-hemoglobin(NMHb)was detected by LC-MS/MS.Pathological changes of liver tissue(the left lobe of liver)were observed by H&E staining.2.Forty-four male C57BL/6 mice(8 weeks old)were randomly assigned to four groups,with eleven mice in each group.Different concentrations of DMF were administered by gavage at 150,450 and 1350 mg/kg of body weight for 28 consecutive days.The control group was administrated 0.9%saline in a similar manner.On day 29,overnight-fasted animals were sacrificed.All mice were anesthetized using 1%pentobarbital sodium salt injection.Blood samples were collected for serum biochemical assays.To detect the metabolites of DMF,NMHb was detected by LC-MS/MS.After collection of blood,the liver was immediately excised and weighed.The left lobe of liver was fixed in 10%formalin for the histopathological examination,apoptotic analysis and immuno-fluorescence staining.The remaining part was snap frozen in liquid nitrogen and then stored at-80 ?.The tissue was used for the RT-PCR and western blot analysis.The mRNA expression of GRP94,GRP78,CHOP,Caspase-12,Bcl-2,Bax and CYP2E1 of liver tissue were measured by RT-PCR.The protein expression of IREla,p-IRE1?,GRP94,GRP78,CHOP,Caspase-12,Bcl-2,Bax,and CYP2E1 were detected by western blot.The expression level of ER-associated apoptotic protein Caspase-12 and metabolic enzyme CYP2E1 were also determined by immuno-fluorescence staining.Results:1.Repeated DMF exposure led to significant suppression of body weight gain,as well as increases in liver indexes both in male mice and female mice.DMF exposure significantly increased serum level of ALT on the 14th day and 28th day in male mice,and increased ALT on the 7th day,28th day in female mice.AST were significantly increased on each point after DMF administration both in male and female mice.Morphology evaluation show a time-dependent increase in vacuolation of hepatocytes.Repeated DMF treatment significantly increased the concentration of NMHb in a time-dependent manner both in male mice and female mice.2.The body weight gain was significantly suppressed after repeated DMF administrations for 28 consecutive days in comparison with that of the control group(P<0.05).The liver indexes of mice exposed to DMF significantly increased compared with that of the control group(P<0.05).The increase of liver index exhibited a dose-dependent manner(P<0.05).DMF exposure caused the elevation of serum ALT and AST levels.DMF significantly increased serum ALT levels at the concentrations of 450 mg/kg and 1350 mg/kg,respectively.The serum AST levels were elevated in the 1350 mg/kg group.The levels of these two transaminases also increased in a dose-dependent manner(P<0.05).Moreover,repeated DMF treatment significantly increase the concentration of NMHb in a dose-dependent manner.Histopathological examination showed hepatocellular edema and vacuolization around the portal space in the 150 mg/kg group;Widespread hepatic swelling accompanied by a few instances of eosinophilic cell were observed in 450 mg/kg group.Interestingly,the hepatic swelling was alleviated but the mount of eosinophilic cell was increased significantly in 1350 mg/kg group.TUNEL staining showed evident nuclear condensation and fragmentation were observed in the 450 mg/kg and 1350 mg/kg group.Meanwhile no apoptosis was observed in control group.RT-PCR results showed that the mRNA levels of GRP94,GRP78,CYP2E1,CHOP,Caspase-12,and Bax were up-regulated,while Bcl-2 was down-regulated after DMF exposure.The expressions of these protein were confirmed by western blots or immunofluorescence staining.Conclusion:1.The current study demonstrated that repeated DMF exposure led to liver injury,which characterized by abnormal pathological changes,apoptosis and abnormal liver function.2.Repeated DMF exposure induced prolonged ER stress then triggered the apoptosis signaling pathway,which likely play a critical role in DMF-induced liver injuries.3.Repeated DMF exposure induced the expression of CYP2E1,which resulting in formation of more reactive intermediate then leading to further liver injury. |
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