The Protective Effects And Molecular Mechanisms Of Allyl Methyl Disulfide On Liver Injury Induced By Dimethylformamide

ObjectivesN,N-dimethylformamide(DMF)is a widely used organic solvent.Along with the increase in production and consumption,occupational DMF poisoning cases are frequently reported.Both acute and chronic exposure to DMF can lead to severe liver damage.Although the molecular mechanism of DMF-induced hepatotoxicity is not fully understood,inflammation and oxidative stress have been suggested to play important roles in DMF-induced hepatotoxicity.Garlic is a kind of medicinal and edible substances rich in various organic sulfides,among which allyl methyl disulfide(AMDS),allyl sulfide(DAS)and diallyl disulfide(DADS)are three active ingredients in garlic.AMDS,DAS and DADS have been shown to alleviate liver damage caused by various foreign chemicals.This study aimed to investigate the antagonistic effect of AMDS,DAS,and DADS on the hepatotoxicity induced by DMF,and to explore the molecular mechanism by focusing on inflammasome and oxidative stress,so as to explore new drug for the prevention and treatment of occupational DMF poisoning.Methods1.80 SPF-grade male ICR mice were randomly divided into 8 groups with 10 in each,i.e.control group,DMF group,and 6 organosulfur compounds intervention groups.The mice were acclimated to the feeding environment for 7 d and then were gavaged with AMDS(25 mg/kg bw),AMDS(50 mg/kg bw),DAS(50 mg/kg bw),DAS(100 mg/kg bw),DADS(25 mg/kg bw),DADS(50 mg/kg bw),or an equal volume of corn oil once daily for 5 days.On the 4th day,mice in both the DMF group and the organosulfur compounds intervention group were exposed to 3 g/kg bw of DMF orally,while the control group mice received an equal volume of normal saline.All mice were sacrificed 48 h later.The blood samples and liver tissue were collected.Serum aminotransferase assay and liver pathological HE staining were performed.Based on these results,the most effective organosulfur compound was selected for the following molecular mechanism study.The activities of serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were determined using a commercial kit.The levels of serum inflammatory factors were determined by the Elisa method,while RT-qPCR was performed to determine the mRNA levels of relevant genes.Western blotting,immunofluorescence,immunohistochemistry,and other methods were used to determine the protein levels of related proteins.2.A co-culture model of J774A.1 macrophage and AML12 hepatocyte at a ratio of 9:17 was constructed.The cells were divided into six groups:control group,DMF group(100 mM),AMDS control group(100 μM),DMF+AMDS low-dose intervention group(25 μM),DMF+AMDS medium-dose intervention group(50 μM),and DMF+AMDS high-dose intervention group(100 μM).The levels of lactate dehydrogenase(LDH)and ALT were determined using a commercial kit.ELISA was used to determine the level of tumor necrosis factor α(TNF-α)in cell culture media.The Western blotting method was used to determine the expression of NF-κB and NRF2 signaling pathway-related proteins.Results1.The three garlic-derived organosulfur compounds AMDS,DAS,and DADS could reduce the increase of liver weight and liver coefficient,improve focal necrosis and inflammatory cell infiltration of the liver,and reduce the increase of serum ALT and AST activity.AMDS could reduce the mortality rate of mice induced by DMF.2.AMDS intervention significantly inhibited DMF-induced increases in serum interleukin 1β(IL-1β),interleukin 6(IL-6)and TNF-α levels,neutrophil infiltration,macrophage aggregation and M1-type polarization,and inhibited the increase of the protein expression of key proteins p-p65,NLRP3 and IL-1β in mice induced by DMF,and supressed the increase of the expression of GSDMD,a hallmark protein of pyroptosis.3.AMDS could significantly inhibit DMF-induced reduction of glutathione(GSH)content,increase of malondialdehyde(MDA)level,increase of 4-hydroxynonenal(4-HNE)protein expression,reduction of cytochrome P4502E1(cytochrome P450 2E1,CYP2E1)protein expression and increased levels of protein expression associated with the liver NRF2-KEAP1 signaling pathway.4.In vitro experiments showed that AMDS intervention could inhibit the increase of TNF-α level and ALT and LDH activities of J774A.1/AML12 co-culture model cell culture medium induced by DMF,as well as the increase of expression levels of key protein molecules such as p-IκBα and p-p65 in cell NF-κB signaling pathway.Conclusions1.AMDS could significantly improve DMF-induced acute liver injury in mice.2.AMDS can significantly ameliorate DMF-induced liver inflammation and oxidative stress in mice,which may be related to the inhibition of macrophage M1-type polarization and NF-κB/NLRP3 signaling pathway activation.