The Study Of Lipidomics And Mechanistic Biomarkers In Mice Models Of Apap-Induced Liver Injury And Acute Liver Failure

Background and ObjectiveDrug-induced liver injury(Drug induced liver injury,DILI)is one of the most important drug-induced diseases,as well as the main reason of failure in drug development,warning,or removed from market.The clinical phenotype of DILI is complicated,which can cause a variety of manifestations recovering almost all types of acute,subacute,and chronic liver injury.Some patients with gently DILI can represent mild-to-moderate elevated liver enzymes,while severe cases can lead to liver failure and even death.In the western countries,DILI has exceeded other causes and become the primary cause of acute liver failure.In our country,the clinical incidence of DILI also increased rapidly,which has lead to medical workers and related institutions included the CFDA attach much attention and recognition on heaptotoxicity.However,the current understanding of the pathogenesis,clinical diagnosis and treatment of DILI and severe DILI is limited.Fundamental research of DILI is also limited due to the lack of effective animal models.Acetaminophen(APAP)is the most common inherent drug-induced type DILI and it is also the common medicine which was used to build a stable DILI animal model.Our study established stable APAP-induced liver injury(AILI)and APAP-induced acute liver failure(Acetaminophen-induced liver failure,AILF)animal models by different doses of APAP.The aim of present study is to analyze of the differences in lipidomics and mechanistic biomarkers between the models of AILI and AILF,and explore the efficient biomarker to predict the outcome of different-degree APAP-induced liver injury.MethodsOur study established stable APAP-induced liver injury(AILI)and APAP-induced acute liver failure(Acetaminophen-induced liver failure,AILF)animal models by different doses of APAP.LC-MS technique was employed to test and analyze the differences and characteristics of lipidomics in plasma and liver among the control group,AILI group and AILF group.Then we use multi-dimensional statistical analysis methods such as PCA,PLS-DA and OPLS-DA to preliminary evaluate efficiency of the built model,and if the differences in lipid composition could distinguish different groups.Further,our study used T test and VIP(VIP>1),and other screening criteria to analyze difference expression in liver and plasma lipid molecules,Looking for potential lipid molecules which can forecast prognosis.Our study also used RT-PCR,western blot to test the difference of transcription and expression of genes and proteins related to the metabolism and synthesis of PC and PE,which was to explore the mechanism of different changes of PC and PE in AILI models.By EILSA,RT-PCR and Western Blot techniques,we studied the differently expression of mechanistic biomarkers between AILI and AILF group and aimed to explore the potential value of mechanistic biomarkers in predicting prognosis of disease.We analyzed the database of mRNA and miRNA in APAP-induced liver injured mice by using bioinformatics analysis technology and verified Egr 1 early significant changes in the AILI model.Our research sythentic overexpression and interference adenovirus,which could overexpress and interfere the expression of Egr-1,to analyze the role of Egr-1 in pathogenesis of AILI,then we use bioinformatics analysis to find out an downstream target gene of Egr-1-Gadd45,and we used CRISP/Cas9 technical to knockout gene Gadd45 a in hepatocellular carcinoma cell line and judged whether an Egr 1 play adaptive and protective role through regulating Gadd45 in AILI animal models.Results1.This research built stable and repeatable mice models of AILI and AILF through the abdominal injection of different doses of APAP.Compared with control group,the animal model of AILI and AILF group transaminase were significantly increased(P < 0.05),AILI group peaked plasma ALT at 6 h,while AILF group were still increasing at12 h(P < 0.05);AILF group compared with AILI group,at 12 h,AILF group had a significantly higher serum ALT levels AILI group(P < 0.05).Liver pathology results showed that the control group normal liver cell have structure and form,without hepatic sinus congestion;AILI and AILF group compared with control group,both groups represented the typical morphological changes: liver cell necrosis around the central vein;The pathology of AILF group has classic representative Massive Hepatic necrosis,which was the main characteristic of AHF.2.AILI and AILF group compared with control group,the metabolism of lipid molecules differ significantly.At 1h,in AILI group,TAG50: 1,TAG 50: 4 and TAG 50: 5 decreased significantly compared with AILF group suggested that those lipids may have potential value to predict the outcome;PE 38: 6,PE 34: 2,PE 36: 4,PE 38: 4,FFA 22: 6,TAG 48: 2,TAG 48: 3 and TAG 54: 5 changed remarkable at 3h compared with control,and the group can be clearly distinguished AILI and AILF group,therefore it may be also possible to