Biomarker Discovery In Valproic Acid Induced Hepatotoxicity Based On Lipidomics And Relative Mechanism Study

Objective: Valproic acid(VPA)is a broad-spectrum antiepileptic drug that can treat multiple types of epilepsy by single or combination therapy.Hepatotoxicity is one of the serious adverse reactions of VPA.Hepatitis-like syndrome,hyperammonemia and nonalcoholic fatty liver disease(NAFLD)are considered to be the main clinical manifestations of hepatotoxicity.Therefore,it is important to determine the biomarkers(Biomarker)of VPA-induced hepatotoxicity for the relative mechanism study.Lipids play an important role in cell function,and exploring changes in lipid expression profiles associated with disease progression will provide evidence for elucidation of chemical foreign body-induced toxicity and its pathogenesis.At present,lipidomics has become one of the main methods for screening biomarkers in vivo,providing a basis for early diagnosis and treatment of diseases,thereby improving the effectiveness of treatment.This study will screen the differential lipid expression profiles of serum in children with VPA hepatotoxic epilepsy and hepatocytes.Using molecular biology techniques to explore the mechanisms by which VPA causes disorders of liver lipid metabolism.At the same time,a pharmacokinetic model of VPA population with comprehensive investigation of genetic factors and clinical factors was established in children with epilepsy,and a clinical drug regimen was formulated to guide individualized medication.Methods: 1.Identification of lipids in children with VPA hepatotoxicity based on liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF/MS)with non-targeted lipidomics method,and the identified lipid components were statistically analyzed by multivariate statistical analysis method(PLS-DA)to obtain differentially expressed lipid components between the groups.Correlation analysis and receiver operating characteristic curve(ROC)analysis were performed on differential components and liver function indicators(ALT).2.Detection of hepatotoxicity of different concentrations of VPA by MTT method and liver enzyme level.Based on the non-targeted lipidomics method of LC-Q-TOF/MS,the lipids of hepatic hepatocytes were identified,and the identified lipid components were statistically analyzed by multivariate statistical analysis(PLS-DA).Analysis revealed differential expression of lipid components between the groups.Lipidomics was used to analyze the effect of VPA on lipid components in hepatocytes under high-fat conditions.3.Oil red O staining and TAG kit to detect intracellular lipid levels.The effect of VPA on various lipid metabolism/transport related genes in hepatocytes was detected by Real-time PCR.4.Western blot was used to detect the effects of VPA at different concentrations on lipid metabolism-related protein expression;siRNA was used to interfere with the expression of CD36 and DGAT2,and then the expression of CD36 and DGAT2 and the lipid level in VPA cells were detected.5.PPARγ selective inhibitors(GW9662)were used to investigate the role of PPARγ in VPA lipid accumulation.Western blot was used to detect the expression of CD36 and DGAT2.The effects of PPARγ selective agonists(rosiglitazone)on CD36 and DGAT2 were investigated.6.AMPK selective inhibitor(Compound C)was used to investigate the effects of AMPK on PPARγ and downstream proteins,and ERK pathway inhibitor(U0126)was used to investigate the effects of ERK on AMPK,PPARγ and downstream proteins.7.Western blot was used to detect the effect of VPA at different concentrations on the expression of insulin pathway related proteins,and PI3K/Akt pathway inhibitor(LY294002)was used to investigate the effect of Akt pathway on PPARγ and downstream proteins.8.The effect of VPA on PPARγ and related proteins in hepatocytes under high-fat conditions was detected by Western blot.9.The PPK model of VPA in children with epilepsy was established by NONMEM.The effects of physiological,clinical and genetic factors on clearance rate of VPA(CL/F)were quantitatively investigated.The stability and accuracy of the model were evaluated,and the clinical medication program was formulated to guide individual medication.Results: 1.Biomarker discovery in valproic acid induced hepatotoxicity based on lipidomics.A