Protective Effect Of Allyl Methyl Disulfide On Concanavalin A-induced Acute Liver Injury In Mice And Its Mechanism

BackgroundAutoimmune hepatitis(AIH)is a T-cell-mediated liver disease that occurs globally in all ethnic groups.The incidence and morbidity rate of AIH is increasing in worldwide.The specific etiology of AIH is not clear,but it is believed to be caused by the interaction of immune,genetic and environmental factors.Furthermore,the onset of AIH is insidious and the therapeutic effect is not ideal.The animal model of Concanavalin A(ConA)is one of the most widely used to study AIH in vivo in recent years.Allyl methyl disulfide(AMDS)is an organosulfur compound in garlic.One study found that AMDS has a protective effect on liver injury induced by acetaminophen,but whether AMDS can prevent AIH has not been reported.Inflammation is the main manifestation of AIH,and the activation of macrophage plays a central role.Nuclear factor kappa B(NF-κB)is an important transcription factor involved in inflammatory response and a key factor in the activation of M1-type macrophages,which plays an important role in promoting the classical activation of macrophages.Meanwhile,NF-κB is also the central mediator of the initiation signal of NLRP3 inflammasome activation.Aims1.The objective was to select the most sensitive mouse strain to hepatic injury induced by ConA.2.The acute toxicity of AMDS was evaluated,and the dose of AMDS with no obvious toxic effect on mice was determined according to the oral LD50 of AMDS.3.To study the protective effect of AMDS on hepatic injury induced by ConA in vivo and in vitro,and the molecular mechanism was explored centered on the regulation of macrophage polarization and activation of NLRP3 inflammasome.Methods1.ICR mice,Balb/c mice and C57BL/6J mice were divided into male control group,male ConA-treated group,female control group and female ConA-treated group,respectively.Mice in the ConA-treated group were injected with 12 mg/kg ConA through tail vein,while mice in the control group were injected with the same volume of normal saline.The mice were sacrificed 8 hours later.To evaluate the sensitivity and stability of different strains and genders to ConA-induced liver injury,we calculated the liver coefficient,detect the expression levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),lactate dehydrogenase(LDH)in serum and examined liver pathology by H&E staining in liver sections.2.Screening the safe dose of repeated administration of AMDS:using the C57BL/6J mice screened in the first part that are sensitive to ConA.The acute toxicity evaluation of AMDS was conducted according to Horn’s method.C57BL/6J mice were randomly divided into 4 groups:AMDS 100 mg/kg group,215 mg/kg group,464 mg/kg group and 1000 mg/kg group.Changes in normal activity and body weight were monitored daily for 14 days,and the time for signs of toxicity or death were recorded.According to the number of dead animals in each dose group,the transoral LD50 of AMDS was obtained by consulting the table.In the safety evaluation of repeated administration of AMDS,dose design was conducted according to 1/10 LD50,1/5 LD50 and 2/5 LD50.C57BL/6J male mice were randomly divided into 4 groups according to the AMDS transoral LD50:control group,AMDS low-dose group(25 mg/kg),AMDS medium-dose group(50 mg/kg),and AMDS high-dose group(100 mg/kg).The mice were given intragastric administration for 7 consecutive days,after that,the mice were killed 24 hours after the last administration.Then determining the safe dose of repeated administration of AMDS by blood routine,blood biochemistry(liver function,kidney function,blood glucose,blood lipid,etc.)and organ coefficients of major organs(liver,spleen,kidney,testis).3.Protective effect and mechanism of AMDS pretreatment on ConA-induced acute liver injury in mice:C57BL/6J male mice were randomly divided into 5 groups:control group,AMDS(50 mg/kg)group,ConA group,ConA+AMDS 25 mg/kg group and ConA+AMDS 50 mg/kg group,with 8 mice in each group.Four groups were given AMDS intragastric administration for 7 consecutive days,while the control group was administration with the same volume of corn oil.On the last day,AMDS was administered for 2 hours and ConA was injected into caudal vein.The control group was injected with the same volume of normal saline.After 8 hours,the blood samples were collected and centrifuged to obtain serum.The protective effect of AMDS on ConA-induced liver injury in mice was evaluated by measuring the activities of ALT,AST,LDH in serum and H&E staining in liver sections.Detecting cell proliferation markers,neutrophil markers and macrophage polarization markers by immunohistochemistry.We detected the polarization of macrophages,the expression levels of NLRP3 inflammasome pathway,NF-κB signaling pathway and apoptosis pathway in mouse liver by immunofluorescence,qPCR and western blot to explore the possible mechanism of AMDS preventing ConA-induced liver injury in mice.4.It was further verified by in vitro experiment:ConA was exposed to mouse mononuclear macrophage J774A.1 and normal liver cells AML 12 to establish AIH model.The cytotoxicity of ConA and AMDS to the two kinds of cells was detected by CCK-8 detection,in order to screen out the safe dose.Corresponding to the animal model,the cells were divided into 6 groups:control group,AMDS(50 μM)group,ConA group,ConA+AMDS 12.5 μM group,ConA+AMDS 25 μM group and ConA+AMDS 50μM group.ELISA was used to detect interleukin-6(IL-6),tumor necrosis factor α(TNF-α),interleukin-1 beta(IL-1β)and LDH levels in cell culture-medium.The NLRP3 pathway-related protein expression levels were detected by western blot.Finally,in order to clarify the role of macrophages in AMDS on ConA-induced hepatocyte injury,J774A.1 cells were given AMDS in advance and then treated with ConA for 24 hours.Cell supernatant was collected as conditioned medium and AML 12 cells were treated for 24 hours.The mRNA levels of inflammatory factors TNF-α and inducible nitric oxide synthase(iNOS)were detected by qPCR.Results1.AIH animal model was established successfully in ICR,Balb/c and C57BL/6J strain mice,among which C57BL/6J male mice had the most serious liver injury,and the variability among the mice was the least,which was the most sensitive strain to liver injury induced by ConA.2.According to Horn’s method,the transoral LD50 of AMDS was 271 mg/kg for males,and female LD50 is 316 mg/kg.AMDS 25,50 and 100 mg/kg had no obvious toxic effect on mice after 7 days of administration.3.AMDS pretreatment significantly antagonized the increase of ConA-induced liver injury marker enzymes in mice,hepatocyte necrosis and neutrophil infiltration.AMDS intervention significantly reduced the release of inflammatory factors TNF-α,IL-6 and IL-1β induced by ConA exposure,inhibited the polarization of Ml-type macrophages and the activation of NLRP3 and NF-κB pathways.4.AMDS intervention can significantly improve the morphology of J774A.1 macrophages induced by ConA,the release of inflammatory cytokines TNF-α and IL-6,and inhibit the activation of NLRP3 pathway in macrophages.AMDS intervention and ConA exposure had no significant effect on AML 12 hepatocytes.The supernatant obtained from J774A.1 pretreated by AMDS and then exposed to ConA was used as conditioned medium for AML 12.It was found that AMDS could significantly inhibit the mRNA levels of Inos and Tnfa in AML12 cells.ConclusionsAMDS displays potent protective effects against acute liver injury in mice induced by ConA,possibly through alleviating hepatic neutrophil infiltration,inhibiting M1-type macrophage polarization and NLRP3 inflammasome activation.