predict the disease earlier prognosis by those above lipid molecules.3.By comparing the change in phospholipid molecules AILI and control groups,we established a series of screening methods to screen the potential PC and PE and we finally found that PC 32: 2,PC 34: 3,PC 34: 2,PE 34: 2,PE 36: 3,PE 38:4,PE 38: 6,7 phospholipid molecules significantly increased in AILI animal models and may have potential diagnostic value of AILI.Compared to the control group,besides Pla2g12 a,other phospholipase genes(PLA1(Pla1a),PLA2(Pla2g6,Pla2g7,Pla2g12 b,Pla2g15,Pnpla2,Pnpla7,Pnpla8),PLC(Plcg1,Plcxd2),PLD(Pld3,Pld4))transcriptional levels in AILI group were significantly decreased(P<0.05)compared with control group.Compared with the control group,the transcription of phospholipid biosynthesis-related gene-Pemt declined in AILI group,while transcription and expression of Chka were significantly increased,and the transcription of Chka peaked at 6h,the protein expression fof Chka peaked at 12 h.4.The research results related mechanistic biomarker showed: compared with the control group,ROS in liver of AILI and AILF group began to rise at 1h,each time point the level of ROS in liver could be seen significantly increased(P <0.05);moreover,at 1h,the production of ROS in AILF group significantly higher than AILI group.COX1 in AILI group was significantly higher than control and AILF group at 6h,while the control group and AILF group showed no significant increase.Compared with the control group,at 3h,CYTB,COX2 and ATP8 could be seen an significant decrease in AILI and AILF group.At 6h in AILI group and AILF group the transcriptional level of COX1,ND1,ND5 and ATP8 significantly reduced.At 12 h,other subunits of NADH were significantly reduced in both AILI and AILF groups.When compared AILI group and AILF group,at 6h,COX1 in AILI group was significantly higher than AILF group,while the transcription level of ATP6 in AILF decreased more remarkable than that in AILI group.When 12 h,liver AILF group’s CYTB,COX2,ATP8 and ND3,5,6 significantly decreased and the level of that was much lower than the decrease of those genes in AILI group.Plasma biomarkers related to mitochondrial damage,GLDH increased significantly early in the AILI group,GLDH in AILF group was also increase,but the extent in AILF group is significantly lower than AILI group;SDH in plasma in AILF group at each time point were significantly higher in the control group and AILI group,while SDH in AILI group had no significant increase compared with control.Biological markers associated necrosis,HMGB-1 in AILI group was significantly higher than the control group,while no significant change in AILF group.CK-18 M65 in plasma of AILI group and AILF group significantly increased compared that in control,M30 levels in both group were lower than the control group;moreover the M65 in plasma levels of AILF group were significantly higher than that in AILI group.Inflammatory cytokines TNF-a,IFN-r,IL-6 and IL-10 rose marked in both groups compared that in the control group,and inflammatory factors in AILF group increased more significantly that AILI group.Transcription and protein expression of CyclinD1 and PCNA in the liver of AILI and AILF group increased significantly compared to the control group,which was biological makers associated with the hepatocytes proliferation.5.Transcriptional level of Egr-1 in AILI animal models and plasma levels were significantly higher in 1h and peaked in 6h.Immunohistochemistry showed that Egr-1 expression in the liver was positive.Three subtypes of Gadd45 which is the potential target gene of Egr-1 had similar transcription spectrum in AILI animal models;we overexpressed the transcriptional level of Egr-1 in animal models and primary hepatocytes,after APAP treatment,the transaminase and LDH in cell supernatant reduced significantly than that in positive control;primary liver cell after interference the transcription of Egr-1 in primary hepatocytes,and after APAP treatment,LDH levels in cell supernatants were significantly higher than that in positive control;We knockout the Gadd45 a by CRISP / Cas9 technical in Hepatoma cell line,after APAP-treated,the level of LDH rose remarkable compared with normal Hepatoma cell line.ConclusionsDifference of lipid metabolism compositions can effectively distinguish among various groups,the change of lipid composition may have potential to predict the prognosis of APAP-induced liver injury with different degree.Preliminary exploration of changes of PC and PE in AILI animal models may be associated with the change of transcription and expression of phospholipase,phospholipid synthesis transcriptional gene.Mitochondrial damage makers(ROS,mtDNA,GLDH and SDH),necrosis makers(CK18-M65,HMGB-1),inflammatory makers(TNF-a,IFN-γ,IL-6 and IL-10)and hepatocytes proliferation makers(Cyclin D1,PCNA)and other biomarkers have different changes in both groups of AILI and AILF and may have potential prognostic value.Egr-1 in AILI animal models play adaptive protection by upregulating Gadd45 a.