Study On The Effect And Mechanism Of Oxymatrine-Induced Hepatotoxicity:TNF-α/ROS Mediated JNK And ER Stress Pathway

Objective:Oxymatrine(OMT)is one of alkaloids compounds,extracted from traditional Chinese medicine leguminous plants such as Sophora flavescen,Radix Sophorae Tonkinensis et al.We aim to explore the toxic effect of OMT on the liver and its possible molecular mechanism in vivo and in vitro,providing theoretical basis for its clinical application.Methods1.Forty male Institue for Cancer Research(ICR)mice were randomly grouped:the control group,40,160 and 320 mg/kg OMT-treated group,OMT was oral administration once daily for seven days.Then liver index,liver function,histopathology and makers of oxidative stress were assessed.The expression of tumor necrosis factor α(TNF α)and interleukin 6 were assayed by immunohistochemical staining.TNF α,tumor necrosis factor receptor 1(TNFR1),TNF receptor associated structure domain(TRADD),stress activated protein kinase phosphorylation(p-JNK)and Caspase-8/-9/-3 protein expression levels were detected by Western blot.2.The cell vitality of normal human liver cell(L02)expored to OMT in vitro was detected by MTT method under different concentrations and different time points,cell morphology observed by optical microscope,cell apoptosis detected by Annexin V-PI double staining and Hoechst 33342 fluorescent staining respectively by flow cytometry and fluorescence microscope.The Bcl-2,Bax and Caspase-8/-9/-3 levels were detected by Western blot.Superoxide dismutase(SOD)and malonaldehyde(MDA)were assessed Reactive oxygen species(ROS)was tested by fluorescent probe DCFH-DA dyeing.3.Pre-treatment with Z-vad-FMK,NAC,SP600125,4-PBA and TM in L02 cells divided into control group,inhibitors group,OMT group,inhibitors+OMT group.then Detecting the cell vitality,observing the morphological changes,The expression levels of p-JNK,Caspase 3,endoplasmic reticulum stress(ERS)related protein immunoglobulin heavy chain binding protein(Bip),enhancer binding protein homologous protein(CHOP)and Caspase 4 protein were detected by Western blot.Results:1.Compared with the control group,body weight decreased(P<0.05)and liver coefficient were increased in OMT 160 and 320 mg/kg-treated mice(P<0.05).In the OMT 320 mg/kg group,liver function index of ALT,AST,ALP were increased(P<0.05),lipid peroxide(MDA)content increased(P<0.01).Expression of TNF alpha were increased(P<0.01)with the vast majority of liver cells disorganized and local inflammatory cell infiltration,Compared with the control group,the expression of TNFR1,TRADD and p-JNK were up-regulated in OMT 320mg/kg group mice liver(P<0.05),pro-Caspase-9 and-3 were down-regulated(P<0.05),the cleavage of pro-Caspase-8 was inhibited.these results indicated that OMT at a dose of 320 mg/kg has toxic effect on mice liver,destroying the structure of liver cells and promoting the release of TNF alpha,and then activation of JNK-Caspase pathway.2.Exposed to the concentration of 0-30 mM OMT,respectively 8,16,24 and 48h,survival rate of L02 cells were decreased in concentration and time depended manner.Four time points of half inhibitory concentration(IC50)were 20.1,17.6,13.5,3.6 mM.With increasing concentration,the number of cells reduced gradually,adhered not tightly,shriveled,fall off.Intracellular ROS levels rise,intracellular SOD enzyme activity decreased(P<0.01).MDA content increased(P<0.05).The nucleus into dense thick the bright blue dye with Hoechst 33342 staining in OMT-treated group,appearing pyknosis,broken part,cytoplasm condensed apoptosis phenomenon and apoptosis rate increased.The Bcl-2 significantly decreased(P<0.05),Bax levels was on the rise(_P<0.05),Pro-Caspase-8/-9/-3 all showed a trend of decline(P<0.05).Z-VAD-fmk weakened OMT-induced cellular damage and partly improved the survival rate.3.In addition,p-JNK expression levels rose in OMT-treated L02 cells(P<0.05).The ERS markers Bip and CHOP expression increased(P<0.05),pro-Caspase-4 enzyme cleaved,activation Caspase-4 expression increased(P<0.05).4.Pretreatment with SP600125 or NAC in L02 cells,compared with OMT group,SP600125 or NAC improved the number of adherent cells,reduced membrane bubble phenomenon and the morphology of the nucleus budding of irregular protrusions,NAC effect is more obvious,the survival rate increased(P<0.05).SP600125 or NAC reduced JNK phosphorylation and caspase 3 cleavage.The Bip,CHOP and activation of caspase-4 still has no obvious difference with SP600125.Interestingly,NAC made the CHOP protein expression increased(P<0.05),but significantly lower Caspase-4(P<0.05),suggesting inhibition of ROS generation alleviated OMT-induced caspase-4 pathway activation.In addition,OMT treatment with 4-PBA weakened inhibitory effect of OMT in L02 cells,TM vice versa.4-PBA also reduced the levels of JNK phosphorylation and cleaved-Caspase 3,and had no obvious effect the expression of Bip.Conclusion:The study illustrated that OMT had toxic effects on the liver,cause liver tissue morphology change and structural damage,promote TNF a release and elevate ROS level,cause oxidative stress,then induce ERS,activation of apoptotic proteins JNK,CHOP and Caspase-4.In a word,TNF α/ROS mediated JNK phosphorylation and ERS related pathways was involved in OMT-induced apoptosis,its hepatotoxicity could be alleviated by blocking this pathway.