The Mechanism Of Oxidative Stress In The Pathogenesis Of VPA-induced Hepatotoxicity

Epilepsy is a commonly chronic neurological disorder.Currently,the main treatment of epilepsy is drug therapy.Valproic acid(VPA)is a broad-spectrum antiepileptic drug used for the treatment of generalized or partial seizures.Although VPA is effective and generally tolerated,it is usually associated with side effects,in which hepatotoxicity is the most severe circumstance.Approximately 61% of patients treated with VPA monotherapy had been diagnosed with NAFLD,but the disease also progresses into more advanced stages including non-alcoholic steatohepatitis(NASH),fibrosis,and eventually cirrhosis and hepatocellular carcinoma(HCC).For this reason,it is meaningful to clarify the underlying mechanism and initiate intervention treatment for VPA-induced hepatotoxicity.It is generally believed that the VPA-caused hepatotoxicity is related to its active metabolites.VPA undergoes extensive biotransformation,including glucuronidation conjugation,mitochondrial ?-oxidation,and cytochrome P450(CYP)mediated ?-oxidation.Among them,4-ene-VPA and 2,4-diene-VPA,which were catalyzed by P450 ?-oxidation have been linked to VPA-caused hepatotoxicity.They inhibit the mitochondrial fatty acid ?-oxidation and interrupt the homeostasis of mitochondrial respiratory chain,leading to the accumulation of reactive oxygen stress(ROS)and caused direct damage to cellular macromolecules such as DNA,protein,and lipids.However,the endogenous 4-ene-VPA and 2,4-diene-VPA are not adequate to cause the hepatotoxicity,suggesting that metabolites can't explain VPA-caused hepatotoxicity.In fact,more and more evidences confirm that oxidative stress plays an important role in the pathogenesis of VPA-associated hepatotoxicity.But the source of ROS and its mechanism responsible for VPA-associated hepatotoxicity remain elusive.Therefore,we established VPA-caused hepatotoxicity models in patients with epilepsy,LO2 cells,and mice.Then,we systematic investigated the underlying mechanism about VPA-induced hepatotoxicity via oxidative stress pathway.Oxidative stress occurs when the balance between oxidants and antioxidants is disrupted and results in excessive accumulation of reactive oxidative stress(ROS).Glutathione S-transferases(GSTs),catalase(CAT),superoxide dismutase(SOD),and glutathione peroxidase(GPX)plays an important role in detoxification of ROS and reactive metabolites.Individual susceptibility to VPA-caused hepatotoxicity might result from genetic deficiencies in the antioxidant enzymes.Hence,the first step of this study was to comprehensively investigate the associations of GST mu1(GSTM1),GST theta 1(GSTT1),CAT C-262 T,GPX Pro200 Leu and SOD2 Val16 Ala polymorphisms with VPA-induced liver dysfunction in patients with epilepsy.The results showed carriers of CAT C-262 T had an increased risk of developing abnormal liver function compared with noncarriers in VPA-treated patients.Meanwhile,Logistic regression analyses indicate that the CAT C-262 T genotype is a significant genetic risk factor for VPA-induced liver dysfunction.It showed that individual susceptibility to VPA-induced liver dysfunction at least,might partly result from genetic deficiencies in CAT C-262 T,suggesting that ROS is an important factor in the pathogenesis of VPA-associated hepatotoxicity.Cytochrome P450 2E1(CYP2E1)has been known as a potentially important factor of ROS overproduction.It had been implicated in the development of NAFLD.Previous studies indicated that the elevated expression of CYP2E1 increased the VPAassociated hepatotoxicity.However,the effect of CYP2E1 on VPA-caused ROS generation and liver steatosis needs to be carefully elucidated.For this reason,we next focus on the underlying mechanism of ROS in VPA-induced hepatotoxicity.We observed that VPA treatment induced hepatic steatosis and enhanced ROS levels,and ROS scavenger,N-acetyl-L-cysteine(NAC)reversed these changes both in vitro and in vivo.Then,we found the enhanced m RNA,protein expression and enzymatic activity of CYP2E1 in VPA-treated mice and LO2 cells.Importantly,VPA-induced ROS accumulation and hepatic steatosis were attenuated when CYP2E1 was inhibited using CYP2E1 inhibitor,diallyl sulfide(DAS)or in CYP2E1-knockdown cell line,suggesting that CYP2E1 plays a critical role in ROS production following hepatic steatosis.Furthermore,to clarify the molecular mechanisms of ROS induced hepatic steatosis,we measured the gene expression related to lipid uptake,lipogenesis and lipid transport.These results showed that the m RNA levels of cluster of differentiation 36(CD36),a fatty acid translocase protein and distinct diacylglycerol acyltransferase 2(DGAT2)were significantly upregulated in mice and LO2 cells after VPA treatment,while this effect was alleviated by NAC and DAS.Meanwhile,time course experiments demonstrated that the change of CYP2E1 level occurred earlier than that of ROS,CD36 and DGAT2,suggesting the correlation among “CYP2E1-ROS-lipid metabolism related proteins”.Taken together,we conclude that VPA treatment enhances the expression and activity of CYP2E1,leading to ROS formation,CD36 and DGAT2 overproduction and hepatic steatosis in mice and LO2 cells.In addition,some studies indicated that ferroptosis was associated with the production of ROS and pathogenesis of NAFLD.Then,we investigated the role of ferroptosis in VPA-induced hepatotoxicity in mice.The results revealed that ferroptosis was associated with VPA-caused hepatotoxicity.On the one hand,the expression of GPX4 was inhibited by VPA treatment,which decreased the ROS detoxification and caused the lipid peroxidation.On the other hand,VPA caused the accumulation of ferrous iron via mediated the iron responsive elements system(IREs),then resulted in iron overload and ferroptosis which exacerbate VPA-induced hepatotoxicity.In summary,our study found that VPA-caused ROS was the result of the inhibition of antioxidant enzymes,elevated of CYP2E1 expression and increase of ferrous iron.VPA induce hepatotoxicity via abnormal liver function,CYP2E1 caused accumulation of ROS increased the expression of CD36 and DGAT2 leading to the hepatic steatosis and ferroptosis.The study provides a novel insight into VPA-induced hepatotoxicity and guide rational drug use in clinics.