total of 176 lipids were identified by using lipidomics under positive and negative ion modes.After screening for different lipid components,20 lipid components with significant differences were screened,including LPC,SM,Cer and TAG.Compared with children with normal liver function(NLF),serum LPCs,SMs and Cers levels in children with abnormal liver function(ALF)were significantly lower,while TAGs levels were significantly higher.Compared with NLF group,eight longchain TAGs were significantly up-regulated in ALF group(Fold change > 1.5),and positively correlated with hepatotoxicity(ALT level).Pearson correlation analysis and ROC curve showed that the screened lipid components had good predictive effect.MTT assay and liver enzyme(ALT,AST)tests suggest that VPA has hepatotoxicity.A total of 191 lipid components were identified in hepatocytes using lipidomics techniques.Long-chain TAGs increased significantly in the VPA group,and the TAGs level increased with the increase of VPA dosage.The number of carbon and double bonds of TAGs in VPA group showed a positive correlation with the increase of TAGs.The use of oleic acid to induce high-fat state hepatocytes,combined with VPA,can lead to increased accumulation of TAGs in hepatocytes.This indicates that VPA can cause accumulation of long-chain TAGs in hepatocytes.2.Mechanism of valproic acid leading to disruption in hepatic lipid metabolism.Hepatocyte oil red O staining and TAG detection confirmed that VPA can induce lipid accumulation in cells.Real-time PCR results showed that VPA can affect the expression levels of various lipid metabolism/transport related genes,which in turn leads to changes in various lipid levels in cells.Western blot analysis showed that VPA up-regulated the expression of CD36 and DGAT2 in a dose-dependent manner,and the corresponding siRNA could significantly inhibit the expression of CD36 and DGAT2 mRNA and protein,and inhibit the accumulation of TAG in hepatocytes.It is indicated that CD36 and DGAT2 play an important role in the accumulation of TAG caused by VPA.At the same time,VPA can significantly up-regulate the expression of PPARγ in hepatocytes.PPARγ inhibitor(GW9662)can inhibit the upregulation of CD36 and DGAT2 by VPA,and inhibit the lipid accumulation of VPA on hepatocytes.The PPARγ agonist(rosiglitazone)was able to up-regulate the expression of CD36 and DGAT2.This study found that VPA can significantly up-regulate the expression of pAMPK and p-ERK.AMPK inhibitor(Compound C)significantly inhibited the upregulation of p-AMPK induced by VPA,while inhibiting the protein expression levels of VPA-induced PPARγ,CD36 and DGAT2.The use of ERK inhibitor(U0126)inhibited VPA-induced up-regulation of p-AMPK,PPARγ,CD36,and DGAT2.VPA also significantly up-regulated the expression of PI3 K,p-Akt,and the expression of IRβ and IRS-1 in hepatocytes.LY294002 can inhibit the upregulation of PPARγ,CD36 and DGAT2 by VPA.In OA-induced high-fat hepatocyte,VPA up-regulated PPARγ and CD36 more significantly,while DGAT2 decreased expression in high-fat state.3.Population pharmacokinetics of valproic acid in children with epilepsy based on genetic factors.This study was the first to systematically evaluate the effects of genetic polymorphisms on VPA PK,including CYPs,UGTs,ABC transporters,nuclear receptors,and other related genes.The CL/F of LEPR 668A>G mutant homozygous(GG)and mutant heterozygous(AG)patients was found to be 22.6% and 17.2% lower than that of wild homozygous(AA)patients,respectively.The established PPK model can better describe the PK parameters of VPA,and quantitatively explain the effects of body weight,daily dose,combined CBZ and LEPR gene polymorphism on VPA CL/F.Conclusion: 1.VPA can cause the accumulation of long-chain TAG in hepatocytes,aggravating the steatosis in hepatocytes under high-fat conditions.Long chain TAG can be used as a potential biomarker for VPA hepatotoxicity.2.VPA may regulate the expression of PPARγ through the AMPK pathway and the Akt pathway,thereby promoting the expression of CD36 and DGAT2,leading to lipid accumulation in hepatocytes.3.In the children with epilepsy,a pharmacokinetic model of VPA population based on genetic factors and clinical factors was established to guide clinical individualized medication and reduce adverse reactions.